OBJECTIVEThe aim of this study is to determine whether the SUMO4 M55V polymorphism is associated with susceptibility to type 2 diabetes mellitus (T2DM).

METHODSA meta-analysis was performed to detect the potential association of the SUMO4 M55V polymorphism and susceptibility to T2DM under dominant, recessive, co-dominant (homogeneous and heterogeneous), and additive models.

RESULTSA total of eight articles including 10 case-control studies, with a total of 2932 cases and 2679 controls, were included in this meta-analysis. The significant association between the SUMO4 M55V polymorphism and susceptibility to T2DM was observed in the dominant model (GG + GA versus AA: OR = 1.21, 95% CI = 1.05-1.40, P = 0.009), recessive model (GG versus GA + AA: OR = 1.29, 95% CI = 1.07-1.356, P = 0.010), homozygous model (GG versus AA: OR = 1.41, 95% CI = 1.06-1.56, P = 0.001), and additive model (G versus A: OR = 1.18, 95% CI = 1.08-1.29, P = 0.001), and marginally significant in the heterozygous model (GA versus AA: OR = 1.16, 95% CI = 0.98-1.36, P = 0.080). In subgroup analyses, significant associations were observed in the Chinese population under four genetic models excluding the heterozygous model, whereas no statistically significant associations were observed in the Japanese population under each of the five genetic models.

CONCLUSIONThe meta-analysis demonstrated that the G allele of the SUMO4 M55V polymorphism could be a susceptible risk locus to T2DM, mainly in the Chinese population, while the association in other ethnic population needs to be further validated in studies with relatively large samples.

Diabetes Mellitus, Type 2 ; epidemiology ; genetics ; Genetic Predisposition to Disease ; epidemiology ; genetics ; Humans ; Small Ubiquitin-Related Modifier Proteins ; genetics ; metabolism

OBJECTIVETo investigate whether progesterone receptor B (PRB) can be sumoylated by SUMO-2/3 and the effect of sumoylation on PRB transcriptional activity.

METHODSSUMO-2/3 cDNA was amplified from MCF-7 cDNA and cloned into the eukaryotic expression vector pcDNA3-FLAG. The plasmid pXJ40-myc-PRB was cotransfected with pcDNA3FLAG-SUMO2, pcDNA3FLAG-SUMO3 or the mock control into 293T cells, and PRB sumoylation was detected by immunoprecipitation and Western blotting. The effect of PRB sumoylation on its transcriptional activity was determined using reporter luciferase assay.

RESULTSpcDNA3FLAG-SUMO2 and pcDNA3FLAG-SUMO3 vectors were successfully constructed. SUMO-2/3 could bind covalently to PRB and increase its transcriptional dependent on the presence of progesterone.

CONCLUSIONPRB can be sumoylated by SUMO-2/3 and its function is regulated by this modification.

Animals ; Cell Line ; Humans ; Plasmids ; genetics ; Receptors, Progesterone ; genetics ; metabolism ; Small Ubiquitin-Related Modifier Proteins ; genetics ; metabolism ; Transcription, Genetic ; Transfection ; Ubiquitination ; Ubiquitins ; genetics ; metabolism

Zinc finger protein 133 (ZNF133) is composed of a Kruppel-associated box (KRAB) domain and 14 contiguous zinc finger motifs. ZNF133 is regarded as a transcriptional repressor because the KRAB domain has potent repressor activity and the zinc finger motifs usually act in binding to DNA. However, we found that the zinc finger motifs of ZNF133 also possessed transcriptional repressor activity. By two-hybrid screening assay, we found that the zinc finger motifs of ZNF133 interacted with protein inhibitor of activated STAT1 (PIAS1). PIAS1 enhanced the transcriptional repression activity of ZNF133 through the zinc finger motifs. This effect of PIAS1 was relieved by an inhibitor of the histone deacetylases (HDACs). These results demonstrate that the transcriptional repressor activity of ZNF133 is regulated by both the KRAB domain and the zinc finger motifs, and that the repressive effect by zinc finger motifs is mediated by PIAS1.
Cell Line ; DNA-Binding Proteins/*metabolism ; Histone Deacetylases/antagonists & inhibitors/metabolism ; Humans ; Protein Binding ; Protein Inhibitors of Activated STAT/*metabolism ; Protein Structure, Tertiary ; Repressor Proteins/*metabolism ; Small Ubiquitin-Related Modifier Proteins/*metabolism ; Transcription, Genetic ; Two-Hybrid System Techniques ; Zinc Fingers

The cDNA of human FGF-21 was subcloned into the pSUMO expression vector and the fusion protein was induced to express in Escherichia coli Rosetta (DE3). The recombinant hFGF-21 was expressed in soluble form in the pSUMO expression system. The recombinant fusion protein was purified by Ni-NTA column. The purified recombinant protein was dialyzed against PBS for re-nature. To obtain pure and active recombinant protein, the fusion protein was subjected to cleavage with SUMO protease I. To examine glucose regulation activity of hFGF-21, 3T3-L1 pre-adipocytes were differentiated into adipocytes, glucose up-take activity of hFGF-21 was examined by glucose oxidase and peroxidase (GOD-POD) assay. Compared with no stimulation control, the recombinant hFGF-21 treatment led to a significant increase in glucose consumption of adipocytes and a significant decrease in concentration of glucose in the medium (P < 0.05, P < 0.001).
3T3-L1 Cells ; Adipocytes ; metabolism ; Animals ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; metabolism ; Fibroblast Growth Factors ; genetics ; metabolism ; pharmacology ; Glucose ; metabolism ; Humans ; Mice ; Plasmids ; Recombinant Fusion Proteins ; genetics ; metabolism ; pharmacology ; Small Ubiquitin-Related Modifier Proteins ; genetics ; metabolism ; Solubility

Nowadays, SUMO fusion system is important for recombinant protein production in Escherichia coli, yet a few aspects remain to be improved, including the efficacy for vector construction and protein solubility. In this study, we found the SUMO gene Smt3 (Sm) of Saccharomyces cerevisiae conferred an unexpected activity of constitutive prokaryotic promoter during its PCR cloning, and the gene coding regions of SUMOs in most species had a sigma70-dependent prokaryotic promoter embedded, through the prediction via the BPROM program developed by Softberry. By combining the characters of Sm promoter activity and the Stu I site (added at the 3'-terminal of Sm), and introducing a His-tag and a hyper-acidic solubility-enhancing tag, we further constructed a set of versatile vectors for gene cloning and expression on the basis of Sm'-LacZa fusion gene. Experimentally started from these vectors, several target genes were subcloned and expressed through blue-white screening and SDS-PAGE analysis. The results manifest a few of expectable advantages such as rapid vector construction, highly soluble protein expression and feasible co-expression of correlated proteins. Conclusively, our optimized SUMO fusion technology herein could confer a large potential in E. coli protein expression system, and the simultaneously established co-expression vector systems could also be very useful in studying the protein-protein interactions in vivo.
Amino Acid Sequence ; Base Sequence ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Lac Operon ; genetics ; Molecular Sequence Data ; Promoter Regions, Genetic ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Saccharomyces cerevisiae Proteins ; biosynthesis ; genetics ; Small Ubiquitin-Related Modifier Proteins ; biosynthesis ; genetics ; Sumoylation

Autophagy is a self-degradation system of cellular components through an autophagosomal-lysosomal pathway. Over the last 15 yr, yeast genetic screens led to the identification of a number of genes involved in the autophagic pathway. Most of these autophagy genes are present in higher eukaryotes and regulate autophagy process for cell survival and homeostasis. Significant progress has recently been made to better understand the molecular mechanisms of the autophagy machinery. Especially, autophagy process, including the regulation of autophagy induction through mTOR and the nucleation and elongation in autophagosome formation through class III phosphatidylinositol 3-kinase complex and ubiquitin-like conjugation systems, became evident. While many unanswered questions remain to be answered, here, we summarize the recent process of autophagy with emphasis on molecules and their protein complexes along with advanced molecular mechanisms that regulate the autophagy machinery.
Autophagy/genetics/*physiology ; Carrier Proteins/genetics/metabolism ; Class III Phosphatidylinositol 3-Kinases/genetics/metabolism ; Humans ; Intracellular Signaling Peptides and Proteins/genetics/metabolism ; Membrane Proteins/genetics/metabolism ; Microtubule-Associated Proteins/genetics/metabolism ; Models, Biological ; Protein-Serine-Threonine Kinases/genetics/metabolism ; Small Ubiquitin-Related Modifier Proteins/genetics/metabolism

Fibroblast growth factor -21 (FGF-21) is a recently discovered metabolic regulation factor, regulating glucose and lipid metabolism and increasing insulin sensitivity. FGF-21 is expected to be a potential anti-diabetic drug. Expression of FGF-21 as inclusion bodies has advantages for high yield and purity, but the bioactivity of the protein is almost totally lost after denature and renature. That is why FGF-21 is currently expressed in soluble form. As a result, the yield is considerably low. In this study, we used SUMO vector to express SUMO-human FGF-21 (SUMO-hFGF-21) in form of inclusion body. We optimized the culture conditions to increase the yield of the bioactive human fibroblast growth factor-21. We applied the hollow fiber membrane filtration column to enrich the bacteria, wash, denature and renature inclusion bodies. After affinity and gel filtration chromatography, we examined the hypoglycemic activity of FGF-21 by the glucose uptake assay in HepG2 cells. We also detected the blood glucose concentration of type 2 diabetic db/db model mice after short or long-term treatment. The results show that the yield of ihFGF-21 was 4 times higher than that of shFGF-21. The yield was 20 mg/L for ihFGF-21 vs. 6 mg/L for shFGF-21. The purity of ihFGF-21 was above 95%, while shFGF-21 was 90%. Compared with the traditional method of extracting inclusion bodies, the production cycle was about three times shortened by application of hollow fiber membrane filtration column technology, but the bioactivity did not significantly differ. This method provides an efficient and cost-effective strategy to the pilot and industrial production of hFGF-21.
Animals ; Bacteria ; metabolism ; Diabetes Mellitus, Experimental ; drug therapy ; Disease Models, Animal ; Fibroblast Growth Factors ; biosynthesis ; Genetic Vectors ; Glucose ; metabolism ; Hep G2 Cells ; Humans ; Hypoglycemic Agents ; isolation & purification ; Inclusion Bodies ; metabolism ; Mice ; Recombinant Fusion Proteins ; biosynthesis ; Small Ubiquitin-Related Modifier Proteins ; biosynthesis

SUMOylation is recently found to function as a targeting signal for the degradation of substrates through the ubiquitin-proteasome system. RNF4 is the most studied human SUMO-targeted ubiquitin E3 ligase. However, the relationship between SUMO proteases, SENPs, and RNF4 remains obscure. There are limited examples of the SENP regulation of SUMO2/3-targeted proteolysis mediated by RNF4. The present study investigated the role of SENP3 in the global protein turnover related to SUMO2/3-targeted ubiquitination and focused in particular on the SENP3 regulation of the stability of Sp1. Our data demonstrated that SENP3 impaired the global ubiquitination profile and promoted the accumulation of many proteins. Sp1, a cancer-associated transcription factor, was among these proteins. SENP3 increased the level of Sp1 protein via antagonizing the SUMO2/3-targeted ubiquitination and the consequent proteasome-dependent degradation that was mediated by RNF4. De-conjugation of SUMO2/3 by SENP3 attenuated the interaction of Sp1 with RNF4. In gastric cancer cell lines and specimens derived from patients and nude mice, the level of Sp1 was generally increased in parallel to the level of SENP3. These results provided a new explanation for the enrichment of the Sp1 protein in various cancers, and revealed a regulation of SUMO2/3 conjugated proteins whose levels may be tightly controlled by SENP3 and RNF4.
Animals ; Cysteine Endopeptidases ; genetics ; metabolism ; Gene Expression Regulation, Neoplastic ; Humans ; Immunoenzyme Techniques ; Immunoprecipitation ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Prognosis ; Protein Processing, Post-Translational ; Proteolysis ; RNA, Messenger ; genetics ; Real-Time Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction ; Small Ubiquitin-Related Modifier Proteins ; antagonists & inhibitors ; genetics ; metabolism ; Sp1 Transcription Factor ; genetics ; metabolism ; Stomach Neoplasms ; genetics ; metabolism ; pathology ; Sumoylation ; Tumor Cells, Cultured ; Ubiquitination ; Ubiquitins ; antagonists & inhibitors ; genetics ; metabolism ; Xenograft Model Antitumor Assays