METHODS AND COMPOSITIONS FOR INCREASING THE SUPPRESSIVE FUNCTION OF REGULATORY T-CELLS (TREGS)

Abstract

Methods and compositions for increasing the suppressive function of regulatory T-cells (Tregs) are provided.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a Continuation of U.S. application Ser. No. 15/757,516, filed Mar. 5, 2018, which is a National Stage application under 35 U.S.C. .sctn. 371 of International Application No. PCT/US2016/050215, having an International Filing Date of Sep. 2, 2016, which claims the benefit of priority under 35 U.S.C. .sctn. 119(e) to U.S. Provisional Application Ser. No. 62/214,680, filed Sep. 4, 2015, the disclosures of which are incorporated herein by reference in their entirety.

TECHNICAL FIELD

[0003] This disclosure generally relates to T-cells and methods of engineering T-cells to reduce or eliminate graft-vs.-host-disease.

BACKGROUND

[0004] Regulatory T-cells (Tregs) play a critical role in preventing and treating autoimmune and alloimmune reactions, including graft-versus-host disease (GVHD) and solid organ transplant rejection, allergies and responses to foreign antigens (e.g., microbes, protein replacement therapy for deficiency disorders). Two recent clinical trials demonstrated that, in patients undergoing hematopoietic stem cell transplantation, adoptive transfer of Tregs significantly reduced the incidence of grades II-IV GVHD. While Tregs significantly reduced GVHD severity, they did not eliminate GVHD. One potential way to augment Treg-mediated inhibition of GVHD is to increase Treg suppressive potency. It was previously shown that Treg-specific inhibition of protein kinase C-theta (PKC-.theta.) enhances Treg function. However, it is unclear whether PKC-.theta. inhibition can boost Treg function in a systemic inflammatory condition like GVHD. Furthermore, the mechanism by which PKC-.theta. inhibition augments Treg function is unknown.

SUMMARY

[0005] In one aspect, a method of reducing or eliminating the vimentin protein and/or the RLTPR protein and/or the PKC-.theta. protein in Treg cells in provided. Such a method typically includes contacting the Treg cells with a vimentin-specific and/or a RLTPR-specific and/or a PKC-.theta.-specific inhibitory nucleic acid molecule.

[0006] In some embodiments, the vimentin-specific inhibitory nucleic acid molecule is complementary to at least a portion of the sequence shown in SEQ ID NO: 1. In some embodiments, the RLTPR-specific inhibitor nucleic acid is complementary to at least a portion of the sequence shown in SEQ ID NO: 5. In some embodiments, the PKC-.theta.-specific inhibitory nucleic acid molecule is complementary to at least a portion of the sequence shown in SEQ ID NO: 9. Representative vimentin-specific inhibitory nucleic acid molecules have the sequence shown in SEQ ID NO: 13, 14, 15 or 16.

[0007] In some embodiments, the vimentin-specific and/or the RLTPR-specific and/or the PKC-.theta.-specific inhibitory nucleic acid molecule is a RNAi nucleic acid molecule. In some embodiments, the vimentin-specific and/or the RLTPR-specific and/or the PKC-.theta.-specific inhibitory nucleic acid molecule is an antisense nucleic acid molecule. In some embodiments, the vimentin-specific and/or the RLTPR-specific and/or the PKC-.theta.-specific inhibitor nucleic acid molecule is a siRNA nucleic acid molecule.

[0008] In some embodiments, the Treg cells are contacted in vitro. In some embodiments, the Treg cells are contacted in situ. In some embodiments, the Treg cells are contacted in vivo in an individual who has received or is receiving a bone marrow transplant.

[0009] In some embodiments, the Treg cells exhibit a phenotype of at least one of the following: reduced PKC-.theta. auto-phosphorylation at Ser676; improved ability to suppress CD4+ and CD8+ Tcon proliferation; increased surface expression of Nrp1; increased surface expression of Lag3; increased basal and maximal oxygen consumption rate (OCR); increased BoDipy.sub.C1-C12 uptake; increased expression of CD71; increased expression of CD98; increased expression of CPT1a; or reduced activity of mTORC2, compared to Tregs that lack the vimentin-specific and/or the RLTPR-specific and/or the PKC-.theta.-specific inhibitory nucleic acid molecule, respectively.

[0010] In another aspect, a method of increasing or augmenting the suppressor cell potency of Treg cells is provided. Typically, such a method includes reducing or eliminating vimentin and/or RLTPR and/or PKC-.theta. in the Treg cells.

[0011] In some embodiments, reducing or eliminating the vimentin and/or the RLTPR and/or the PKC-.theta. in the Treg cells comprising contacting the Treg cells with a moiety selected from the group consisting of a nucleic acid, a nuclease, an antibody, a ligand, a peptide, a drug, a chemical, or a small molecule. Representative nucleic acids include, without limitation, a vimentin-specific and/or a RLTPR-specific and/or a PKC-.theta.-specific inhibitory nucleic acid molecule. In some embodiments, the vimentin-specific and/or the RLTPR-specific and/or the PCK-.theta.-specific inhibitory nucleic acid molecule is selected from the group consisting of a RNAi nucleic acid molecule, an antisense nucleic acid molecule, and a siRNA nucleic acid molecule.

[0012] In some embodiments, the vimentin-specific inhibitory nucleic acid molecule is complementary to at least a portion of the sequence shown in SEQ ID NO: 1. In some embodiments, the RLTPR-specific inhibitory nucleic acid molecule is complementary to at least a portion of the sequence shown in SEQ ID NO: 5. In some embodiments, the PKC-.theta.-specific inhibitory nucleic acid molecule is complementary to at least a portion of the sequence shown in SEQ ID NO: 9.

[0013] In some embodiments, the method is performed in vitro. In some embodiments, the method is performed in situ. In some embodiments, the method is performed on an individual who has received or is receiving a bone marrow transplant.

[0014] In some embodiments, the Treg cells in which the vimentin, and/or RLTPR and/or PCK-.theta. has been reduced or eliminated exhibit a phenotype of at least one of the following: reduced PKC-.theta. auto-phosphorylation at Ser676; improved ability to suppress CD4+ and CD8+ Tcon proliferation; increased surface expression of Nrp1; increased surface expression of Lag3; increased basal and maximal oxygen consumption rate (OCR); increased BoDipy.sub.C1-C12 uptake; increased expression of CD71; increased expression of CD98; increased expression of CPT1a; or reduced activity of mTORC2, compared to Tregs in which vimentin, RLTPR and/or PCK-.theta., respectively, is not reduced or eliminated.

[0015] In still another aspect, a method of disrupting the structural integrity or the metabolic activity of Treg cells is provided. Typically, such a method includes reducing or eliminating vimentin and/or RLTPR and/or PKC-.theta. in the Treg cells.

[0016] In yet another aspect, a method of screening for compounds that increasing or augmenting the suppressor cell potency of Treg cells is provided. Typically, such a method includes contacting Treg cells with a test compound and determining whether or not the structural integrity or metabolic activity of the cell is disrupted. Disruption of the structural integrity or metabolic activity of the cell can be determined, for example, by detecting a reduction or elimination of vimentin and/or RLTPR and/or PKC-.theta. in the Treg cells. A test compound that disrupts the structural integrity or metabolic activity of the cell is indicative of a compound that increases or augments the suppressor cell potency of Treg cells.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and compositions of matter belong. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods and compositions of matter, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

DESCRIPTION OF DRAWINGS

[0018] FIG. 1 shows that PKC-.theta. inhibition and vimentin siRNA treatment increase Treg function.

[0019] Panel A shows acute GVHD. Lethally irradiated BALB/c mice were given 10e6 C57BL/6 bone marrow only (BM), or BM with 2e6 CD4+25-/CD8+25-Tcon cells without (BM+T) or with 1e6 Tregs treated with either DMSO (DMSO) or 10 .mu.M AEB071 (AEB071) for 30 minutes. Compared to DMSO, Tregs treated with AEB071 significantly increased recipient survival (p=0.0036).

[0020] Panel B shows Treg activation. Tregs were treated with DMSO or AEB071 as above, and activated overnight with plate bound anti-CD3/28 and IL-2. AEB071 treatment significantly increased the surface expression of Neuropilin-1.

[0021] Panel C shows Treg activation. Tregs were treated with DMSO or AEB071 as above, and activated overnight with plate bound anti-CD3/28 and IL-2. AEB071 treatment significantly increased the surface expression of Lymphocyte activation gene 3 (Lag3).

[0022] Panel D shows Treg metabolic activity. Tregs were treated with DMSO or AEB071 and activated as above. Oxygen consumption rate (OCR) analysis revealed that AEB071 treatment significantly increased baseline Treg OCR (time 0-19 min) and maximal OCR (time=53-70 min).

[0023] Panel E shows Treg suppression. Tregs were transfected with vimentin siRNA (vim) or control (GFP), and activated for 36 hours. CFSE-labeled Tcon were mixed with T-cell depleted splenocytes and soluble anti-CD3 mAb. Treg:Tcon ratios of 1:1-1:27 were plated and CFSE dilution assessed after 3 days.

[0024] FIG. 2A shows that vimentin is highly enriched in Tregs compared to conventional CD4+ T-cells (CD4 Tcon).

[0025] FIG. 2B shows that, compared to the transfection control (top panel), transfection with GFP-siRNA using the transfection protocol described herein yielded 50-60% or greater transfection of Tregs.

[0026] FIG. 2C shows that, compared to the control GFP-siRNA (GFP), when utilizing vimentin siRNA (vim siRNA) and the transfection protocol described herein, vimentin levels were knocked down by 15-30%.

[0027] FIG. 3A shows that, in standard in vitro suppression assays, vimentin siRNA-transfected Tregs (Vim) were able to suppress proliferation of CD4 conventional T-cells significantly better than control GFP-siRNA transfected Tregs (GFP). Treg:Tcon ratios of 1:1-1:27 are represented with 1:1, 1:3 etc. denotations in the x-axis labels.

[0028] FIG. 3B shows that, in standard in vitro suppression assays, vimentin siRNA-transfected Tregs (Vim) were able to suppress proliferation of CD8 conventional T-cells significantly better than control GFP-siRNA transfected Tregs (GFP). Treg:Tcon ratios of 1:1-1:27 are represented with 1:1, 1:3 etc. denotations in the x-axis labels.

[0029] FIG. 4A are histograms showing a symmetrical distribution of the labeling of the samples.

[0030] FIG. 4B is a scatter plot showing the consistency between labeled samples.

[0031] FIG. 5, Panels A-D, show that RLTPR siRNA significantly decreased the amount of RLTPR protein in Treg cells.

[0032] FIG. 6A is a graph showing that RLTPR siRNA increased suppression of CD4+ T-cells in vitro.

[0033] FIG. 6B is a graph showing that RLTPR siRNA increased suppression of CD8+ T-cells in vitro.

[0034] FIG. 7A is a graph showing that RLTPR siRNA resulted in a significant increase in the expression of Foxp3 in Treg cells in vitro (p<0.05).

[0035] FIG. 7B is a graph showing that RLTPR siRNA had no significant effect on the expression of CD25 in Treg cells in vitro.

[0036] FIG. 7C is a graph showing that RLTPR siRNA significantly increased the expression of neuropilin-1 (Nrp1) in Treg cells in vitro (p<0.0001).

[0037] FIG. 8A are photographs of the results of experiments in which Tregs were pre-treated with DMSO (control), AEB071, or vimentin siRNA, then activated. PKC-.theta. and vimentin staining were analyzed by confocal microscopy. Data show one experiment representative of 4 independent experiments.

[0038] FIG. 8B is a representative histogram of PKC-.theta. phosphorylated at Ser676 after vimentin siRNA treatment, and a graph showing median fluorescent intensity (MFI) quantification. Data show one experiment representative of 2 independent experiments. n=4 replicates/group. Bars show mean.+-.SEM. *, p<0.05; **, P<0.01; ***, P<0.0001; ****, p<0.00001 (Student's t-tests or one-way ANOVA with Tukey's post-test).

[0039] FIG. 8C is a graph showing percent in vitro suppression of CD4+ Tcon proliferation by control and vimentin siRNA-treated Tregs in a standard in vitro Treg suppression assay. 1:1-1:9 indicates Treg:Tcon ratio. Data show one experiment representative of 3 independent experiments. n=4 replicates/group. Bars show mean.+-.SEM. *, p<0.05; **, P<0.01; ***, P<0.0001; ****, p<0.00001 (Student's t-tests or one-way ANOVA with Tukey's post-test).

[0040] FIG. 8D shows a representative histogram of Neuropilin-1 expression, and graphs showing MFI quantifications of Nrp1, Foxp3 and CD25. Data show one experiment representative of 3 independent experiments. n=4 replicates/group. Bars show mean.+-.SEM. *, p<0.05; **, P<0.01; ***, P<0.0001; ****, p<0.00001 (Student's t-tests or one-way ANOVA with Tukey's post-test).

[0041] FIG. 9A is a graph showing the percent survival of recipients. Tregs were transfected with either control (non-targeting) or vimentin siRNA. Recipient mice were given BM alone, BM+ Tcon (BM+T), or BM+ Tcon+ Tregs; Tregs pre-treated with control or vimentin siRNA. Data show one experiment representative of 3 independent experiments. n=5 mice/group/experiment. Bars show mean.+-.SEM. Survival differences analyzed by log-rank test. *, p<0.05; **, P<0.01; ***, P<0.0001; ****, p<0.00001 (Student's t-tests or one-way ANOVA with Tukey's post-test).

[0042] FIG. 9B is a graph showing the clinical GVHD scores (0=no disease, 10=most severe disease) for recipients. Tregs were transfected with either control (non-targeting) or vimentin siRNA. Recipient mice were given BM alone, BM+ Tcon (BM+T), or BM+ Tcon+ Tregs; Tregs pre-treated with control or vimentin siRNA. Data show one experiment representative of 3 independent experiments. n=5 mice/group/experiment. Bars show mean.+-.SEM. Survival differences analyzed by log-rank test. *, p<0.05; **, P<0.01; ***, P<0.0001; ****, p<0.00001 (Student's t-tests or one-way ANOVA with Tukey's post-test).

[0043] FIG. 9C is a graph showing basal and maximal oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measured after transfection. Tregs were transfected with either control (non-targeting) or vimentin siRNA. Data show one experiment representative of 3 independent experiments. n=5 replicates/group. Bars show mean.+-.SEM. Survival differences analyzed by log-rank test. *, p<0.05; **, P<0.01; ***, P<0.0001; ****, p<0.00001 (Student's t-tests or one-way ANOVA with Tukey's post-test).

[0044] FIG. 9D is a representative histogram showing BoDipyC1-C12 uptake and a graph showing median fluorescent intensity (MFI) quantification of splenic Tregs from recipients on D4 after transplant. Tregs were transfected with either control (non-targeting) or vimentin siRNA, and recipients were given BM+ Tcon+ Tregs. Data show one experiment representative of 3 independent experiments. n=4 replicates/group. Bars show mean.+-.SEM. Survival differences analyzed by log-rank test. *, p<0.05; **, P<0.01; ***, P<0.0001; ****, p<0.00001 (Student's t-tests or one-way ANOVA with Tukey's post-test).

[0045] FIG. 9E is a graph showing quantification of Glut1 and CPT1a MFI from flow cytometry analysis of splenic Tregs from recipients on D4 after transplant. Tregs were transfected with either control (non-targeting) or vimentin siRNA, and recipients were given BM+ Tcon+ Tregs. Data show one experiment representative of 3 independent experiments. n=4 replicates/group. Bars show mean.+-.SEM. Survival differences analyzed by log-rank test. *, p<0.05; **, P<0.01; ***, P<0.0001; ****, p<0.00001 (Student's t-tests or one-way ANOVA with Tukey's post-test).

[0046] FIG. 9F are representative histograms showing Akt phosphorylation at Ser473 and Foxo3a phosphorylation (at Ser253) after control and vimentin siRNA transfection, and graphs showing corresponding MFI quantifications. Tregs were transfected with either control (non-targeting) or vimentin siRNA. Data show one experiment representative of 2 independent experiments. n=4 replicates/group. Bars show mean.+-.SEM. Survival differences analyzed by log-rank test. *, p<0.05; **, P<0.01; ***, P<0.0001; ****, p<0.00001 (Student's t-tests or one-way ANOVA with Tukey's post-test).

[0047] FIG. 10A is a graph showing the quantification of vimentin median fluorescent intensity (MFI) from flow cytometry analysis of purified Tregs and CD4+ Tcon. Data show one experiment representative of 4 independent experiments. n=4 replicates/group. Bars show mean.+-.SEM. *, p<0.05; **, P<0.01; ***, P<0.0001; ****, p<0.00001 (Student's t-tests).

[0048] FIG. 10B is a graph showing the percent in vitro suppression of CD8+ Tcon proliferation in a standard in vitro Treg suppression assay. Data show one experiment representative of 3 independent experiments. n=4 replicates/group. Bars show mean.+-.SEM. *, p<0.05; **, P<0.01; ***, P<0.0001; ****, p<0.00001 (Student's t-tests).

[0049] FIG. 10C is a graph showing MFI quantification of Lag3 expression in purified Tregs from flow cytometry analysis. Data show one experiment representative of 3 independent experiments. n=4 replicates/group. Bars show mean.+-.SEM. *, p<0.05; **, P<0.01; ***, P<0.0001; ****, p<0.00001 (Student's t-tests).

[0050] FIG. 10D is a graph showing recipient weights from mice given only BM, BM+ Tcon (BM+T), or BM+ Tcon+ Tregs. Data show one experiment representative of 3 independent experiments. n=5 mice/group/experiment. Bars show mean.+-.SEM. *, p<0.05; **, P<0.01; ***, P<0.0001; ****, p<0.00001 (Student's t-tests).

[0051] FIG. 10E are graphs showing quantifications of alpha4beta7 and CCR9 MFI, and graphs showing percent of alpha4beta7 and CCR9 positive Tregs after transfection. Data show one experiment representative of 2 independent experiments. n=4 replicates/group. Bars show mean.+-.SEM. *, p<0.05; **, P<0.01; ***, P<0.0001; ****, p<0.00001 (Student's t-tests).

[0052] FIG. 10F are graphs showing the quantification of CD71 and CD98 MFI from splenic Tregs from recipient mice transplanted with BM+ Tcon+ Tregs on D4 after transplant. Data show one experiment representative of 2 independent experiments. n=4 replicates/group. Bars show mean.+-.SEM. *, p<0.05; **, P<0.01; ***, P<0.0001; ****, p<0.00001 (Student's t-tests).

[0053] FIG. 10G are graphs showing quantifications of S6 and 4E-BP1 phosphorylation after transfection. Data show one experiment representative of 2 independent experiments. n=4 replicates/group. Bars show mean.+-.SEM. *, p<0.05; **, P<0.01; ***, P<0.0001; ****, p<0.00001 (Student's t-tests).

DETAILED DESCRIPTION

[0054] The present disclosure provides evidence that disrupting cytoskeletal structure in Tregs results in a release of structural components from physical constraints that is then able to increase suppressor cell potency. This effect can be direct (e.g., the molecule or compound released from the structural components within the Treg cells can exert this effect) or indirect (e.g., a molecule or compound that, upon binding to a binding partner such as, without limitation, adapter molecules, signaling molecules, enzymes, or molecules involved in degradation, recycling, mobility, metabolism, and/or differentiation, results in a release of one or more physical constraints in the Treg cells) or a combination thereof. Simply by way of example, and without being bound by any particular mechanism, such physical constraints within the Treg cells may occur at a cellular level (e.g., on the membrane of the cell, involving one or more cell-to-cell communication mechanisms) or at an intracellular level (e.g., on one or more organelles). This is the first evidence that there are structurally-based processes that occur within Treg cells that regulate their suppressor potency.

[0055] This phenomenon (e.g., the occurrence of structurally-based processes within Treg cells that regulate their suppressor potency) can be used in methods of increasing or augmenting the function (e.g., suppressor potency) of Treg cells. As described herein, increasing or augmenting the function of Treg cells can be accomplished by reducing or eliminating any of the vimentin protein, the RLTPR protein, or the PKC-.theta. protein, or a combination thereof.

[0056] Vimentin is a type III intermediate filament protein, and is the major cytoskeleton protein in mesenchymal cells. Vimentin plays a significant role in maintaining the position of organelles within the three-dimensional cell, but, at the same time, vimentin is a dynamic protein that allows for some of the structural flexibility exhibited by cells. The human vimentin nucleic acid sequence is shown in SEQ ID NO: 1, and the encoded protein is shown in SEQ ID NO: 2. The mouse vimentin nucleic acid sequence is shown in SEQ ID NO: 3, and the encoded protein is shown in SEQ ID NO: 4.

[0057] RLTPR is also known as CARMIL2 and is an adaptor protein that links PKC-.theta. with CD28. RLTPR knock-out mice exhibit a phenotype similar to CD28 knock-out mice, underscoring the importance of RLTPR in CD28/PKC-.theta. signaling (see, Liang, 2013, Nat. Immunol., 14(8):858-66). In addition, RLTPR also links protein kinase C-theta (PKC-.theta.) with the intermediate filament, vimentin (see, Liang, 2009, Mol. Biol. Cell., 20(24):5290-305). The human RLTPR nucleic acid sequence is shown in SEQ ID NO: 5, and the encoded protein is shown in SEQ ID NO: 6. The mouse RLTPR nucleic acid sequence is shown in SEQ ID NO: 7, and the encoded protein is shown in SEQ ID NO: 8.

[0058] Protein kinase C-theta (PKC-.theta.; also known as PRKCQ) is a member of the PKC family of serine- and threonine-specific protein kinases. PKC-.theta. is a calcium-independent and phospholipid-dependent protein kinase. The human PKC-.theta. nucleic acid sequence is shown in SEQ ID NO: 9, and the encoded protein is shown in SEQ ID NO: 10. The mouse PKC-.theta. nucleic acid sequence is shown in SEQ ID NO: 11, and the encoded protein is shown in SEQ ID NO: 12.

[0059] A nucleic acid encoding vimentin from human is shown in SEQ ID NO: 1, and a nucleic acid encoding vimentin from mouse is shown in SEQ ID NO: 3. In addition, a nucleic acid encoding RLTPR from human is shown in SEQ ID NO: 5, and a nucleic acid encoding RLTPR from mouse is shown in SEQ ID NO: 7. Further, a nucleic acid encoding PKC-.theta. from human is shown in SEQ ID NO:9, and a nucleic acid encoding PKC-.theta. from mouse is shown in SEQ ID NO:11. Unless otherwise specified, nucleic acids referred to herein can refer to DNA and RNA, and also can refer to nucleic acids that contain one or more nucleotide analogs or backbone modifications. Nucleic acids can be single stranded or double stranded, and linear or circular, both of which usually depend upon the intended use.

[0060] As used herein, an "isolated" nucleic acid molecule is a nucleic acid molecule that is free of sequences that naturally flank one or both ends of the nucleic acid in the genome of the organism from which the isolated nucleic acid molecule is derived (e.g., a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease digestion). Such an isolated nucleic acid molecule is generally introduced into a vector (e.g., a cloning vector, or an expression vector) for convenience of manipulation or to generate a fusion nucleic acid molecule, discussed in more detail below. In addition, an isolated nucleic acid molecule can include an engineered nucleic acid molecule such as a recombinant or a synthetic nucleic acid molecule.

[0061] The sequence of the vimentin polypeptide from human is shown in SEQ ID NO: 2, and the sequence of the vimentin polypeptide from mouse is shown in SEQ ID NO: 4. In addition, the sequence of the RLTPR polypeptide from human is shown in SEQ ID NO: 6, and the sequence of the RLTPR polypeptide from mouse is shown in SEQ ID NO: 8. Further, the sequence of the PKC-.theta. polypeptide from human is shown in SEQ ID NO:10, and the sequence of the PKC-.theta. polypeptide from mouse is shown in SEQ ID NO:12. As used herein, a "purified" polypeptide is a polypeptide that has been separated or purified from cellular components that naturally accompany it. Typically, the polypeptide is considered "purified" when it is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, or 99%) by dry weight, free from the polypeptides and naturally occurring molecules with which it is naturally associated. Since a polypeptide that is chemically synthesized is, by nature, separated from the components that naturally accompany it, a synthetic polypeptide is "purified."

[0062] Nucleic acids can be isolated using techniques well known in the art. For example, nucleic acids can be isolated using any method including, without limitation, recombinant nucleic acid technology, and/or the polymerase chain reaction (PCR). General PCR techniques are described, for example in PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleic acid techniques include, for example, restriction enzyme digestion and ligation, which can be used to isolate a nucleic acid. Isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule or as a series of oligonucleotides.

[0063] Polypeptides can be purified from natural sources (e.g., a biological sample) by known methods such as DEAE ion exchange, gel filtration, and hydroxyapatite chromatography. A polypeptide also can be purified, for example, by expressing a nucleic acid in an expression vector. In addition, a purified polypeptide can be obtained by chemical synthesis. The extent of purity of a polypeptide can be measured using any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0064] Nucleic acids can be detected using any number of amplification techniques (see, e.g., PCR Primer: A Laboratory Manual, 1995, Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; and U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; and 4,965,188) with an appropriate pair of oligonucleotides (e.g., primers). A number of modifications to the original PCR have been developed and can be used to detect a nucleic acid. Nucleic acids also can be detected using hybridization.

[0065] Polypeptides can be detected using antibodies. Techniques for detecting polypeptides using antibodies include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. An antibody can be polyclonal or monoclonal. An antibody having specific binding affinity for a polypeptide can be generated using methods well known in the art. The antibody can be attached to a solid support such as a microtiter plate using methods known in the art. In the presence of a polypeptide, an antibody-polypeptide complex is formed.

[0066] Detection (e.g., of an amplification product, a hybridization complex, or a polypeptide) is oftentimes accomplished using detectable labels. The term "label" is intended to encompass the use of direct labels as well as indirect labels. Detectable labels include enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.

[0067] A construct, sometimes referred to as a vector, containing a nucleic acid (e.g., a coding sequence or a RNAi nucleic acid molecule) is provided. Constructs, including expression constructs (or expression vectors), are commercially available or can be produced by recombinant DNA techniques routine in the art. A construct containing a nucleic acid can have expression elements operably linked to such a nucleic acid, and further can include sequences such as those encoding a selectable marker (e.g., an antibiotic resistance gene). A construct can encode a chimeric or fusion polypeptide (i.e., a first polypeptide operatively linked to a second polypeptide). Representative first (or second) polypeptides are those that can be used in purification of the other (i.e., second (or first), respectively) polypeptide including, without limitation, 6.times.His tag or glutathione S-transferase (GST).

[0068] Expression elements include nucleic acid sequences that direct and regulate expression of nucleic acid coding sequences. One example of an expression element is a promoter sequence. Expression elements also can include introns, enhancer sequences, response elements, or inducible elements that modulate expression of a nucleic acid. Expression elements can be of bacterial, yeast, insect, mammalian, or viral origin, and vectors can contain a combination of elements from different origins. As used herein, operably linked means that a promoter or other expression element(s) are positioned in a vector relative to a nucleic acid in such a way as to direct or regulate expression of the nucleic acid (e.g., in-frame).

[0069] Constructs as described herein can be introduced into a host cell. Many methods for introducing nucleic acids into host cells, both in vivo and in vitro, are well known to those skilled in the art and include, without limitation, electroporation, calcium phosphate precipitation, polyethylene glycol (PEG) transformation, heat shock, lipofection, microinjection, and viral-mediated nucleic acid transfer. As used herein, "host cell" refers to the particular cell into which the nucleic acid is introduced and also includes the progeny or potential progeny of such a cell. A host cell can be any prokaryotic or eukaryotic cell. For example, nucleic acids can be introduced into bacterial cells such as E. coli, or into insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.

[0070] A number of methods are known in the art that can be used to reduce or eliminate vimentin and/or RLTPR and/or PKC-.theta. polypeptides. For example, RNA interference (RNAi) nucleic acid molecules, nucleases (e.g., CRISPR, TALENs, megaTALs, meganucleases, zinc finger nucleases); antibodies (e.g., Fab, Fab2, chimeric, humanized); or ligands, peptides, drugs, chemicals, or small molecules that competitively bind vimentin or RLTPR or PKC-.theta., that down-regulate vimentin or RLTPR or PKC-.theta. expression (transcription of DNA into RNA or translation of RNA into protein), that increase vimentin or RLTPR or PKC-.theta. degradation, or that cause intracellular depletion (e.g., by secretion) of vimentin or RLTPR or PKC-.theta., can be used to reduce or eliminate vimentin and/or RLTPR and/or PKC-.theta..

[0071] RNA interference (RNAi), also referred to as post-transcriptional gene silencing (PTGS), is known in the art and, as indicated herein, can be used to reduce or eliminate vimentin and/or RLTPR and/or PKC-.theta. polypeptides. RNAi is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules. Without being bound by theory, it appears that, in the presence of an antisense RNA molecule that is complementary to an expressed message (i.e., a mRNA), the two strands anneal to generate long double-stranded RNA (dsRNA), which is digested into short (<30 nucleotide) RNA duplexes, known as small interfering RNAs (siRNAs), by an enzyme known as Dicer. A complex of proteins known as the RNA Induced Silencing Complex (RISC) then unwinds siRNAs, and uses one strand to identify and thereby anneal to other copies of the original mRNA. RISC cleaves the mRNA within the complementary sequence, leaving the mRNA susceptible to further degradation by exonucleases, which effectively silences expression of the encoding gene.

[0072] Several methods have been developed that take advantage of the endogenous machinery to suppress the expression of a specific target gene and a number of companies offer RNAi design and synthesis services (e.g., Life Technologies, Applied Biosystems). In some instances, the use of RNAi can involve the introduction of long dsRNA (e.g., greater than 50 bps) or siRNAs (e.g., 12 to 23 bps) that have complementarity to the target gene, both of which are processed by the endogenous machinery. Alternatively, the use of RNAi can involve the introduction of a small hairpin RNA (shRNA); shRNA is a nucleic acid that includes the sequence of the two desired siRNA strands, sense and antisense, on a single strand, connected by a "loop" or "spacer" nucleic acid. When the shRNA is transcribed, the two complementary portions anneal intra-molecularly to form a "hairpin," which is recognized and processed by the endogenous machinery. Irrespective of the particular type used (e.g., dsRNA, siRNA or shRNA), such RNAi nucleic acid molecules can be referred to as "specific inhibitory nucleic acid molecules" (e.g., a vimentin-specific inhibitory nucleic acid molecule, a RLTPR-specific inhibitory nucleic acid molecule, a PKC-.theta.-specific inhibitory nucleic acid molecule).

[0073] A RNAi nucleic acid molecule as described herein includes a nucleic acid molecule that is complementary to at least a portion of a target mRNA (i.e., a vimentin or a RLTPR or a PKC-.theta. mRNA); this nucleic acid molecule typically is referred to as an "antisense strand". Generally, the antisense strand includes at least 12 contiguous nucleotides of the DNA sequence (e.g., the vimentin nucleic acid sequence shown in SEQ ID NO: 1 or 3; the RLTPR nucleic acid sequence shown in SEQ ID NO: 5 or 7; or the PKC-.theta. nucleic acid sequence shown in SEQ ID NO: 9 or 11); it would be appreciated that the antisense strand has the "RNA equivalent" sequence of the DNA (e.g., uracils instead of thymines; ribose sugars instead of deoxyribose sugars).

[0074] A RNAi nucleic acid molecule can be, for example, 12 to 500 nucleotides in length (e.g., 12 to 50, 12 to 45, 12 to 30, 15 to 47, 15 to 38, 15 to 29, 16 to 53, 17 to 44, 17 to 38, 18 to 36, 19 to 49, 20 to 60, 20 to 40, 25 to 75, 25 to 100, 28 to 85, 30 to 90, 12 to 100, 12 to 300, 12 to 450, 15 to 70, 15 to 150, 16 to 275, 17 to 74, 17 to 162, 17 to 305, 18 to 60, 18 to 75, 18 to 250, 18 to 400, 20 to 35, 20 to 60, 20 to 80, 20 to 175, 20 to 225, 20 to 325, 20 to 400, 20 to 475, 25 to 45, 25 to 65, 25 to 100, 25 to 200, 25 to 250, 25 to 300, 25 to 350, 25 to 400, 25 to 450, 30 to 280, 35 to 250, 200 to 500, 200 to 400, 250 to 450, 250 to 350, or 300 to 400 nucleotides in length).

[0075] In some embodiments, the antisense strand (e.g., a first nucleic acid) can be accompanied by a "sense strand" (e.g., a second nucleic acid), which is complementary to the antisense strand. In the latter case, each nucleic acid (e.g., each of the sense and antisense strands) can be between 12 and 500 nucleotides in length (e.g., between 12 to 50, 12 to 45, 12 to 30, 14 to 47, 15 to 38, 16 to 29, 17 to 53, 17 to 44, 17 to 38, 18 to 36, 19 to 49, 20 to 60, 20 to 40, 25 to 75, 25 to 100, 28 to 85, 30 to 90, 12 to 100, 13 to 300, 14 to 450, 16 to 70, 16 to 150, 16 to 275, 17 to 74, 17 to 162, 17 to 305, 18 to 60, 18 to 75, 18 to 250, 18 to 400, 20 to 35, 20 to 60, 20 to 80, 20 to 175, 20 to 225, 20 to 325, 20 to 400, 20 to 475, 25 to 45, 25 to 65, 25 to 100, 25 to 200, 25 to 250, 25 to 300, 25 to 350, 25 to 400, 25 to 450, 30 to 280, 35 to 250, 200 to 500, 200 to 400, 250 to 450, 250 to 350, or 300 to 400 nucleotides in length).

[0076] In some embodiments, a spacer nucleic acid, sometimes referred to as a loop nucleic acid, can be positioned between the sense strand and the antisense strand. In some embodiments, the spacer nucleic acid can be an intron (see, for example, Wesley et al., 2001, The Plant J., 27:581-90). In some embodiments, although not required, the intron can be functional (i.e., in sense orientation; i.e., spliceable) (see, for example, Smith et al., 2000, Nature, 407:319-20). A spacer nucleic acid can be between 20 nucleotides and 1000 nucleotides in length (e.g., 25-800, 25-600, 25-400, 50-750, 50-500, 50-250, 100-700, 100-500, 100-300, 250-700, 300-600, 400-700, 500-800, 600-850, or 700-1000 nucleotides in length).

[0077] In some embodiments, a construct can be produced by operably linking a promoter to a DNA region, that, when transcribed, produces an RNA molecule capable of forming a hairpin structure; and a DNA region involved in transcription termination and polyadenylation. It would be appreciated that the hairpin structure has two annealing RNA sequences, where one of the annealing RNA sequences of the hairpin RNA structure includes a sense sequence identical to at least 15 consecutive nucleotides of a vimentin or a RLTPR or a PKC-.theta. nucleotide sequence, and where the second of the annealing RNA sequences includes an antisense sequence that is identical to at least 15 consecutive nucleotides of the complement of the vimentin or the RLTPR or the PKC-.theta. nucleotide sequence. In addition, as indicated herein, the DNA region can include an intron (e.g., a functional intron). When present, the intron generally is located between the two annealing RNA sequences in sense orientation such that it is spliced out by the cellular machinery (e.g., the splicesome). Such a construct can be introduced into one or more plant cells to reduce the phenotypic expression of a vimentin or a RLTPR or a PKC-.theta. nucleic acid (e.g., a nucleic acid sequence that is normally expressed in a Treg cell).

[0078] In some embodiments, a construct (e.g., an expression construct) can include an inverted-duplication of a segment of a target nucleic acid sequence, where the inverted-duplication includes a nucleotide sequence substantially identical to at least a portion of the target nucleic acid and the complement of a portion of the target nucleic acid. It would be appreciated that a single promoter can be used to drive expression of the inverted-duplication nucleic acid, and that the inverted-duplication typically contains at least one copy of the portion of the target nucleic acid in the sense orientation. Such a construct can be introduced into one or more Treg cells to delay, inhibit or otherwise reduce the expression of the target nucleic acid in the Treg cells.

[0079] Representative siRNA nucleic acid molecules directed toward vimentin are shown in SEQ ID NOs: 13, 14, 15, and 16. It would be appreciated by the skilled artisan that the region of complementarity, between the antisense strand of the RNAi and the mRNA or between the antisense strand of the RNAi and the sense strand of the RNAi, can be over the entire length of the RNAi nucleic acid molecule, or the region of complementarity can be less than the entire length of the RNAi nucleic acid molecule. For example, a region of complementarity can refer to, for example, at least 12 nucleotides in length up to, for example, 500 nucleotides in length (e.g., at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 28, 30, 35, 49, 50, 60, 75, 80, 100, 150, 180, 200, 250, 300, 320, 385, 420, 435 nucleotides in length up to, e.g., 30, 35, 36, 40, 45, 49, 50, 60, 65, 75, 80, 85, 90, 100, 175, 200, 225, 250, 280, 300, 325, 350, 400, 450, or 475 nucleotides in length). In some embodiments, a region of complementarity can refer to, for example, at least 12 contiguous nucleotides in length up to, for example, 500 contiguous nucleotides in length (e.g., at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 28, 30, 35, 49, 50, 60, 75, 80, 100, 150, 180, 200, 250, 300, 320, 385, 420, 435 nucleotides in length up to, e.g., 30, 35, 36, 40, 45, 49, 50, 60, 65, 75, 80, 85, 90, 100, 175, 200, 225, 250, 280, 300, 325, 350, 400, 450, or 475 contiguous nucleotides in length).

[0080] It would be appreciated by the skilled artisan that complementary can refer to, for example, 100% sequence identity between the two nucleic acids. In addition, however, it also would be appreciated by the skilled artisan that complementary can refer to, for example, slightly less than 100% sequence identity (e.g., at least 95%, 96%, 97%, 98%, or 99% sequence identity). In calculating percent sequence identity, two nucleic acids are aligned and the number of identical matches of nucleotides (or amino acid residues) between the two nucleic acids (or polypeptides) is determined. The number of identical matches is divided by the length of the aligned region (i.e., the number of aligned nucleotides (or amino acid residues)) and multiplied by 100 to arrive at a percent sequence identity value. It will be appreciated that the length of the aligned region can be a portion of one or both nucleic acids up to the full-length size of the shortest nucleic acid. It also will be appreciated that a single nucleic acid can align with more than one other nucleic acid and hence, can have different percent sequence identity values over each aligned region.

[0081] The alignment of two or more nucleic acids to determine percent sequence identity can be performed using the computer program ClustalW and default parameters, which allows alignments of nucleic acid or polypeptide sequences to be carried out across their entire length (global alignment). Chenna et al., 2003, Nucleic Acids Res., 31(13):3497-500. ClustalW calculates the best match between a query and one or more subject sequences (nucleic acid or polypeptide), and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a query sequence, a subject sequence, or both, to maximize sequence alignments. For fast pairwise alignment of nucleic acid sequences, the default parameters can be used (i.e., word size: 2; window size: 4; scoring method: percentage; number of top diagonals: 4; and gap penalty: 5); for an alignment of multiple nucleic acid sequences, the following parameters can be used: gap opening penalty: 10.0; gap extension penalty: 5.0; and weight transitions: yes. For fast pairwise alignment of polypeptide sequences, the following parameters can be used: word size: 1; window size: 5; scoring method: percentage; number of top diagonals: 5; and gap penalty: 3. For multiple alignment of polypeptide sequences, the following parameters can be used: weight matrix: blosum; gap opening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps: on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg, and Lys; and residue-specific gap penalties: on. ClustalW can be run, for example, at the Baylor College of Medicine Search Launcher website or at the European Bioinformatics Institute website on the World Wide Web.

[0082] The skilled artisan also would appreciate that complementary can be dependent upon, for example, the conditions under which two nucleic acids hybridize. Hybridization between nucleic acids is discussed in detail in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Sections 7.37-7.57, 9.47-9.57, 11.7-11.8, and 11.45-11.57). Sambrook et al. disclose suitable Southern blot conditions for oligonucleotide probes less than about 100 nucleotides (Sections 11.45-11.46). The Tm between a nucleic acid that is less than 100 nucleotides in length and a second nucleic acid can be calculated using the formula provided in Section 11.46. Sambrook et al. additionally disclose Southern blot conditions for oligonucleotide probes greater than about 100 nucleotides (see Sections 9.47-9.54). The Tm between a nucleic acid greater than 100 nucleotides in length and a second nucleic acid can be calculated using the formula provided in Sections 9.50-9.51 of Sambrook et al.

[0083] The conditions under which membranes containing nucleic acids are prehybridized and hybridized, as well as the conditions under which membranes containing nucleic acids are washed to remove excess and non-specifically bound probe, can play a significant role in the stringency of the hybridization. Such hybridizations and washes can be performed, where appropriate, under moderate or high stringency conditions. For example, washing conditions can be made more stringent by decreasing the salt concentration in the wash solutions and/or by increasing the temperature at which the washes are performed. Simply by way of example, high stringency conditions typically include a wash of the membranes in 0.2.times.SSC at 65.degree. C.

[0084] In addition, interpreting the amount of hybridization can be affected, for example, by the specific activity of the labeled oligonucleotide probe, by the number of probe-binding sites on the template nucleic acid to which the probe has hybridized, and by the amount of exposure of an autoradiograph or other detection medium. It will be readily appreciated by those of ordinary skill in the art that although any number of hybridization and washing conditions can be used to examine hybridization of a probe nucleic acid molecule to immobilized target nucleic acids, it is more important to examine hybridization of a probe to target nucleic acids under identical hybridization, washing, and exposure conditions. Preferably, the target nucleic acids are on the same membrane. A nucleic acid molecule is deemed to hybridize to a nucleic acid, but not to another nucleic acid, if hybridization to a nucleic acid is at least 5-fold (e.g., at least 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, or 100-fold) greater than hybridization to another nucleic acid. The amount of hybridization can be quantified directly on a membrane or from an autoradiograph using, for example, a PhosphorImager or a Densitometer (Molecular Dynamics, Sunnyvale, Calif.).

[0085] A construct (also known as a vector) containing a RNAi nucleic acid molecule is provided. Constructs, including expression constructs, are described herein and are known to those of skill in the art. Expression elements (e.g., promoters) that can be used to drive expression of a RNAi nucleic acid molecule are known in the art and include, without limitation, constitutive promoters such as, without limitation, the cassava mosaic virus (CsMVM) promoter, the cauliflower mosaic virus (CaMV) 35S promoter, the actin promoter, or the glyceraldehyde-3-phosphate dehydrogenase promoter, or tissue-specific promoters such as, without limitation, root-specific promoters such as the putrescine N-methyl transferase (PMT) promoter or the quinolinate phosphosibosyltransferase (QPT) promoter. It would be understood by a skilled artisan that a sense strand and an antisense strand can be delivered to and expressed in a target cell on separate constructs, or the sense and antisense strands can be delivered to and expressed in a target cell on a single construct (e.g., in one transcript). As discussed herein, a RNAi nucleic acid molecule delivered and expressed on a single strand also can include a spacer nucleic acid (e.g., a loop nucleic acid) such that the RNAi forms a small hairpin (shRNA).

[0086] Treg cells can be contacted in vitro, in situ, or in vivo with any of the moieties discussed herein (e.g., nucleic acids, nucleases, antibodies, ligands, peptides, drugs, chemicals, or small molecules) using any number of methods known to those skilled in the art. For example, Treg cells can be contacted with any of the moieties discussed herein (e.g., a nucleic acid (e.g., a vimentin-specific and/or a RLTPR-specific and/or a PKC-.theta.-specific inhibitory nucleic acid molecule; e.g., one or more RNAi molecules)) in vitro, in situ, or in vivo.

[0087] For example, one or more nucleic acids can be attached to or contained within a carrier such as, without limitation, liposomes, nanoparticles, or antibodies. Such carriers can be delivered to an individual (e.g., a patient) using routine cellular therapies, and such carriers can be targeted to Treg cells using one or more Treg targeting moieties such as, for example, cytokines that preferentially activate Tregs such as IL2; or the use of one or more moieties that specifically binds to a ligand that is preferentially expressed by Tregs such as neuropillin-1, lag3, TIGIT, CD39, CD73, IL10R, ST2, PD-1, CTLA4, CD49d, GITR, GARP, FR4.

[0088] The methods described herein can be applied to an individual who has received or is receiving a bone marrow transplant or a solid organ transplant. Alternatively, the methods described herein can be applied to an individual in order to treat or mitigate the symptoms of an autoimmune disease, or to induce tolerance to one or more foreign antigens (for example, in cases of enzyme therapy, gene therapy, antibody therapy, or drug therapy). Further, the methods described herein can be applied to an individual in order to treat or mitigate the symptoms of one or more allergic reactions.

[0089] Following contact with one or more of the moieties described herein, the Treg cells (e.g., Treg cells in which the vimentin, and/or RLTPR and/or PCK-.theta. has been reduced or eliminated) typically exhibit at least one of the following phenotypes (relative to Tregs in which vimentin, RLTPR and/or PCK-.theta. is not reduced or eliminated (e.g., relative to Tregs that lack the vimentin-specific and/or the RLTPR-specific and/or the PKC-.theta.-specific inhibitory nucleic acid molecule)): reduced PKC-.theta. auto-phosphorylation at Ser676; improved ability to suppress CD4+ and CD8+ Tcon proliferation; increased surface expression of Nrp1; increased surface expression of Lag3; increased basal and maximal oxygen consumption rate (OCR); increased BoDipy.sub.C1-C12 uptake; increased expression of CD71; increased expression of CD98; increased expression of CPT1a; or reduced activity of mTORC2.

[0090] In accordance with the present invention, there may be employed conventional molecular biology, microbiology, biochemical, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. The invention will be further described in the following examples, which do not limit the scope of the methods and compositions of matter described in the claims.

EXAMPLES

Example 1-PKC-.theta. Inhibition and Treg Function

[0091] Using a mouse MHC class I/II disparate acute GVHD model, it was found that freshly isolated Tregs treated for 30 minutes with 10 .mu.M of the clinically available PKC-.theta. inhibitor, AEB071, suppressed GVHD mortality (FIG. 1A) and severity significantly better than DMSO-treated Tregs. As Tregs exert much of their protective effect against GVHD early in the course of the disease, proliferation of GVHD-causing conventional T-cells (Tcon) on D4 after transplant was analyzed. A significant reduction in Tcon proliferation in mice given AEB071 treated Tregs was observed compared to DMSO treated Tregs. Multi-photon microscopy on D4 was performed after transplant using TE.alpha.-GFP Tcon, CD11c-eYFP antigen presenting cells (APCs) and wild-type Tregs. Compared to DMSO, AEB071-treated Tregs significantly increased Tcon velocity and displacement from APCs. Increased velocity and displacement are indicative of decreased Tcon-APC interactions, suggesting reduced priming when AEB071-treated Tregs are present.

[0092] AEB071 vs DMSO treatment of Tregs resulted in augmented expression of the suppressive molecules, Neuropilin-1 (Nrp1) and Lymphocyte activation gene 3 (Lag3), after in vitro activation (FIGS. 1B and 1C) and in Tregs isolated from acute GVHD mice. Antibody blockade of Nrp1 and Lag3 in in vitro trans-well suppression assays reduced the effect of AEB071 treatment, suggesting that these molecules play a role in enhancing Treg function after PKC-.theta. inhibition. Flow cytometry analysis of phosphorylated proteins in activated Tregs revealed that PKC-.theta. inhibition resulted in reduced phosphorylation of the mTORC2 target, FoxO3a, but not mTORC1 targets, S6 and 4E-BP1. In addition, the mTORC2-specific phosphorylation site on Akt, serine 473, was reduced, whereas the mTORC1-specific phosphorylation site, threonine 308, was unaltered. Together, these data suggest reduced mTORC2 activity. Reduced phosphorylation increases FoxO3a nuclear translocation, which may result in increased Nrp1 and Lag3 expression, since FoxO3a has binding sites in both gene promoters. As both mTORC1 and mTORC2 are involved in T-cell metabolism, the effect of AEB071 treatment on Treg oxygen consumption rate (OCR) was investigated. Compared to DMSO, AEB071 treatment significantly increased Treg baseline and maximal OCRs after activation (FIG. 1D). Increased OCR has been associated with increased Treg function.

[0093] To identify additional alterations in phosphorylated proteins after PKC-.theta. inhibition, a phosphoproteomic screen was performed using in vitro-expanded human Tregs treated with AEB701 or DMSO. Significant alterations in phosphorylation sites on 72 proteins was observed, including reduced phosphorylation of an adaptor molecule that links PKC-.theta. to the intermediate filament, vimentin. It was found that vimentin is highly upregulated in Tregs compared to Tcon and that, in Tregs, vimentin interacts with PKC-.theta. after activation. AEB071 treatment reduced the interaction between vimentin and PKC-.theta.. As with AEB071 treatment, vimentin siRNA significantly increased Treg suppression in vitro compared to control-transfected Tregs (FIG. 1E), and augmented expression of Nrp1 and Lag3. AEB071-treatment of vimentin siRNA transfected Tregs did not further augment Treg function, suggesting an overlapping mechanism.

[0094] In summary, the data presented herein demonstrates that PKC-.theta. interacts with mTORC2 and vimentin to modulate multiple aspects of Treg function, and that a brief incubation of Tregs with a PKC-.theta. inhibitor or a reduction in vimentin protein levels may be viable methods to enhance the efficacy of Treg therapeutics.

Example 2--T-Cell Purification from Lymph Nodes

[0095] Lymph nodes were harvested into a gentle MACS C tube containing PBS with at least 2% FBS. The tissue was homogenized one or two times on the Miltenyi GentleMACS dissociator using the Spleen-1 protocol, inverting the tube between runs.

[0096] The tissue was spun at 1200 RPM for 10 minutes at 4.degree. C. The supernatant was removed and the pellet was resuspended in MACS buffer (PBS containing 2% FBS and 1 mM EDTA). The solution was filtered through a 70 .mu.M cell strainer into a 50 mL tube. The original C-tube was washed with additional MACS buffer and put through the strainer. The cells were counted at least three different times, and resuspended at 100.times.10e6 cells/mL in a 50 mL tube.

[0097] FCS was added to the cells at 50 .mu.L/mL and a biotin-labeled antibody (e.g., anti-CD4, CD8, CD19, B220, CD11b, CD11c, anti-CD25, anti-NK1, anti-gamma delta TCR) was added and incubated for 10 minutes at room temperature. RapidSphere Magnetic Particles were added at 35 .mu.L/mL and incubated for 10 minutes at room temperature. If the volume of cells after the RapidSpheres were added was between 1 and 10 mL, then 25 mL of MACS buffer was added and the sample was mixed gently. If the volume of cells after the RapidSpheres were added was between 11 and 40 mL, then 50 mL of MACS buffer was added and the sample was mixed gently. The samples were incubated on the magnet for 5 minutes at room temperature.

[0098] The negative fraction (e.g., the purified cells) were pipetted off and transferred to a different 50 mL tube. Cells were stained with anti-CD4, anti-CD8, and anti-CD25 antibodies to test for purity, and then counted at least three times. If the purity is not ideal, the sample can be placed in the magnet for another 5 minutes and the purity re-checked.

Example 3--T-Cell Purification from Spleen

[0099] The spleen was gently harvested into a MACS C tube containing PBS and at least 2% FBS. The tissue was homogenized 1 to 2 times on the Miltenyi GentleMACS dissociator using the Spleen-1 protocol, inverting the tube between runs. The tubes were spun at 1200 RPM for 10 minutes at 4.degree. C.

[0100] The supernatant was removed and resuspended in 0.5-1 mL ACK lysis buffer (per spleen), and incubated for 1 minute. The C-tube was filled with MACS/PBS+2% FBS, and the solution was filtered through a 100 .mu.M cell strainer into a 50 mL tube. The C-tube was washed with additional MACS buffer and put through the strainer. The sample was spun at 1200 rpm for 10 minutes at 4.degree. C., the supernatant was removed, and the cells resuspended in MACS buffer. The cells were counted as least three times, and resuspended at 100.times.10e6 cells/mL in a fresh 50 mL tube.

[0101] FCS was added at 50 .mu.L/mL, antibodies labeled with biotin were added (anti-CD19, B220, CD11b, CD11c, anti-CD4, CD8, CD25, NK1.1, DX5, gamma delta TCR), and incubated for 10 minutes at room temperature. RapidSphere Magnetic Particles were added at 55 .mu.L/mL, and the sample was incubated for 10 minutes at room temperature. If the volume of cells after adding the RapidSpheres was between 1 and 10 mL, then 25 mL of MACS buffer was added and the sample gently mixed. If the volume of cells after adding the RapidSpheres was between 11 and 40 mL, then 50 mL of MACS buffer was added and the sample gently mixed. The samples were incubated on the magnet for 5 minutes at room temperature.

[0102] The negative fraction (e.g., purified cells) was pipetted off and transferred to another 50 mL tube. The cells were stained with anti-CD4, anti-CD8, and anti-CD25 to test for purity, then counted. If the purity is not ideal, the cells were placed in contact with the magnet for another 5 minutes, and their purity checked.

Example 4--T-Cell Purification Using CD25 Positive Selection

[0103] Cells were resuspended at 100.times.10e6 cells/mL. A selection antibody (anti-CD25 labeled with phycoerythrin (PE)) was added at 0.1 .mu.l/10 e6 cells (2 .mu.g/mL), and the sample was incubated for 10 minutes at 4.degree. C. The cells were washed one time with MACS buffer, spun at 1500 RPM for 5 minutes at 4.degree. C., and the supernatant was removed.

[0104] Cells were resuspended at 0.8 mL/100.times.10e6 cells, and 10 .mu.l/10e6 of anti-PE micro beads were added and incubated for 15 minutes at 4.degree. C. Cells were washed one time with MACS buffer, spun at 1500 RPM for 5 minutes at 4 C, and the supernatant was removed. Cells were resuspended in a volume to bring the cells to 200.times.10e6 cells/mL.

[0105] A MS or LS column were placed on a magnet with a filter on top. The filter was washed with 500 .mu.L MS or 2 mL LS buffer, and the column was loaded with the cells. The column was washed five times with 500 .mu.L MS or 3 mL LS buffer. The column was removed from the magnet and the column was loaded with 1 mL MS or 5 mL LS buffer. The column was flushed with a plunger into a 15 mL tube. The cells were spun down, resuspended in 1 mL, and the steps above were repeated with a second MS/LS column. The cells were stained with anti CD4/8 to test for purity and counted at least three times.

Example 5--Amaxa Transfection (Nucleofection) Protocol for Mouse T-Cells

[0106] The number of cells was calculated to determine the number of cuvettes needed (e.g., 2-5.5 M cells per cuvette). After determining the number of cuvettes needed, 2 mL of fully supplemented Amaxa media with 300 IU/mL recombinant human IL-2 for each cuvette was warmed in a 12 well warm plate. After the cells are counted, they are pelleted by centrifugation at 1500 RPM for 5 minutes at 30.degree. C.

[0107] As much of the supernatant as possible was removed, and cells were re-suspended in 100 .mu.L of room temperature Amaxa Nucleofector solution per sample. Each sample contained about 2-5.5.times.10e6 healthy CD4+ T-cells in 100 .mu.L of RT Nucleofector Solution. It would be appreciated that transfection will kill cell that are not healthy. To ensure healthy cells, the cells were rested for 2-4 hours to overnight in RPMI-c with 300 IU/mL recombinant human IL-2 at 37.degree. C. prior to transfection.

[0108] Cell solutions were aliquoted into Amaxa cuvettes as follows: 10 .mu.L of vim siRNA was added to a final concentration of 5 or 5 .mu.L of Amaxa GFP was added to a final concentration of 2.5 .mu.g per sample, then 100 .mu.L of cells were added to each cuvette, and each cuvette was capped. A mock control cuvette using no GFP/siRNA also was included.

[0109] Cuvettes were loaded into the Amaxa Nucleofector II machine and transfected using Nucleofector Program X-001 for mouse CD4 T-cells. After transfection, the transfer pipettes provided by Amaxa were used to mix about 200-300 .mu.L of warmed media with the cell solution in the cuvette, and the cells were gently transferred into a 12 well plate. The cells were allowed to rest in the incubator for 4-5 hours.

[0110] While the cells were resting, the same volume of new Amaxa media supplemented with 300 IU/mL rhIL-2 was warmed. After 4-5 hours, the cells were removed from each well, and spun down at 1500 RPM for 5 minutes at 37.degree. C. As much of the supernatant as possible was removed, and the pellet was re-suspended in the warmed media containing rhIL-2. Cells were placed in a 12 well plate that had been coated with 10 .mu.g/mL anti-CD3/28 the day before the experiment and allowed to sit overnight at 37.degree. C., and spun for 5 min at 500 RPM at 37.degree. C. The cells were placed in an incubator.

[0111] 24 hours later, 1 ml of fresh media, with an additional 300 IU/mL of recombinant human IL-2, was added to each well. 48 hours later, the cells were ready for use. The cells were collected from the wells, spun down at 1500 RPM for 5 minutes at 30.degree. C., and counted. Samples were taken to check transfection efficiency.

Example 6--Vimentin siRNA Transfection Methods

[0112] Regulatory T-cells (Tregs) were purified from lymph nodes and spleens of C57Bl/6 mice using a two-step process: Step 1: CD4 negative selection--selection of CD4+ T-cells was accomplished using eBioscience biotinylated Ab (anti-CD19, B220, CD8, NK1.1, gamma delta TCR) and StemCell technologies streptavidin RapidSpheres; and Step 2: CD25 positive selection--after CD4+ negative selection, CD25 positive selection was completed using eBiosceince anti-CD25 PE Ab and Miltenyi anti-PE microbeads.

[0113] Once purified, Tregs were brought to a volume of about 1e6/mL in complete media with 300 IU/mL recombinant human IL-2, and the cells were rested for 2 hours at 37.degree. C. in an incubator in a 24-well plate. After resting, Tregs were counted and split into 2 groups: one for transfection of control GFP siRNA and one for transfection of vimentin siRNA. After being split, Tregs were diluted in Amaxa Nucleofection Solution at a concentration of 5.5e6/100 .mu.L and contacted with either 2.5 .mu.g of control GFP plasmid or 5 .mu.M vimentin siRNA per 100 .mu.L.

[0114] A mixture of 4 siRNA oligonucleotides were combined in equal ratios such that the final concentration of the siRNA mixture is 5 .mu.M. The siRNA oligonucleotides that were used have the following sequences: siRNA 1: CCA GAG AGA GGA AGC CGA A (SEQ ID NO: 13); siRNA 2: AGG AAG AGA UGG CUC GUC A (SEQ ID NO: 14); siRNA 3: GUC UUG ACC UUG AAC GGA A (SEQ ID NO: 15); and siRNA 4: AAG CAG GAG UCA AAC GAG U (SEQ ID NO: 16).

[0115] Cells were then placed in Lonza cuvettes (100 .mu.L per cuvette) and electroporated using program X-001 in a Lonza Nucleofector II machine. After transfection, cells were placed in 2 mL of warmed complete Amaxa media (5% FBS, Pen/Strep, 10 .mu.L/mL of Lonza media supplement) in a 12-well plate and incubated at 37.degree. C. for 4 hours. After 4 hours, cells were removed from the wells, spun down (1500 RPM for 5 minutes), then diluted in 2 mL of warmed Amaxa complete media supplemented with 300 IU/mL recombinant human IL-2 and plated on a 12-well placed coated with anti-CD3 and anti-CD28 (10 .mu.g/mL of each antibody). 24 hours after transfection (2D), 1 mL of Lonza complete media with 300 IU rhIL-2 was added to each well. 48 hours after transfection (3D), cells were removed from the wells, counted and used in functional studies.

Example 7--Vimentin siRNA Results

[0116] The experiments described herein demonstrated that vimentin siRNA significantly decreased vimentin levels in Tregs and that vimentin siRNA treatment increased the in vitro suppressive function in Tregs.

[0117] FIG. 2A shows that vimentin is highly enriched in Tregs compared to conventional CD4+ T-cells (CD4 Tcon). FIG. 2B shows that, compared to the transfection control (top panel), transfection with GFP-siRNA using the transfection protocol described herein yielded 50-60% or more transfection of Tregs. FIG. 2C shows that, compared to control GFP-siRNA (GFP), vimentin levels were knocked down by 15-30% in the presence of vimentin siRNA (vim siRNA). In total, the protocol described herein yielded 50-60% Tregs transfected with siRNA, and a 15-30% reduction in vimentin levels in transfected cells.

[0118] In standard in vitro suppression assays, vimentin siRNA-transfected Tregs (Vim) were able to suppress proliferation of both CD4 conventional T-cells (FIG. 3A) and CD8 conventional T-cells (FIG. 3B) significantly better than the control GFP-siRNA-transfected Tregs (GFP). Treg: Tcon ratios of 1:1-1:27 are represented with the 1:1, 1:3, etc., denotations in the x-axis labels.

Example 8--Suppression Assay

[0119] On day 3 after transfection, CD4/8 Tcon and T-cell depleted splenoctyes were isolated for a suppression assay. CD4/CD8 Tcon were purified from spleen of CD45.1 C57BL/6 mice using negative selection with eBioscience biotinylated Ab (anti-CD19, B220, NK1.1, gamma-delta TCR) and StemCell technologies streptavidin RapidSphere technology. Once isolated, Tcon were labeled with CFSE (2.5 .mu.M) for 5 minutes at room temperature with constant agitation.

[0120] Responder Tcon were provided with 0.75 .mu.g/mL soluble anti-CD3 mAb. This 0.75 .mu.g/mL was equivalent to a 3.times. concentration such that the final concentration of anti-CD3, once Teff were mixed with stimulator splenocytes and Tregs, was 0.25 .mu.g/mL.

[0121] Splenocytes from 1 CD45.1 C57BL/6 mouse were depleted of T-cells and NK cells using eBioscience biotinylated mAb (anti-CD4, CD8, NK1.1, gamma delta TCR) and StemCell technologies streptavidin RapidSphere technology. These TCD splenocytes were used as stimulator cells.

[0122] Ratios of 0:1, 1:1, 1:3, 1:9 and 1:27 Treg:Teff were made. Four replicates of each ratio were made and plated in a 96-well, round bottom plate. After 3 days in culture, the cells from each well were harvested, washed with PBS and then stained with antibodies for flow cytometry analysis of CFSE dilution (proliferation) of CD4 and CD8 Tcon. The antibodies used were as follows: CD4--BV510, CD8--PE-ef610, CD25--BV605, Nrp1--PerCP-ef710, Lag3--APC, Fixable viability dye--APC-ef780, Foxp3--PE-Cy7, and Vimentin--PE.

Example 9--Phosphoproteomic Screen with Human Treg Cells

[0123] Utilizing mass spectroscopy, a total of 12,452 phosphorylation sites were quantified from human Tregs. In order to determine significant differences between DMSO-treated and AEB071-treated Tregs, two experiments were performed concurrently:

[0124] Forward experiment: This experiment utilized heavy isotope-labeled Tregs treated with AEB071 and medium isotope-labeled Tregs treated with DMSO.

[0125] Reverse experiment: medium isotope-labeled Tregs treated with AEB071 and heavy isotope-labeled Tregs treated with DMSO.

[0126] After processing the raw spectra with Max Quant software, the positively identified phosphopeptides were analyzed using Persus software. Contaminating peptides and reverse database hits were filtered out and the peptide intensity values and normalized H/M ratios were log 2 transformed. The significances of the individual H/M ratios calculated using the Significance B test with a false discovery rate of 0.05 for the forward and reverse experiment separately.

[0127] Relying only on the Sig B test, the overlap between the two data sets was low with only 15% overlap. In order to improve the overlap between the two data sets, the data was filtered based on fold change (<1.5), consistency of ratios, phospho site localization probability (<75%), and the MaxQuant score (<75). By filtering the results, the overlap improved to 60%. See Table 1.

TABLE-US-00001 TABLE 1 Sig B Sig B Significant Category Test Only Test and Filters Forward Experiment 380 96 Reverse Experiment 448 92 Either For or Rev 723 117 Both 105 72 (105/772 = 15%) (72/117 = 62%)

[0128] Histograms show a nice symmetrical distribution centered at 0 (Log 2 transformed data), indicating equal amounts of samples and labeling overall. See FIG. 4A. The scatter plot provides an indication of the consistency between samples. The red crosses are consistent between samples and, for the most part, cluster in the upper left quadrant as expected, since the inhibitor should decrease the levels of phosphorylation. See FIG. 4B.

[0129] The results of the phosphoproteomic screen pointed toward the PKC-.theta. adapter and interaction partner, RLTPR. The RLTPR protein had significantly reduced phosphorylation at the PKC-theta consensus site: IGVSRGS(ph)GGAEGK (SEQ ID NO:17), with the phosphorylation site on the serine at residue 1226 indicated by the (ph) after the amino acid.

Example 10--RLTPR siRNA Transfection Methods and Results

[0130] Tregs were contacted with either 2.5 .mu.g of control GFP plasmid or 1 .mu.M, 5 .mu.M, or 10 .mu.M RLTPR siRNA per 100 .mu.L. Cells were then placed in Lonza cuvettes (100 .mu.L per cuvette) and electroporated using program X-001 in a Lonza Nucleofector II machine. After transfection, cells were placed in 2 mL of warmed complete Amaxa media (5% FBS, Pen/Strep, 10 .mu.L/mL of Lonza media supplement) in a 12-well plate and incubated at 37.degree. C. for 4 hours. After 4 hours, cells were removed from the wells, spun down (1500 RPM for 5 minutes), then diluted in 2 mL of warmed Amaxa complete media supplemented with 300 IU/mL recombinant human IL-2 and plated on a 12-well placed coated with anti-CD3 and anti-CD28 (10 .mu.g/mL of each antibody). 24 hours after transfection (2D), 1 mL of Lonza complete media with 300 IU rhIL-2 was added to each well. 48 hours after transfection (3D), cells were removed from the wells and counted. The cells contacted with the 10 .mu.M of RLTPR siRNA were used in functional studies.

[0131] FIGS. 5A-5D show that RLTPR siRNA significantly decreased RLTPR protein levels in Tregs. Results also demonstrated that RLTPR siRNA treatment (at 10 .mu.M) increased in vitro suppressive function (see FIGS. 6A and 6B) and augmented Nrp1 (see FIG. 7C).

Example 11--PKC-.theta. Inhibition Alters PKC-.theta. Localization and Vimentin Interaction

[0132] Since PKC-.theta. localization and function appear to be linked, experiments were performed to understand whether PKC-.theta. inhibition modulated PKC-.theta. localization. Consistent with previous work, PKC-.theta. accumulated at the distal pole of control Tregs after 5 minutes of anti-CD3/CD28 mAb activation (FIG. 8A, top). It was also found that PKC-.theta. was tightly associated with the intermediate filament, vimentin (FIG. 8A, top), a molecule that was found to be more highly expressed in Tregs compared to CD4+ Tcons (FIG. 10A). In contrast to controls, in AEB071-pre-treated Tregs, PKC-.theta. was dispersed throughout the cell after activation, and the overlap between PKC-.theta. and vimentin was reduced (FIG. 8A, middle). Since PKC-.theta. inhibition dispersed PKC-.theta. and reduced PKC-.theta./vimentin overlap, experiments were performed to determine whether modifying vimentin levels with siRNA might result in comparable changes. Indeed, vimentin knockdown by as little as 30% resulted in a similar pattern as AEB071: dispersed PKC-.theta., and reduced PKC-.theta./vimentin overlap (FIG. 8A, bottom). These data demonstrate that inhibition of PKC-.theta. or vimentin alters PKC-.theta. localization and increases Treg function, and that PKC-.theta./vimentin interactions may be important for PKC-.theta. trafficking.

Example 12--Vimentin siRNA Reduces PKC-.theta. Activity and Augments Treg Function

[0133] To better characterize whether PKC-.theta./vimentin interactions were facilitating PKC-.theta. function, PKC-.theta. activity and Treg suppressive function were analyzed after treatment with vimentin siRNA. Using phosphoflow, it was noted that, similar to treatment with AEB071, PKC-.theta. auto-phosphorylation at Ser676 was significantly reduced in vimentin siRNA-treated Tregs compared with controls (FIG. 8B). Furthermore, vimentin siRNA-treated Tregs were significantly better at suppressing CD4+ and CD8+ Tcon proliferation in standard in vitro suppression assays compared to control Tregs (FIG. 8C, FIG. 10B). This increased Treg function correlated with a significant increase in surface expression of Nrp1 (FIG. 8D) and Lag3 (FIG. 10C). All other Treg suppressive molecules remained unchanged (FIG. 8D, and other data not shown).

[0134] To determine whether vimentin siRNA treatment would augment the ability of Treg to suppress GVHD, control- and vimentin siRNA-treated Tregs were compared using the GVHD model. Recipients given vimentin siRNA-treated Tregs had significantly increased survival and reduced GVHD severity compared with controls (FIG. 9A-B, FIG. 10D). As with AEB071 treatment, vimentin siRNA did not increase GI homing molecule expression (FIG. 10E). In combination, these results suggest that vimentin plays a key role in coordinating PKC-.theta. activity, and that vimentin knockdown in Tregs results in a similar functional enhancement as direct PKC-.theta. inhibition.

Example 13--Vimentin siRNA Augments Treg Metabolic Activity and Reduces mTORC2 Function

[0135] Since PKC-.theta. inhibition modulated Treg metabolism and mTORC2 function, experiments were performed to determine whether vimentin siRNA might have a similar effect. As with PKC-.theta. inhibition, treatment with vimentin siRNA significantly increased basal and maximal OCR, but did not alter ECAR (FIG. 9C). In GVHD, vimentin siRNA-treated Treg also had increased BoDipy.sub.C1-C12 uptake, and augmented expression of CD71, CD98, and CPT1a, but not Glut1 (FIG. 9D-E, FIG. 10F). BoDipy.sub.C1-C12 uptake was also increased in vitro. Since mTORC2 signaling was reduced after PKC-.theta. inhibition, it was hypothesized that mTORC2 activity also could be reduced after treatment with vimentin siRNA. Consistent with this hypothesis, phosphoflow analysis demonstrated reduced phosphorylation of Akt at Ser473 and Foxo3a (FIG. 9F), but no change in the phosphorylation of S6 or 4E-BP-1 (FIG. 10G), in vimentin siRNA-treated Tregs versus controls. Together, these data demonstrate that reducing vimentin levels alters Treg metabolism (e.g., vimentin siRNA increases Treg function) and mTORC2 activity in a manner similar to PKC-.theta. inhibition.

[0136] It is to be understood that, while the methods and compositions of matter have been described herein in conjunction with a number of different aspects, the foregoing description of the various aspects is intended to illustrate and not limit the scope of the methods and compositions of matter. Other aspects, advantages, and modifications are within the scope of the following claims.

[0137] Disclosed are methods and compositions that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that combinations, subsets, interactions, groups, etc. of these methods and compositions are disclosed. That is, while specific reference to each various individual and collective combinations and permutations of these compositions and methods may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular composition of matter or a particular method is disclosed and discussed and a number of compositions or methods are discussed, each and every combination and permutation of the compositions and the methods are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed.

Sequence CWU 1

1

1719659DNAHomo sapiens 1tttgtgttac ataattgcct ttcatttgaa ctcattaatc aaattggggt ttttaagcaa 60cacctaatta attctttaac tggctcatat tataccttta atgacttcca ccagggtaaa 120aaccactgat cactgagttc tattttgaaa ctacggacgt cgagtttcct ctttcaccca 180gaattttcag atcttgttta aaaagttggg tgtggtttca tggggggagg gggaagagcg 240agaggagacc agagggacgg gggcggggac tctgcaagaa aaaccttccc ggtgcaatcg 300tgatctggga ggcccacgta tggcgcctct ccaaaggctg cagaagtttc ttgctaacaa 360aaagtccgca cattcgagca aagacaggct ttagcgagtt attaaaaact taggggcgct 420cttgtccccc acagggcccg accgcacaca gcaaggcgat ggcccagctg taagttggta 480gcactgagaa ctagcagcgc gcgcggagcc cgctgagact tgaatcaatc tggtctaacg 540gtttccccta aaccgctagg agccctcaat cggcgggaca gcagggcgcg gtgagtcacc 600gccggtgact aagcgacccc acccctctcc ctcgggcttt cctctgccac cgccgtctcg 660caactcccgc cgtccgaagc tggactgagc ccgttaggtc cctcgacaga acctcccctc 720cccccaacat ctctccgcca aggcaagtcg atggacagag gcgcgggccg gagcagcccc 780cctttccaag cgggcggcgc gcgaggctgc ggcgaggcct gagccctgcg ttcctgcgct 840gtgcgcgccc ccaccccgcg ttccaatctc aggcgctctt tgtttctttc tccgcgactt 900cagatctgag ggattcctta ctctttcctc ttcccgctcc tttgcccgcg ggtctccccg 960cctgaccgca gccccgagac cgccgcgcac ctcctcccac gcccctttgg cgtggtgcca 1020ccggacccct ctggttcagt cccaggcgga cccccccctc accgcgcgac cccgcctttt 1080tcagcacccc agggtgagcc cagctcagac tatcatccgg aaagccccca aaagtcccag 1140cccagcgctg aagtaacggg accatgccca gtcccaggcc ccggagcagg aaggctcgag 1200ggcgccccca ccccacccgc ccaccctccc cgcttctcgc taggtcccta ttggctggcg 1260cgctccgcgg ctgggatggc agtgggaggg gaccctcttt cctaacgggg ttataaaaac 1320agcgccctcg gcggggtcca gtcctctgcc actctcgctc cgaggtcccc gcgccagaga 1380cgcagccgcg ctcccaccac ccacacccac cgcgccctcg ttcgcctctt ctccgggagc 1440cagtccgcgc caccgccgcc gcccaggcca tcgccaccct ccgcagccat gtccaccagg 1500tccgtgtcct cgtcctccta ccgcaggatg ttcggcggcc cgggcaccgc gagccggccg 1560agctccagcc ggagctacgt gactacgtcc acccgcacct acagcctggg cagcgcgctg 1620cgccccagca ccagccgcag cctctacgcc tcgtccccgg gcggcgtgta tgccacgcgc 1680tcctctgccg tgcgcctgcg gagcagcgtg cccggggtgc ggctcctgca ggactcggtg 1740gacttctcgc tggccgacgc catcaacacc gagttcaaga acacccgcac caacgagaag 1800gtggagctgc aggagctgaa tgaccgcttc gccaactaca tcgacaaggt gcgcttcctg 1860gagcagcaga ataagatcct gctggccgag ctcgagcagc tcaagggcca aggcaagtcg 1920cgcctggggg acctctacga ggaggagatg cgggagctgc gccggcaggt ggaccagcta 1980accaacgaca aagcccgcgt cgaggtggag cgcgacaacc tggccgagga catcatgcgc 2040ctccgggaga agtaaggctg cgcccatgca agtagctggg cctcgggagg gggctggagg 2100gagaggggaa cgcccccccg gcccccgcga gagctgccac gcccttgggg atgtggccgg 2160ggggaggcct gccagggaga cagcggagag cggggctgtg gctgtggtgg cgcagccccg 2220cccagaaccc agaccttgca gttcgcattt cctcctctgt ccccacacat tgcccaagga 2280cgctccgttt caagttacag atttcttaaa actaccactt tgtgtgcagt tgaaggccct 2340tgggcacaat gagagccagt cctccaaact ttcagaaagt ttcctgcccc ttctggcagg 2400ctgccaatca ccgggcggga gaaggaagga ggggaaggcg gtggagggag cgagacaaag 2460ggatggtccc tcgggggcgg ggatggcggg gctgtcctgt aggtctgtgc ggccaccgtg 2520attgcccctc tgcgcggtgc ccgaagtccc gctgaaacct gccgagggca gcaggtctga 2580aagctgcagg cgctagttgc gcggaggtgg cgcagctgct ctggaggcgc agagcgaata 2640cgtggtgttt gggtgtggcc gccccgcccc tggcggtttc ctcgttcccc tttggttaat 2700gcgcaactgt ttcagattgc aggaggagat gcttcagaga gaggaagccg aaaacaccct 2760gcaatctttc agacaggttt gtagactctc ttcccactcg cagccgcctg accccaccca 2820acacaaccca cgagcaattc taaaagttgc ttaactcacg tctaaaaagt gcaaaacttc 2880agggctgcgc gtaaagccct ctagtggcgg gaagaccaca ggttggagct tctcatgatt 2940agaaaaatat taataaaacc ccttgagcga tttttttttt ttttttgaga cggagtctta 3000ctctgtcgcc caggctggag tgcagtggcg agatcttggc tcactgcatc ctccgcctcc 3060cgggttcaag cgatccttga atgatttcta agcagttcct tgggacataa agaaaaatct 3120tttaactttt tactttgttt cccaaatgtt gcacagtttt gcaacacatt gaccttctgg 3180tttcgaacgg ttacaatttt agattgtggt ttgccaaagt caagttgctt aatttttaca 3240aggccacaaa aagcgcaatt atgccctgca gtttaaaatg gaaaacgtgt tggaagataa 3300gaaaacttag tttccaactg gaatggagcc agcaagtttc ttttcttctt tgcaaattct 3360attgtgtcat taaagttcga tggaagtatc actatgcaca actattttgt gatctaataa 3420gggtgaaaag gagccatctg tccccttggc taaggggtat taatggtttc tatgggcttc 3480actatggaat gtagatacag acattctggc aaatgtggtg gctctggaca gaaataatag 3540gagtctttgt attcccaggg aagctttgca acaggctaca ttcttactga atatgtaatg 3600atgtaagcac ggttctaatt ggacacaagt atttgctaac atccgttatc taatatctgg 3660cccagacttg agaagtaggt aatgtaaaaa gtttttaaag ctacaagcat acctcacatt 3720ttaaaagtcc tttcttgatt gggttcttgt gttctttagc actcttgcca taaaaaataa 3780taacagtaat aaacccaagg ctgaaaaact gaattttaac taaagggttt ttgtgcgtgt 3840tttttttttt tttcaccaaa attagatgga cttacagaat ttttaactta aaattggaat 3900ccaaaagcca gaagatcccc attatagttt atagttgtat tccctggaat atttactggg 3960attaactgca aagcactctc agatgaatag tgtagtataa cattttgaaa ctgaaataca 4020tttaccaaat taatttaacc acagcaatgt gtgtggttca ttttagtcct tgagcatttt 4080tgattatcat acctgtcatg ttttctgcag tgtagtgagt taacataaaa caacatcaat 4140acaaaagatc ctctgtttcg agattaagca aaattcctca ttctcttcaa tgtgatagaa 4200taccacattg atctttcttt ggaggttagt aaaatatctt ttatgtattt ttcagggctt 4260aacaagtaaa aatcaatgtt ttcatcaagt ctgatctttt tgtcacccac tcttcattca 4320tttttccact aaggtgatag aaaagtctca acagtttaag accgtaaggc tatgaactcc 4380aaatataatt gctgacaaga taagcaatcc tcacgcatcc ttttgagagg aaataaaatc 4440ttagttgcaa gattacatat tctgatttgg aatgctgagc tttttaaatg gaaatataga 4500aggacggctg aatcagcaaa aatcctttat gtagtttcat tctttgcaag cttgaccagt 4560cattctgaaa caggctaact gaactgatac agtggcaagt gaaaaagaca tgcctttaca 4620ggatgagtca aaggagtttt agaagaaaaa tccaccagag aaagccaagc aaatacagtt 4680cagagttaca tttcttttcc attttttcct gaactgaatc tttggcatgc atatcctgaa 4740ttgggttatt gaatataaat ctagccttgt acaatggatg ccagatgact acatatttgc 4800tttggagcct aaggataagt ttcaaaagat ttgagtggag aagaaaagct aaaactcttg 4860aagcacaagt ttctgttctc catgtactca agtgtacatg aagttgtgaa aatttgtcca 4920cctctatcat catgttattc catgaaatta caaaacaaat cttaaaaatg ttgtggcata 4980gattttctag atttaaaaag taattaaatt aaatgaatta ctttattttt tgagacagag 5040tgtcactctg ttgcccaggc tggagtgcag tggcactatg ttggctcact gcaacctctg 5100cctcctgggt tgaagaaatt ctcctgcctc aacctcccaa gtagctggga ctacaggcat 5160gtgccaccac acccagctaa tttttgtatt tttggtagag acggggtttc gccatgttgg 5220ctaggctggt ctcgaactcc tgacctcaag tgatccaccc gtctcagcct cccaaagtgc 5280tgggattaca ggcataagcc accatgacca gccttaaaaa gtaattttaa aatatcactg 5340gtaaaatgtg gattcagtca tgattgagtg cagtttacca tgtgtgtgga catttattta 5400ttttaaaatt gtctgatcac caccttgagt aaaacacaag cagtcacaat taaaatatat 5460tagtgagcag gagaaagcac agcatattat agcactgaat gatttataaa cctattccag 5520ggtcataaaa tgtgtcaacg gcttttctat agtaaggaga ctaggttcag atggttaatc 5580taagacaaat aaatgagata agccatacac ttttacatcc tccatgtcct gtcttttctc 5640tgttcaaaat aggatgttga caatgcgtct ctggcacgtc ttgaccttga acgcaaagtg 5700gaatctttgc aagaagagat tgcctttttg aagaaactcc acgaagaggt tagtggagtg 5760actttcgggg aatgaatgag ggtaaggcag cccccacggt tggcagagct gaccgtctgt 5820ctgttctttt tgcaggaaat ccaggagctg caggctcaga ttcaggaaca gcatgtccaa 5880atcgatgtgg atgtttccaa gcctgacctc acggctgccc tgcgtgacgt acgtcagcaa 5940tatgaaagtg tggctgccaa gaacctgcag gaggcagaag aatggtacaa atccaaggta 6000ggaaacaaat cagtgcggct tcaaccaaag aaaagcattg tgttctcaaa accccatacc 6060tgtgtgtgat tcctaaatat cctctagctc caatgcaaag ctggctttga cttcttgctc 6120atattgtgtt tgccaccaca gcctccccac cactcacatc acctccttta tttatttatt 6180tattttctta tttatttatg agacagagtc ttgctctact gcccaggctg gagtgcagtg 6240gcaacatctt ggcacactgc aacctccgcc tcccaggttc aagtgattct cctgcctcag 6300ccccctaaga gctggaacca caggcaagca ccaccatgcc cggctaattt ttgtattttt 6360agtagagatg gggtttcacc atgttgacca ggcttctctc aaactcctga cctcaggtga 6420tccaccctcc tcagcctccc aaaatgttgg gattacaggc atgcgccacc acgcctggcc 6480acatcacctc cttcagaata gcagactctc ttccccctaa ccttgcctcc aagtaaaccc 6540caatgccata cctttgacct ccactgtgtt gaaatgagca ctgtagagtg aactctgaaa 6600atactaatgt cagtactcca ctgctctttc cctggctttc aaaacagaaa tttaaaccta 6660tactggaaga cattcagtga gaaatatgat tttttttttc taagagagtc aaaagacttg 6720aatgtgagca atctacattt ctgttttctt cccaacagtt tgctgacctc tctgaggctg 6780ccaaccggaa caatgacgcc ctgcgccagg caaagcagga gtccactgag taccggagac 6840aggtgcagtc cctcacctgt gaagtggatg cccttaaagg aaccgtgagt accaaccctg 6900cagtaaaaga gggaaaataa tgacccattc tgctgactag gctcatgatg atacctgaac 6960aaaatgttga gtgagtaaaa atgtatatca taatgcaaag aaaatgagtt atcaagacag 7020actcaaaagg gacttcatgg aactcttgaa ggttttagct tgcctatatc attgcttcta 7080atatgaagga cttggtactc gcattctcca cctaaaatta gagtggtcgc catttgccgc 7140taatggaaat tattgcagaa ggtctgtaaa tggttctggg aacagctggg tttttctgag 7200aaataacacc agacatcttt ctcaccccct gcagaatgag tccctggaac gccagatgcg 7260tgaaatggaa gagaactttg ccgttgaagc tgctaactac caagacacta ttggccgcct 7320gcaggatgag attcagaata tgaaggagga aatggctcgt caccttcgtg aataccaaga 7380cctgctcaat gttaagatgg cccttgacat tgagattgcc acctacagga agctgctgga 7440aggcgaggag agcaggtagg gaactcagac ttggatgcgt gaactaatgg tgaccatttg 7500ttaggccctg tgccactggg ctctaagcag tgtcacattt aatctttaga aagtttcttt 7560gaggtaactg ctttccactt tttgtagagg aggaatttga attgagagag agtaagtgac 7620ttgctgaaaa agggttaatc aacagcagag ctgggatttg aacccataac tctgtcaaag 7680cctccactcc taactcctgt tcatgctcct gtggagaaaa tgcttgtagt aacatatttt 7740aaatgtacta acaagaccag tcatgggaaa atgtttctga gacaaatctc tagtttatga 7800tttaaaacag tacgttttct tacgtgacga aaacaaaaag tgtgttaatt tgttcccagt 7860ggttgaagtt atttgccaac aattttactg tttctcttca tctgtttata ggatttctct 7920gcctcttcca aacttttcct ccctgaacct gaggggtaag cattttattt ccctttagga 7980aaaacgtcag ctgcttgtaa ccactgtgtt tatgtcaaag cattcatttt ttttaggata 8040tctgaaaaaa tgccatataa gagaaaactc tataaaacat ctataatttt cgaacccaag 8100tacactcttg cattctatgc tttaagttaa atgcaaactc ctttttcctt cttcctgctg 8160caagtactat ctcatcctga tgctcaagag tgtcagggcc tgggtttcca aacagagact 8220accctaaaat tatttggcga gtagtacttt acacaattgc ctctccccca caaatcataa 8280ttgtttcagt aaaatggtta cttggttttt ccaagaaaaa actcgttttt actcattttt 8340ggcctgtttg tttatttaga aactaatctg gattcactcc ctctggttga tacccactca 8400aaaaggacac ttctgattaa gacggttgaa actagagatg gacaggttgg tatcttttaa 8460ggaaaaaata gggtaatctc agacaggagt tgatatattt taaaatcagt gaatctgaat 8520ctcagataca gctggctaat ttgagaggtt caggtttcat tcatgcctac taaaaaaaga 8580ataggcttct tcttccagca gtacacacag ccaactaatt atttggctcc tggatgtgaa 8640gttgagatag cagtcttcct gtgctccaga attagtgatt tgctttggtg cttaatttga 8700agtgggagta agcttcctta aaccacttcc taaagcagct acatgaaaca gcttcactag 8760actacctcaa tatgaggaat gttttgatcc tggacatatg gtgtcttcct acctccatac 8820tttatagatt cctaaaccca tctatataat acaagcatgt gccatacgat catttagttt 8880cttattacct ccctatgcca ggaaagaaat agttgcaatt tattgtagtc atcatgaaat 8940cttcccttgc acataaattt aaaatgtacc tgctgcacat tttaatatgt cttaattgct 9000tttaaacttg gctgtattgt gtacaactat tataccatct tttataaaca cagtttttta 9060agaaatttct ttttgtaagt tacaacattc cactggatcc ttatattgcc tgtagtggaa 9120gagggtcttg tgtgtctgcc ccttctagtt ttcactcatg cagaagcaac ataaccttct 9180gatttgcaca ataaattaca tatatttagc aggattttta tttgccgtga tatataggat 9240aatttagtct ttggcatgtg gcattatatt tattttggtt ttttttttta aacaggttat 9300caacgaaact tctcagcatc acgatgacct tgaataaaaa ttgcacacac tcagtgcagc 9360aatatattac cagcaagaat aaaaaagaaa tccatatctt aaagaaacag ctttcaagtg 9420cctttctgca gtttttcagg agcgcaagat agatttggaa taggaataag ctctagttct 9480taacaaccga cactcctaca agatttagaa aaaagtttac aacataatct agtttacaga 9540aaaatcttgt gctagaatac tttttaaaag gtattttgaa taccattaaa actgcttttt 9600tttttccagc aagtatccaa ccaacttggt tctgcttcaa taaatctttg gaaaaactc 96592466PRTHomo sapiens 2Met Ser Thr Arg Ser Val Ser Ser Ser Ser Tyr Arg Arg Met Phe Gly1 5 10 15Gly Pro Gly Thr Ala Ser Arg Pro Ser Ser Ser Arg Ser Tyr Val Thr 20 25 30Thr Ser Thr Arg Thr Tyr Ser Leu Gly Ser Ala Leu Arg Pro Ser Thr 35 40 45Ser Arg Ser Leu Tyr Ala Ser Ser Pro Gly Gly Val Tyr Ala Thr Arg 50 55 60Ser Ser Ala Val Arg Leu Arg Ser Ser Val Pro Gly Val Arg Leu Leu65 70 75 80Gln Asp Ser Val Asp Phe Ser Leu Ala Asp Ala Ile Asn Thr Glu Phe 85 90 95Lys Asn Thr Arg Thr Asn Glu Lys Val Glu Leu Gln Glu Leu Asn Asp 100 105 110Arg Phe Ala Asn Tyr Ile Asp Lys Val Arg Phe Leu Glu Gln Gln Asn 115 120 125Lys Ile Leu Leu Ala Glu Leu Glu Gln Leu Lys Gly Gln Gly Lys Ser 130 135 140Arg Leu Gly Asp Leu Tyr Glu Glu Glu Met Arg Glu Leu Arg Arg Gln145 150 155 160Val Asp Gln Leu Thr Asn Asp Lys Ala Arg Val Glu Val Glu Arg Asp 165 170 175Asn Leu Ala Glu Asp Ile Met Arg Leu Arg Glu Lys Leu Gln Glu Glu 180 185 190Met Leu Gln Arg Glu Glu Ala Glu Asn Thr Leu Gln Ser Phe Arg Gln 195 200 205Asp Val Asp Asn Ala Ser Leu Ala Arg Leu Asp Leu Glu Arg Lys Val 210 215 220Glu Ser Leu Gln Glu Glu Ile Ala Phe Leu Lys Lys Leu His Glu Glu225 230 235 240Glu Ile Gln Glu Leu Gln Ala Gln Ile Gln Glu Gln His Val Gln Ile 245 250 255Asp Val Asp Val Ser Lys Pro Asp Leu Thr Ala Ala Leu Arg Asp Val 260 265 270Arg Gln Gln Tyr Glu Ser Val Ala Ala Lys Asn Leu Gln Glu Ala Glu 275 280 285Glu Trp Tyr Lys Ser Lys Phe Ala Asp Leu Ser Glu Ala Ala Asn Arg 290 295 300Asn Asn Asp Ala Leu Arg Gln Ala Lys Gln Glu Ser Thr Glu Tyr Arg305 310 315 320Arg Gln Val Gln Ser Leu Thr Cys Glu Val Asp Ala Leu Lys Gly Thr 325 330 335Asn Glu Ser Leu Glu Arg Gln Met Arg Glu Met Glu Glu Asn Phe Ala 340 345 350Val Glu Ala Ala Asn Tyr Gln Asp Thr Ile Gly Arg Leu Gln Asp Glu 355 360 365Ile Gln Asn Met Lys Glu Glu Met Ala Arg His Leu Arg Glu Tyr Gln 370 375 380Asp Leu Leu Asn Val Lys Met Ala Leu Asp Ile Glu Ile Ala Thr Tyr385 390 395 400Arg Lys Leu Leu Glu Gly Glu Glu Ser Arg Ile Ser Leu Pro Leu Pro 405 410 415Asn Phe Ser Ser Leu Asn Leu Arg Glu Thr Asn Leu Asp Ser Leu Pro 420 425 430Leu Val Asp Thr His Ser Lys Arg Thr Leu Leu Ile Lys Thr Val Glu 435 440 445Thr Arg Asp Gly Gln Val Ile Asn Glu Thr Ser Gln His His Asp Asp 450 455 460Leu Glu46538515DNAMus musculus 3ctctgccact cttgctccgg gaccccagag accccagcgc tcctacgatt cacagccacc 60gcgccctcat tcccttgttg cagtttttcc agccgcagca agccagccca ccttcgaagc 120catgtctacc aggtctgtgt cctcgtcctc ctaccgcagg atgttcggtg gctccggcac 180atcgagccgg cccagctcca accggagcta tgtgaccacg tccacacgca cctacagtct 240gggcagcgca ctgcgcccca gcactagccg cagcctctat tcctcatccc ccggtggcgc 300ctatgtgacc cggtcctcgg cagtgcgcct gcggagcagc gtgccgggcg tgcggctgct 360tcaagactcg gtggacttct cgctggccga cgccatcaac actgagttca agaacacccg 420caccaacgag aaggtagaac tgcaggagct gaatgaccgc tttgccaact acatcgacaa 480ggtgcgcttc ctcgagcagc agaacaaaat cctgctggct gagctcgagc agctcaaggg 540ccagggcaag tcgcgcctgg gcgacctgta cgaggaggag atgcgggagc tgcgccggca 600ggtggatcag ctcaccaacg acaaggcccg tgtcgaggtg gagcgggaca acctggccga 660ggacatcatg cggctgcgag agaagtaagg cctgacctac gcgagtggtg gggtgtggga 720gggaggggag tcctgggcct tgcactgggg ctgccacgcc cttggggatg tggctggggt 780ggcggttgtc tggaggatag cagaggaagc gggccccacc ctgaacctaa actttgcagt 840gaacatttct cttttcgcct cactcaagca cttccattga actttttaga aagtttctta 900ggtccttctt ctgggcttac agttaccaga caggaggaaa gaatgggtgt gaagggttcc 960agacaaagag atggcaaagg catctgggat attgggacag gggattggct gtaattgtaa 1020gggaagggca gtgattgtct tttccagtgt gccccaactc cagctaacac ctgcagagag 1080ctgccacctc gacaagttgc agtaactact gtgggtggag gtgtcacagc agctctggac 1140tcaaggcttg gcggcatttg tggttgggtg tggccgcccc gcccctggcg gttttccagt 1200tcccctttga ttaatgtgct gctgcttcag attgcaggag gagatgctcc agagagagga 1260agccgaaagc accctgcagt cattcagaca ggtttgtaga cttctttttc acatgcagcc 1320agcagcccaa tggagccaac ccgcgagcgg ttctaaaagt tacttccctt aggttttaaa 1380aggtgcagaa cttcacgtct gcttgtaaag ccccctggcg gcggaaagcg cacagattga 1440aaaactccac tatttagggc agtctttgga acatgaaggc tttattttct gcttcgtttc 1500cagatttttc tgatccccca cacccccgaa acatttctcc tttcgttttt tgaaacatat 1560cgacattcgg atattgaatg gttacaattt gattacattg tggattgtga aggtgaaatt 1620ggctaacttt cacaaggtca taaaaaccac agttgcttcc tccagttgga aatgtagaat 1680actctggagg atgagaaagt ttaactggaa ttaagccaag ttccctttct tccttcgaag 1740ttcggcactg cgtgaaagtt agcagaagag tcattgcaca accatttttg aaatctaaca 1800agggtgaaaa gaagccatct gttcccgtag ctcaagggtg ttaaaggttt ctatgggctt 1860cactatggaa tgtagatagg gacgttctgg caaatgcggt ggctctggac agaaagaata 1920ggggcctttg tgttcccaga gcagctttgc aacaggctac attcttattg aatatgtaat 1980gatgtaacca cagttccaat tggacacaag tatttgctaa catccattat cttgtgtctg 2040ggccaggctt gtgaggtatg ccgtgtacaa agttcttaaa gctaaagtcc tccctcacat 2100ttggaaaggc ccccttttag gggggtttgt agactcatta gcaccctcca caaacaaaat 2160taaaccctaa actgaagaat ggagtatttt atggagggag gatttgtttg tttgtgtttg 2220ttccagggtt aggcattacc ttagcatccc gaagtggaca gatggccagt tcagtcttgt 2280ttggaacact tgttaggatc aacagccaaa ctctccagat gaatgttcta aggtagcatt 2340ctgaaggcat ttgcatcctt aatggcagta gacgaatatg cagccccttt cagtcactgg 2400gcattttcag tcatcaccaa aggaatgttt tctacaaagc actgagttaa tacacaccat 2460ctcttatttg tggactaaaa attgtttcat tcattcattt aaatgaaatg acacatttag

2520ctgaactttt tcgagaaggt tagtccaata tcatttatat attttttgat ggcttagcag 2580aagtcagaaa tgaatgatac tactatttaa gttcttatat tcttttttaa attagtattt 2640tctttgggac tgatagagta actcaacatt atagttataa actatactgc aaatacagtt 2700gcttacaagg gaagtttcca aacatttcct tgaaaggaaa caacatttca catgcaagat 2760tgaacagtct gtcttaaaat agatcacttt ataaaaactc tgcagagaag tttaagtata 2820gtttcattgt aagtgttttg atctgtcatc ctaaaacaga caaagtcatt gaaataacac 2880tgtgacagct gggacaggca gaaataccct ataggctgag ccagggtgtt ttacacatta 2940aaaaaattag agaaggcaaa acaatatagt tcaggattat atttcttttc cttcttgccc 3000gaggagcata tctctgcaaa tgcaagacct ggattttgaa caatgaatta ctacttgata 3060cgaggctagc ctgggaacct ggatgccaga taactgagtg cttattctaa attccaaaga 3120taagcatctg aaggtttgaa tacaggaaac aaaaaagagc atgcatatat gttaaccctt 3180ctgagctcaa ttttaaatga tttttaaaaa atatgtaatg tgtttgtgtc tgtgtgagtg 3240tataggacat ttctgcaggc ttgcatgagg ccagaagagg gcacctgatg ccctggagtt 3300ggaatcataa accttcatga actgctccat gtgggtgctg ggaaccaaac taaaatcctc 3360tgaaagaaca gcattcttta ctactaagcc tcactttaga aaacatttta attttatttt 3420tgaagctctc atttttatat tacgtgtact caagcaaaca tgaagttgga caatctcatc 3480aactactatc atcacaaaat tacagactta ttaaatgtga cagaggttta aaactgaaaa 3540tgaatttttc catatagttc accataccat gtcattgtgc atatacatgt gtttattttt 3600aacttgtcta ctagttagct tcaatacaac caaatggttt cctattcatt tttaaagcta 3660agaatgaata ttggaatgtg accctaaatg atttataaat ttatcaaggg tcatgaaatt 3720ttcagcacct tttctacaat aaggagaatg ggtttacatg gttaattttt gaacaaataa 3780ataaataaat aaataaataa ataaataaat aaataagggg ctgtagagac aggtcaatgg 3840ttaaaagcac tggctactct tttagggcac ctggaattca atttgcagaa cccacagggc 3900tgcccacact cttctccagt tccagggcat cttttgctct cttattgctt ttgtggatac 3960cacatgctcg tggcatactt atatacatgc aggcaaaaca cctttataca caacataaag 4020tgaaaaaggg attaatataa ataagttaga taaaggacag actttatcac cctccacatc 4080atcttttctc ttttcgaaat aggatgttga caatgcttct ctggcacgtc ttgaccttga 4140acggaaagtg gaatccttgc aggaagaaat tgcctttttg aagaaactgc acgatgaagt 4200aagtgacacc aatttctgtg ggtgaacaaa gctgcgaccg tgtagtctac gacctctctg 4260tgtctgttct ttctccagga gatccaggag ctgcaggccc agattcagga acagcatgtc 4320cagatcgatg tggacgtttc caagcctgac ctcactgctg ccctgcgtga tgtgcgccag 4380cagtatgaaa gcgtggctgc caagaacctc caggaggccg aggaatggta caagtccaag 4440gtatgaaatg agccagagtg gcaagaacct ctagctctca gacacgtgcc tctaattcca 4500ttagtaacct ccagctctac tcagaagctg gcttaaagct tggaccgcca ttgttgtttc 4560acagcacccc ttccccctca tctccattca cagtttcctt tagtatggca gactcccttt 4620ccactcattg tcttccctga cttcaaagaa taccttactc tcatgctctt gactgccacc 4680acttggaaag cattgcagta gagcaaatac tgtttgaaaa cgcaatgctg gtgttctgct 4740tctctttccc tggttttcca aaagacgttc agacaaattc taggaaacac acaggcagga 4800tgaagatgcc tctttgttct aagtgagtga aaactcatgg aagtgaacaa tctctacttc 4860ttttcttttc ttttcttttc ttttcttttc ttttcttttc ttttcttttc ttttctttct 4920cctcctcctc ctccttctcc tcctcctcct cctcctcctc ttcttcttct tctacttctt 4980cttcttcttc ttcttcttct tcttcttctt cttcttcttc ttcttcttct tcttcttctt 5040cttcttcttc ttcttcttcg actaagctca caaccaaaaa caacctctat ttctgattcc 5100ttcttgacag tttgctgacc tctctgaggc tgccaaccgg aacaacgatg ccctgcgcca 5160ggccaagcag gagtcaaacg agtaccggag acaggtgcag tcactcacct gtgaagtgga 5220tgcccttaaa ggcactgtga gtactacagc agcaaagggg gggaaatacc gtgctcagta 5280ctattctgct gactaggtta acaatgagac ctgtacaaat aaattaaaca cacacggaca 5340cacacacaca cacttatcca aagagaatga attatcaaga tagttttaag gattatttaa 5400tgagtacctt gaaagtctta gtttacatat gttattgatt gtaataagag gggttttaga 5460ggttactcct gtaacctcat cactctggag aatgaggcag ggggattata aattcacagt 5520taagctaggc tgcatgtaaa agtgttattt catcaaagca aaacatggaa ctgggtgctt 5580gtgttctctg ttcaaagcta gtgggcacct accattaaaa gaattactgt accttattta 5640tgaatatttt gggggacagc cgggttttct gggaaatcac atgagaagcc atttttgctt 5700tctgcagaac gagtccctgg agcgccagat gcgtgagatg gaagagaatt ttgcccttga 5760agctgctaac taccaggaca ctattggccg cctgcaggat gagatccaaa acatgaagga 5820agagatggct cgtcaccttc gtgaatacca agatctgctc aatgttaaga tggccctgga 5880cattgagatc gccacctaca ggaagctgct ggaaggcgag gagagcaggt atagaaggca 5940aacgtgcatg tgtgaactaa ctcacagtcg tctttctagg aagatctcat ttaacccaga 6000caccatatat atgaggaact taacgttgca gagagtaact tgctgtggtc agagataatt 6060aatagcagag tagatttgaa ctcacagcca tgcaaaagac aatacgttct ccctctataa 6120attcttctgg ttcatgtttc tgtgtggaaa aaaatatctt ccgaaattta aatgggccaa 6180tgagaccaag catgggtaga aagttcttaa acaagtcact aacagcaata gattttcatt 6240tggtggaaac aagaactgcg aaaacttgtt cctgcttctt gaagtgcttt gttctcaagt 6300tttttgtgtg tttctttctc tttttatagg atttctctgc ctctgccaac cttttcttcc 6360ctgaacctga gaggtaagta cattgtttca tcttatggga aaaaaatgtc acaaggctgt 6420caccagtcac tctgcctgta gcatagcttc cacattttta aaaatatttt ttattacgta 6480ttttcctcaa ttacatttcc aatgctatcc caaaagtccc ccataccctc ccccccactc 6540ctctacccac ccactcccac agtaattctt gattgaactg tgcttttcca aagaaaattc 6600aatgaaacat gatttctctt ttataaatac atgtgtatag ccattttatt ttctgaatat 6660ttcttttggg cttctgaggt tctgatagag ttaagtcact ttctcgcttc cgtttcttct 6720ctcgaaacct tccatgtact gccccctgac atatatggct gtcttttcag ttcatggctt 6780ccttttgttt aaatgctgtt acacacacac acacacacac acacacacac acacacacac 6840acactgtgta ttcttacata catgagtaca atggctcatt ctgtataata ttacttacat 6900gtatgctttc aggctcgacc atctggtccc ttttttatta tagttctcat gactgctgga 6960gaccaaaccc agggcaatca gctaagctaa acccacagag ctccacccct agcctgatac 7020cctcagtttc aaactcgagt acattcttgc ctttgctacc ctgttaagta tagactcagt 7080ctacttcctg ctaatggcaa agtcagtgaa ctactacctc agcctgaatg aaggagccct 7140taggtgctgt gtactcaaat ggatagtgct atgtaggatt atgtatgagt agcactttaa 7200ataaccataa aaataaagtc atgatgcttt gggtaaaatg gtaatgtttt aaactttttc 7260ttatgttact gccttttttt tttttttttt tttttaattc agaaactaac ctggagtcac 7320ttcctctggt tgacacccac tcaaaaagaa cactcctgat taagacggtt gagaccagag 7380atggacaggt cagtattttt tagttaaaat atgggaaagt ttaaaagaag gaatgatgaa 7440taaactatac aaccacttta aatatgtgtt ggtccccttt aagtcagcaa gcagatatat 7500cagatatggc agctaactta aggagtcccc gtggatactg gatgaatagc ccccaattcc 7560accaatgcaa gaaccaacag tctatttggt cttgattagg acagtaaact tcctgcacac 7620catagtctgt gcactaaaca ggagtaaaac acccttaagg catctcttac attggctgca 7680ttaaagtgtg tcacttaatg acatccgtga tatcagggat agtttgacat tggacataca 7740gttctctctc agtgattgtc acctgcttat atagtacgag gacccatatg tggtcaatca 7800ctttcttgtt gccactccat gccagggaag acatagttac aatgagttat aattataaaa 7860gctcagcctt gcatgaggat ataaaataca tttgccatac ctctacatat gccttagttg 7920cttttaagct ttgtttggaa ttgcaaaggc caacttccta aacacagttt caaagtattt 7980tttgtcacac ctttccactg gatacttgta ttgtctgcag gaagaggcca ccctgcctat 8040gcttctcctt agggcctcgt tctgcagagt cagcataaac tcccagtctg ctcaaccaat 8100tctgtctgat tagggccata tctccttacc atgagttgat ttttgtttct tttttgagca 8160ggtgatcaat gagacttctc agcatcacga tgaccttgaa taaaaattgc acacacttgg 8220tgcaacagtg cagtaccagc aagaaggaaa aaaaaatcgt atcttaggaa aacagctttc 8280aagtgccttt actgcagttt ttcaggagcg caagatagat ttggaataga aagaagctca 8340gcacttaaca actgacaccc caaaagacgt agaaaaggtt tacaaaataa tctagtttac 8400gaagaaatct tgtgctagaa tactttttaa agtatttttg aataccatta aaactgcttt 8460tttccagtaa atatctgacc aacttgttac tgcttcaata aatcttcaaa aatac 85154466PRTMus musculus 4Met Ser Thr Arg Ser Val Ser Ser Ser Ser Tyr Arg Arg Met Phe Gly1 5 10 15Gly Ser Gly Thr Ser Ser Arg Pro Ser Ser Asn Arg Ser Tyr Val Thr 20 25 30Thr Ser Thr Arg Thr Tyr Ser Leu Gly Ser Ala Leu Arg Pro Ser Thr 35 40 45Ser Arg Ser Leu Tyr Ser Ser Ser Pro Gly Gly Ala Tyr Val Thr Arg 50 55 60Ser Ser Ala Val Arg Leu Arg Ser Ser Val Pro Gly Val Arg Leu Leu65 70 75 80Gln Asp Ser Val Asp Phe Ser Leu Ala Asp Ala Ile Asn Thr Glu Phe 85 90 95Lys Asn Thr Arg Thr Asn Glu Lys Val Glu Leu Gln Glu Leu Asn Asp 100 105 110Arg Phe Ala Asn Tyr Ile Asp Lys Val Arg Phe Leu Glu Gln Gln Asn 115 120 125Lys Ile Leu Leu Ala Glu Leu Glu Gln Leu Lys Gly Gln Gly Lys Ser 130 135 140Arg Leu Gly Asp Leu Tyr Glu Glu Glu Met Arg Glu Leu Arg Arg Gln145 150 155 160Val Asp Gln Leu Thr Asn Asp Lys Ala Arg Val Glu Val Glu Arg Asp 165 170 175Asn Leu Ala Glu Asp Ile Met Arg Leu Arg Glu Lys Leu Gln Glu Glu 180 185 190Met Leu Gln Arg Glu Glu Ala Glu Ser Thr Leu Gln Ser Phe Arg Gln 195 200 205Asp Val Asp Asn Ala Ser Leu Ala Arg Leu Asp Leu Glu Arg Lys Val 210 215 220Glu Ser Leu Gln Glu Glu Ile Ala Phe Leu Lys Lys Leu His Asp Glu225 230 235 240Glu Ile Gln Glu Leu Gln Ala Gln Ile Gln Glu Gln His Val Gln Ile 245 250 255Asp Val Asp Val Ser Lys Pro Asp Leu Thr Ala Ala Leu Arg Asp Val 260 265 270Arg Gln Gln Tyr Glu Ser Val Ala Ala Lys Asn Leu Gln Glu Ala Glu 275 280 285Glu Trp Tyr Lys Ser Lys Phe Ala Asp Leu Ser Glu Ala Ala Asn Arg 290 295 300Asn Asn Asp Ala Leu Arg Gln Ala Lys Gln Glu Ser Asn Glu Tyr Arg305 310 315 320Arg Gln Val Gln Ser Leu Thr Cys Glu Val Asp Ala Leu Lys Gly Thr 325 330 335Asn Glu Ser Leu Glu Arg Gln Met Arg Glu Met Glu Glu Asn Phe Ala 340 345 350Leu Glu Ala Ala Asn Tyr Gln Asp Thr Ile Gly Arg Leu Gln Asp Glu 355 360 365Ile Gln Asn Met Lys Glu Glu Met Ala Arg His Leu Arg Glu Tyr Gln 370 375 380Asp Leu Leu Asn Val Lys Met Ala Leu Asp Ile Glu Ile Ala Thr Tyr385 390 395 400Arg Lys Leu Leu Glu Gly Glu Glu Ser Arg Ile Ser Leu Pro Leu Pro 405 410 415Thr Phe Ser Ser Leu Asn Leu Arg Glu Thr Asn Leu Glu Ser Leu Pro 420 425 430Leu Val Asp Thr His Ser Lys Arg Thr Leu Leu Ile Lys Thr Val Glu 435 440 445Thr Arg Asp Gly Gln Val Ile Asn Glu Thr Ser Gln His His Asp Asp 450 455 460Leu Glu465512566DNAHomo sapiens 5gggcgggcag gtgggactcg gcccccctcc cacaaccccg ctcccgggca agctctcgag 60ccgcgaggcc ggggcgggga ggggccgggc cgggggcggc ctggcaggaa gcggcgcgca 120ccttccgccg ccggaggagc aggtggctgc cgtgcgggtc tgggccccag gcttcctgtg 180tgcgcgctcg tcctctgctg tttcccgccg gagctcgttg ggcctccccg gcccgcccgg 240cccatggccc agacccccga cggcatctcc tgtgagctcc gaggtaagcg ctggcccttc 300ctgccttctt ggccgggagg aagtagtgca gccccaaaat cagaggccca cccagcgccc 360cagagggcct ggtcagaggt cctccctgct ccccaggagg gcaggcgccc cagatcctct 420cccctccttc ctcgctggcc gcagataagc cccagggccc cagcctggca cagactgtgg 480gcaccagtgg cttctgtgca aggactgaag tcacccagca ggctgccctt ccacaggcga 540gatcaccagg ttcctgtggc ccaaagaggt ggagctgctg ctgaaaacct ggctacccgg 600ggagggtgct gtgcaaaacc atgtcctggt atggggcagg ggacagagcc ggggaggcgg 660ctgtggcccc acagaaagag cctcttgctc tgcccaggat atcagactgt ctttccccag 720gcactgctac gatggagagc ctacctgctg cacaccacct gcctcccgct gagggtgagt 780cccagggcct ggccacaccc ccgcccgcca gcagctacct tggcaggggg ctgcatctgc 840agagtggtcc ttcttggcct tgtgggccct gactttgcac cagcactggg ctctgggcag 900ccgacaggag gggagtccag gcagatctgg ctctgcccca ggctgcccaa aggactggac 960ttccatgagg gcggggctgg gggcttggtc ccagctgatc taggcccttt ctaggtggac 1020tgcacgttca gctacctgga ggtccaggcc atggcgctgc aggagacacc ccctcaggtg 1080agacacctag taccctacct gggcctgcag cctggtcttc atgctaccac catcacctgc 1140gcccatgtgt ggagccgagg cctagtagtg ccccttccct aggtcacctt tgagctggag 1200tccctgcgtg agctggtcct ggagtttcct ggtgtggccg ccctggaaca gctggcccag 1260cacgtggctg cagccatcaa gaaggtcttc cctcgctcga cccttgggtg aggcctggca 1320aattcgaggg gctggcaggg gaggagggag tgcatgagaa gggctgcttc ccatcccaga 1380ggctggaagt cctttgcccc cttctcctta accccctacc aggaagctat tccggaggcc 1440cacaccagcc tccatgctgg ctcggctgga gagaagcagc ccctcggagt ccactgaccc 1500ctgcagcccc tgtggtaagg gtgaaggcag agccacaggc cttccagcct gccccacctc 1560tgcctcccca gacccgcaag ctgggggggc cccaaactga acagagccat tcaggtccca 1620gccaccacct agtctgtggg tctggtcttc ctccatcgct gatgttttgt ctctctcctt 1680ttccacatcg cacccctatc ccctccccag gtggcttctt ggagacatac gaggctctgt 1740gtgactacaa tggcttccct ttccgagagg agattcagtg ggtgagggta gggccctttt 1800gaagggctct ggagacatct ggtcccccat gtgtgctgag cccctccctg ccccttgtgg 1860ccccaggcga actgtaagca tctccttctc acccaggacg tggacaccat ttaccatcgc 1920cagggctgcc gccatttcag cctgggagac ttcagccacc tcggcagtcg gtgtgtggcc 1980tgccaggatg ggagaggagg aagatcccgg ggcccatatc cctgggcctc agtttctcca 2040tggaggggct gctgggcctg ggacctggct ggagggccct gagctctgcc tctgttccca 2100ccctcccacc agggacctgg ccttgagtgt ggctgccctg tcctacaacc tgtggttccg 2160gtgcctctcc tgtgtggaca tgaagctggt gagggggttc ggggtaaggg cagggagggg 2220ccaagggtgt gggccagggt gcagcccgct gagggccagg gtgcagcccg tgagccgccg 2280ccctctgctt ctcagagcct tgaggtctca gaacagattc tgcacatgat gagtcagtca 2340tcacacctgg aggagctggt gctggagacc tgcagcctga gggggtgagg gggacagggc 2400agggcttgga gaggagagtc tggaggcttg ggactggggg ctagtggcct gggaggggtt 2460ggcaaaccag gggcagaatc tcatgcctgg ggtctggtcc ccagagactt tgtccgacga 2520ctggcccagg cgctggcggg acactcaagc tctgggctgc gggagctcag cctcgcgggg 2580aacctgctgg atgaccgagg tatgactgag cctggggacc gcaggggtgg gcagcaggag 2640gaggtgagac ccacgtggct gttcccaccc cccaaggcat gactgcactc agcagacacc 2700tcgagcgttg tccaggagcc ctgaggagac tcagcctggc ccagacaggg ttgacaccgc 2760gaggtaggct ggatgaggga gggggtgagg ggaggggggt gggggtctgt ccaactgctg 2820agtgaccccg acccaccacc aggaatgagg gctctgggcc gggcactggc caccaatgcc 2880gccttcgact ccaccctgac ccacctggac ctttctggga atcctggggc gctgggggcc 2940tccgaggaca gtggggtgag tggctgtctt cagggtggga gcttggggtt gctcataagc 3000cctgggtagg cgatccccca ctccatcgca cccctgtcct ccctccaggg cctctatagc 3060ttcctgagcc gtcctaacgt actgtcgttc ctgaatctcg caggcaccga cactgccctg 3120gacactgtga gggggtgctc cgtgggggga tggatgaccg gcagggcgga ctggagggcg 3180ggacggggag ggctcggtcc ccccgcgggt gtagccaaca gcctcccccc gcagctcttc 3240gcagcggtat cccgaggctg ctgcaccagc cttacccacc tcgacgcttc gaggaacgtc 3300ttctcccgca cgtaaggggg acctgtcggg gccgggggag gctgctggaa gccgcctcct 3360tgcggcccca ggcccacctt cccacttccc ccaggaagtc ccgcgccgcg ccggccgcgc 3420tgcagctctt cctcagccgc gcgcggacgc tgcggcacct gggcctggcg ggctgcaagc 3480tgccgcccga cgcgctcagg tcagtgtcgg accccggcca cgcccccgcg ggcgctccca 3540ccctgccctg gccttcgccc ctccccgctc ctgcttctgt cgctcccaca acctccccca 3600gatcctggcc ctgcctcctt cgttcgcacc ctggagcccc ctgtcccagc tcccgccacc 3660ccgtgtaacg tcctctccca gagccctttt ggatggcctc gcgctcaaca cgcacctccg 3720cgacctgcac ctggacctca gcgcttgcga ggtgagcgcc ggcccccaga agagaccaca 3780cattgggaga ggcgctggga ggcggaaggg cagggccgtg ggccgcctgc ccctccccac 3840tcgcggccta agtgggtccc acttcccacc tcccacctcc cacatacagc tgcgctcggc 3900cggcgcccag gtgatacaag acttagtgtg cgacgcaggc gctgtgagct ccctggatct 3960ggcggataac ggtgaggctg caggagagcc catcctcgca tcatccactc gattcccaat 4020ccccacccta cccttgcaac ttcgcctcgt gcgtgacccg agtcaccccc aggcttcggc 4080tcagacatgg tgactctggt gctggccatc gggagaagcc ggtccctgag acatgtggcg 4140cttggaagga acttcaacgt ccggtgcaag tgagccccca ccctactcct gggcctccca 4200gacaacaccc caccacccct gtcccccaca actgcggccc ctgcccacag ggagaccctg 4260gacgacgtcc tgcaccggat tgtccagctc atgcaggacg acgattgtgt gagttcacgg 4320gaccttgcag ggcctcgggc aattagacca ctttggtcct cctttctctt gttccctcag 4380accctgtgac ctgccctcac tgacccctga ctccagccat caatggcttt ctcttaaccc 4440cagcctcttc agtctctgtc ggtggctgag tctcggctga agctgggtgc cagcgtccta 4500ctccgggccc tagccaccaa tcctaacctg accgcgctgg atatcagcgg caacgccatg 4560ggggacgcgg gcgccaagtt gctggccaag gcgctgcggg tcaactcgag gctccggtgg 4620gcggggtcag aggggtggga ccagcgggca gggggcgcgg tggagaggag ggcaccgggc 4680taaggggagg gactgaatga ggcggagcaa atggagcagg ctgacgaggc gaatggacta 4740ggccgagggt tgggtggggc gttgggaagc tccgtccccg actgaagcca ggcccggccc 4800aggtctgtgg tctgggaccg gaaccacaca tctgctttgg gtctgctgga cgtggcgcag 4860gcgctggagc agaaccacag cctgaaggcc atgcctctgc cactgaacga cgtggcccag 4920gcgcagcgca gccgcccgga actgacagca cgtgcagtcc atcaggtggg cgtccccctc 4980ttcccttgcc cttctctgca cggtaactcc gtccctcggc atttctcaat accccttgcc 5040cctagatcca agcctgtctc ttgaggaaca accgcgcaga ccctgcctct tctgaccaca 5100cgacccgcct tcagccactt ggtctggtct cagacccctc agagcaggtc aatgtcccct 5160ccccaaccac gagaggtagg gcatgaggag acctgtggca tccgggagcc tccatgagtc 5220agagggtctg actttcctgg ggccagggtg cctgagccct gctgtcccac acaaagtccc 5280tctccttgat ctggaggcgg ctaaacaccc cttaaatagc tctccaaata acacccttgg 5340ctgcctaacg ttaagcctgt aaacaggctg tcctgttaga gaggaatttt tatgtccttc 5400ctcaattttc ccccaattgt agccccatct ctgtcctcac atatacgtga ctcccctccc 5460ctctccattg tgtgcccaaa cccagctgct ccttcactgt cttcatctcc ctccactacc 5520ttatctgttg gagatgtttc tacctctcca ttcccaaata cctcttcttt cctaagcttc 5580agagcccaaa ggtcttaatc cagacatcct gctgggcttc atgctctgca cttctccaag 5640tgatgtcatc gtcgctcaca aaccctgttg tctaccctgg agcccaatcc cactggtgcc 5700cacagccacc cagtgctgtg gacagaggtt ggggcaccct tccaggtctc tcctggtcac 5760actggagccc tcataggcct ccttcgtcct gcctctagac caaccttcat acatgccact 5820ccttaccccc acaccaatta tcccttgctc ctcctggtcc ctcctccagg gctactcact 5880cccttttcta cccctctctc agtctatcct ctgtgtggac agactgagct tctaaaacac 5940agacatgatt tacaactcct ttccttccta ttctgggcac ttaggaagtg tatataaagt 6000ccttaaaatg aaccaaagca acagatagtt ccttccctcc ttgaacagat agggttccaa 6060agtggctggt ctaagggtgt cagcttcagg acctccctct gttaaagagc ctgggaggaa 6120ggcaggcagg atctgggaga ctgggagggg gctaggctga gactgatccc catttcgccc 6180caggaagtga

atgaattgtg tcagtcggtg caggagcatg tggagctgct gggctgtggg 6240gctgggcccc agggtgaagc cgctgtgcgc caggccgagg atgccatcca aaatgccaac 6300ttctctctca gcgtgagcac tccccctcct gctacaagga cccttcccct cttagtcagg 6360tgtcagctcg caactgcttt tcctctttgg ccctcagatt ctccccattc tatatgaagc 6420tggaagctcc ccaagccatc actggcagct tgggcagaag ctggagggcc ttctgagaca 6480ggtgggcgag gtctgccgcc aggacatcca ggtgagaggg tactcctgcc ccaaccccac 6540ctccgtttgc aggtttgact cccatccttt agttttaact gtgatatccc ctgactcaat 6600attctgttgg agttggagcc tcaagccagc agcctggtgt ctggccacat cctcaccatg 6660ctctgactga cctttgtcta ggacttcact caggccacac tggacacagc aaggagcctc 6720tgcccacaga tgctgcaggg atccagctgg agggagcagc tagagggggt cctggcaggc 6780tcgaggggcc tcccggagct gctcccagag cagctgctgc aagatgcctt cactaggctc 6840aggtaggctg gatggggctg ggctgggcaa ggctgagcaa agccagccca ttaacccctg 6900tcctctcctg cccttaggga catgcggcta tcaatcacgg ggaccttggc agagagcatt 6960gtggctcagg ctttggcagg cctgagtgca gcccgggatc agctggtgag ggggagatgt 7020gcacacactt tcgtgcaaac acacacatgc agtgacttgg ccatctccct tgcaggggct 7080ctgtaatgtc ttctggggtc atgtagccca gggctatttc agggtcccct tagttagcat 7140caatgggatc atggctgagg ccctacctgc catctttggc tgcttggtat tgcaggtgga 7200gagtctggct cagcaggcaa cagtgacaat gccccctgcc ctaccagcac cggatggagg 7260tgagcccagc ctccttgagc ctggggaatt ggaaggtctt ttcttccccg aggagaagga 7320agaggagaag gagaaggtaa gtggttttag aacacggggc atggcactcc cagtcttccc 7380atcttgctgt ggagtgtgga aggtgaaagg cagctctttt gggttggtgc tctcctaccc 7440caggctgagt ttgtgccctc cccaccagga tgacagtcct ccacagaaat ggcctgagct 7500cagccacggt cttcacctgg tccccttcat tcacagtaag tcagggcctt gggggaggga 7560gtttacgtgg gtgggctgaa gctccattag acttggggac ccggggacct ggatgaactc 7620attcagcctt gtctgggtac ccaccagccc ttgactgggc caggtcgggc cccttgtgca 7680acctgcaaag ctggggtctg ctgtggtcag cccggggcgg gacctgggcg gatctgggac 7740aggacaccgg ctcagctcgc ctccccgcgc ccctttccct accccaggga cgcgcatgct 7800gggcggcggc gcggagccgc gcgcgctcgg ggccgcgctc agcaccacgg acagcggcgg 7860cggcggggga ggggcggagg ggcagagggg cggggggacg ggcggcgtag ccgggcccct 7920ccccgcgccc tgggcgggtc ccccgccgcc ctagcgcctc cgccgccacc tccccgggct 7980cggcgctcgg tgctcttctg gtgctgtccc ctcaggtgct gctgaggaag cggagccgga 8040gcccgagctg gcggctccgg gagaagatgc agagccgcag gcggggccgt ccgcgcgcgg 8100ctctccgagc cctgccgccc ctgggccccc ggccggcccg ctgccccgca tggacctgcc 8160actggcgggg cagcccctgc gccatccgac ccgggcccgg ccgcggccgc gccgccagca 8220ccaccaccgc ccgccgccgg ggggccccca ggtgagcacc cttcccccac tccggagcgc 8280gtggaattgg ggatcacggg tggcccggcc gctcctcatg ggtggtagcg gtcaaggaga 8340gggggtggtg ggggcccaag gccacctggt cgtgcggccg cggtcacatc ctggcttctt 8400cctgaccacc ccccacccca ggccatgcct gtgcgggacc atgggtgtgc gggttccgtt 8460cggggtgtgc ctgggtgtgg gactccgtct cggggcggcc cgcggcctcc gtggctgcgc 8520gagagagggt gtccgtcctc cctccctccc ccggggctgc tgagagatgc cgggcagccg 8580ggtagccgag ccgcggggcc aagcctgcgt ctgctgcgcg tccggcggcg gcgcgtgtgg 8640ggagatgcgt gtgtgggctg caagcccgcg ggggcagcgg gcactggcgg agggcgggga 8700ggagccggct tgaggccccc cagagggtct gacgcagcag ccggcgccac tgagccggca 8760gcaggccggg tggagtgggg gtggggtggg ggacactgca gggaactgtt cggagcagag 8820ctggtggcaa gtgggaacgg gtgaccccgg gggcacgtga gggctaggtt tgctggtgac 8880cgggtggctc gggcgtgtag gatactctgt gggacaagga cgggtgtgga ctgggtgtgc 8940gggagccagc agcgagtgag gagtgcgtga aatctggagt ctctgtccag cagcaggaca 9000gtaggaggct ggtatcagca gcccctaggg tcaccccagt ctggaatcct ggagttattc 9060agtccaggct gccggccggg gggggggggg ggtagaagcc agagttgcac tcaaccctga 9120cccctgaccc catgatgccc cccaggtacc cccagccttg ccgcaggaag ggaatgggct 9180cagtgcccgc gtggacgagg gcgtggagga attcttctcc aaaaggctga tccagcagga 9240tcgcctgtga gtgaggggca tctgctgggg gtgggagagg gtgggatgcc tgatgggctt 9300tctcgctcac tgctgggtgt ccccacagct gggcccccga ggaggacccg gccactgagg 9360ggggcgccac tcctgtcccc cgtacactgc gaaagaagct gggcaccctc tttgccttca 9420agaagcctcg ttcaacgcgg ggtccacgga ctgatctaga gaccagccct ggggcagctc 9480cccgaacccg aaaaactaca tttggcgacc tactgcggcc gccaacccgt cccagccgtg 9540gtgaggagct tggtggggct gagggggaca ccagcagccc tgaccctgcc ggcaggagcc 9600gacctcgcta cacaagagat agcaaggcct actcgatgat actgctgcct gccgaggagg 9660aggcaacgct gggtgccaga cccgacaagg tgaggcttgc tgatgggggg tggtgaaggc 9720gcttctggga cccagggcgc ggactgtctc caactcgagc atctctgtcc ctagcggcgg 9780cccctggagc ggggagaaac agaactggct ccatcctttg aacagcgggt acaagtaatg 9840ctgcagagga taggcgtcag ccgaggcagc gggggtgccg aaggcaagag gaagcaagtg 9900agttgagggg acctgagcat gaagtgagag ggcagatggc atgctgtggg tgacataagt 9960gaccaagatg gaggagactc atgacagata ggtctaattg tcagttctac tccattctcc 10020tcgaaatggt tacagattcg acctttcttc cctcttggct ttgacccagg cacaggccac 10080cagcatctcc tctttggacc tgagaaagtc ttttctctgc aggtctaata gaaatctgag 10140gcctgggcca ggcgccatgg ctcacgcctg taatcccgac actttgggag gctgaggcag 10200gtggattacc tgaggtcagg agttaagaga ccaacctggc caacatggtg aaaccctgtc 10260tctactaaaa atacaaaaat tagccgggtg tggcggtggg cacttgtagt cccagctact 10320tgggagggtg aggcacgaga attgcttgaa cctgggagac aggttgcagt gagcctagat 10380cgtgccacta cacactagcc tggacaacaa gagtgagact catctcaaaa aaagaaaaag 10440aaaaaaatct gaccttgtca cttgtgcttg aaatagcaag tcctccaggg gcttcctctc 10500aagacactgt tcagactcca caacctgctt cagagccctg tggggtcttg gccctggcca 10560ccttaaaacc tcactgcttt cctcccctgg tgcaaatcag agcctccaca cacgtccctg 10620ggtggtctgg atgtcctccc tgcctcgctt ccagtctcta attgtgggtt acgccaggtc 10680cctcctcagc catccctgca gtgcccatgg ttcttccctg actctcctct gtgggccacc 10740ctgctaatgt ctctctcatt agagtcccaa agaaccccag ccctccatac catccattca 10800atatcttaaa aaacaaaaaa caaaaacaaa caaaaaacat gaagtcggcc tgaatttaga 10860aatattcaag tgtgcaaaga gttctaagag ggctgtactg ggtttgccat gttcttgtcc 10920agcccatggt gggcattcag tggcaggatt ataagaacaa gaacgaacaa aaggctaggg 10980tgtggggagc gggcagggct ggaatctgat tgccctgttt cctgcagagc aaagatggcg 11040agatcaagaa agctggctca gatggtaagt gggaccctgg ggtgtggcag tacatttgcc 11100aggaggtgtc cttatagaca gcccccaagg cacttgctct ccttggggag gaaggggaga 11160ggccaggttt ttcttcccct cacctccaag gtctggtacc tgagtcccca gctccgcctt 11220gcaggtgaca ttatggacag ttccacggag gcccctccca tctcgatcaa gtcccgcacc 11280cactctgtgt ctgctggtga gtgagggcca ctgtgtgtgt tggtagtggg agcagggaca 11340ggcaggagtt gggtcagact gttgttcaga cctatcgcca atgcctgacc aggtcttggc 11400tgacaccttt ctccctacag acccttcctg cagacctggc ccagggagcc aggggcctga 11460gtctgccacc tggaagacac tggggcagca gttgaatgcg gagctcagga gccgtggttg 11520gggccaacag gatggtccag gccctccctc ccctggtcaa agcccaagtc cctgcagaac 11580cagcccctcc ccagacagcc tgggcctccc agaggaccct tgcttgggcc ccagaaatga 11640aggtaggcag gcacctgatt ccccacccca atcctggcct cggggctgga ggagttcctg 11700ctgggaactc agctcctcta aggaccctga gggtgggagg caagggctgg agtgggggca 11760gctgttggaa tacccctgat tccctggcct ccctcagatg gccagctgag gccgaggcct 11820ctctcggcag ggcggcgagc agtgtctgtg catgaggacc agctccaggc ccctgctggt 11880gaggggagac acctccacgt gtgcttgaat gcaggagatg tgggagggtg ggcttctggg 11940gctcccttca tagctttggt ccttggaggg agccaccagt gtgtgaggtc tcaagaaatc 12000agggagccag aagaccaggt gcaagggttt gacagcaagc ccttccaact agcactggct 12060ccacttcccg aagagcagcc tctgccaggg gtgaggacgc agggacgggg gatgctctga 12120aggcagcagt gtgtgtgagt gcatgcttat gtgcactgga ggtggaagag aggggcaggg 12180gatggacaga ccccaagcct tagcaaccca ccccaagcct ttctgtgtcc cttagaacgg 12240cccctgaggc tgcagcgctc ccccgtcctc aaacgcaggc caaaactcga ggcacctcca 12300tccccaagcc taggtaagag ggggtccagg ccagctggga gggtggcagg actgcttagc 12360ccagccctga cccttcctct ctctccctcc ctcccctcac aggatctggc cttggaaccg 12420agcctctgcc cccacagccc acagagccct ccagccctga gcggagccca ccctccccag 12480ccacagacca aagaggcggc ggccccaatc cctgatcctc tcctctcctg ccgcatgaga 12540ttattttatt aaaaaactca aaggaa 1256661435PRTHomo sapiens 6Met Ala Gln Thr Pro Asp Gly Ile Ser Cys Glu Leu Arg Gly Glu Ile1 5 10 15Thr Arg Phe Leu Trp Pro Lys Glu Val Glu Leu Leu Leu Lys Thr Trp 20 25 30Leu Pro Gly Glu Gly Ala Val Gln Asn His Val Leu Ala Leu Leu Arg 35 40 45Trp Arg Ala Tyr Leu Leu His Thr Thr Cys Leu Pro Leu Arg Val Asp 50 55 60Cys Thr Phe Ser Tyr Leu Glu Val Gln Ala Met Ala Leu Gln Glu Thr65 70 75 80Pro Pro Gln Val Thr Phe Glu Leu Glu Ser Leu Arg Glu Leu Val Leu 85 90 95Glu Phe Pro Gly Val Ala Ala Leu Glu Gln Leu Ala Gln His Val Ala 100 105 110Ala Ala Ile Lys Lys Val Phe Pro Arg Ser Thr Leu Gly Lys Leu Phe 115 120 125Arg Arg Pro Thr Pro Ala Ser Met Leu Ala Arg Leu Glu Arg Ser Ser 130 135 140Pro Ser Glu Ser Thr Asp Pro Cys Ser Pro Cys Gly Gly Phe Leu Glu145 150 155 160Thr Tyr Glu Ala Leu Cys Asp Tyr Asn Gly Phe Pro Phe Arg Glu Glu 165 170 175Ile Gln Trp Asp Val Asp Thr Ile Tyr His Arg Gln Gly Cys Arg His 180 185 190Phe Ser Leu Gly Asp Phe Ser His Leu Gly Ser Arg Asp Leu Ala Leu 195 200 205Ser Val Ala Ala Leu Ser Tyr Asn Leu Trp Phe Arg Cys Leu Ser Cys 210 215 220Val Asp Met Lys Leu Ser Leu Glu Val Ser Glu Gln Ile Leu His Met225 230 235 240Met Ser Gln Ser Ser His Leu Glu Glu Leu Val Leu Glu Thr Cys Ser 245 250 255Leu Arg Gly Asp Phe Val Arg Arg Leu Ala Gln Ala Leu Ala Gly His 260 265 270Ser Ser Ser Gly Leu Arg Glu Leu Ser Leu Ala Gly Asn Leu Leu Asp 275 280 285Asp Arg Gly Met Thr Ala Leu Ser Arg His Leu Glu Arg Cys Pro Gly 290 295 300Ala Leu Arg Arg Leu Ser Leu Ala Gln Thr Gly Leu Thr Pro Arg Gly305 310 315 320Met Arg Ala Leu Gly Arg Ala Leu Ala Thr Asn Ala Ala Phe Asp Ser 325 330 335Thr Leu Thr His Leu Asp Leu Ser Gly Asn Pro Gly Ala Leu Gly Ala 340 345 350Ser Glu Asp Ser Gly Gly Leu Tyr Ser Phe Leu Ser Arg Pro Asn Val 355 360 365Leu Ser Phe Leu Asn Leu Ala Gly Thr Asp Thr Ala Leu Asp Thr Val 370 375 380Arg Gly Cys Ser Val Gly Gly Trp Met Thr Gly Arg Ala Asp Trp Arg385 390 395 400Ala Gly Arg Gly Gly Leu Gly Pro Pro Ala Gly Val Ala Asn Ser Leu 405 410 415Pro Pro Gln Leu Phe Ala Ala Val Ser Arg Gly Cys Cys Thr Ser Leu 420 425 430Thr His Leu Asp Ala Ser Arg Asn Val Phe Ser Arg Thr Lys Ser Arg 435 440 445Ala Ala Pro Ala Ala Leu Gln Leu Phe Leu Ser Arg Ala Arg Thr Leu 450 455 460Arg His Leu Gly Leu Ala Gly Cys Lys Leu Pro Pro Asp Ala Leu Arg465 470 475 480Ala Leu Leu Asp Gly Leu Ala Leu Asn Thr His Leu Arg Asp Leu His 485 490 495Leu Asp Leu Ser Ala Cys Glu Leu Arg Ser Ala Gly Ala Gln Val Ile 500 505 510Gln Asp Leu Val Cys Asp Ala Gly Ala Val Ser Ser Leu Asp Leu Ala 515 520 525Asp Asn Gly Phe Gly Ser Asp Met Val Thr Leu Val Leu Ala Ile Gly 530 535 540Arg Ser Arg Ser Leu Arg His Val Ala Leu Gly Arg Asn Phe Asn Val545 550 555 560Arg Cys Lys Glu Thr Leu Asp Asp Val Leu His Arg Ile Val Gln Leu 565 570 575Met Gln Asp Asp Asp Cys Pro Leu Gln Ser Leu Ser Val Ala Glu Ser 580 585 590Arg Leu Lys Leu Gly Ala Ser Val Leu Leu Arg Ala Leu Ala Thr Asn 595 600 605Pro Asn Leu Thr Ala Leu Asp Ile Ser Gly Asn Ala Met Gly Asp Ala 610 615 620Gly Ala Lys Leu Leu Ala Lys Ala Leu Arg Val Asn Ser Arg Leu Arg625 630 635 640Ser Val Val Trp Asp Arg Asn His Thr Ser Ala Leu Gly Leu Leu Asp 645 650 655Val Ala Gln Ala Leu Glu Gln Asn His Ser Leu Lys Ala Met Pro Leu 660 665 670Pro Leu Asn Asp Val Ala Gln Ala Gln Arg Ser Arg Pro Glu Leu Thr 675 680 685Ala Arg Ala Val His Gln Ile Gln Ala Cys Leu Leu Arg Asn Asn Arg 690 695 700Ala Asp Pro Ala Ser Ser Asp His Thr Thr Arg Leu Gln Pro Leu Gly705 710 715 720Leu Val Ser Asp Pro Ser Glu Gln Glu Val Asn Glu Leu Cys Gln Ser 725 730 735Val Gln Glu His Val Glu Leu Leu Gly Cys Gly Ala Gly Pro Gln Gly 740 745 750Glu Ala Ala Val Arg Gln Ala Glu Asp Ala Ile Gln Asn Ala Asn Phe 755 760 765Ser Leu Ser Ile Leu Pro Ile Leu Tyr Glu Ala Gly Ser Ser Pro Ser 770 775 780His His Trp Gln Leu Gly Gln Lys Leu Glu Gly Leu Leu Arg Gln Val785 790 795 800Gly Glu Val Cys Arg Gln Asp Ile Gln Asp Phe Thr Gln Ala Thr Leu 805 810 815Asp Thr Ala Arg Ser Leu Cys Pro Gln Met Leu Gln Gly Ser Ser Trp 820 825 830Arg Glu Gln Leu Glu Gly Val Leu Ala Gly Ser Arg Gly Leu Pro Glu 835 840 845Leu Leu Pro Glu Gln Leu Leu Gln Asp Ala Phe Thr Arg Leu Arg Asp 850 855 860Met Arg Leu Ser Ile Thr Gly Thr Leu Ala Glu Ser Ile Val Ala Gln865 870 875 880Ala Leu Ala Gly Leu Ser Ala Ala Arg Asp Gln Leu Val Glu Ser Leu 885 890 895Ala Gln Gln Ala Thr Val Thr Met Pro Pro Ala Leu Pro Ala Pro Asp 900 905 910Gly Gly Glu Pro Ser Leu Leu Glu Pro Gly Glu Leu Glu Gly Leu Phe 915 920 925Phe Pro Glu Glu Lys Glu Glu Glu Lys Glu Lys Asp Asp Ser Pro Pro 930 935 940Gln Lys Trp Pro Glu Leu Ser His Gly Leu His Leu Val Pro Phe Ile945 950 955 960His Ser Ala Ala Glu Glu Ala Glu Pro Glu Pro Glu Leu Ala Ala Pro 965 970 975Gly Glu Asp Ala Glu Pro Gln Ala Gly Pro Ser Ala Arg Gly Ser Pro 980 985 990Ser Pro Ala Ala Pro Gly Pro Pro Ala Gly Pro Leu Pro Arg Met Asp 995 1000 1005Leu Pro Leu Ala Gly Gln Pro Leu Arg His Pro Thr Arg Ala Arg 1010 1015 1020Pro Arg Pro Arg Arg Gln His His His Arg Pro Pro Pro Gly Gly 1025 1030 1035Pro Gln Val Pro Pro Ala Leu Pro Gln Glu Gly Asn Gly Leu Ser 1040 1045 1050Ala Arg Val Asp Glu Gly Val Glu Glu Phe Phe Ser Lys Arg Leu 1055 1060 1065Ile Gln Gln Asp Arg Leu Trp Ala Pro Glu Glu Asp Pro Ala Thr 1070 1075 1080Glu Gly Gly Ala Thr Pro Val Pro Arg Thr Leu Arg Lys Lys Leu 1085 1090 1095Gly Thr Leu Phe Ala Phe Lys Lys Pro Arg Ser Thr Arg Gly Pro 1100 1105 1110Arg Thr Asp Leu Glu Thr Ser Pro Gly Ala Ala Pro Arg Thr Arg 1115 1120 1125Lys Thr Thr Phe Gly Asp Leu Leu Arg Pro Pro Thr Arg Pro Ser 1130 1135 1140Arg Gly Glu Glu Leu Gly Gly Ala Glu Gly Asp Thr Ser Ser Pro 1145 1150 1155Asp Pro Ala Gly Arg Ser Arg Pro Arg Tyr Thr Arg Asp Ser Lys 1160 1165 1170Ala Tyr Ser Met Ile Leu Leu Pro Ala Glu Glu Glu Ala Thr Leu 1175 1180 1185Gly Ala Arg Pro Asp Lys Arg Arg Pro Leu Glu Arg Gly Glu Thr 1190 1195 1200Glu Leu Ala Pro Ser Phe Glu Gln Arg Val Gln Val Met Leu Gln 1205 1210 1215Arg Ile Gly Val Ser Arg Gly Ser Gly Gly Ala Glu Gly Lys Arg 1220 1225 1230Lys Gln Ser Lys Asp Gly Glu Ile Lys Lys Ala Gly Ser Asp Gly 1235 1240 1245Asp Ile Met Asp Ser Ser Thr Glu Ala Pro Pro Ile Ser Ile Lys 1250 1255 1260Ser Arg Thr His Ser Val Ser Ala Asp Pro Ser Cys Arg Pro Gly 1265 1270 1275Pro Gly Ser Gln Gly Pro Glu Ser Ala Thr Trp Lys Thr Leu Gly 1280 1285 1290Gln Gln Leu Asn Ala Glu Leu Arg Ser Arg Gly Trp Gly Gln Gln 1295 1300 1305Asp Gly Pro Gly Pro Pro Ser Pro Gly Gln Ser Pro Ser Pro Cys 1310 1315 1320Arg Thr Ser Pro Ser Pro Asp Ser Leu Gly Leu Pro Glu Asp Pro 1325 1330 1335Cys Leu Gly Pro Arg Asn Glu Asp Gly Gln Leu Arg Pro Arg Pro 1340 1345 1350Leu Ser Ala Gly Arg Arg Ala Val Ser Val His Glu Asp Gln Leu 1355 1360 1365Gln Ala Pro Ala Glu Arg Pro Leu Arg Leu Gln Arg Ser Pro Val 1370 1375 1380Leu Lys Arg Arg Pro Lys Leu Glu Ala Pro Pro Ser Pro Ser Leu 1385 1390 1395Gly Ser Gly Leu Gly Thr Glu Pro Leu Pro Pro Gln Pro Thr Glu 1400 1405

1410Pro Ser Ser Pro Glu Arg Ser Pro Pro Ser Pro Ala Thr Asp Gln 1415 1420 1425Arg Gly Gly Gly Pro Asn Pro 1430 1435712186DNAMus musculus 7ccgagtacgc ggaaggaaaa gtccctgagc gacagcaaca gtcaagattc gaccatcctc 60ccacaacctc ccacccgggg cgaactcagc caacgaagcc ggggcgggga ggggccgggc 120cgggggcggc ctggcaggaa gaagcgcgta ccttccgctg caggaggagc aggtggctgc 180actgctgcct gggcccctgg cttcctgtgt acgctcctgc cggctgcttt ttcccgccag 240agctcattgg gccgtcccgg ccctatggca cagaccccag acgacatatc ctgtgaactg 300cgaggtaagc cctgggagcc cacctggcca tgaagagcct gactccaaac cagacctctc 360ctctcggcgc cttagggaac ttgaccaggg gtttttgttc tggagggagg gccagtgcct 420aaaagtccgc ttcatttctt cctagctgat ccacacatga atacctgggc actcagtcta 480gcacagacct tgggcacaga gtggccctaa tagaggagcc aaagtcaccc agtagactgt 540ctttccacag gcgagatcac caggttcctg tggcccaagg aggcggagct gctgttgaaa 600acctggcttc cccaggaggg tgccgagcaa agccatatcc tggtatggta gccaggagtg 660aagctcagga cagtggccag accctgcaca aagaaacttg atttccctag agtattagtc 720ttttgtctcc ccccaggcac tgcttcgatg gagggcatat ttgctgcaca cctgccttcc 780cctgagggtg agtcccaggc cgggccaaca ccccttcccc ttcagcaagg gactgccttg 840cgcagctgtc ttctttgccc atgctggctt ttactttcca acagcagaca ggagggaagt 900ctgggcaggt ctagctcagg atgccacaaa atcatgggct gtggagagag gacctgggtt 960caattctccc accaccaggt gggctcacaa gtggttggaa ctctagttcg aaggcaccca 1020ccaccgtctt ctggcatctt tgggaaccag gcacacaaga gtggtacaga gacaaacatg 1080ccagcaaaca cccaatagta caatttttaa attttaagaa caagctagga ggtggtggtg 1140cacgccttta gtcctggcac tagggagaca gaggcaggca ggcctggtct acagagtgag 1200ctccaggaca gccaggtcta cacagagaaa ccctgtctca ggaaagagaa agaaagaaag 1260aaagaaagaa agaaagaaag aaagaaagaa agaaagaaag aacgaactag gctcaagaga 1320gtagcaacag tcagttggaa tgactctgat gctttccagg tggactgtac attcagctac 1380ctggaggtcc aggccatggc actacaggaa acaccccctc gggtaagaaa gctggacccc 1440aacttgaaca tacactccgc ttgatcattt attcctacac atatcactct gaatggacag 1500cgcttccacc acacagggat ccaaaaggct cctccttccc caggtcacct ttgagctgga 1560gtccctgcct gaactggtcc tggagtttcc ctgtgtggct gccctcgaac agctggctca 1620gcatgtagct gctgccatca aaaaggtctt ccctcgctca acccttgggt gaggcttaga 1680aaatccaagg ggtggggcag ggcctcagtg aaagcttttc atccttcaag gagctagaag 1740tcccttttgc ctcttctcct tatcccctgc ctaggaagct attccggaag cccacaccct 1800cgtctctgct ggctcgactg gagagaagcc atcccttaga gtccaccata cccagcagtc 1860cgtgcggtga gggccaagtc agagccgcat aaaccttcca gtttgcctcc atgcaatcct 1920agtctcttgg tattgggggg gctccccaaa gtgaacaggg ccacttttat cactgccgcc 1980atctactatg gggctgacct tcccctgtct ctgacttagt gcctgtctgt ttccctatca 2040ctcccccgac acctctctag gtggcttctt ggaaacatac gaagctctgt gcgattacaa 2100tggcttccca ttccgagagg agattcagtg ggtgagggta gggccctttt gggggagttc 2160tggggatgtt tgacgcccct tgtgtgtgct aagcccctct accctttgtg gccccaggct 2220agctgtgact ttttttcttt acatctagga tgtggacact atctaccatc gccagggctg 2280ccgccacttc tgccttggag atttcagcca ctttggcagc cggtatgagg ccagccagga 2340ttgggaggcg agaactccag cacccaaatt cctagtctgt ctgtagaggg gggatgctga 2400ctgagccctg tgtttggaag gccctgagca ctgcattttc tcttccccgc gatagagacc 2460tggctttgag tgtagctgcc ttatcttata acctgtggtt ccggcgcctc tcctgcgagg 2520acatgaagct ggtgagagag tacacacaaa agggagtgcc aagggtgtaa gggtcaaagt 2580tcaacctgat gagtcacgac cctcttgctc tcagagcctg gaggtctcag aacagattct 2640gcacatgacg agtcaatctt cctatttgga ggagctggtg ttggaggctt gtggtctgcg 2700agggtgagag gcctgagctg ggcgtgggaa cctgtgatct gcggagagac tgggaatcag 2760tcttaactgg ggtcttggtc ctcagagact tcgttaggcg actagcccag gccctggcag 2820gacattttaa ttctggactt cgggagctca gcctgtctgg gaacttgctg gatgacagag 2880gtatggcaga atctgggaga cccaaggcag ccgagccgga agaggtgagg ttcacagagc 2940tgctctgtcc acaggtatgg ctgcactcag cagacaccta gagcattgtc caggagcctt 3000gaggagactc agcctagcac agacaggctt gacacctcga ggtgggtggg actggggagg 3060gttgggaggg ctcgaggtaa ggtgagcatg tcttgatact cagccgctcc tcccacccac 3120caccaggaat gagagctcta ggcagggcac tggcaacgaa tgccaccttt gactctaccc 3180tgacccacct ggatctttct gggaaccctg gggcactggg accctcacag gatagtgggg 3240tgagtagcag tttttaaagt cagaaactgg gacggccgag tcagtgggat gccagatcct 3300taggcaaccc ctgcctccct ctctccctcc gtagggcctg tatactttcc tgagccgtcc 3360taacgtcctg gcgtatttga atcttgcagg cactgacgcc acgctaggca cggtaagagt 3420gtggtcatgg gggatgcgtg gcagggcttg gctgcgcctg agcagggaag gaaggagagc 3480ggagcgtggc agagaaagcc agggcttctg gaaggttatc gctaatacca ccttcctctc 3540agctcttcac ggccttagct ggtggctgct gctccagcct cacccatctg gaagcttcaa 3600ggaatatctt ctctcgcatg taaggggcac caggcaggct cggaaggacg aggcttgagg 3660gtatcagctc ccctgagcgt aaatcccact tctgctctcc acccctcacc gcaggaagtc 3720ccaagcggca ccagccgcgc tgcaacgctt ccttggcggt accaggatgc tacggcacct 3780gggcctggcg ggctgcaagc tgccacctga agcgctcagg tcagtcctcc cgtgcagtca 3840caaccttgag cagtgtctct tgacctctac tgcctcaccc ctctatgaca cccataatct 3900ttcgtaaccc caacctgcct ggcgcttatt cgttcgcacc gagtcccgtg ttcccagatc 3960ttgtcacctg tgtgttaccc ctctctcagg gcccttctag aaggtcttgc actcaacact 4020cagatccatg atctgcacct agacctcagc gcgtgtgagg taggtacccg gtcctggttg 4080tgagctgaga gcacgtccac attattgaga gtgaggctgg gaggccttcc ctacccaggc 4140ataatcctga gtgcacccta cctccacctc cagctccgct ccgtgggtgc ccaggtgata 4200caagacttgg tttgtgatgc gggtgccttg agttccttgg atctgtcgga taatggtgag 4260gccgccaggg aacccatcca tccatgctcc tgttatttgt accagatccc cagggtgggc 4320accctgacct ggtaggagtg atttgtgcat cactctggtt acccctaggc tttggctccg 4380acatggtgac actggtgctt gccatcggga ggagccggtc tctgaaacac gtggcccttg 4440gaaggaactt caacgttcgg tgcaagtgag tcccaagctt ccccctgtac ctctcaagaa 4500ggactgcatc acccatttag ttccctaaca gcagcccctt tccacaggga gaccctggac 4560gatgtcctgc atcggatagc ccagctaatg caggatgacg actgtgtgag ttcacagagc 4620cctgtggggg gtcctcaagc aattaacgtc ttgtaatgct tcttatgtat attccttcaa 4680accttctttg ctgaacctga gcccatgtat ccatcagtgc cttcctttta accccagcct 4740ttgcagtcac tatccgtggc tgagtcgcgg ttgaagcagg gtgccagcat cctgatccgg 4800gctttgggca ccaatcctaa actgacagcg ctggatatca gtggcaatgc cataggggat 4860gctggggcca agatgctagc caaggctcta cgcgtcaaca ccaggctacg gtgggtatga 4920tcatgataag ggctgggacc cgcaggagag ggcaagagtt atataataga cgggtgtatg 4980ggtttaaggt atacatggtg atgagacagg tgattggata aggccagagg gttgggcggg 5040gttctgcccc tgctgaagcc tggtggggcc caggtctgtg atctgggacc ggaacaacac 5100atctgctctg ggcctgctgg atgtggcgca agccctggaa cagaaccaca gcctcaagtc 5160catgccgctg ccactgaatg acgtaaccca ggctcatcgc agccggccag aactcacaac 5220tcgagcggtc catcaggtgg gggtccgcgc ttctctctgc ccttttgtgt gtggtgactc 5280caccccctgg cgtttctcac tatctctttg actgcagatc caagcctgtc tctggaggaa 5340caaccaagta gactctactt cggacctcaa gccctgcctt cagcccttag gtctgatttc 5400agaccactca gagcaggtta gtgccccttg ccctaatctt gaatacctga gcacctgagt 5460cagagtctga cttttctgag ccacagtgct gagttctatg tctcaaacaa tttccttttc 5520ttggtcaccg ttcttgaaac agcttcccgt gtaacatttg gctgctttaa gggtttaaat 5580aggctgtcct tgctgtagcc tctttattcg acttcctatt taagggctaa cctctcttta 5640ctgactgtgt ggctagctcc agcagtccta tctctggctt taatttcctt tcattaaccc 5700atctttttgt attattctta cctctgcatt cccaagcctc aattcttttg tttggttttg 5760gtttcttgag acagggtttc tctgtgtagc ctgggatatc cttgaactcc cagagatctg 5820cttgcttctg cctcccaagt gctaggatta aaggcatgtg ccaacactgc ccagcccaag 5880ccttgattga ttgattgatt gattgattga ttgatttgag acagggtttc tctgtgtaat 5940ccttgccgtc ttggaattca ctctgtagac caagctggcc tcgaactcag aaatccgcct 6000gcctctgcct cccaagtgct gggattaaag gcgtgcgcca ccatacacac acacatacac 6060acaactgttc tctgtatgga tggttaaggt gacatctttg aaatcagagt gaccaaagct 6120tctttccttc ctgttttagg cctggagaaa ataatcaagt gatcagatag ggtttccctc 6180acagtttgtc ttcctgtagg aaaaccttgg aagggagaca ggcagaatcg gggagactga 6240tgagaaacaa agggaggact aatccatgac tccctcctgc ctgcccccca ggaggtgaac 6300gagctatgcc agtcagtaca ggagcatatg gagctgctgg gctgtggggc tgggccccag 6360ggtgaggttg ccgtgcacca ggctgaggac gccatccaga atgccaactt ctctctcagt 6420gtaagcaccc tctttcctgc tctgaggacc tttccgctcc caatcatgtg ccagtttgca 6480agtccttttg ttccttggcc tccccagatc ctccccattc tctatgaggc tgggagatcc 6540ccaagccacc actggcagct gcagcagaag ctagagagcc tcctgggtca ggtgggcgag 6600atctgccgcc aggacatcca ggtaagatac tagtcctgcc ccagccgcac ctccccatgc 6660aggcttgacc ctcattccac acgcttgtct gggataggcc cttagtcagc agtggtttaa 6720tgggaactca caccttcaaa gccaaagggg ctgggttttg gcatactgct aacattttgt 6780aacctttgcc caggacttca ctcagaccac cctggatacc acaaggagcc tctgcccaca 6840gatgttgcag acacctggct ggaggaagca gctagaggga gttctggtgg gctccggggg 6900cctcccagag ctgcttccgg aacatctgct gcaagatgcc ttctctaggc tgaggtgagc 6960aagcccaact gggctgggca atgctggaca gagacagcac actaatcctc actgtctcct 7020gcctttaggg acatgcgcct gtcaatcact gggaccctag cagagagcat tgtggctcag 7080gctcttgcag gtcttcatgc agcccgagat cgactggtga gggggaagct ttgaacacat 7140ttcatagagt aacttgttta accctcacgg gcaaccctga aatgacttct ggggtcataa 7200gctagctact tagttctgcc tatctttggg tggcctggta ttacaggtgg agaggctaac 7260tcagcaggca ccagtgacca tggcccctgc tgtaccacca ctgggtggaa atgagctcag 7320cccccttgag actgggggat tggaagagct tttctttccc acggagaagg aagaggagag 7380agaaaaggtg agtgtcttca gaatttcaag cctaggaccc cggctggctt tctccttctt 7440gctctggaat gtagaaggtg aggagcaggt ctgggtcatc ctaaccctag actgaggctg 7500tacccatccc accaggatga gagttcttca tggaaatggc ttgagcctag taactgtttt 7560cacctggtct cctcccttca tggtaagtca ggccctggag taagagagtt taatccaagt 7620ggactgaagc tctgttaaat ctggacactc tttcagccct gtcccgggtg taccctagcc 7680cttccccgag ccaagccagg aagaaagcca gagtcagctg ttctcagaac cctgtgggac 7740cagaacagaa taacactaag acacctcctt ccctacctcc tcctcccctt tccctaaccc 7800caggacgcgc atgcttggcg gctgcacaaa gtcgcgcgca ctcggggccg cgctcagcac 7860cacggacagc ggcgactggg ggaggggcgg gcggcgtagc cgggcccctc cccgcgccct 7920gggcgggtcc ccgccgccct ccctagctcc tccgccacct ccccgggttc gacgctctgt 7980gctcttctgg tgcttttgcc tcaggtgctg ctgaggaagc ggaacgggac cccgagctgg 8040cagctccggg ggaagatgca gagccgcagg ctgggccgtc tgcacgcggc tctccaagcc 8100ccgccgcccc ggggccaccc gccggccctc tgcctcgcat ggacctgcca cccgccgggc 8160aacccctacg ccacccaacc cgagcccgac cgagaccacg tcgccagcac caccaccgcc 8220cgccgccggg gggcccccag gtaagtgctt cccttccccc tcgcccctct tgagggtcat 8280gggtggacca ttcactcttc acttgagata gaagactgga agatgggttg tggaggccca 8340aggccaccgg atcgcgctct atcatctttc tggccaccct catactcagg ccatggcaat 8400aacgggccat ggtgtgtggc tttcctgtga ggagctacta gatggtggac cccctccttg 8460tagtggtctg ctctatgggt gaacatgcga gagcggatgt ccgccttgcc ctccctctcc 8520taaggttgct gaaaaacact gggcagctgg accgcgggac actcctgcgt ctgctgtgta 8580ccagcgggtg gatgtatgtg tgggttgggt actggcaagg gtagtgggca ctggcagaag 8640gagaggagca gctggcctga ggccccctga gggtctgacg cagcagcggc gccactgagc 8700cgggatgaga ggccgggtgg tgtggtgtgg aggtggaagg ttagggtact gcagggaact 8760gttgggagta gaagggatgg catatttgca atgtgcgacc cataggtgtt tgcttagtga 8820cagccaggtg gttcagggta tagcagcctc tggactgggt gggtgcgctg tgacagcaat 8880gaagaatgaa agccagagtg gtacagacag tctccagctc cgggatgtac tcaggccagg 8940ccactgggaa ggagaggatg tctaggaaga gtcacagttc ccagtcctga ctcctaacct 9000cccaatgtct cccaggtgcc cccagccctg cttcaagaag gaaatgggct cactgctcgc 9060gtggatgagg gtgtggagga gttcttctcc aaaaggctga tccagcagga tcacttgtga 9120gtgagggtcg gttgactggg ggcggaggag ggtggaacat ccccgagttt cttcactcac 9180tgatggttat tcctacagct gggccccaga ggaggatcca gccactgagg gtggtgccac 9240tcctgtcccc cgcacacttc gaaagaagct gggtacgctc tttgccttta agaaacctcg 9300ttcaacaagg ggtccgcgac ctgacctgga gaccagccct ggagcagcag ctcgagccag 9360aaaatccaca cttggggatc tcctgcgacc accggcccgt ccaggccgtg gtgaggaacc 9420tggaggggcg gaaggcggca ccagcagccc tgaccctgct cgcagaaatc ggcctcggta 9480cacgcgggaa agcaaggcct actccatgat cctgctacct gctgaggagg aagcagccgt 9540gggtaccaga cctgataagg taaggccagg ggccggggct gaggctcctc aggaacagag 9600agcagtgggg ttctctcacc acttaggcat ctctgtcctc agaggcggcc tctggaacgg 9660ggagacacag aactggctcc atcttttgag cagcgggtac aagtgatgct acagaggatc 9720ggcgtgagcc gggccagtgg gggtgccgag agcaagagga agcaagtgag ttgggggtga 9780actgcggggg caggtggcat gtggtaatct gtgggtgaca tatgtgacca agaagggaga 9840aacacatggt gggaatagat ctaattatca gttacatgtt ctcatccctc aagaggcaag 9900gaggagaact ttcttccttc ttgcttctga cctggttcag atcttctagg gtatagggaa 9960ctcctttttc tgaagaagaa attaacagaa atttgacctt gttttacctg aaagttctcc 10020aggaacttcc ttatccagat gccttatgtt ttttttttgt tttaatattt atgtgagtac 10080actgtccctg ccttctgtct tcagacacac cagaaactag cattggatcc cattacagat 10140agttgccggg agttgaactt gggtcctctg gaagagtagt cagtgctctt agctgctgag 10200ccgtctctcc agtcccttta tccagattcc taatcccgct gtgagatctc acaggacctt 10260ggtactggcc accttagacc tgttttcctt tgaagcctca cttggccatt gatttttatt 10320ctctgactgt cgggtcatgt actggattat acaggctcct ttccagcctt ccctgacacc 10380cttccatggt cacccggata acattactac aatcatggtc tttattagga actacacctc 10440tgtatgccat ccatttaaat agctttccag gaagaacaca tgcagtgaga cctggcctga 10500atgtagtgat gttaaagtgt ggcaagtcat agaactaatt atctgctgga tgtggcggta 10560atctcagcat tcagaaaact ggggcaggag gattgcaagt aagactagcc tgagttgtct 10620acagcaagac tatctcaaaa gcaaagtcta cctgtcatga ccaacatgaa catttttgca 10680tgttagctat ctgtctatcc cagactgtaa ggtcagaggg tggaaactgg ctttaccatg 10740ctctggccag cctatgctat gcattctgta gctgggtttt aacaacatgg acagagacaa 10800ggctagggtc agggcggcag ggctgctgcc tgactcatgt gtttcctaca gagtaaagac 10860ggcgagatca agaaggcagg ctctgatggt gagtaggacc ccacccggga caaggagata 10920tatgttgggg aacaaggtgg cctcagataa cccctaaggc tccctccctc caccactaag 10980gtctgacacc taagcctcta atccttcctt gcaggtgaca ttatggacag ttccacagag 11040acccctccca tctcaatcaa gtcccgtacc cactctgtgt ctgctggtga gtgagggcca 11100ctggatatgt taaaggaggt gcaagggttg ggaggggatc ggactgttgc tcagaaccat 11160tccacagaca ggcttgggtg ttagctgaca ttgtcttctc tacccagatc cctcatgcag 11220acctgggcca ggaggccagg ggcctgagtc tgccacctgg aagacattgg gacagcagct 11280gaatgcagag ctcagaggcc gtggttgggg ccaacaggat ggtccagggc ccccctcccc 11340atgtcccagc ccaagtcccc gaagaaccag ccccgcccca gacatcctga gtctcccaga 11400ggacccctgc ttgggcccta ggaatgaagg taggtaggca ccccactcct aggctaggaa 11460cccgactcat ctgggaaggc caaaggtggg aggcgaaggc tgggtgtggg cggagtatgg 11520gaacgtcctc tgacatctcg gcctcctcta gatggccagc tgaggccgag gcctctttcg 11580gcaggccggc gagcagtgtc tgtgcatgag gaccagctcc aggcccccgc gggtgaggga 11640gacccctctg cctgtgcttg agtgcaggag aaagggggag gtgggctcga tcggtcttgg 11700tccttggaca gcgccacctc ctgaaggccc acctcagcta gggtgggagg acacttgctg 11760gacaatctat gggcgtagca tgtgcatggt tttgcgtgca ctcaaggtgg accagagggt 11820tggggacagc cagtgagttt tttctgtatc ctttttagaa cggcccctcc ggctgcagcg 11880ctcccctgtc ctcaagcgta ggccgaagct cgaggcaccc ccatccccaa gcttaggtga 11940gagtgggcac gggccagctg ggatgctgat acgactgctc agtacaagcc tagcattgtt 12000tctctccata acccttcaca ggctctggcc ttggatccaa gcctcttcct ccgtacccca 12060cagaaccctc cagccctgag cggagccctc cctccccagc cacagaccaa agaggcggcg 12120gccccaaccc ctgaatctct ctcctcctgc cgcatgaaat tattttatta aaaaacttga 12180atgaaa 1218681295PRTMus musculus 8Met Ala Gln Thr Pro Asp Asp Ile Ser Cys Glu Leu Arg Gly Glu Ile1 5 10 15Thr Arg Phe Leu Trp Pro Lys Glu Ala Glu Leu Leu Leu Lys Thr Trp 20 25 30Leu Pro Gln Glu Gly Ala Glu Gln Ser His Ile Leu Ala Leu Leu Arg 35 40 45Trp Arg Ala Tyr Leu Leu His Thr Cys Leu Pro Leu Arg Val Asp Cys 50 55 60Thr Phe Ser Tyr Leu Glu Val Gln Ala Met Ala Leu Gln Glu Thr Pro65 70 75 80Pro Arg Val Thr Phe Glu Leu Glu Ser Leu Pro Glu Leu Val Leu Glu 85 90 95Phe Pro Cys Val Ala Ala Leu Glu Gln Leu Ala Gln His Val Ala Ala 100 105 110Ala Ile Lys Lys Val Phe Pro Arg Ser Thr Leu Gly Lys Leu Phe Arg 115 120 125Lys Pro Thr Pro Ser Ser Leu Leu Ala Arg Leu Glu Arg Ser His Pro 130 135 140Leu Glu Ser Thr Ile Pro Ser Ser Pro Cys Gly Gly Phe Leu Glu Thr145 150 155 160Tyr Glu Ala Leu Cys Asp Tyr Asn Gly Phe Pro Phe Arg Glu Glu Ile 165 170 175Gln Trp Asp Val Asp Thr Ile Tyr His Arg Gln Gly Cys Arg His Phe 180 185 190Cys Leu Gly Asp Phe Ser His Phe Gly Ser Arg Asp Leu Ala Leu Ser 195 200 205Val Ala Ala Leu Ser Tyr Asn Leu Trp Phe Arg Arg Leu Ser Cys Glu 210 215 220Asp Met Lys Leu Ser Leu Glu Val Ser Glu Gln Ile Leu His Met Thr225 230 235 240Ser Gln Ser Ser Tyr Leu Glu Glu Leu Val Leu Glu Ala Cys Gly Leu 245 250 255Arg Gly Asp Phe Val Arg Arg Leu Ala Gln Ala Leu Ala Gly His Phe 260 265 270Asn Ser Gly Leu Arg Glu Leu Ser Leu Ser Gly Asn Leu Leu Asp Asp 275 280 285Arg Gly Met Arg Ala Leu Gly Arg Ala Leu Ala Thr Asn Ala Thr Phe 290 295 300Asp Ser Thr Leu Thr His Leu Asp Leu Ser Gly Asn Pro Gly Ala Leu305 310 315 320Gly Pro Ser Gln Asp Ser Gly Gly Leu Tyr Thr Phe Leu Ser Arg Pro 325 330 335Asn Val Leu Ala Tyr Leu Asn Leu Ala Gly Thr Asp Ala Thr Leu Gly 340 345 350Thr Leu Phe Thr Ala Leu Ala Gly Gly Cys Cys Ser Ser Leu Thr His 355 360 365Leu Glu Ala Ser Arg Asn Ile Phe Ser Arg Met Lys Ser Gln Ala Ala 370 375 380Pro Ala Ala Leu Gln Arg Phe Leu Gly Gly Thr Arg Met Leu Arg His385 390 395 400Leu Gly Leu Ala Gly Cys Lys Leu Pro Pro Glu Ala Leu Arg Ala Leu 405 410 415Leu Glu Gly Leu Ala Leu Asn Thr Gln Ile His Asp Leu His Leu Asp 420 425

430Leu Ser Ala Cys Glu Leu Arg Ser Val Gly Ala Gln Val Ile Gln Asp 435 440 445Leu Val Cys Asp Ala Gly Ala Leu Ser Ser Leu Asp Leu Ser Asp Asn 450 455 460Gly Phe Gly Ser Asp Met Val Thr Leu Val Leu Ala Ile Gly Arg Ser465 470 475 480Arg Ser Leu Lys His Val Ala Leu Gly Arg Asn Phe Asn Val Arg Cys 485 490 495Lys Glu Thr Leu Asp Asp Val Leu His Arg Ile Ala Gln Leu Met Gln 500 505 510Asp Asp Asp Cys Pro Leu Gln Ser Leu Ser Val Ala Glu Ser Arg Leu 515 520 525Lys Gln Gly Ala Ser Ile Leu Ile Arg Ala Leu Gly Thr Asn Pro Lys 530 535 540Leu Thr Ala Leu Asp Ile Ser Gly Asn Ala Ile Gly Asp Ala Gly Ala545 550 555 560Lys Met Leu Ala Lys Ala Leu Arg Val Asn Thr Arg Leu Arg Ser Val 565 570 575Ile Trp Asp Arg Asn Asn Thr Ser Ala Leu Gly Leu Leu Asp Val Ala 580 585 590Gln Ala Leu Glu Gln Asn His Ser Leu Lys Ser Met Pro Leu Pro Leu 595 600 605Asn Asp Val Thr Gln Ala His Arg Ser Arg Pro Glu Leu Thr Thr Arg 610 615 620Ala Val His Gln Ile Gln Ala Cys Leu Trp Arg Asn Asn Gln Val Asp625 630 635 640Ser Thr Ser Asp Leu Lys Pro Cys Leu Gln Pro Leu Gly Leu Ile Ser 645 650 655Asp His Ser Glu Gln Glu Val Asn Glu Leu Cys Gln Ser Val Gln Glu 660 665 670His Met Glu Leu Leu Gly Cys Gly Ala Gly Pro Gln Gly Glu Val Ala 675 680 685Val His Gln Ala Glu Asp Ala Ile Gln Asn Ala Asn Phe Ser Leu Ser 690 695 700Ile Leu Pro Ile Leu Tyr Glu Ala Gly Arg Ser Pro Ser His His Trp705 710 715 720Gln Leu Gln Gln Lys Leu Glu Ser Leu Leu Gly Gln Val Gly Glu Ile 725 730 735Cys Arg Gln Asp Ile Gln Asp Phe Thr Gln Thr Thr Leu Asp Thr Thr 740 745 750Arg Ser Leu Cys Pro Gln Met Leu Gln Thr Pro Gly Trp Arg Lys Gln 755 760 765Leu Glu Gly Val Leu Val Gly Ser Gly Gly Leu Pro Glu Leu Leu Pro 770 775 780Glu His Leu Leu Gln Asp Ala Phe Ser Arg Leu Arg Asp Met Arg Leu785 790 795 800Ser Ile Thr Gly Thr Leu Ala Glu Ser Ile Val Ala Gln Ala Leu Ala 805 810 815Gly Leu His Ala Ala Arg Asp Arg Leu Val Glu Arg Leu Thr Gln Gln 820 825 830Ala Pro Val Thr Met Ala Pro Ala Val Pro Pro Leu Gly Gly Asn Glu 835 840 845Leu Ser Pro Leu Glu Thr Gly Gly Leu Glu Glu Leu Phe Phe Pro Thr 850 855 860Glu Lys Glu Glu Glu Arg Glu Lys Val Leu Leu Arg Lys Arg Asn Gly865 870 875 880Thr Pro Ser Trp Gln Leu Arg Gly Lys Met Gln Ser Arg Arg Leu Gly 885 890 895Arg Leu His Ala Val Ala Glu Lys His Trp Ala Ala Gly Pro Arg Asp 900 905 910Thr Pro Ala Ser Ala Val Tyr Gln Arg Val Asp Val Cys Val Gly Trp 915 920 925Val Pro Pro Ala Leu Leu Gln Glu Gly Asn Gly Leu Thr Ala Arg Val 930 935 940Asp Glu Gly Val Glu Glu Phe Phe Ser Lys Arg Leu Ile Gln Gln His945 950 955 960Phe Trp Ala Pro Glu Glu Asp Pro Ala Thr Glu Gly Gly Ala Thr Pro 965 970 975Val Pro Arg Thr Leu Arg Lys Lys Leu Gly Thr Leu Phe Ala Phe Lys 980 985 990Lys Pro Arg Ser Thr Arg Gly Pro Arg Pro Asp Leu Glu Thr Ser Pro 995 1000 1005Gly Ala Ala Ala Arg Ala Arg Lys Ser Thr Leu Gly Asp Leu Leu 1010 1015 1020Arg Pro Pro Ala Arg Pro Gly Arg Gly Glu Glu Pro Gly Gly Ala 1025 1030 1035Glu Gly Gly Thr Ser Ser Pro Asp Pro Ala Arg Arg Asn Arg Pro 1040 1045 1050Arg Tyr Thr Arg Glu Ser Lys Ala Tyr Ser Met Ile Leu Leu Pro 1055 1060 1065Ala Glu Glu Glu Ala Ala Val Gly Thr Arg Pro Asp Lys Arg Arg 1070 1075 1080Pro Leu Glu Arg Gly Asp Thr Glu Leu Ala Pro Ser Phe Glu Gln 1085 1090 1095Arg Val Gln Val Met Leu Gln Arg Ile Gly Val Ser Arg Ala Ser 1100 1105 1110Gly Gly Ala Glu Ser Lys Arg Lys Gln Ser Lys Asp Gly Glu Ile 1115 1120 1125Lys Lys Ala Gly Ser Asp Gly Asp Ile Met Asp Ser Ser Thr Glu 1130 1135 1140Thr Pro Pro Ile Ser Ile Lys Ser Arg Thr His Ser Val Ser Ala 1145 1150 1155Asp Pro Ser Cys Arg Pro Gly Pro Gly Gly Gln Gly Pro Glu Ser 1160 1165 1170Ala Thr Trp Lys Thr Leu Gly Gln Gln Leu Asn Ala Glu Leu Arg 1175 1180 1185Gly Arg Gly Trp Gly Gln Gln Asp Gly Pro Gly Pro Pro Ser Pro 1190 1195 1200Cys Pro Ser Pro Ser Pro Arg Arg Thr Ser Pro Ala Pro Asp Ile 1205 1210 1215Leu Ser Leu Pro Glu Asp Pro Cys Leu Gly Pro Arg Asn Glu Glu 1220 1225 1230Arg Pro Leu Arg Leu Gln Arg Ser Pro Val Leu Lys Arg Arg Pro 1235 1240 1245Lys Leu Glu Ala Pro Pro Ser Pro Ser Leu Gly Ser Gly Leu Gly 1250 1255 1260Ser Lys Pro Leu Pro Pro Tyr Pro Thr Glu Pro Ser Ser Pro Glu 1265 1270 1275Arg Ser Pro Pro Ser Pro Ala Thr Asp Gln Arg Gly Gly Gly Pro 1280 1285 1290Asn Pro 129593295DNAHomo sapiens 9ccgccagccc cgccagtccc cgcgcagtcc ccgcgcagtc cccgcgcagt cccagcgcca 60ccgggcagca gcggcgccgt gctcgctcca gggcgcaacc atgtcgccat ttcttcggat 120tggcttgtcc aactttgact gcgggtcctg ccagtcttgt cagggcgagg ctgttaaccc 180ttactgtgct gtgctcgtca aagagtatgt cgaatcagag aacgggcaga tgtatatcca 240gaaaaagcct accatgtacc caccctggga cagcactttt gatgcccata tcaacaaggg 300aagagtcatg cagatcattg tgaaaggcaa aaacgtggac ctcatctctg aaaccaccgt 360ggagctctac tcgctggctg agaggtgcag gaagaacaac gggaagacag aaatatggtt 420agagctgaaa cctcaaggcc gaatgctaat gaatgcaaga tactttctgg aaatgagtga 480cacaaaggac atgaatgaat ttgagacgga aggcttcttt gctttgcatc agcgccgggg 540tgccatcaag caggcaaagg tccaccacgt caagtgccac gagttcactg ccaccttctt 600cccacagccc acattttgct ctgtctgcca cgagtttgtc tggggcctga acaaacaggg 660ctaccagtgc cgacaatgca atgcagcaat tcacaagaag tgtattgata aagttatagc 720aaagtgcaca ggatcagcta tcaatagccg agaaaccatg ttccacaagg agagattcaa 780aattgacatg ccacacagat ttaaagtcta caattacaag agcccgacct tctgtgaaca 840ctgtgggacc ctgctgtggg gactggcacg gcaaggactc aagtgtgatg catgtggcat 900gaatgtgcat catagatgcc agacaaaggt ggccaacctt tgtggcataa accagaagct 960aatggctgaa gcgctggcca tgattgagag cactcaacag gctcgctgct taagagatac 1020tgaacagatc ttcagagaag gtccggttga aattggtctc ccatgctcca tcaaaaatga 1080agcaaggccg ccatgtttac cgacaccggg aaaaagagag cctcagggca tttcctggga 1140gtctccgttg gatgaggtgg ataaaatgtg ccatcttcca gaacctgaac tgaacaaaga 1200aagaccatct ctgcagatta aactaaaaat tgaggatttt atcttgcaca aaatgttggg 1260gaaaggaagt tttggcaagg tcttcctggc agaattcaag aaaaccaatc aatttttcgc 1320aataaaggcc ttaaagaaag atgtggtctt gatggacgat gatgttgagt gcacgatggt 1380agagaagaga gttctttcct tggcctggga gcatccgttt ctgacgcaca tgttttgtac 1440attccagacc aaggaaaacc tcttttttgt gatggagtac ctcaacggag gggacttaat 1500gtaccacatc caaagctgcc acaagttcga cctttccaga gcgacgtttt atgctgctga 1560aatcattctt ggtctgcagt tccttcattc caaaggaata gtctacaggg acctgaagct 1620agataacatc ctgttagaca aagatggaca tatcaagatc gcggattttg gaatgtgcaa 1680ggagaacatg ttaggagatg ccaagacgaa taccttctgt gggacacctg actacatcgc 1740cccagagatc ttgctgggtc agaaatacaa ccactctgtg gactggtggt ccttcggggt 1800tctcctttat gaaatgctga ttggtcagtc gcctttccac gggcaggatg aggaggagct 1860cttccactcc atccgcatgg acaatccctt ttacccacgg tggctggaga aggaagcaaa 1920ggaccttctg gtgaagctct tcgtgcgaga acctgagaag aggctgggcg tgaggggaga 1980catccgccag caccctttgt ttcgggagat caactgggag gaacttgaac ggaaggagat 2040tgacccaccg ttccggccga aagtgaaatc accatttgac tgcagcaatt tcgacaaaga 2100attcttaaac gagaagcccc ggctgtcatt tgccgacaga gcactgatca acagcatgga 2160ccagaatatg ttcaggaact tttccttcat gaaccccggg atggagcggc tgatatcctg 2220aatcttgccc ctccagagac aggaaagaat ttgccttctc cctgggaact ggttcaagag 2280acactgcttg ggttcctttt tcaacttgga aaaagaaaga aacactcaac aataaagact 2340gagacccgtt cgcccccatg tgacttttat ctgtagcaga aaccaagtct acttcactaa 2400tgacgatgcc gtgtgtctcg tctcctgaca tgtctcacag acgctcctga agttaggtca 2460ttactaacca tagttattta cttgaaagat gggtctccgc acttggaaag gtttcaagac 2520ttgatactgc aataaattat ggctcttcac ctgggcgcca actgctgatc aatgaaatgc 2580ttgttgaatc aggggcaaac ggagtacaga cgtctcaaga ctgaaacggc cccattgcct 2640ggtctagtag cggatctcac tcagccgcag acaagtaatc actaacccgt tttattctat 2700tcctatctgt ggatgtgtaa atggctgggg ggccagccct ggataggttt ttatgggaat 2760tctttacaat aaacatagct tgtaacttga gatctacaaa tccattcatc ctgattgggc 2820atgaaatcca tggtcaagag gacaagtgga aagtgagagg gaaggtttgc tagacacctt 2880cgcttgttat cttgtcaaga tagaaaagat agtatcattt cacccttgcc agtaaaaacc 2940tttccatcca cccattctca gcagactcca gtattggcac agtcactcac tgccattctc 3000acactataac aagaaaagaa atgaagtgca taagtctcct gggaaaagaa ccttaacccc 3060ttctcgtgcc atgactggtg atttcatgac tcataagccc ctccgtaggc atcattcaag 3120atcaatggcc catgcatgct gtttgcagca gtcaattgag ttgaattaga attccaacca 3180tacattttaa aggtatttgt gctgtgtgta tattttgata aaatgttgtg acttcatggc 3240aaacaggtgg atgtgtaaaa atggaataaa aaaaaaaaaa gagtcaaaaa aaaaa 329510706PRTHomo sapiens 10Met Ser Pro Phe Leu Arg Ile Gly Leu Ser Asn Phe Asp Cys Gly Ser1 5 10 15Cys Gln Ser Cys Gln Gly Glu Ala Val Asn Pro Tyr Cys Ala Val Leu 20 25 30Val Lys Glu Tyr Val Glu Ser Glu Asn Gly Gln Met Tyr Ile Gln Lys 35 40 45Lys Pro Thr Met Tyr Pro Pro Trp Asp Ser Thr Phe Asp Ala His Ile 50 55 60Asn Lys Gly Arg Val Met Gln Ile Ile Val Lys Gly Lys Asn Val Asp65 70 75 80Leu Ile Ser Glu Thr Thr Val Glu Leu Tyr Ser Leu Ala Glu Arg Cys 85 90 95Arg Lys Asn Asn Gly Lys Thr Glu Ile Trp Leu Glu Leu Lys Pro Gln 100 105 110Gly Arg Met Leu Met Asn Ala Arg Tyr Phe Leu Glu Met Ser Asp Thr 115 120 125Lys Asp Met Asn Glu Phe Glu Thr Glu Gly Phe Phe Ala Leu His Gln 130 135 140Arg Arg Gly Ala Ile Lys Gln Ala Lys Val His His Val Lys Cys His145 150 155 160Glu Phe Thr Ala Thr Phe Phe Pro Gln Pro Thr Phe Cys Ser Val Cys 165 170 175His Glu Phe Val Trp Gly Leu Asn Lys Gln Gly Tyr Gln Cys Arg Gln 180 185 190Cys Asn Ala Ala Ile His Lys Lys Cys Ile Asp Lys Val Ile Ala Lys 195 200 205Cys Thr Gly Ser Ala Ile Asn Ser Arg Glu Thr Met Phe His Lys Glu 210 215 220Arg Phe Lys Ile Asp Met Pro His Arg Phe Lys Val Tyr Asn Tyr Lys225 230 235 240Ser Pro Thr Phe Cys Glu His Cys Gly Thr Leu Leu Trp Gly Leu Ala 245 250 255Arg Gln Gly Leu Lys Cys Asp Ala Cys Gly Met Asn Val His His Arg 260 265 270Cys Gln Thr Lys Val Ala Asn Leu Cys Gly Ile Asn Gln Lys Leu Met 275 280 285Ala Glu Ala Leu Ala Met Ile Glu Ser Thr Gln Gln Ala Arg Cys Leu 290 295 300Arg Asp Thr Glu Gln Ile Phe Arg Glu Gly Pro Val Glu Ile Gly Leu305 310 315 320Pro Cys Ser Ile Lys Asn Glu Ala Arg Pro Pro Cys Leu Pro Thr Pro 325 330 335Gly Lys Arg Glu Pro Gln Gly Ile Ser Trp Glu Ser Pro Leu Asp Glu 340 345 350Val Asp Lys Met Cys His Leu Pro Glu Pro Glu Leu Asn Lys Glu Arg 355 360 365Pro Ser Leu Gln Ile Lys Leu Lys Ile Glu Asp Phe Ile Leu His Lys 370 375 380Met Leu Gly Lys Gly Ser Phe Gly Lys Val Phe Leu Ala Glu Phe Lys385 390 395 400Lys Thr Asn Gln Phe Phe Ala Ile Lys Ala Leu Lys Lys Asp Val Val 405 410 415Leu Met Asp Asp Asp Val Glu Cys Thr Met Val Glu Lys Arg Val Leu 420 425 430Ser Leu Ala Trp Glu His Pro Phe Leu Thr His Met Phe Cys Thr Phe 435 440 445Gln Thr Lys Glu Asn Leu Phe Phe Val Met Glu Tyr Leu Asn Gly Gly 450 455 460Asp Leu Met Tyr His Ile Gln Ser Cys His Lys Phe Asp Leu Ser Arg465 470 475 480Ala Thr Phe Tyr Ala Ala Glu Ile Ile Leu Gly Leu Gln Phe Leu His 485 490 495Ser Lys Gly Ile Val Tyr Arg Asp Leu Lys Leu Asp Asn Ile Leu Leu 500 505 510Asp Lys Asp Gly His Ile Lys Ile Ala Asp Phe Gly Met Cys Lys Glu 515 520 525Asn Met Leu Gly Asp Ala Lys Thr Asn Thr Phe Cys Gly Thr Pro Asp 530 535 540Tyr Ile Ala Pro Glu Ile Leu Leu Gly Gln Lys Tyr Asn His Ser Val545 550 555 560Asp Trp Trp Ser Phe Gly Val Leu Leu Tyr Glu Met Leu Ile Gly Gln 565 570 575Ser Pro Phe His Gly Gln Asp Glu Glu Glu Leu Phe His Ser Ile Arg 580 585 590Met Asp Asn Pro Phe Tyr Pro Arg Trp Leu Glu Lys Glu Ala Lys Asp 595 600 605Leu Leu Val Lys Leu Phe Val Arg Glu Pro Glu Lys Arg Leu Gly Val 610 615 620Arg Gly Asp Ile Arg Gln His Pro Leu Phe Arg Glu Ile Asn Trp Glu625 630 635 640Glu Leu Glu Arg Lys Glu Ile Asp Pro Pro Phe Arg Pro Lys Val Lys 645 650 655Ser Pro Phe Asp Cys Ser Asn Phe Asp Lys Glu Phe Leu Asn Glu Lys 660 665 670Pro Arg Leu Ser Phe Ala Asp Arg Ala Leu Ile Asn Ser Met Asp Gln 675 680 685Asn Met Phe Arg Asn Phe Ser Phe Met Asn Pro Gly Met Glu Arg Leu 690 695 700Ile Ser705113313DNAMus musculus 11cttgggtcgc caggcccgcg ccagtccccg ccatccgagc aacagcggcg ctgctctggg 60accgcggccg cgacaccagg gaacaaccat gtcaccgttt cttcgaatcg gtttatccaa 120ctttgactgt gggacctgcc aagcttgtca gggagaggca gtgaacccct actgcgctgt 180gcttgtcaaa gagtatgtgg aatcagaaaa tgggcagatg tacatccaga aaaagccaac 240catgtaccca ccttgggaca gcacctttga cgcccacatt aacaagggaa gggtgatgca 300gatcatcgtg aagggcaaga atgtagacct catctcagaa acaaccgtgg aactctactc 360cctggcggag agatgccgca agaacaatgg gcggacagaa atatggttag agctgaaacc 420tcaaggccga atgctaatga atgcaagata ctttctggaa atgagtgaca caaaggacat 480gagtgagttt gagaatgaag gattctttgc actgcatcag cgccgaggag ccatcaaaca 540ggccaaagtc caccatgtca agtgtcacga gttcacggcc acctttttcc ctcaacccac 600attttgctct gtctgccatg aatttgtctg gggcctgaac aagcagggtt accagtgccg 660acagtgtaat gcagcgattc acaagaagtg cattgataaa gtgatagcca agtgcacagg 720atccgcaatc aatagccgag aaaccatgtt ccataaggag agattcaaga tcgacatgcc 780acacagattc aaagtctaca actacaagag tccaaccttc tgtgagcact gtggtaccct 840gctctggggg ctggcgaggc aaggactcaa atgtgatgca tgtggcatga acgtccacca 900ccgatgccag acaaaggttg ccaatctttg tggtataaac cagaagctaa tggctgaagc 960actagcgatg attgaaagca cccaacaggc tcgctcctta cgagattcag aacacatctt 1020ccgagaaggc ccagttgaaa ttggtctccc atgctccacc aaaaacgaaa ccaggccacc 1080atgcgtacca acacctggga aaagagaacc ccagggcatt tcctgggatt cccctttgga 1140tgggtcaaat aaatcggccg gtcctcctga acccgaagtg agcatgcgca ggacttcact 1200gcagctgaaa ctgaagatcg atgacttcat cctgcacaag atgttgggaa aaggaagttt 1260tggcaaggtc ttcctggcag agttcaagag aaccaatcag tttttcgcaa taaaagcctt 1320aaagaaagat gtggtgttga tggatgatga cgtcgagtgt acaatggtgg aaaagagggt 1380tctgtccttg gcatgggagc atccatttct aacacacatg ttctgcacat tccagaccaa 1440ggaaaatctc tttttcgtga tggagtatct caatggaggc gacttaatgt accacatcca 1500aagttgccac aaatttgatc tttccagagc cacgttttat gctgctgagg tcatccttgg 1560tctgcagttc cttcattcca aaggaattgt ctacagggac ctgaagcttg ataatatcct 1620gttagacaga gatggacata tcaaaatagc agactttggg atgtgcaaag agaacatgct 1680aggagatgcg aagacaaata ctttctgtgg aactcctgac tacattgctc cggagatctt 1740gctgggtcag aagtacaacc attccgtcga ctggtggtcc ttcggggtgc tcgtttatga 1800gatgctgatt ggccagtccc ccttccacgg gcaggacgag gaggagctgt tccactccat 1860ccgcatggac aaccccttct acccgaggtg gctcgaaagg gaggccaagg accttctagt 1920gaagcttttt gtgagagaac ctgagaagag gctgggagtg agaggagaca tccgccagca 1980tcctttgttt cgagagatca actgggaaga gcttgaaagg aaagagattg acccaccctt 2040cagaccaaaa gtgaaatcac catatgactg tagcaatttc gacaaggaat tcctaagtga 2100gaaaccccgg ctatcattcg ccgacagagc

actcatcaac agcatggacc agaacatgtt 2160cagcaacttt tccttcatta acccagggat ggagactctc atttgctcct gaacctcatc 2220tgtctccaga ctggaaggga tttgccttct ctctgggaac tggttcaagt aacacttctg 2280ggggtggggg tctctttttc acgttagaga agaaaagaaa cactgcaaag gcagggagga 2340ctgctgagct ccttgtgtga cttgttacct acagcacaaa ccacgcctac ttcactaatg 2400acatcatccc taatgacatc atcccgttat atctcctgga atctctcaca gcagcccttg 2460aagttagatc attattaact ctagtcattt acttgaaaga tggttcccga tgctgtgaaa 2520gattcgaaat gcagttctgc tcttgcccta gacaacagct gctggttggt gatgaaccaa 2580ggcgcaagtg gaacagattt ctcaagactg gagcagtgat cgcctgttat agaagtcaat 2640tccactcaac cacagagaag gaaccactaa gccacgttga tgtgtgcatg tctgtggaaa 2700tgtcgatgac agaagggagg gaaaggggaa gctctgagca gattgtaatg ggaagctctc 2760caataaacat agcatgaaac ttgaaattta caaatctgtt cattctggct agccccaaaa 2820ttcccaaggc agaggaaagt aaagggcagt gagcttagca gagccctttg tcgccaacag 2880ggaagggtaa ggatgtcgcc tacgtggaac atcttataca cacagaagga aagtataacc 2940aacaagggca gggtggttta cagctgccaa tcaaacctgc cctcccccct ctgttctcag 3000ttgatctctc tgtcagcgta ggtaggcact cattaccatc ctcccatcat acaagaaata 3060aaatgcatga ctcttctaag ataaagaaaa ccaatccctt atcacgttgt tcccagtgat 3120ttgatggcaa ataagtccct ccttaggcat cctgcaagac aacccaaccc atgcatgcta 3180tttgcagtag tcagtcctgt tgagttagag tcctaactat acacaatatc gtgcgatgtt 3240tatatatgtt gatgagatgt tgtgatgata acgtggatat gtaaaaggga ataaaagaag 3300aaagaaagat gcc 331312707PRTMus musculus 12Met Ser Pro Phe Leu Arg Ile Gly Leu Ser Asn Phe Asp Cys Gly Thr1 5 10 15Cys Gln Ala Cys Gln Gly Glu Ala Val Asn Pro Tyr Cys Ala Val Leu 20 25 30Val Lys Glu Tyr Val Glu Ser Glu Asn Gly Gln Met Tyr Ile Gln Lys 35 40 45Lys Pro Thr Met Tyr Pro Pro Trp Asp Ser Thr Phe Asp Ala His Ile 50 55 60Asn Lys Gly Arg Val Met Gln Ile Ile Val Lys Gly Lys Asn Val Asp65 70 75 80Leu Ile Ser Glu Thr Thr Val Glu Leu Tyr Ser Leu Ala Glu Arg Cys 85 90 95Arg Lys Asn Asn Gly Arg Thr Glu Ile Trp Leu Glu Leu Lys Pro Gln 100 105 110Gly Arg Met Leu Met Asn Ala Arg Tyr Phe Leu Glu Met Ser Asp Thr 115 120 125Lys Asp Met Ser Glu Phe Glu Asn Glu Gly Phe Phe Ala Leu His Gln 130 135 140Arg Arg Gly Ala Ile Lys Gln Ala Lys Val His His Val Lys Cys His145 150 155 160Glu Phe Thr Ala Thr Phe Phe Pro Gln Pro Thr Phe Cys Ser Val Cys 165 170 175His Glu Phe Val Trp Gly Leu Asn Lys Gln Gly Tyr Gln Cys Arg Gln 180 185 190Cys Asn Ala Ala Ile His Lys Lys Cys Ile Asp Lys Val Ile Ala Lys 195 200 205Cys Thr Gly Ser Ala Ile Asn Ser Arg Glu Thr Met Phe His Lys Glu 210 215 220Arg Phe Lys Ile Asp Met Pro His Arg Phe Lys Val Tyr Asn Tyr Lys225 230 235 240Ser Pro Thr Phe Cys Glu His Cys Gly Thr Leu Leu Trp Gly Leu Ala 245 250 255Arg Gln Gly Leu Lys Cys Asp Ala Cys Gly Met Asn Val His His Arg 260 265 270Cys Gln Thr Lys Val Ala Asn Leu Cys Gly Ile Asn Gln Lys Leu Met 275 280 285Ala Glu Ala Leu Ala Met Ile Glu Ser Thr Gln Gln Ala Arg Ser Leu 290 295 300Arg Asp Ser Glu His Ile Phe Arg Glu Gly Pro Val Glu Ile Gly Leu305 310 315 320Pro Cys Ser Thr Lys Asn Glu Thr Arg Pro Pro Cys Val Pro Thr Pro 325 330 335Gly Lys Arg Glu Pro Gln Gly Ile Ser Trp Asp Ser Pro Leu Asp Gly 340 345 350Ser Asn Lys Ser Ala Gly Pro Pro Glu Pro Glu Val Ser Met Arg Arg 355 360 365Thr Ser Leu Gln Leu Lys Leu Lys Ile Asp Asp Phe Ile Leu His Lys 370 375 380Met Leu Gly Lys Gly Ser Phe Gly Lys Val Phe Leu Ala Glu Phe Lys385 390 395 400Arg Thr Asn Gln Phe Phe Ala Ile Lys Ala Leu Lys Lys Asp Val Val 405 410 415Leu Met Asp Asp Asp Val Glu Cys Thr Met Val Glu Lys Arg Val Leu 420 425 430Ser Leu Ala Trp Glu His Pro Phe Leu Thr His Met Phe Cys Thr Phe 435 440 445Gln Thr Lys Glu Asn Leu Phe Phe Val Met Glu Tyr Leu Asn Gly Gly 450 455 460Asp Leu Met Tyr His Ile Gln Ser Cys His Lys Phe Asp Leu Ser Arg465 470 475 480Ala Thr Phe Tyr Ala Ala Glu Val Ile Leu Gly Leu Gln Phe Leu His 485 490 495Ser Lys Gly Ile Val Tyr Arg Asp Leu Lys Leu Asp Asn Ile Leu Leu 500 505 510Asp Arg Asp Gly His Ile Lys Ile Ala Asp Phe Gly Met Cys Lys Glu 515 520 525Asn Met Leu Gly Asp Ala Lys Thr Asn Thr Phe Cys Gly Thr Pro Asp 530 535 540Tyr Ile Ala Pro Glu Ile Leu Leu Gly Gln Lys Tyr Asn His Ser Val545 550 555 560Asp Trp Trp Ser Phe Gly Val Leu Val Tyr Glu Met Leu Ile Gly Gln 565 570 575Ser Pro Phe His Gly Gln Asp Glu Glu Glu Leu Phe His Ser Ile Arg 580 585 590Met Asp Asn Pro Phe Tyr Pro Arg Trp Leu Glu Arg Glu Ala Lys Asp 595 600 605Leu Leu Val Lys Leu Phe Val Arg Glu Pro Glu Lys Arg Leu Gly Val 610 615 620Arg Gly Asp Ile Arg Gln His Pro Leu Phe Arg Glu Ile Asn Trp Glu625 630 635 640Glu Leu Glu Arg Lys Glu Ile Asp Pro Pro Phe Arg Pro Lys Val Lys 645 650 655Ser Pro Tyr Asp Cys Ser Asn Phe Asp Lys Glu Phe Leu Ser Glu Lys 660 665 670Pro Arg Leu Ser Phe Ala Asp Arg Ala Leu Ile Asn Ser Met Asp Gln 675 680 685Asn Met Phe Ser Asn Phe Ser Phe Ile Asn Pro Gly Met Glu Thr Leu 690 695 700Ile Cys Ser7051319RNAArtificial Sequencesynthetic oligonucleotide 13ccagagagag gaagccgaa 191419RNAArtificial Sequencesynthetic oligonucleotide 14aggaagagau ggcucguca 191519RNAArtificial Sequencesynthetic oligonucleotide 15gucuugaccu ugaacggaa 191619RNAArtificial Sequencesynthetic oligonucleotide 16aagcaggagu caaacgagu 191713PRTArtificial Sequencesynthetic oligopeptide 17Ile Gly Val Ser Arg Gly Ser Gly Gly Ala Glu Gly Lys1 5 10

Claims

1. A method of reducing or eliminating the vimentin protein and/or the RLTPR protein and/or the PKC-.theta. protein in Treg cells, comprising: contacting the Treg cells with a vimentin-specific and/or a RLTPR-specific and/or a PKC-.theta.-specific inhibitory nucleic acid molecule.

2. The method of claim 1, wherein the vimentin-specific inhibitory nucleic acid molecule is complementary to at least a portion of the sequence shown in SEQ ID NO: 1.

3. The method of claim 1, wherein the RLTPR-specific inhibitor nucleic acid is complementary to at least a portion of the sequence shown in SEQ ID NO: 5.

4. The method of claim 1, wherein the PKC-.theta.-specific inhibitory nucleic acid molecule is complementary to at least a portion of the sequence shown in SEQ ID NO: 9.

5. The method of claim 1, wherein the vimentin-specific and/or the RLTPR-specific and/or the PKC-.theta.-specific inhibitory nucleic acid molecule is a RNAi nucleic acid molecule.

6. The method of claim 1, wherein the vimentin-specific and/or the RLTPR-specific and/or the PKC-.theta.-specific inhibitory nucleic acid molecule is an antisense nucleic acid molecule.

7. The method of claim 1, wherein the vimentin-specific and/or the RLTPR-specific and/or the PKC-.theta.-specific inhibitor nucleic acid molecule is a siRNA nucleic acid molecule.

8. The method of claim 1, wherein the vimentin-specific inhibitory nucleic acid molecule has the sequence shown in SEQ ID NO: 13, 14, 15 or 16.

9. The method of claim 1, wherein the Treg cells are contacted in vitro.

10. The method of claim 1, wherein the Treg cells are contacted in situ.

11. The method of claim 1, wherein the Treg cells are contacted in vivo in an individual who has received or is receiving a bone marrow transplant.

12. The method of claim 1, wherein the Treg cells exhibit a phenotype of at least one of the following: reduced PKC-.theta. auto-phosphorylation at Ser676; improved ability to suppress CD4+ and CD8+ Tcon proliferation; increased surface expression of Nrp1; increased surface expression of Lag3; increased basal and maximal oxygen consumption rate (OCR); increased BoDipy.sub.C1-C12 uptake; increased expression of CD71; increased expression of CD98; increased expression of CPT1a; or reduced activity of mTORC2, relative to Tregs that lack the vimentin-specific and/or the RLTPR-specific and/or the PKC-.theta.-specific inhibitory nucleic acid molecule.

13. A method of increasing or augmenting the suppressor cell potency of Treg cells, comprising: reducing or eliminating vimentin and/or RLTPR and/or PKC-.theta. in the Treg cells.

14. The method of claim 13, wherein reducing or eliminating the vimentin and/or the RLTPR and/or the PKC-.theta. in the Treg cells comprising contacting the Treg cells with a moiety selected from the group consisting of a nucleic acid, a nuclease, an antibody, a ligand, a peptide, a drug, a chemical, or a small molecule.

15. The method of claim 14, wherein the nucleic acid is a vimentin-specific and/or a RLTPR-specific and/or a PKC-.theta.-specific inhibitory nucleic acid molecule.

16. The method of claim 15, wherein the vimentin-specific and/or the RLTPR-specific and/or the PCK-.theta.-specific inhibitory nucleic acid molecule is selected from the group consisting of a RNAi nucleic acid molecule, an antisense nucleic acid molecule, and a siRNA nucleic acid molecule.

17. The method of claim 15, wherein the vimentin-specific inhibitory nucleic acid molecule is complementary to at least a portion of the sequence shown in SEQ ID NO: 1.

18. The method of claim 15, wherein the RLTPR-specific inhibitory nucleic acid molecule is complementary to at least a portion of the sequence shown in SEQ ID NO: 5.

19. The method of claim 15, wherein the PKC-.theta.-specific inhibitory nucleic acid molecule is complementary to at least a portion of the sequence shown in SEQ ID NO: 9.

20. The method of claim 13, wherein the method is performed in vitro.

21. The method of claim 13, wherein the method is performed in situ.

22. The method of claim 21, wherein the method is performed on an individual who has received or is receiving a bone marrow transplant.

23. The method of claim 13, wherein the Treg cells in which the vimentin, and/or RLTPR and/or PCK-.theta. has been reduced or eliminated exhibit a phenotype of at least one of the following: reduced PKC-.theta. auto-phosphorylation at Ser676; improved ability to suppress CD4+ and CD8+ Tcon proliferation; increased surface expression of Nrp1; increased surface expression of Lag3; increased basal and maximal oxygen consumption rate (OCR); increased BoDipy.sub.C1-C12 uptake; increased expression of CD71; increased expression of CD98; increased expression of CPT1a; or reduced activity of mTORC2, relative to Tregs in which vimentin, RLTPR and/or PCK-.theta. is not reduced or eliminated.