KR20130096338A - Screening method using mg53-ube2h interaction for the treatment of type 2 diabetes - Google Patents

Abstract

PURPOSE: A screening method of a treatment medicine for type 2 diabetes using interaction of MG53 and UBE2H is provided to be able to effectively select and develop a treatment medicine for type 2 diabetes by screening materials inhibiting the interaction of UBE2H and MG53. CONSTITUTION: A screening method of a treatment medicine for type 2 diabetes comprises (a) a step of treating a cell expressing mitsugumin 53 (MG53) and ubiquitin-conjugating enzyme E2H (UBE2H) with a candidate compound; and (b) a step of determining whether or not the interaction of MG53 and UBE2H is inhibited in the cell. The interaction of MG53 and UBE2H is detected by using an antibody specific to MG53 and UBE2H. The interaction of MG53 and UBE2H is detected with the immunoblotting, immune precipitation or immunostaining.

Description

Screening method using MG53-UBE2H interaction for the treatment of type 2 diabetes}

The present invention relates to a method for screening a type 2 diabetes treatment agent by determining a drug that inhibits ubiquitin-conjugating enzyme E2H (UBE2H) and MG53 (mitsugumin 53) interaction as a type 2 diabetes treatment. Agents that inhibit the interaction of MG53 and UBE2H can treat type 2 diabetes by increasing the amount of IRS-1 and increasing insulin sensitivity.

Diabetes is a group of metabolic disorders characterized by hyperglycemia. Chronic hyperglycemia causes complications such as macrovascular complications, microvascular complications, diabetic neuropathy and kidney disease if not treated properly. Diabetes is largely divided into two types. Type 1 diabetes mellitus is caused by a deficiency of insulin, a glucose-regulating hormone in the blood, and is mainly caused by young people in their teens and 20s. It is also called diabetes. Type 2 diabetes mellitus occurs mainly after the age of 40 and accounts for most of the diabetic patients in Korea. Unlike type 1, it is called adult-type diabetes and the cause of the disease is not clear yet, but it is known that it is caused by genetic factors and environmental factors involved. As a etiology of type 2 diabetes, both insulin secretion in pancreatic beta cells and defects in insulin action (insulin resistance) in target cells are observed.

The treatment of diabetes is generally combined with medication, diet, and exercise therapy. The goal is to prevent and delay diabetic complications by continuously maintaining blood sugar to normal levels. However, until now, the cure for diabetes has not been established, and oral hypoglycemic agents have been reported in various side effects, and thus, there is an interest in developing safer type 2 diabetes treatment. Type 2 diabetes is caused by a variety of problems in carbohydrates, lipid metabolism, and insulin signaling systems, and therefore, the development of new therapeutics requires the discovery of new target proteins that generally regulate these signaling and metabolic processes.

On the other hand, the present inventors (Korean Patent Registration No. 10-1048316) in the previous study, first identified that MG53 is a negative regulator of muscle differentiation, the overexpression of MG53 inhibits muscle formation and inhibiting the expression of MG53 muscle Since it promotes formation, it has been disclosed that drugs or gene therapies targeting MG53 can be developed as anabolic or cardiac enhancers by promoting differentiation and hypertrophy of skeletal muscle.

Then, the inventors confirmed that the MG53 knockout mice had a 30% increase in the size of soleus muscle, an increase in the amount of IRS-1, amplified insulin signaling, and no insulin resistance even when fed a high fat diet. In the process of identifying the mechanism by which MG53 acts on muscle differentiation and insulin signaling, the UBE2H-MG53-IRS-1 complex was discovered. In addition, interaction of UBE2H with the action of MG53 is essential, and drugs that inhibit UBE2H and MG53 interaction can be developed as a type 2 diabetes treatment by increasing the amount of IRS-1 and increasing insulin sensitivity. The present invention was completed by confirming.

One object of the invention is (a) treating a candidate compound to cells expressing MG53 and UBE2H; And (b) determining whether the interaction of MG53 and UBE2H in the cells is inhibited.

Another object of the present invention is to (a ') treating a candidate compound to a mixture of isolated MG53 and isolated UBE2H; And (b ') determining whether the interaction of MG53 and UBE2H is inhibited.

The present invention relates to a method for screening a type 2 diabetes treatment agent by determining a drug that inhibits UBE2H and MG53 interaction as a type 2 diabetes treatment agent.

In the present invention, in the process of identifying the mechanism by which MG53 acts on muscle differentiation and insulin signal transduction, the interaction of MG53 is essential to the interaction with UBE2H, and the agent that inhibits UBE2H and MG53 interaction is the amount of IRS-1. It was found for the first time that it can be developed as a type 2 diabetes treatment by increasing insulin sensitivity.

First, in the present invention, mRNA and protein expression levels of UBE2H gradually increase together with MG53 in the process of C2C12 myoblast differentiation (Fig. 1a-d), and through in vitro binding assay, MG53 strongly binds to UBE2H. It was confirmed (Fig. 2a). Molecular interactions between MG53 and UBE2H include reciprocal endogenous immunoprecipitation in C2C12 myotubes and exogenous immunoprecipitation in HEK 293 cells overexpressing MG53 and UBE2H. Reconfirmed by (FIGS. 2B and 3A). Thus, it can be seen that UBE2H and MG53 interact.

In addition, the degree of C2C12 cell muscle differentiation was increased by knocking down UBE2H by myosin heavy chain immunofluorescence, muscle differentiation index, and immunotaxel analysis, and the differentiation of C2C12 myoblasts was increased (Fig. 2C-E) and C2C12 muscle. It was confirmed that IRS-1 expression was increased in myotubes and ubiquitination was inhibited (FIGS. 2E and F). To determine whether inhibition of MG53 was caused by UBE2H knockdown during muscle differentiation, HA-MG53 was cotransfected into C2C12 myoblasts with si-control and si-UBE2H, followed by differentiation into HA and myosin metabolism. Slow immunofluorescence and muscle differentiation indices were measured, indicating that muscle differentiation induced by MG53 was inhibited by UBE2H knockdown (FIG. 2G, h). In addition, MG53-induced IRS ubiquitination was increased in HEK 293 cells overexpressed with UBE2H (FIG. 3B). These results support that UBE2H is an E2 enzyme for MG53 and targets IRS-1.

In addition, the inventors examined the effect of MG53 to control the size of the muscle fiber in the MG53 knockout mice, the weight of the sole of the MG53 knockout mice is about 27% increased compared to wild-type mice (Fig. 4a) , The area of the longitudinal section was significantly increased (Fig. 4b, c).

In addition, to demonstrate that MG53 inhibits insulin signal transduction by inducing IRS-1 ubiquitination and degradation as well as IGF-1 signaling, IRS-1, Akt and insulin induced by insulin in MG53-overexpressed C2C12 myoblasts As a result of confirming ERK1 / 2 phosphorylation, IRS-1, Akt and ERK1 / 2 activity by insulin was markedly decreased by MG53 overexpression (FIG. 5A). These results indicate that MG53 negatively regulates insulin signaling as well as IGF-1 signaling. In addition, after insulin injection into MG53 knockout mice, insulin signal transduction in the sole, calf and plantar muscles of the MG53 knockout mice resulted in increased insulin-induced IRS-1, Akt and ERK1 / 2 activity compared to wild-type mice. The amount of IRS-1 expression was markedly increased (FIGS. 5B-D).

In addition, high sugar high fat diet MG53 knockout mice had an increased glucose disposal rate in glucose and insulin load experiments (Figs. 6a, b), and compared to triglycerides in blood and free fat compared to wild type mice fed a high sugar high fat diet. Fatty acid and total cholesterol contents were significantly reduced (FIG. 6C-E), and blood levels of insulin and leptin were decreased (FIG. 6F, g). These results indicate that the diabetic phenotype induced by the high sugar high fat diet was improved by MG53 knockout.

In summary, it can be seen that MG53 is a target protein for the treatment of type 2 diabetes, and furthermore, the type 2 diabetes treatment agent can be effectively selected using the interaction with UBE2H, which is essential for the action of MG53.

Thus, in one aspect,

(a) treating a candidate compound with a cell expressing MG53 and UBE2H; And

(b) determining whether the interaction of MG53 and UBE2H in said cells is inhibited,

The present invention relates to a method for screening a type 2 diabetes treatment.

As another aspect, the present invention

(a ') treating the candidate compound with a mixture of isolated MG53 and isolated UBE2H; And

(b ') determining whether the interaction of MG53 and UBE2H is inhibited,

The present invention relates to a method for screening a type 2 diabetes treatment.

Hereinafter, the present invention will be described in more detail.

The screening method of the present invention comprises the step of treating candidate compounds with MG53 and UBE2H, wherein the candidate compounds are treated with cells expressing MG53 and UBE2H, or the candidate compounds with a mixture of isolated MG53 and isolated UBE2H. You can do it directly.

As used herein, the term "MG53 (mitsugumin 53)" is a protein that acts as a negative regulator of muscle differentiation, also called TRIM72 (tripartite motif-containing 72), RING region, B box, Coiled-coil region and SPRY region It consists of. The base sequence of the mouse MG53 gene and the amino acid sequence of the protein can be obtained from NCBI registration numbers NM_001079932 and NP_001073401, and the amino acid sequence of the mouse MG53 protein is shown by SEQ ID NO: 1. The nucleotide sequence of the human MG53 gene and the amino acid sequence of the protein can be obtained from NCBI registration numbers NM_001008274 and NP_001008275, and the amino acid sequence of the human MG53 protein is shown by SEQ ID NO: 2.

In the present invention, the term "UBE2H (ubiquitin-conjugating enzyme E2H)" is one of the ubiquitin binding enzyme E2 enzyme involved in the ubiquitination of the protein, the nucleotide sequence of the gene and amino acid sequence of the protein is NCBI registration number NM_003344 and NP_003335 The amino acid sequence of the UBE2H protein is shown in SEQ ID NO: 3.

As used herein, the term "candidate compound" means a candidate substance that is expected to inhibit the interaction of MG53 and UBE2H. Such candidate substances may include not only a single compound such as an organic or inorganic compound but also a complex of a protein, a carbohydrate, a nucleic acid molecule (RNA, DNA, etc.) and a polymer compound such as lipid and a plurality of compounds.

As used herein, the term "treatment" means not only the candidate compound directly contacts any one or more of MG53 and UBE2H, but also the substance affects the cell membrane and the signal generated from the cell membrane contacts any one or more of MG53 and UBE2H. Includes or affects. Thus, in the present invention, the candidate compound includes not only substances that are permeable to cell membranes but also substances that are impermeable to cell membranes. At this time, the candidate compound is treated within the effective amount range. The term "effective amount" means an amount sufficient to induce the reaction. Since the accurate result can not be obtained at an effective amount or less, it is preferable to measure the inhibitory ability within an effective amount range.

When the candidate compound is treated to cells expressing MG53 and UBE2H, the present invention

(a) treating a candidate compound with a cell expressing MG53 and UBE2H; And (b) determining whether the interaction of MG53 and UBE2H in said cells is inhibited.

The cells expressing MG53 and UBE2H are originally cells containing MG53 and UBE2H, or may be prepared by transfecting cells with a vector expressing MG53 and UBE2H intentionally. In addition, in the case of the vectors expressing MG53 and UBE2H, one vector contains different promoters for MG53 and UBE2H, which may be expressed from one vector, respectively, and the vector expressing MG53 and the vector expressing UBE2H may be co-expressed. It can also be prepared by transfection.

The cells include all cells of animal origin, such as humans or cows, goats, pigs, mice, rabbits, hamsters, rats, guinea pigs, and the like, primary cells, secondary cells, immortalized cells and the like can be used. In addition, recombinant vectors comprising MG53 and / or UBE2H genes can be used to use cells engineered to stably or temporarily overexpress MG53 and / or UBE2H in cells.

Furthermore, detection of substances that inhibit the interaction of MG53 and UBE2H can be performed in vivo using experimental animals such as mice, rabbits, hamsters, sagittal mice, guinea pigs, as well as at the cellular level.

In the step (b) of the present invention, whether the interaction of MG53 and UBE2H is inhibited, it can be confirmed by applying techniques commonly used in the art to determine whether the reaction between protein-protein or protein-compound. . For example, immunological assays, fluorescence assays, yeast two-hybrids, search for phage display peptide clones that bind MG53 or UBE2H, high throughput screening (HTS) using natural and chemical libraries, Screening methods using drug hit HTS or cell-based screening may be used, but the present invention is not limited thereto.

In one preferred embodiment, antibodies specific for MG53 or UBE2H can be used to detect whether the interaction of MG53 and UBE2H is inhibited. For example, the amount of the formed antigen-antibody complex formed can be compared with the amount of the antigen-antibody complex formed in the control group not treated with the candidate substance to determine the inhibition. Absolute or relative differences in the amount of antigen-antibody complex formation can be ascertained by molecular biology or histological analysis including, but not limited to, immunostaining, immunoprecipitation and immunostaining.

In the detection method using the antigen-antibody reaction, the formation amount of the antigen-antibody complex can be quantitatively measured through the signal label of the detection label. In the present invention, the term "detection label" is to be detected by means of spectroscopic, photochemical, biochemical, immunochemical, chemical or other physicochemical means ≪ / RTI > Such detection labels include, but are not limited to, enzymes, minerals, ligands, luminescent materials, microparticles, redox molecules, and radioactive isotopes.

In another preferred embodiment, after connecting the reporter protein to MG53 or UBE2H, respectively, it is possible to detect whether the interaction of MG53 and UBE2H is inhibited by the signal size of the reporter protein.

The reporter protein may be a luciferase, a chloramphenicol acetyltransferase, a beta-glucuronidase, a beta-galactosidase, an alkaline phosphatase, or a fluorescent protein, wherein the fluorescent protein is a green fluorescent a fluorescent protein, a red fluorescent protein, a blue fluorescent protein, a yellow fluorescent protein, a cyan fluorescent protein, an enhanced green fluorescent protein, , An enhanced red fluorescent protein, an enhanced blue fluorescent protein, an enhanced yellow fluorescent protein, or an enhanced blue fluorescent protein.

To express the reporter protein in a form linked to MG53 and UBE2H, respectively, an expression vector operably linked with a gene encoding MG53 and a reporter gene, and an expression vector operably linked with a gene encoding UBE2H and a reporter gene Co-transfection.

When the reporter protein is a fluorescent protein, it is possible to determine whether the interaction of MG53 and UBE2H is inhibited by measuring the fluorescence size of the reporter protein using a fluorescence microscope, and it is known when a luminescent enzyme such as luciferase is used as the reporter protein. Luciferase activity measurement or luciferase luminescence can determine whether the interaction of MG53 and UBE2H is inhibited. In addition, as a reporter protein, an enzyme that uses a chloramphenicol acetyltransferase, beta-glucuronidase, beta-galactosidase, alkaline phosphatase and the like to convert a substrate into a detectable light emitting product In this case, it can be confirmed whether the interaction between MG53 and UBE2H is inhibited by the optical signal obtained by the enzymatic reaction using the chromogenic substrate.

On the other hand, when the candidate compound is treated with a mixture of separated MG53 and separated UBE2H, the present invention

(a ') treating the candidate compound with a mixture of isolated MG53 and isolated UBE2H; And (b ') determining whether the interaction of MG53 and UBE2H is inhibited.

In the present invention, the term "isolated" means a protein expressed and isolated in a cell, and is separable by applying common separation techniques known in the art. A method for separating and purifying a protein expressed in a cell is not particularly limited. Isolated MG53 and isolated UBE2H can react directly with the candidate compound in vitro and not in cells.

The method of checking whether the interaction of MG53 and UBE2H is inhibited is as described above.

Agents that inhibit the interaction of MG53 and UBE2H obtained by the screening method of the present invention can treat type 2 diabetes by increasing the amount of IRS-1 and increasing insulin sensitivity.

The present invention can effectively screen and develop Type 2 diabetes therapeutics by screening for substances that inhibit UBE2H and MG53 interactions.

Figure 1a shows the results of confirming the expression level of MBP-MG53, UBE2H, UBE2R1, UBE2D2, UBE2E1, UBE2C, UBE2M and UBE2N in the process of muscle differentiation of C2C12 cells by SDS-PAGE.
FIG. 1B shows mRNA expression levels of UBE2H, UBE2R1, UBE2D2, UBE2E1, UBE2L3, UBE2L6, UBE2C, UBE2M, UBE2N, Cav-3, MyHC, MyoD and GAPDH for 5 days from the start of muscle differentiation of C2C12 cells One result is shown. GAPDH represents the loading control.
1C shows the results of standardizing the real-time quantitative PCR results for UBE2H mRNA for GAPDH mRNA results for 5 days from the start of muscle differentiation of C2C12 cells (* p <0.01, ** p <0.05).
FIG. 1D shows the results of confirming the expression of UBE2H, MG53, Mgn, IRS-1 and Cav-3 by immunoassay for 5 days from the start of muscle differentiation of C2C12 cells. Actin represents the loading control.
2A shows the results of in vitro protein binding assays with MG53 and various E2 enzymes (UBE2H, UBE2R1, UBE2D2, UBE2E1, UBE2C, UBE2M and UBE2N).
Figure 2b shows the results confirming the molecular binding between MG53 and UBE2H by reciprocal endogenous immunoprecipitation in C2C12 cells on the 4th day of differentiation start.
Figure 2c-e after suppressing the expression of UBE2H using siRNA in C2C12 myoblasts, the degree of muscle differentiation was confirmed by immunofluorescence (c), muscle differentiation index (d), immunosampling method for differentiation marker protein Indicates.
FIG. 2F shows the results of confirming the ubiquitination of IRS-1 by immunoprecipitation by treating MG132 after suppressing the expression of UBE2H using siRNA in C2C12 myoblasts.
Figure 2g-h shows the results of overexpressing UBE2H siRNA and MG53 in C2C12 myoblasts, the degree of differentiation through immunofluorescence (g) and the number of nuclei in HA-positive cells (h).
FIG. 3A shows the results of confirming the molecular interaction between MG53 and UBE2H by reciprocal immunoprecipitation in HEK293 cells co-transfected with Myc-Mg53 and Flag-UBE2H.
3b shows immunoprecipitation and anti-His using anti-Flag antibody when MG132 was treated for 12 hours with HEK293 cells co-transfected with Flag-IRS-1, HA-MG53 WT, Myc-UBE2H and His-Ubiquitin. The result of confirming IRS-1 ubiquitination by the immunotaxine method using an antibody is shown.
FIG. 4A shows the results of a comparison of soleus muscle weight in wild type mice and MG53 knockout mice.
Figures 4b and c show the results of confirming the shape of the longitudinal section and the distribution of the longitudinal area of the sole of the MG53 knockout mouse by immunofluorescence.
Figure 5a shows the result of confirming the phosphorylation of the signaling substances by immuno-tax method after insulin treatment of C2C12 myoblasts overexpressing MG53.
FIG. 5B shows the immunoassay method for the protein amount of insulin signaling material and tyrosine phosphorylation of IRS-1 in gastrocnemius muscle and plantaris muscle after vascular injection of wild type mice and MG53 knockout mice. The result confirmed is shown.
5C and D show the results of statistical analysis of IRS-1 expression, IRS-1 phosphorylation and Akt phosphorylation in soleus, calf and plantar muscles of wild type mice and MG53 knockout mice (* p <0.01 and ** p). <0.05).
Figures 6a and b show the results of the glucose load test (a) and insulin load test (b) in MG53 knockout mice fed high sugar-high fat diet for 10 weeks. AUC represents the area under the curve.
Figure 6c to g quantify the concentration of triglycerides (c), free fatty acids (d), total cholesterol (e), insulin (f) and leptin (g) in the blood of the MG53 knockout mice inducing obesity and diabetes The analyzed results are shown (* p <0.01 and ** p <0.05).
Figure 7 illustrates the mechanism by which MG53 regulates muscle differentiation and insulin signaling.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  1. Preparation for Animal Experiments

MG53 ^-/- mice were prepared as previously described (Lee, CS et al., Cell Death Differ 17, 1254-65, 2010). For backcross, MG53 ^+/− mice were bred for 7 generations with C57BL / 6 mice and incubated in a 12: 12h light-dark photoperiod condition in a plastic cage free of water and food. Type 2 diabetes was induced with a 10-week high sugar-high-fat diet (Research diet, D12330), and glucose tolerance and insulin tolerance tests were performed as previously described (Yadav, H. et al., Cell Metab 14, 67-79, 2011). Triglycerides, free fatty acids, and total cholesterol were measured using a colorimetric assay kit obtained from Biovision. Blood levels of insulin and leptin were determined using the Bio-Plex Pro ^™ Mouse Diabetes Assay Kit (Bio-Rad). The experimental animals were treated according to the Principles of Laboratory Animal Protection (NIH Publication No. 85-23, revised 1985) and followed the protocol approved by Korea University Animal Experiment Ethics Committee.

Example  2. Cell culture

After C2C12 cells were purchased from ATCC, 37 ° C., 5% (v) in DMEM medium (Dulbecco's modified Eagle's medium) supplemented with 2% (v / v) penicillin / streptomycin and 10% (v / v) fetal calf serum. / v) incubated in a CO ₂ incubator. Confluent C2C12 myoblasts were cultured in DMEM medium supplemented with these antibiotics and 2% (v / v) horse serum to differentiate into myotubes (microtubes). Every 48 hours, fresh muscle cells were supplied with fresh DMEM containing 2% of horse serum. Mouse embryonic fibroblasts (MEFs) were obtained from MG53 ^{+ / +} and MG53 ^{− / −} embryos at day 12.5 of development. The head and internal organs were separated from the embryos and the remaining sites were ground and dispersed in trypsin / EDTA at 37 ° C. for 30 minutes. Trypsinized tissue was passed through a 100 μM cell strainer and the filtrate was centrifuged at 1000 × g. Cell pellets were resuspended in growth medium.

Example  3. Immunotaxine immunoblotting , IB ), Immunoprecipitation ( immunoprecipitation , IP ) And Immunofluorescence  ( immunofluorescence assay , IFA )

Immunotaxin and immunofluorescence were performed as previously described (Kim, KB et al. Proteomics 6, 2444-53, 2006). For immunoprecipitation, cells were treated with 20 mM Tris-HCl (pH 7.4), 137 mM NaCl, 1 mM MgCl ₂ , 1 mM CaCl ₂ , 20 mM NaF, 10 mM Na ₄ P ₂ O ₇ , 1 mM Na ₃ VO ₄ , 1% (v / v) was dissolved in a buffer containing NP-40, 1 mM PMSF and protease inhibitor cocktail (Roche). Total cell lysates (50 μl protein) were incubated with specific antibodies for 90 minutes and then incubated with 50 μl of Protein A-Sepharose or G-Agarose beads (50%) slurry for 90 minutes. Thereafter, immunoprecipitates were analyzed by immunoassay method. For immunosampling of skeletal muscle protein, muscle fibers were dissolved in a low salt buffer containing 10 mM KH ₂ PO ₄ (pH 7.4), a protease inhibitor cocktail. The antibodies used for immunotaxine, immunoprecipitation and immunofluorescence are listed in Tables 1 and 2.

protein manufacturer Host (clone number) Experimental Method (Dilution Rate) Flag Sigma Mouse Monoclonal (M2) IB (1: 1,000), IFA (1: 100) Santa cruz
Biotechnology Rabbit Polyclonal IP (1: 100), IB (1: 1,000) Myhc Developmental Studies Hybridoma Bank Mouse Monoclonal (MF-20) IB (1: 1,000), IFA (1: 100) IRS-1 Millipore Rabbit Polyclonal IP (1: 200), IB (1: 1,000) Transduction laboratories Mouse, Monoclonal (6 / IRS-1) IB (1: 1,000) Actin (beta) Santa cruz
Biotechnology Mouse Monoclonal (C4) IB (1: 1,000) Actin (alpha) Santa cruz
Biotechnology Mouse Monoclonal (? -SR1) IB (1: 1,000) pAkt (T308) Cell signaling Rabbit Polyclonal IB (1: 1,000) Akt Millipore Mouse Monoclonal (skb1) IB (1: 1,000) pY
(Tyrosine phosphorylation) Transduction laboratories Mouse Monoclonal (PY20) IB (1: 1,000) Myc Santa cruz
Biotechnology Mouse monoclonal
(Clone 90000000000) IB (1: 1,000) Rabbit Polyclonal IP (1: 100), IB (1: 1,000) His Santa cruz
Biotechnology Mouse Monoclonal (AD1.1.10) IP (1: 100), IB (1: 1,000) Ubiquitin Cell signaling Mouse Monoclonal (P4D1) IB (1: 1,000) HA Santa cruz
Biotechnology Rabbit Polyclonal IP (1: 100), IFA (1: 100) Mouse Monoclonal (F-7) IB (1: 1,000) Myod Santa cruz
Biotechnology Mouse Monoclonal (5.8A) IB (1: 1,000) ERK Santa cruz
Biotechnology Rabbit Polyclonal IB (1: 1,000) pERK Santa cruz
Biotechnology Mouse Monoclonal (E-4) IB (1: 1,000) β-Dystroglycan Santa cruz
Biotechnology Mouse Monoclonal (4F7) IFA (1: 100) MBP NEB Mouse monoclonal IB (1: 5,000)

Antibody name manufacturer host Experimental Method (Dilution Rate) HRP-conjugated Anti-mouse IgG Pierce Goat polyclonal IB (1: 20,000-40,000) HRP-conjugated Anti-mouse IgM Pierce Goat polyclonal IB (1: 20,000) HRP-conjugated Anti-Rabbit IgG Pierce Goat polyclonal IB (1: 20,000-40,000) Alexa Fluor 488-conjugated Anti-mouse IgG Invitrogen Goat polyclonal IFA (1: 100) FITC-conjugated Anti-mouse IgG Abcam Rabbit polyclonal IFA (1: 100) Rhodamine-conjugated Anti-Rabbit IgG Abcam Goat polyclonal IFA (1: 100)

Example  4. Histology ( histology )

Skeletal muscle was fixed in 4% paraformaldehyde, frozen in frozen 2-methylbutane and then cleaved with cryomicrotome. Skeletal muscle sections were stained with anti-β-distoglycan antibody to the muscle fiber membrane (sarcolemma) and DAPI to the nucleus. The cross-sectional area of muscle fibers in muscle fibers was determined by Multigauge software (Fuji Photo Film Co.). Nine separate slides from six animals in each group were used.

Example  5. Muscle differentiation index ( myogenic index ) Measure

Differentiated C2C12 cells or MEFs were stained with anti-myosin heavy chain antibody and DAPI, and cell images were observed with fluorescence microscopy (Olympus). The ratio of the number of nuclei in myosin heavy chain-positive myoblasts to the total number of nuclei at the stained sites is indicated by the muscle differentiation index.

Example  6. RNA  Interference ( RNA interference )

SiRNA oligomers that target MG53 (si-MG53) or siRNA oligomers that target UBE2H (si-UBE2H), and scrambled oligomers (si-control) were prepared in Ambion. The target sequence of MG53 is 5'-AAGCACGCCUCAAGACACAGCtt-3 '(SEQ ID NO: 4) and the target sequence of UBE2H is 5'-CUAUGAUCUUACCAAUAUAtt-3' (SEQ ID NO: 5). si-control, si-MG53 or si-UBE2H were transfected into C2C12 myoblasts using electroporation according to the manufacturer's (Digital Bio) protocol.

Example  7. In vitro binding analysis ( In vitro binding assay )

Human MG53 gene (residues 7-470) was cloned into pMAL-c2x vector (NEB). E. coli C41 (DE3) was used as a host for MBP-MG53 expression. Expressed proteins were incubated in amylose resin previously equilibrated with phosphate-buffered saline (PBS) buffer. Resin was washed with PBS and MBP-fused protein was eluted from resin using elution buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM DTT and 10 mM maltose). For further purification, the eluted protein was loaded on a HiTrap Q Fast Flow column equilibrated with buffer (50 mM Tris-HCl, pH 8.0 and 1 mM DTT). MBP-MG53 was eluted with 150-250 mM NaCl. Purified protein was stored at -80 ° C until use. Pull-down assay for 1 hour at 4 ° C. using 1 ml of assay buffer (1X PBS and 1 mM DTT) containing 20 μl of amylose resin, 50 μg of MBP-MG53 and 100 μg of histidine-tagged E2 enzyme. Was performed. Histidine-tagged E2 enzyme was purchased from Boston Biochem and LifeSensors. To remove unbound E2 enzyme, the resin was washed with assay buffer at least five times. To elute the bound MBP-MG53: E2 enzyme complex, 35 μl of elution buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM DTT and 10 mM maltose) was incubated in the resin for 1 minute. After centrifugation, 30 μl of each eluate was mixed with 5 × SDS sample buffer and heated for 3 minutes. The interaction between MG53 and E2 enzymes was measured by immunotaxon method with anti-histidine and anti-MBP antibodies.

Example  8. RT - PCR

CDNA was synthesized from DNase1 treated RNA (1 μg) by reverse transcription using a random hexamer primer and M-MLV reverse transcriptase (Invitrogen). PCR was initially performed in the 24-38 cycle range and 2 μl of 1: 4-diluted cDNA (12.5 ng / 50 μl PCR reaction) performed 28-36 cycles. Primers used for the RT-PCR reaction are as follows.

gene Forward (5 '-> 3') Reverse (5 '-> 3') SEQ ID NO: MG53 TCCCTGTTGTCAGGCATCTAC TTCTTCCACACCTGGAATTTG 6,7 Myod CTCCTTTGAGACAGCAGACGACTT AAATCGCATTGGGGTTTGAGCCTG 8,9 Myhc AGAAGGAGGAGGCAACTTCTG ACATACTCATTGCCGACCTTG 10,11 α-actin GAAGAGCTATGAGCTGCCTGA CTCATCGTACTCCTGCTTGCT 12,13 UBE2H AAGGAACACCATATGAAGGCG GCGTACTTCTGGATGTACTCTTTAA 14,15 UBE2R1 TGCCAAGCTCGCAGAAGGCG CCACCGCTCTGAGGGCAGCT 16,17 UBE2D2 GCCCTCAGCTCGTCTGATCCG TGATAGGGGCTGTCATTTGGCCC 18,19 UBE2E1 TGCCTGGTTAATAGTTGCTGTTGCT CACTGCAGTTTGGCGGAGGGT 20,21 UBE2L3 AAGGGGCAGGTCTGTCTGCC CCTGCTTTGCGGGGTGTCTGA 22,23 UBE2L6 AGCTGCCGCCATACCTTCGC GCAAGTTCCAGACGCACAGGCT 24,25 UBE2C AAACCGCGACCCAGCTGCTG GCCCTGGGTGTCCACGTTGG 26,27 UBE2M AGGGCAGCAGCAAGAAGGCG GGCAGACGTTGCCCTCGAGG 28,29 UBE2N ATGTGGTCATTGCTGGCCCCC GCAGAACAGGAGAAGTGGTGTACAC 30,31 GAPDH TGGCAAATTCCATGGCACC AGAGATGATGACCCTTTTG 32,33

Example  9. Real time quantitative PCR  ( Quantitative real - time PCR )

Real-time quantitative PCR analysis was performed in the presence of Lightcycler 480 SYBR Green I Master Mix (Roche Diagnostics GmbH) using single stranded cDNA and gene-specific oligonucleotides. Lightcycler PCR conditions were as follows: 35-45 cycles of initial denaturation at 95 ° C. for 10 minutes, followed by denaturation at 95 ° C. for 10 seconds, annealing at 57 ° C. for 10 seconds and extension at 72 ° C. for 30 seconds. Melting curves were analyzed for each PCR product for quality control.

Example  10. Chemical crosslinking ( Chemical cross - linking )

HEK 293 cells were collected with octyl-β-D-glucopyranoside in PBS. Total cell lysates were mixed with glutaraldehyde and incubated at 37 ° C. for 20 minutes. Treatment with 1.5 M Tris-HCl (pH 7.4) stopped the crosslinking reaction and separated the protein on SDS-PAGE.

Example  11. Statistical Analysis Statistical analysis )

Statistics are expressed as standard error of the mean (SEM). Two-tailed Student's test was used to calculate p values.

Result 1. MG53 On by IRS -One Ubiquitination UBE2H Is required

In order to determine what E2 ligase is required for IRS-1 ubiquitination via MG53, the mRNA and protein levels of various E2 enzymes were examined during differentiation of C2C12 myoblasts. First, the maltose binding protein (MBP) tagged MG53 and His (histidine) tagged E2 enzymes UBE2H, UBE2R1, UBE2D2, UBE2E1, UBE2C, UBE2M and UBE2N proteins used in FIG. 2A were coomassie-stained on an SDS-PAGE gel. Confirmed by (Fig. 1a). As a result, it was confirmed that the expression level of UBE2H increased. MRNA differentiation of UBE2H, UBE2R1, UBE2D2, UBE2E1, UBE2L3, UBE2L6, UBE2C, UBE2M, UBE2N, Cav-3, MyHC and MyoD for 5 days from the beginning of muscle differentiation of C2C12 myoblasts was determined by real-time quantitative PCR It was confirmed that the mRNA expression level of UBE2H gradually increased as the progression was performed (FIG. 1B). Among these, UBE2H mRNA expression was normalized based on real-time quantitative PCR results for GAPDH mRNA results. Could be (FIG. 1C). In addition, as a result of confirming the expression of UBE2H, MG53, Mgn, IRS-1 and Cav-3 for 5 days from the start of muscle differentiation of C2C12 cells by immunotaxy method, it was confirmed that UBE2H protein expression level gradually increased as muscle differentiation progressed. (FIG. 1D).

In addition, in vitro protein binding assays with MG53 and various E2 enzymes (UBE2H, UBE2R1, UBE2D2, UBE2E1, UBE2C, UBE2M and UBE2N) were performed to demonstrate that UBE2H and MG53 directly interact. To this end, MBP-MG53 and His-E2 enzymes were mixed, immobilized on amylose resin, and analyzed by immunotaxy. As a result, it was confirmed that UBE2H and MG53 had protein-protein binding (FIG. 2a). In addition, molecular interactions between MG53 and UBE2H were reconfirmed by reciprocal endogenous immunoprecipitation in C2C12 cells at day 4 of differentiation (FIG. 2B).

In addition, C2C12 cells were treated with si-control and si-UBE2H for 24 hours at 50 nM for 24 hours to knock down UBE2H and differentiate for 48 hours. Myocyte differentiation pattern was analyzed by DACI staining for myosin heavy chain immunoblot (FIG. 2C) The expression of muscle differentiation marker protein (FIG. 2d) and the expression level of muscle differentiation marker protein (FIG. 2e) through the immunostocking method showed that the muscle differentiation was enhanced by UBE2H knockdown and the expression level of IRS-1 was increased in particular. This shows that UBE2H is involved in the degradation of IRS-1. In addition, the ubiquitinization of IRS-1 was confirmed by endogenous immunoprecipitation by treating MG132 for 12 hours in C2C12 myoblasts treated with si-UBE2H and knocking down UBE2H. As a result, UBE2H knockdown resulted in ubiquitin of IRS-1. Confirmation was inhibited (FIG. 2F).

In addition, si-UBE2H or si-control on C2C12 myoblasts were treated simultaneously with HA-MG53 and differentiated for 48 hours, and analyzed by HA and MyHC immunofluorescence using DAPI to investigate the degree of muscle differentiation (FIG. 2g), three independent micrographs of the number of nuclei in HA-positive cells (FIG. 2H) showed that ubiquitination of IRS-1 did not occur and muscle differentiation occurred without UBE2H in the presence of MG53. It can be confirmed that it happens well.

In addition, 2 μg of each of Myc-Mg53 and Flag-UBE2H was co-transfected into HEK293 cells for 48 hours, and then the molecular interactions between MG53 and UBE2H were monitored by reciprocal immunoprecipitation, resulting in MG53 and UBE2H. Interactions of (Fig. 3a), Flag-IRS-1, HA-MG53 WT, Myc-UBE2H and His-Ubiquitin co-transfected HEK293 cells, and after 12 hours of MG132 10 μM, As a result of confirming IRS-1 ubiquitination by immunoprecipitation using anti-Flag antibody and immunotaxin method using anti-His antibody, it was confirmed that IRS-1 ubiquitination was strongly induced by UBE2H overexpression in HEK293 cells (FIG. 3b).

Therefore, it can be seen from the data that UBE2H is required for MG53 to induce ubiquitination of IRS-1.

Result 2. MG53 Knockout  Mice show hypertrophy and insulin sensitivity of skeletal muscle

Because MG53 negatively regulates IGF signaling, MG53 knockout mice are expected to have more developed muscles, actually the soleus (soleus) in 8-week-old male wild-type mice and MG53 knockout mice (n = 7 in each group). muscle) was isolated and the weight was examined, it was confirmed that the weight of the soleus muscle in the MG53 knockout mice increased by about 30% (Fig. 4a). In addition, as a result of examining the shape and the distribution of the longitudinal area of the sole of the MG53 knockout mouse after DAPI-staining and beta-distoglycan immunofluorescence staining, the proportion and distribution of the longitudinal area of the MG53 knockout mouse was high (Fig. 4b and 4c).

Next, to demonstrate that MG53 negatively regulates insulin signaling as well as IGF signaling by inducing ubiquitination of IRS-1, after overexpression of MG53 in C2C12 myoblasts using adenovirus vectors, 6 The serum was kept free for hours. Insulin 100 nM was treated by time period (0, 5, 10 minutes), and the expression of MG53, pAkt, Akt, pERK1 / 2, ERK1 / 2 and actin was measured by immunoassay to investigate the signaling by insulin. . Tyrosine phosphorylation of IRS-1 was confirmed by immunotaxy using an anti-phosphate-tyrosine antibody after immunoprecipitation. As a result, it was confirmed that MG53 interferes with phosphorylation of IRS-1, Akt, and Erk (FIG. 5A). In addition, 8-week-old wild-type mice and MG53 knockout mice were injected with insulin (10 min, 5 U / kg), and 5 min later, gastrocnemius muscle and plantaris muscle were isolated and insulin signaling Materials pAkt, total Akt, pERK1 / 2, total ERK1 / 2 and protein amount of actin and tyrosine phosphorylation of IRS-1 were confirmed by immunotrophography. Tyrosine phosphorylation of IRS-1 was confirmed by immunoprecipitation using anti-IRS-1 and immunotaxine using anti-phosphate-tyrosine antibody. As a result, it was confirmed that the expression level of IRS-1 was increased in the muscles of knockout mice compared to wild type mice, and phosphorylation of IRS-1, Akt, and Erk by insulin was better (FIG. 5B). In addition, IRS-1 expression, IRS-1 phosphorylation, and Akt phosphorylation in soleus, calf and plantar muscles of 8-week-old wild-type and MG53 knockout mice were analyzed. Expression levels were increased and it was confirmed that phosphorylation of IRS-1 and Akt by insulin was better (FIGS. 5c and d).

Result 3. MG53 Knockout  Mice do not show insulin resistance

Four-week-old male wild-type mice and MG53 knockout mice (n = 6 in each group) inducing high-sugar-fat obesity and diabetes (n = 6 in each group) were tested for glucose tolerance and insulin tolerance tests, respectively. ) Showed faster blood glucose reduction in MG53 knockout mice compared to wild-type mice (FIGS. 6A and B). In addition, as a result of measuring the concentrations of triglycerides and free fatty acids, total cholesterol, insulin and leptin in the blood of the MG53 knockout mice induced with obesity, MG53 knockout mice compared with wild type mice were found Concentrations were similar to normal mice (FIGS. 6C-6G). This strongly demonstrates that MG53 is a target protein for the treatment of type 2 diabetes, and further supports the effective selection of the type 2 diabetes treatment using interaction with UBE2H, which is essential for the action of MG53.

<110> Korea University Research and Business Foundation <120> Screening method using MG53-UBE2H interaction for the treatment          of type 2 diabetes <130> DPP20117102KR <160> 33 <170> Kopatentin 1.71 <210> 1 <211> 477 <212> PRT <213> Mus musculus <220> <221> PEPTIDE (222) (1) .. (477) <223> MG53 (Genbank accession No. NP_001073401) <400> 1 Met Ser Ala Ala Pro Gly Leu Leu Arg Gln Glu Leu Ser Cys Pro Leu   1 5 10 15 Cys Leu Gln Leu Phe Asp Ala Pro Val Thr Ala Glu Cys Gly His Ser              20 25 30 Phe Cys Arg Ala Cys Leu Ile Arg Val Ala Gly Glu Pro Ala Ala Asp          35 40 45 Gly Thr Val Ala Cys Pro Cys Cys Gln Ala Pro Thr Arg Pro Gln Ala      50 55 60 Leu Ser Thr Asn Leu Gln Leu Ser Arg Leu Val Glu Gly Leu Ala Gln  65 70 75 80 Val Pro Gln Gly His Cys Glu Glu His Leu Asp Pro Leu Ser Ile Tyr                  85 90 95 Cys Glu Gln Asp Arg Thr Leu Val Cys Gly Val Cys Ala Ser Leu Gly             100 105 110 Ser His Arg Gly His Arg Leu Leu Pro Ala Ala Glu Ala Gln Ala Arg         115 120 125 Leu Lys Thr Gln Leu Pro Gln Gln Lys Met Gln Leu Gln Glu Ala Cys     130 135 140 Met Arg Lys Glu Lys Thr Val Ala Val Leu Glu His Gln Leu Val Glu 145 150 155 160 Val Glu Glu Thr Val Arg Gln Phe Arg Gly Ala Val Gly Glu Gln Leu                 165 170 175 Gly Lys Met Arg Met Phe Leu Ala Ala Leu Glu Ser Ser Leu Asp Arg             180 185 190 Glu Ala Glu Arg Val Arg Gly Asp Ala Gly Val Ala Leu Arg Arg Glu         195 200 205 Leu Ser Ser Leu Asn Ser Tyr Leu Glu Gln Leu Arg Gln Met Glu Lys     210 215 220 Val Leu Glu Glu Val Ala Asp Lys Pro Gln Thr Glu Phe Leu Met Lys 225 230 235 240 Phe Cys Leu Val Thr Ser Arg Leu Gln Lys Ile Leu Ser Glu Ser Pro                 245 250 255 Pro Pro Ala Arg Leu Asp Ile Gln Leu Pro Val Ile Ser Asp Asp Phe             260 265 270 Lys Phe Gln Val Trp Lys Lys Met Phe Arg Ala Leu Met Pro Ala Leu         275 280 285 Glu Glu Leu Thr Phe Asp Pro Ser Ser Ala His Pro Ser Leu Val Val     290 295 300 Ser Ser Ser Gly Arg Arg Val Glu Cys Ser Asp Gln Lys Ala Pro Pro 305 310 315 320 Ala Gly Glu Asp Thr Arg Gln Phe Asp Lys Ala Val Ala Val Val Ala                 325 330 335 Gln Gln Leu Leu Ser Gln Gly Glu His Tyr Trp Glu Val Glu Val Gly             340 345 350 Asp Lys Pro Arg Trp Ala Leu Gly Val Met Ala Ala Asp Ala Ser Arg         355 360 365 Arg Gly Arg Leu His Ala Val Pro Ser Gln Gly Leu Trp Leu Leu Gly     370 375 380 Leu Arg Asp Gly Lys Ile Leu Glu Ala His Val Glu Ala Lys Glu Pro 385 390 395 400 Arg Ala Leu Arg Thr Pro Glu Arg Pro Pro Ala Arg Ile Gly Leu Tyr                 405 410 415 Leu Ser Phe Ala Asp Gly Val Leu Ala Phe Tyr Asp Ala Ser Asn Pro             420 425 430 Asp Val Leu Thr Pro Ile Phe Ser Phe His Glu Arg Leu Pro Gly Pro         435 440 445 Val Tyr Pro Ile Phe Asp Val Cys Trp His Asp Lys Gly Lys Asn Ala     450 455 460 Gln Pro Leu Leu Leu Val Gly Pro Glu Gln Glu Gln Ala 465 470 475 <210> 2 <211> 477 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE (222) (1) .. (477) <223> MG53 (Genbank accession No. NP_001008275) <400> 2 Met Ser Ala Ala Pro Gly Leu Leu His Gln Glu Leu Ser Cys Pro Leu   1 5 10 15 Cys Leu Gln Leu Phe Asp Ala Pro Val Thr Ala Glu Cys Gly His Ser              20 25 30 Phe Cys Arg Ala Cys Leu Gly Arg Val Ala Gly Glu Pro Ala Ala Asp          35 40 45 Gly Thr Val Leu Cys Pro Cys Cys Gln Ala Pro Thr Arg Pro Gln Ala      50 55 60 Leu Ser Thr Asn Leu Gln Leu Ala Arg Leu Val Glu Gly Leu Ala Gln  65 70 75 80 Val Pro Gln Gly His Cys Glu Glu His Leu Asp Pro Leu Ser Ile Tyr                  85 90 95 Cys Glu Gln Asp Arg Ala Leu Val Cys Gly Val Cys Ala Ser Leu Gly             100 105 110 Ser His Arg Gly His Arg Leu Leu Pro Ala Ala Glu Ala His Ala Arg         115 120 125 Leu Lys Thr Gln Leu Pro Gln Gln Lys Leu Gln Leu Gln Glu Ala Cys     130 135 140 Met Arg Lys Glu Lys Ser Val Ala Val Leu Glu His Gln Leu Val Glu 145 150 155 160 Val Glu Glu Thr Val Arg Gln Phe Arg Gly Ala Val Gly Glu Gln Leu                 165 170 175 Gly Lys Met Arg Val Phe Leu Ala Ala Leu Glu Gly Ser Leu Asp Arg             180 185 190 Glu Ala Glu Arg Val Arg Gly Glu Ala Gly Val Ala Leu Arg Arg Glu         195 200 205 Leu Gly Ser Leu Asn Ser Tyr Leu Glu Gln Leu Arg Gln Met Glu Lys     210 215 220 Val Leu Glu Glu Val Ala Asp Lys Pro Gln Thr Glu Phe Leu Met Lys 225 230 235 240 Tyr Cys Leu Val Thr Ser Arg Leu Gln Lys Ile Leu Ala Glu Ser Pro                 245 250 255 Pro Pro Ala Arg Leu Asp Ile Gln Leu Pro Ile Ile Ser Asp Asp Phe             260 265 270 Lys Phe Gln Val Trp Arg Lys Met Phe Arg Ala Leu Met Pro Ala Leu         275 280 285 Glu Glu Leu Thr Phe Asp Pro Ser Ser Ala His Pro Ser Leu Val Val     290 295 300 Ser Ser Ser Gly Arg Arg Val Glu Cys Ser Glu Gln Lys Ala Pro Pro 305 310 315 320 Ala Gly Glu Asp Pro Arg Gln Phe Asp Lys Ala Val Ala Val Val Ala                 325 330 335 His Gln Gln Leu Ser Glu Gly Glu His Tyr Trp Glu Val Asp Val Gly             340 345 350 Asp Lys Pro Arg Trp Ala Leu Gly Val Ile Ala Ala Glu Ala Pro Arg         355 360 365 Arg Gly Arg Leu His Ala Val Pro Ser Gln Gly Leu Trp Leu Leu Gly     370 375 380 Leu Arg Glu Gly Lys Ile Leu Glu Ala His Val Glu Ala Lys Glu Pro 385 390 395 400 Arg Ala Leu Arg Ser Pro Glu Arg Arg Pro Thr Arg Ile Gly Leu Tyr                 405 410 415 Leu Ser Phe Gly Asp Gly Val Leu Ser Phe Tyr Asp Ala Ser Asp Ala             420 425 430 Asp Ala Leu Val Pro Leu Phe Ala Phe His Glu Arg Leu Pro Arg Pro         435 440 445 Val Tyr Pro Phe Phe Asp Val Cys Trp His Asp Lys Gly Lys Asn Ala     450 455 460 Gln Pro Leu Leu Leu Val Gly Pro Glu Gly Ala Glu Ala 465 470 475 <210> 3 <211> 183 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE &Lt; 222 > (1) .. (183) <223> UBE2H (Genbank accession No. NP_003335) <400> 3 Met Ser Ser Pro Ser Pro Gly Lys Arg Arg Met Asp Thr Asp Val Val   1 5 10 15 Lys Leu Ile Glu Ser Lys His Glu Val Thr Ile Leu Gly Gly Leu Asn              20 25 30 Glu Phe Val Val Lys Phe Tyr Gly Pro Gln Gly Thr Pro Tyr Glu Gly          35 40 45 Gly Val Trp Lys Val Arg Val Asp Leu Pro Asp Lys Tyr Pro Phe Lys      50 55 60 Ser Pro Ser Ile Gly Phe Met Asn Lys Ile Phe His Pro Asn Ile Asp  65 70 75 80 Glu Ala Ser Gly Thr Val Cys Leu Asp Val Ile Asn Gln Thr Trp Thr                  85 90 95 Ala Leu Tyr Asp Leu Thr Asn Ile Phe Glu Ser Phe Leu Pro Gln Leu             100 105 110 Leu Ala Tyr Pro Asn Pro Ile Asp Pro Leu Asn Gly Asp Ala Ala Ala         115 120 125 Met Tyr Leu His Arg Pro Glu Glu Tyr Lys Gln Lys Ile Lys Glu Tyr     130 135 140 Ile Gln Lys Tyr Ala Thr Glu Glu Ala Leu Lys Glu Gln Glu Glu Gly 145 150 155 160 Thr Gly Asp Ser Ser Ser Glu Ser Ser Met Ser Asp Phe Ser Glu Asp                 165 170 175 Glu Ala Gln Asp Met Glu Leu             180 <210> 4 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> si-MG53 <400> 4 aagcacgccu caagacacag c 21 <210> 5 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> si-UBE2H <400> 5 cuaugaucuu accaauaua 19 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer for MG53 <400> 6 tccctgttgt caggcatcta c 21 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for MG53 <400> 7 ttcttccaca cctggaattt g 21 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer for MyoD <400> 8 ctcctttgag acagcagacg actt 24 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for MyoD <400> 9 aaatcgcatt ggggtttgag cctg 24 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer for MyHC <400> 10 agaaggagga ggcaacttct g 21 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for MyHC <400> 11 acatactcat tgccgacctt g 21 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer for a-actin <400> 12 gaagagctat gagctgcctg a 21 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for a-actin <400> 13 ctcatcgtac tcctgcttgc t 21 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer for UBE2H <400> 14 aaggaacacc atatgaaggc g 21 <210> 15 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for UBE2H <400> 15 gcgtacttct ggatgtactc tttaa 25 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for UBE2R1 <400> 16 tgccaagctc gcagaaggcg 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for UBE2R1 <400> 17 ccaccgctct gagggcagct 20 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer for UBE2D2 <400> 18 gccctcagct cgtctgatcc g 21 <210> 19 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for UBE2D2 <400> 19 tgataggggc tgtcatttgg ccc 23 <210> 20 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer for UBE2E1 <400> 20 tgcctggtta atagttgctg ttgct 25 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for UBE2E1 <400> 21 cactgcagtt tggcggaggg t 21 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for UBE2L3 <400> 22 aaggggcagg tctgtctgcc 20 <210> 23 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for UBE2L3 <400> 23 cctgctttgc ggggtgtctg a 21 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for UBE2L6 <400> 24 agctgccgcc ataccttcgc 20 <210> 25 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for UBE2L6 <400> 25 gcaagttcca gacgcacagg ct 22 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for UBE2C <400> 26 aaaccgcgac ccagctgctg 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for UBE2C <400> 27 gccctgggtg tccacgttgg 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for UBE2M <400> 28 agggcagcag caagaaggcg 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for UBE2M <400> 29 ggcagacgtt gccctcgagg 20 <210> 30 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer for UBE2N <400> 30 atgtggtcat tgctggcccc c 21 <210> 31 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for UBE2N <400> 31 gcagaacagg agaagtggtg tacac 25 <210> 32 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer for GAPDH <400> 32 tggcaaattc catggcacc 19 <210> 33 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for GAPDH <400> 33 agagatgatg acccttttg 19

Claims (8)

(a) treating candidate compounds to cells expressing MG53 (mitsugumin 53) and UBE2H (ubiquitin-conjugating enzyme E2H); And
(b) determining whether the interaction of MG53 and UBE2H in said cells is inhibited,
Screening for Type 2 Diabetes Therapeutics.
(a ') treating the candidate compound with a mixture of isolated MG53 and isolated UBE2H; And
(b ') determining whether the interaction of MG53 and UBE2H is inhibited,
Screening for Type 2 Diabetes Therapeutics.
The method of claim 1 or 2, wherein the interaction of MG53 and UBE2H is detected using an antibody specific for MG53 or UBE2H.
The method of claim 3, wherein the interaction between MG53 and UBE2H is detected by immunotaxine, immunoprecipitation, or immunostaining.
The method of claim 1 or 2, wherein the MG53 and UBE2H are each reporter protein linked.
The method of claim 5, wherein the reporter protein is chloramphenicolacetyltransferase, beta-glucuronidase, luciferase, beta-galactosidase or fluorescent protein.
The method of claim 5, wherein the fluorescent protein is a green fluorescent protein (red fluorescent protein), red fluorescent protein (red fluorescent protein), blue fluorescent protein (blue fluorescent protein), yellow fluorescent protein (yellow fluorescent protein), navy blue fluorescent protein ( cyan fluorescent protein, enhanced green fluorescent protein, enhanced red fluorescent protein, enhanced blue fluorescent protein, enhanced yellow fluorescent protein or enhanced indigo fluorescent protein.
The method of claim 5, wherein the interaction of MG53 and UBE2H is detected by the signal magnitude of the reporter protein.

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