SUMO-specific protease SENP3 enhances MDM2-mediated ubiquitination of PARIS/ZNF746 in HeLa cells.

The transcriptional repressor PARIS, a substrate of the ubiquitin E3 ligase parkin, represses the expression of the transcriptional co-activator, PGC-1α gene, and is involved in several pathological processes, including neurodegenerative disease and cancers. We have previously shown that SUMOylation of PARIS play an important role in its transcriptional repression activity. In addition, RNF4-mediated ubiquitination of SUMO2/3-conjugated PARIS is required for the control of PARIS-mediated transcriptional repression in HeLa cells that lack parkin expression. However, little is known about how PARIS ubiquitination and degradation are regulated in parkin-deficient cells. Here, we report that the deSUMOylase SENP3 interacted with PARIS and enhanced the ubiquitination of PARIS independently of its SUMOylation in HeLa cells. SENP3-enhanced PARIS ubiquitination mainly contributed to its proteasomal degradation, and required the oncogenic E3 ubiquitin ligase MDM2. MDM2 knockdown by small interfering RNA or expression of a dominant-negative MDM2 mutant inhibited the ubiquitination of PARIS. We further found that MDM2 activation via the PI3K/AKT pathway was involved in PARIS ubiquitination. Taken together, these results suggest that PARIS ubiquitination through SENP3-mediated MDM2 activation may control its functions in parkin-deficient cells.

RNF4-mediated SUMO-targeted ubiquitination relieves PARIS/ZNF746-mediated transcriptional repression.

The transcriptional repressor PARIS, which is a substrate of the ubiquitin E3 ligase parkin, represses the expression of the transcriptional co-activator, PGC-1α. However, little is known about how its repression activity is regulated. We have previously shown that PARIS is SUMOylated, and this SUMOylation plays an important role in regulating its transcriptional repression activity. In this study, we demonstrated that PARIS SUMOylation induced its ubiquitination and subsequent proteasomal degradation, which was mediated by the SUMO-targeted ubiquitin ligase RNF4. Reporter gene assays revealed that co-expression of SUMO3 and RNF4 relieved PARIS-mediated transcriptional repression. Conversely, the SUMO E3 ligase PIASy inhibited the RNF4-mediated ubiquitination of PARIS and blocked the RNF4-mediated relief of PARIS-mediated transcriptional repression. These results suggest that RNF4 regulates PARIS ubiquitination to control its transcriptional repression activity.

SUMOylation of the KRAB zinc-finger transcription factor PARIS/ZNF746 regulates its transcriptional activity.

Parkin-interacting substrate (PARIS), a member of the family of Krüppel-associated box (KRAB)-containing zinc-finger transcription factors, is a substrate of the ubiquitin E3 ligase parkin. PARIS represses the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), although the underlying mechanisms remain largely unknown. In the present study, we demonstrate that PARIS can be SUMOylated, and its SUMOylation plays a role in the repression of PGC-1a promoter activity. Protein inhibitor of activated STAT y (PIASy) was identified as an interacting protein of PARIS and shown to enhance its SUMOylation. PIASy repressed PGC-1a promoter activity, and this effect was attenuated by PARIS in a manner dependent on its SUMOylation status. Co-expression of SUMO-1 with PIASy completely repressed PGC-1a promoter activity independently of PARIS expression. PARIS-mediated PGC-1a promoter repression depended on the activity of histone deacetylases (HDAC), whereas PIASy repressed the PGC-1a promoter in an HDAC-independent manner. Taken together, these results suggest that PARIS and PIASy modulate PGC-1a gene transcription through distinct molecular mechanisms.

Identification of chromosome 3q28 and ALPK1 as susceptibility loci for chronic kidney disease in Japanese individuals by a genome-wide association study.

BACKGROUND: Although genome-wide association studies (GWASs) have implicated several genes in the predisposition to chronic kidney disease (CKD) in Caucasian or African American populations, the genes that confer susceptibility to CKD in Asian populations remain to be identified definitively. We performed a GWAS to identify genetic variants that confer susceptibility to CKD in Japanese individuals. METHODS: 3851 Japanese individuals from three independent subject panels were examined. Subject panels A, B, and C comprised 252, 910, and 190 individuals with CKD and 249, 838, and 1412 controls, respectively. A GWAS for CKD was performed in subject panel A. RESULTS: Five single nucleotide polymorphisms (SNPs) at chromosome 3q28, ALPK1, FAM78B, and UMODL1 were significantly (false discovery rate<0.05) associated with CKD by the GWAS. The relation of these five SNPs and of an additional 22 SNPs at these loci to CKD was examined in subject panel B, revealing that rs9846911 at 3q28 was significantly associated with CKD in all individuals and that rs2074381 and rs2074380 in ALPK1 were associated with CKD in individuals with diabetes mellitus. These three SNPs were further examined in subject panel C, revealing that rs2074381 and rs2074380 were significantly associated with CKD. For subject panels B and C combined, rs9846911 was significantly associated with CKD in all individuals and rs2074381 and rs2074380 were associated with CKD in diabetic individuals. CONCLUSIONS: Chromosome 3q28 may be a susceptibility locus for CKD in Japanese individuals, and ALPK1 may be a susceptibility gene for CKD in such individuals with diabetes mellitus.

Mitotic kinase Aurora-B is regulated by SUMO-2/3 conjugation/deconjugation during mitosis.

The small ubiquitin-related modifier (SUMO) system of higher eukaryotes plays important roles in normal cell division, especially in chromosome segregation. However, only a few mitotic SUMO substrates have been identified in mammals. Here, we show that the mitotic kinase Aurora-B can be modified by SUMO. The E3 SUMO-protein ligase PIAS3 [protein inhibitor of activated STAT (signal transducer and activator of transcription)] dramatically enhanced poly-SUMO-2/3 conjugation of Aurora-B, whereas the SUMO-specific isopeptidase SENP2 (Sentrin/SUMO-specific protease) specifically deconjugated SUMO from Aurora-B. The Lys-202 residue on human Aurora-B was preferentially modified by SUMO, and enhancement of SUMOylation in cells facilitated Aurora-B autophosphorylation, which is essential for its activation. Conversely, SENP2-mediated deSUMOylation of Aurora-B down-regulated its autophosphorylation in cells and also impaired its re-activation in Aurora inhibitor VX-680-treated mitotic cells. Poly-SUMO-2 conjugation of Aurora-B occurred during the M phase of the cell cycle, and both SUMO-2 and PIAS3 were localized adjacent to Aurora-B in the kinetochores in early mitosis. Based on these results, we propose that Aurora-B is a novel mitotic SUMO substrate and that its kinase activity is fine-tuned by the SUMO system.

The nucleolar SUMO-specific protease SMT3IP1/SENP3 attenuates Mdm2-mediated p53 ubiquitination and degradation.

SUMO (small ubiquitin-like modifier) modification plays multiple roles in several cellular processes. Sumoylation is reversibly regulated by SUMO-specific proteases. SUMO-specific proteases have recently been implicated in cell proliferation and early embryogenesis, but the underlying mechanisms remain unknown. Here, we show that a nucleolar SUMO-specific protease, SMT3IP1/SENP3, controls the p53-Mdm2 pathway. We found that SMT3IP1 interacts with p53 and Mdm2, and desumoylates both proteins. Overexpression of SMT3IP1 in cells resulted in the accumulation of Mdm2 in the nucleolus and increased stability of the p53 protein. In addition, SMT3IP1 bound to the acidic domain of Mdm2, which also mediates the p53 interaction, and competed with p53 for binding. Increasing expression of SMT3IP1 suppressed Mdm2-mediated p53 ubiquitination and subsequent proteasomal degradation. Interestingly, the desumoylation activity of SMT3IP1 was not necessary for p53 stabilization. These results suggest that SMT3IP1 is a new regulator of the p53-Mdm2 pathway.

Cadmium interferes with the degradation of ATF5 via a post-ubiquitination step of the proteasome degradation pathway.

ATF5 is a member of the CREB/ATF family of transcription factors. In the current study, using a transient transfection system to express FLAG epitope fusion proteins of ATF5, we have shown that CdCl(2) or NaAsO(3) increases the protein levels of ATF5 in cells, and that cadmium stabilizes the ATF5 protein. Proteasome inhibitors had a similar effect to cadmium on the cellular accumulation of ATF5. Proteasome inhibition led to an increase in ubiquitinated ATF5, while cadmium did not appear to reduce the extent of ATF5 ubiquitination. ATF5 contains a putative nuclear export signal within its N-terminus. We demonstrated that whereas deletion of N-terminal region resulted in a increase of ATF5 levels, this region does not appear to be involved in the ubiquitination of ATF5. These results indicate that ATF5 is targeted for degradation by the ubiquitin-proteasome pathway, and that cadmium slows the rate of ATF5 degradation via a post-ubiquitination mechanism.

SMT3IP1, a nucleolar SUMO-specific protease, deconjugates SUMO-2 from nucleolar and cytoplasmic nucleophosmin.

Sumoylation is reversibly regulated by SUMO-specific proteases. We characterized a nucleolar SUMO-specific protease, SMT3IP1, which has a preference for SUMO-2/3. To elucidate SMT3IP1 function, we screened for its interacting proteins that may be its substrates or regulate its activity. By using yeast two-hybrid screening, we identified nucleophosmin (NPM) as an SMT3IP1-binding partner. SMT3IP1 could preferentially remove SUMO-2 from sumoylated NPM. A catalytically inactive SMT3IP1 mutant increased intracellular accumulation of SUMO-2-modified NPM in a dominant-negative manner. Sumoylation of cytoplasmic mutated NPM was markedly elevated in an ARF-dependent manner. Despite the divergence in their localization, ectopic expression of SMT3IP1 could desumoylate a SUMO-2-modified NPM mutant. Additionally, genotoxic drugs caused the dissociation of NPM from nucleolar co-localization with SMT3IP1, but did not affect desumoylation of NPM by SMT3IP1. Our findings suggest that SMT3IP1-mediated desumoylation might control NPM physiological functions at both the nucleolus and other subcellular compartments.

Sumoylation of CoREST modulates its function as a transcriptional repressor.

It is emerging that covalent modifications of many transcription factors and co-factors by the small ubiquitin-like modifier (SUMO) can have a key role in modulating their transcriptional regulation. As SUMO modification is often associated with transcriptional repression, we studied whether it was involved in modulating the repressive activity of CoREST. We showed that CoREST can be modified by SUMO-1 at lysine 294. PIASxbeta interacted with CoREST in vitro and in vivo, and functions as an E3-ligase to mediate its sumoylation. Furthermore, SENP1 mediated the desumoylation of CoREST. Interestingly, mutation of the CoREST sumoylation site compromised its ability as a corepressor. These results demonstrate that SUMO-1 modification modulates the transcriptional repression by CoREST and is needed for its full repressive activity.

PIASy controls ubiquitination-dependent proteasomal degradation of Ets-1.

The ETS transcription factor Ets-1 (E26 transformation-specific-1) plays a critical role in many physiological processes including angiogenesis, haematopoietic development and tumour progression. Its activity can be regulated by post-translational modifications, such as phosphorylation. Recently, we showed that Ets-1 is a target for SUMO (small ubiquitin-like modifier) modification and that PIASy [protein inhibitor of activated STAT (signal transducer and activator of transcription) Y], a specific SUMO-E3 ligase for Ets-1, represses Ets-1-dependent transcription. In the present study, we demonstrated that Ets-1 is degraded by the proteasome and that overexpression of PIASy increased the stability of endogenous and ectopically expressed Ets-1 protein by preventing proteasomal degradation. Moreover, knockdown of the endogenous PIASy expression by RNA interference reduced the protein level of endogenous Ets-1. The proteasome inhibitor MG132 reversed this effect. Deletion analysis showed that the TAD (transcriptional activation domain), which has been identified as the interaction domain with PIASy, was also required for Ets-1 ubiquitination and proteasomal degradation. However, the Ets-1 stabilization by PIASy was not due to reduced ubiquitination of Ets-1. Our results suggested that PIASy controls Ets-1 function, at least in part, by inhibiting Ets-1 protein turnover via the ubiquitin-proteasome system.

Relations of participation motives with number of golf rounds played and will to continue playing golf.

In a sample of 256 golfers, ages 24 to 76 years, the relations of participation motives with number of golf rounds played and will to continue playing golf were examined. Multiple regression analysis clarified that lifelong sport was an influential motive.

Cross-cultural comparison of motivation to learn in physical education: Japanese vs Swedish schoolchildren.

The present study compared differences between Japanese and Swedish schoolchildren in learning motivation-related variables in physical education. The subjects were 1,562 Japanese fifth and sixth graders (776 boys and 786 girls) ranging in age from 10 to 12 years and 573 Swedish fifth graders (306 boys and 267 girls) from 10 to 13 years (M = 11.4, SD = 0.5). They completed three questionnaires to evaluate the children's learning motivation, factors supporting motivation to learn, and preferences of learning behavior. The questionnaires were taken from Nishida's Diagnosis of Learning Motivation in Physical Education Test, a multidimensional and comprehensive test that measures learning motivation. A 2 x 2 (country by sex) multivariate analysis of variance indicated both Swedish boys and girls scored significantly higher than the Japanese children on most subscales. Results were discussed in relation to differences in the sports environment and culture of the two countries.

Identification of hypo- and hypermethylated genes related to atherosclerosis by a genome-wide analysis of DNA methylation.

Epigenetic modification, particularly changes in DNA methylation at gene promoters, is implicated in the pathogenesis of atherosclerosis. However, the analysis of DNA methylation in atherosclerosis has been limited to a few selected candidate genes. In this study, we therefore performed a genome-wide analysis of DNA methylation in the atherosclerotic human aorta. A total of 48 post-mortem human aortic intima specimens were examined. To avoid the effects of interindividual variation, we performed intraindividual paired comparisons between atheromatous plaque lesions and corresponding plaque-free tissue for 24 subjects. Bisulfite-modified genomic DNA was analyzed for DNA methylation with a specific microarray (Illumina HumanMethylation450 BeadChip). We compensated for multiple comparisons by applying Bonferroni's correction for statistical significance of association. DNA methylation was significantly (P<1.03x10⁻7) reduced at 15 CpG sites in 14 genes and increased at 30 CpG sites in 22 genes in atheromatous plaque compared with plaque-free intima. Three of the hypomethylated genes [Drosophila headcase (HECA), early B-cell factor 1 (EBF1) and nucleotide-binding oligomerization domain containing 2 (NOD2)] and three of the hypermethylated genes [human mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), zinc finger E-box binding homeobox 1 (ZEB1) and FYN] were previously been implicated in atherosclerosis. The overexpression of HECA, EBF1 or NOD2 or the suppression of MAP4K4, ZEB1 or FYN expression in cultured HEK293 cells resulted in significant (P<4.80x10⁻7) changes in the expression of atherosclerosis-related genes, as determined with an expression microarray (Illumina HumanHT-12 v4 Expression BeadChip). Our findings suggested that HECA, EBF1 and NOD2 were significantly hypomethylated, whereas MAP4K4, ZEB1 and FYN were hypermethylated, in atheromatous plaque lesions compared with plaque-free intima. Epigenetic mechanisms may thus contribute to the pathogenesis of atherosclerosis.

Association of a polymorphism of BTN2A1 with myocardial infarction in East Asian populations.

OBJECTIVE: We have performed a genome-wide association study (GWAS) to identify genetic variants that confer susceptibility to myocardial infarction (MI) in Japanese and Korean populations. METHODS: A total of 17,447 Japanese or Korean individuals from four independent subject panels was examined. Japanese subject panels A, B, and C comprised 134 individuals with MI and 137 controls, 1431 individuals with MI and 3161 controls, and 643 individuals with MI and 1347 controls, respectively, whereas the Korean population comprised 1880 individuals with MI and 8714 controls. A GWAS for MI was performed in Japanese subject panel A with the use of the Affymetrix GeneChip Human Mapping 500K Array Set. RESULTS: Seventy single nucleotide polymorphisms (SNPs) significantly (P<1.0×10(-7)) associated with MI by the GWAS were examined further in Japanese subject panel B, revealing two SNPs (rs6929846 of BTN2A1, rs2569512 of ILF3) to be significantly (P<0.0007) associated with MI. The rs6929846 SNP of BTN2A1, but not rs2569512 of ILF3, was also significantly associated with MI in Japanese subject panel C. However, the association of neither rs6929846 nor rs2569512 with MI was replicated in the Korean population. CONCLUSION: BTN2A1 may be a susceptibility gene for MI in Japanese individuals.

Posttranslational regulation of Abcc2 expression by SUMOylation system.

The ATP-binding cassette transporter family C 2 (Abcc2) is a member of efflux transporters involved in the biliary excretion of organic anions from hepatocytes. Posttranslational regulation of Abcc2 has been implicated, although the molecular mechanism is not fully understood. In the present study, we performed yeast two-hybrid screening to identify novel protein(s) that particularly interacts with the linker region of Abcc2 located between the NH(2)-terminal nucleotide binding domain and the last membrane-spanning domain. The screening resulted in the identification of a series of small ubiquitin-like modifier (SUMO)-related enzymes and their substrates. In yeast experiments, all of these interactions were abolished by substituting the putative SUMO consensus site in the linker region (IKKE) in Abcc2 to IRKE. In vitro SUMOylation experiments confirmed that the Abcc2 linker was a substrate of Ubc9-mediated SUMOylation. It was also found that the IKKE sequence is the target of SUMOylation, since a mutant with IKKE is substituted by IRKE was not SUMOylated. Furthermore, we demonstrated for the first time that Abcc2, endogenously expressed in rat hepatoma-derived McARH7777 cells, is SUMOylated. Suppression of endogenous Ubc9 by small interfering RNA resulted in a selective 30% reduction in Abcc2 protein expression in the postnuclear supernatant, whereas subcellular localization of Abcc2 confirmed by semiquantitative immunofluorescence analysis was minimally affected. This is the first demonstration showing the regulation of ABC transporter expression by SUMOylation.

Proinflammatory gene polymorphisms and ischemic stroke.

Despite recent advances in acute stroke therapy, stroke remains the leading cause of severe disability and the third leading cause of death, after heart disease and cancer, in Western countries and Japan. The identification of biomarkers of stroke risk is thus important both for risk prediction and for intervention to avert future events. Although genetic linkage analyses of families and sib-pairs as well as candidate gene and genome-wide association studies have implicated several loci and candidate genes in predisposition to ischemic or hemorrhagic stroke, the genes that contribute to genetic susceptibility to these conditions remain to be identified definitively. Given that vascular inflammation has been recognized as an important mechanism of atherosclerotic disease, proinflammatory genes may play pivotal roles in the pathogenesis of ischemic stroke. In this review, we summarize candidate genes that have been implicated in common forms of ischemic stroke by linkage analyses and association studies. We also review in more detail studies that have revealed an association of ischemic stroke with polymorphisms of proinflammatory genes of particular interest (LTA, IL6, and ALOX5AP) as well as with polymorphisms at chromosomal region 9p21.3, which has recently been identified as a susceptibility locus for coronary heart disease. Such studies may provide insight into the function of implicated genes as well as into the role of genetic factors in the development of ischemic stroke.

Molecular genetics of myocardial infarction.

Myocardial infarction (MI) is an important clinical problem because of its large contribution to mortality. The main causal and treatable risk factors for MI include hypertension, hypercholesterolemia or dyslipidemia, diabetes mellitus, and smoking. In addition to these risk factors, recent studies have shown the importance of genetic factors and interactions between multiple genes and environmental factors. Disease prevention is an important strategy for reducing the overall burden of MI, with the identification of markers for disease risk being key both for risk prediction and for potential intervention to lower the chance of future events. Although genetic linkage analyses of families and sib-pairs as well as candidate gene and genome-wide association studies have implicated several loci and candidate genes in predisposition to coronary heart disease (CHD) or MI, the genes that contribute to genetic susceptibility to these conditions remain to be identified definitively. In this review, we summarize both candidate loci for CHD or MI identified by linkage analyses and candidate genes examined by association studies. We also review in more detail studies that have revealed the association with MI or CHD of polymorphisms in MTHFR, LPL, and APOE by the candidate gene approach and those in LTA and at chromosomal region 9p21.3 by genome-wide scans. Such studies may provide insight into the function of implicated genes as well as into the role of genetic factors in the development of CHD and MI.

Repression of E1AF transcriptional activity by sumoylation and PIASy.

E1AF is a member of the Ets transcriptional factor family, and it plays a crucial role in tumor metastasis. However, the molecular mechanisms regulating its activity are not well characterized. In this study, we show that E1AF is sumoylated at four lysine residues, both in vivo and in vitro. Replacement of these lysines by arginine enhanced the transcriptional activity of E1AF, suggesting that sumoylation negatively regulates E1AF activity. We further demonstrated that PIASy enhanced sumoylation of E1AF as a specific SUMO-E3 ligase. In addition, PIASy repressed the transcriptional activity of both the wild-type and sumoylation defective mutants. However, the C342A mutant of PIASy, which abrogates SUMO-E3 ligase activity, had a significantly decreased ability to repress E1AF activity. Taken together, our results indicate that PIASy negatively regulates E1AF-mediated transcription by both E1AF sumoylation in a dependent and independent fashion.

PIASy-mediated repression of the Ets-1 is independent of its sumoylation.

The transcription factor Ets-1 is involved in many physiological processes, including angiogenesis, hematopoietic development, and tumor progression, and its activity can be regulated by interactions with other proteins and post-translational modifications, such as phosphorylation. Here, we show that Ets-1 is a target for SUMO modification both in vivo and in vitro. Mutational analysis reveals that sumoylation of Ets-1 occurs at two lysine residues at amino acid positions 15 and 227, which lie within previously identified synergy control motifs. Replacement of sumoylation site lysines with arginine or overexpression of SENP1, a desumoylation enzyme, enhances the transactivation ability of Ets-1. Furthermore, we identify PIASy as a novel interaction partner and a specific SUMO-E3 ligase of Ets-1. PIASy represses the Ets-1-dependent transcription, and its repression is independent of the sumoylation status of Ets-1, but it is dependent on the sumoylation of other factors.

Interactions between PIAS proteins and SOX9 result in an increase in the cellular concentrations of SOX9.

We have identified PIAS1 (protein inhibitor of activated STAT-1), -3, -xalpha, and -xbeta as SOX9-associated polypeptides using the Gal4-based yeast two-hybrid system and a cDNA library derived from a chondrocytic cell line. These PIAS proteins were shown to interact directly with SOX9 in two-hybrid, co-immunoprecipitation, and electrophoretic mobility shift assays. SOX9 was sumoylated in cotransfection experiments with COS-7 cells using PIAS and SUMO-1 (small ubiquitin-like modifier-1) expression vectors. SOX9 was also sumoylated in vitro by PIAS proteins in the presence of SUMO-1, the SUMO-activating enzyme, and the SUMO-conjugating enzyme. In COS-7 cells, PIAS proteins stimulated the SOX9-dependent transcriptional activity of a Col2a1 promoter-enhancer reporter. This increase in reporter activity was paralleled by an increase in the cellular levels of SOX9. Cotransfection with a SUMO-expressing vector further enhanced the transcriptional activity of this SOX9-dependent Col2a1 reporter in COS-7 cells, and this additional activation was inhibited in the presence of either SUMO-1 mutants or PIAS RING domain mutants or by coexpression of a desumoylation enzyme. Immunofluorescence microscopy of SOX9-transfected COS-7 cells showed that the subnuclear distribution of SOX9 became more diffuse in the presence of PIAS1 and SUMO-1. Our results suggest that, by controlling the cellular concentrations of SOX9, PIAS proteins and sumoylation may be part of a major regulatory system of SOX9 functions.