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

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

TGF-beta induces IgA class switching by B cells. We previously reported that Smad3 and Smad4, pivotal TGF-beta signal-transducing transcription factors, mediate germline (GL) alpha transcription induced by TGF-beta1, resulting in IgA switching by mouse B cells. Post-translational sumoylation of Smad3 and Smad4 regulates TGF-beta-induced transcriptional activation in certain cell types. In the present study, we investigated the effect of sumoylation on TGF-beta1-induced, Smad3/4-mediated GLalpha transcription and IgA switching by mouse B cell line, CH12F3-2A. Overexpression of small ubiquitin-like modifier (SUMO)-1, SUMO-2 or SUMO-3 did not affect TGF-beta1-induced, Smad3/4-mediated GLalpha promoter activity, expression of endogenous GLalpha transcripts, surface IgA expression, and IgA production. Next, we tested the effect of the E3 ligase PIASy on TGF-beta1-induced, Smad3/4-mediated GLalpha promoter activity. We found that PIASy overexpression suppresses the GLalpha promoter activity in cooperation with histone deacetylase 1. Taken together, these results suggest that SUMO itself does not affect regulation of GLalpha transcription and IgA switching induced by TGF-beta1/Smad3/4, while PIASy acts as a repressor.
Animals ; B-Lymphocytes* ; Cell Line ; Histone Deacetylase 1 ; Immunoglobulin A ; Immunoglobulin Class Switching ; Mice ; Small Ubiquitin-Related Modifier Proteins* ; SUMO-1 Protein* ; Sumoylation ; Transcription Factors ; Transcriptional Activation ; Transforming Growth Factor beta ; Transforming Growth Factor beta1 ; Ubiquitin-Protein Ligases

OBJECTIVETo investigate the association between small ubiquitin-related modifier-1 (SUMO-1) gene rs6709162, rs7599810, rs7580433 polymorphism and non-syndromic oral clefting (NSOC).

METHODSOur study consisted of 208 Ningxia NSOC patients, their parents (189 fathers and 176 mothers), 172 nuclear families (patients and their parents), and 284 normal controls. DNA was extracted and polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) was used to identify rs6709162, rs7599810, rs7580433 genotypes of the samples. The data was analyzed by case-control analysis, family based associated test (FBAT), and transmission disequilibrium test (TDT).

RESULTSCase-control study found that TT genotype's frequency was significantly different in cleft lip and cleft palate group compared with the control group at rs7599810 of SUMO-1 (P=0.01, P=0.01). TDT test showed that rs7599810's T allele had over-transmitted (P=0.00) in cleft lip and palate group. FBAT analysis revealed that distribution of rs7599810's TT genotype and T allele was significantly different (P=0.00, P=0.00). TDT test showed that rs6709162's C allele in cleft palate and cleft lip and palate patients had over-transmitted (P=0.00, P=0.01). rs7580433's G allele in cleft lip group had over-transmitted (P=0.05).

CONCLUSIONSUMO-1 gene polymorphism is associated with NSOC.

Case-Control Studies ; Cerebellar Ataxia ; Cleft Lip ; Cleft Palate ; Female ; Genotype ; Humans ; Intellectual Disability ; Male ; Polymerase Chain Reaction ; Polymorphism, Genetic ; SUMO-1 Protein ; genetics ; Ubiquitins

The ubiquitin-related modifier Urm1 can be covalently conjugated to lysine residues of other proteins, such as yeast Ahp1 and human MOCS3, through a mechanism involving the E1-like protein Uba4 (MOCS3 in humans). Similar to ubiquitination, urmylation requires a thioester intermediate and forms isopeptide bonds between Urm1 and its substrates. In addition, the urmylation process can be significantly enhanced by oxidative stress. Recent findings have demonstrated that Urm1 also acts as a sulfur carrier in the thiolation of eukaryotic tRNA via a mechanism that requires the formation of a thiocarboxylated Urm1. This role is very similar to that of prokaryotic sulfur carriers such as MoaD and ThiS. Evidence strongly supports the hypothesis that Urm1 is the molecular fossil in the evolutionary link between prokaryotic sulfur carriers and eukaryotic ubiquitin-like proteins. In the present review, we discuss the dual role of Urm1 in protein and tRNA modification.
Animals ; Humans ; Models, Biological ; RNA, Transfer ; metabolism ; Sulfur ; metabolism ; Ubiquitin ; metabolism ; Ubiquitins ; metabolism

OBJECTIVETo explore the scavenging action of tenuigenin (TEN) on intracerebral amyloid β protein (Aβ) aggregation and the abnormal phosphorylated tau protein and its mechanism in Alzheimer's disease (AD) rats' brain.

METHODSAβ1-40 was injected into the right CA1 region hippocampus to establish the AD model. Successfully modeled rats were divided into the model group, the low, middle, high TEN group. Rats were administered with TEN (18.5, 37.0, 74.0 mg/kg) by gastrogavage. Besides, a sham-operation group was set up. Expression levels of Aβ1-40 and Tau p-Ser262 were detected by immunohistochemistry. Expression levels of ubiquitin (Ub) and Ub-protein ligase E3 were measured by Western blotting.The content of 26S proteasome was detected by ELISA.

RESULTSImmunohistochemical results showed that the number of Aβ and Tau p-Ser262 positively reacted neurons significantly increased in model group, when compared with the sham-operation group (P < 0.01). Results of Western blot showed expression levels of ubiquitinated protein were up-regulated and those of Ub-protein ligase E3 were down-regulated in the model group (P < 0.01). ELISA results showed that the content of 26S proteasome significantly decreased in AD rats' brain (P < 0.01). Compared with the model group, expression levels of Aβ1-40, Tau p-Ser262, and Ub significantly decreased; expression levels of Ub-protein ligase E3 apparently increased; the content of 26S proteasome significantly increased in each TEN treatment group (P < 0.05, P < 0.01). Best effect was shown in 37.0 mg/kg and 74.0 mg/kg TEN groups.

CONCLUSIONSUb proteasome pathway (UPP) participated in the occurrence of AD. TEN could obviously reduce intracere- bral Aβ1-40 accumulation and abnormal tau phosphorylation.

Alzheimer Disease ; metabolism ; Amyloid beta-Peptides ; Animals ; Drugs, Chinese Herbal ; pharmacology ; Hippocampus ; metabolism ; Neurons ; metabolism ; Phosphorylation ; Proteasome Endopeptidase Complex ; metabolism ; Rats ; Ubiquitin-Protein Ligases ; metabolism ; Ubiquitins

To provide technical support for spider silk functional modification, we developed a simple and efficient functional platform via intein trans-splicing. Small ubiquitin-related modifier protein (SUMO) was fused to the recombinant spider silk protein (W2CT) by peptide bond via S0 split intein Ssp DnaB trans-splicing, resulting in a protein SUMOW2CT. However, incorporation of exogenous protein led to mechanical property defect and lower fiber yield, and also slowed down the fiber assembly velocity but no obvious differences in supercontraction and chemical resistance when compared with fibers from W2CT (W). SUMO protease digestion showed positive results on the fibers, indicating that the SUMO protein kept its native conformation and bioactive. Above all, this work provides a technical support for spider silk high simply and efficient functionalized modification.
Animals ; Inteins ; Protein Splicing ; Recombinant Proteins ; chemistry ; Silk ; chemistry ; Small Ubiquitin-Related Modifier Proteins ; chemistry ; Spiders ; Trans-Splicing

Objective To prepare rabbit polyclonal antibody against mouse IQ and ubiquitin-like domain-containing protein (IQUB) and detect its expression in the mouse testis. Methods Full-length coding sequence of IQUB was inserted into the pET-30a(+) vector to construct pET-30a-IQUB recombinant prokaryotic plasmid. Transformation of pET-30a-IQUB plasmid into E. coli BL21 was performed, and protein expression was induced with isopropyl-beta-D-thiogalactoside (IPTG). The protein was purified through histidine-tagged fusion protein purification column, then denatured by treatment of urea with gradient concentration. New Zealand rabbits were immunized with the denatured protein to produce IQUB polyclonal antibody. Antibody titer was detected by ELISA, and Western blot analysis and immunofluorescence assay were employed to validate the effectiveness and specificity of IQUB antibody. Results pET-30a-IQUB recombinant plasmid was constructed, and protein expression of IQUB was induced successfully with IPTG. The titer of IQUB polyclonal antibody reached 1:1 000 000. The antibody specifically recognized the endogenous IQUB protein of testis in the wild-type adult mouse. IQUB was expressed in spermatogenic cells of different stages. It was localized in the acrosome and flagellum of mature sperms. Conclusion The highly specific rabbit anti-mouse IQUB polyclonal antibody is successfully prepared, which can be used for Western blot and immunofluorescence histochemistry.
Male ; Rabbits ; Animals ; Mice ; Ubiquitins ; Escherichia coli/genetics* ; Isopropyl Thiogalactoside ; Antibodies ; Enzyme-Linked Immunosorbent Assay

OBJECTIVETo identify the specific protein interactions involved in Bat3-mediated apoptosis.

METHODSTandem affinity purification (TAP) was utilized to investigate Bat3-protein interactions, during which full-length human Bat3 fused with Strep2 and FLAG tag as a bait was used to screen the specific protein-protein interactions. The isolated proteins were identified with mass spectrometry.

RESULTSTAP studies showed that Ubl4A was identified as a Bat3-binding partner. Further investigation using co-immunoprecipitation confirmed that Bat3 was associated with Ubl4A.

CONCLUSIONTAP was successfully established and is suitable for isolating the binding partners of Bat3.

Cell Line ; Humans ; Molecular Chaperones ; isolation & purification ; Protein Binding ; Ubiquitins ; isolation & purification

ISG15 is a 15kD ubiquitin-like protein (UBL) induced by interferon (IFN). ISG15 can be covalently attached to proteins, which is called ISGylation process. ISGylation system contains ISG15, UBE1L, UBCH8 and HERC5 proteins, which are all essential for ISGylation. ISG15 and ISGylation system have been found to have anti-viral effects. A better understanding of how ISG15 mediates the anti-viral activity will provide insights for new anti-viral drugs development and new therapeutic strategies. The mechanisms underlying the ISG15 mediated anti-viral response have been explored extensively in recent years. This minireview summarized the research advances of how ISG15 mediated the anti-viral effects against different kinds of viruses.
Animals ; Cytokines ; physiology ; HIV Infections ; immunology ; Humans ; Influenza, Human ; immunology ; Ubiquitins ; physiology ; Virus Diseases ; immunology