UBE2I (UBC9), a SUMO-conjugating enzyme, localizes to nuclear speckles and stimulates transcription in mouse oocytes.
ABSTRACT: Sumoylation is a posttranslational modification in which SUMO (small ubiquitin-related modifier) proteins are covalently attached to their substrates. In vertebrates, developmental roles for sumoylation have been studied, but the function of sumoylation during mammalian oocyte growth and maturation is not known. As a prelude to conducting studies on the role of sumoylation during oocyte development, we analyzed the temporal and spatial pattern of expression of UBE2I, a SUMO-conjugating E2 enzyme. Immunocytochemical analysis of UBE2I revealed a punctate nuclear staining pattern, with transcriptionally quiescent, fully grown, GV-intact oocytes having larger UBE2I-containing bodies than transcriptionally active, meiotically incompetent growing oocytes. Inhibiting transcription in incompetent oocytes resulted in an increase in the size of the UBE2I-containing bodies. Overexpression of either wild-type UBE2I or catalytically inactive UBE2I resulted in an increase in the size of the UBE2I-containing bodies but also an increase in BrUTP incorporation, suggesting that transcriptional activation by UBE2I is independent of its catalytic activity. Although UBE2I-containing bodies did not completely colocalize with SUMO1 or SUMO2 and SUMO3, which were localized mainly on the nuclear membrane and in the nucleoplasm, UBE2I strikingly colocalized with SFRS2, which is a component of nuclear speckles and critical for mRNA processing. These results suggest a novel function for UBE2I and therefore sumoylation in gene expression.
Project description:Multiple myeloma (MM) is a plasma cell neoplasm that proceeds through a premalignant state of monoclonal gammopathy of unknown significance; however, the molecular events responsible for myelomagenesis remain uncharacterized. To identify cellular pathways deregulated in MM, we addressed that sumoylation is homologous to ubiquitination and results in the attachment of the ubiquitin-like protein Sumo onto target proteins. Sumoylation was markedly enhanced in MM patient lysates compared with normal plasma cells and expression profiling indicated a relative induction of sumoylation pathway genes. The Sumo-conjugating enzyme Ube2I, the Sumo-ligase PIAS1, and the Sumo-inducer ARF were elevated in MM patient samples and cell lines. Survival correlated with expression because 80% of patients with low UBE2I and PIAS1 were living 6 years after transplantation, whereas only 45% of patients with high expression survived 6 years. UBE2I encodes the sole Sumo-conjugating enzyme in mammalian cells and cells transfected with a dominant-negative sumoylation-deficient UBE2I mutant exhibited decreased survival after radiation exposure, impaired adhesion to bone marrow stroma cell and decreased bone marrow stroma cell-induced proliferation. UBE2I confers cells with multiple advantages to promote tumorigenesis and predicts decreased survival when combined with PIAS1. The sumoylation pathway is a novel therapeutic target with implications for existing proteasomal-based treatment strategies.
Project description:Cardiac proteins are subject to continuous stress and these intrinsic and extrinsic factors, both physiological and pathological can lead to protein misfolding. If the protein quality control (PQC) pathways are in any way compromised or their activities diminished, intracellular aggregates can form and a proteotoxic environment is generated, which contributes to cardiac disease and heart failure. We studied the role that SUMO post-translational modification plays in a proteotoxic cardiac environment. SUMOylation can have an integral role in controlling flux through the ubiquitin-proteasome system, and expression of the SUMO (small ubiquitin-like modifier) E2 enzyme UBE2I/UBC9 improves cardiac PQC. Our data focus on using gain- and loss-of-function approaches to modify UBE2I levels and measure the effects on cardiomyocyte autophagic flux. UBE2I expression does have an impact on macroautophagy/autophagy as increased SUMOylation results in increased autophagy. We show that increased SUMOylation is cardioprotective and can decrease morbidity in proteotoxic cardiac disease.
Project description:Sumoylation is an important post-translational modification in which SUMO (small ubiquitin-related modifier) proteins are bonded covalently to their substrates. Studies on the roles of sumoylation in cell cycle regulation have been emerging in both mitosis from yeast to mammals and meiosis in budding yeast, but the functions of sumoylation in mammalian meiosis, especially in oocyte meiotic maturation are not well known. Here, we examined the localization and expression of SUMO-1 and SUMO-2/3, the two basic proteins in the sumoylation pathway and investigated their roles through over-expression of Senp2 during mouse oocyte maturation. Immunofluorescent staining revealed differential patterns of SUMO-1 and SUMO-2/3 localization: SUMO-1 was localized to the spindle poles in prometaphase I, MI and MII stages, around the separating homologues in anaphase I and telophase I stages of first meiosis, while SUMO-2/3 was mainly concentrated near centromeres during mouse oocyte maturation. Immunoblot analysis uncovered the different expression profiles of SUMO-1 and SUMO-2/3 modified proteins during mouse oocyte maturation. Over-expression of Senp2, a SUMO-specific isopeptidase, caused changes of SUMO-modified proteins and led to defects in MII spindle organization in mature eggs. These results suggest that the SUMO pathway may play an indispensable role during mouse oocyte meiotic maturation.
Project description:The negatively regulating zinc finger protein (NZFP) is an essential transcription repressor required for early development during gastrulation in Xenopus laevis. In this study, we found that NZFP interacts with the small ubiquitin-like modifier (SUMO) conjugation E2 enzyme, Ubc9, and contains three putative SUMO conjugation sites. Studies with NZFP mutants containing mutations at the putative SUMO conjugation sites showed that these sites were able to be modified independently with SUMO. NZFP was found to be localized in the same nuclear bodies with SUMO-1. However, sumoylation of NZFP did not play a role either in the translocation of NZFP into the nucleus or on nuclear body formation. While wild type NZFP showed significant transcriptional repression, SUMO-conjugation site mutants manifested a decrease in transcriptional repression activity which is reversely proportional to the amount of sumoylation. The sumoylation defective mutant lost its TBP binding activity, while wild type NZFP interacted with TBP and inhibited transcription complex formation. These results strongly suggest that the sumoylation of NZFP facilitates NZFP to bind to TBP and the NZFP/TBP complex then represses the transcription of the target gene by inhibiting basal transcription complex formation.
Project description:BACKGROUND: The Tax protein encoded by Human T-lymphotropic virus type 1 (HTLV-1) is a powerful activator of the NF-?B pathway, a property critical for HTLV-1-induced immortalization of CD4? T lymphocytes. Tax permanently stimulates this pathway at a cytoplasmic level by activating the I?B kinase (IKK) complex and at a nuclear level by enhancing the binding of the NF-?B factor RelA to its cognate promoters and by forming nuclear bodies, believed to represent transcriptionally active structures. In previous studies, we reported that Tax ubiquitination and SUMOylation play a critical role in Tax localization and NF-?B activation. Indeed, analysis of lysine Tax mutants fused or not to ubiquitin or SUMO led us to propose a two-step model in which Tax ubiquitination first intervenes to activate IKK while Tax SUMOylation is subsequently required for promoter activation within Tax nuclear bodies. However, recent studies showing that ubiquitin or SUMO can modulate Tax activities in either the nucleus or the cytoplasm and that SUMOylated Tax can serve as substrate for ubiquitination suggested that Tax ubiquitination and SUMOylation may mediate redundant rather than successive functions. RESULTS: In this study, we analyzed the properties of a new Tax mutant that is properly ubiquitinated, but defective for both nuclear body formation and SUMOylation. We report that reducing Tax SUMOylation and nuclear body formation do not alter the ability of Tax to activate IKK, induce RelA nuclear translocation, and trigger gene expression from a NF-?B promoter. Importantly, potent NF-?B promoter activation by Tax despite low SUMOylation and nuclear body formation is also observed in T cells, including CD4? primary T lymphocytes. Moreover, we show that Tax nuclear bodies are hardly observed in HTLV-1-infected T cells. Finally, we provide direct evidence that the degree of NF-?B activation by Tax correlates with the level of Tax ubiquitination, but not SUMOylation. CONCLUSIONS: These data reveal that the formation of Tax nuclear bodies, previously associated to transcriptional activities in Tax-transfected cells, is dispensable for NF-?B promoter activation, notably in CD4? T cells. They also provide the first evidence that Tax SUMOylation is not a key determinant for Tax-induced NF-?B activation.
Project description:PML nuclear bodies (NBs) are nuclear structures that have been implicated in processes such as transcriptional regulation, genome stability, response to viral infection, apoptosis, and tumor suppression. PML has been found to be essential for the formation of the NBs, as these structures do not form in Pml null cells, although PML add back fully rescues their formation. However, the basis for such a structural role of PML is unknown. We demonstrate that PML contains a SUMO binding motif that is independent of its SUMOylation sites and is surprisingly necessary for PML-NB formation. We demonstrate that the PML RING domain is critical for PML SUMOylation and PML-NB formation. We propose a model for PML-NB formation whereby PML SUMOylation and noncovalent binding of PML to SUMOylated PML through the SUMO binding motif constitutes the nucleation event for subsequent recruitment of SUMOylated proteins and/or proteins containing SUMO binding motifs to the PML NBs.
Project description:This study examined whether small ubiquitin-related modifier-1 (SUMO-1) regulates apoptosis signal-regulating kinase 1 (ASK 1). ASK 1 interacted with SUMO-1 in vitro as well as in BOSC 23 cells. Endogenous ASK 1-SUMO-1 interaction was disrupted following H(2)O(2) signal. SUMO-1 overexpression suppressed the self-oligomerization, kinase activity and apoptotic potential of ASK 1, whereas SUMO-1 depletion potentiated such activities. SUMO-1(Delta C 6), a sumoylation-incompetent mutant lacking carboxy-terminal six amino acids, suppressed AS 1 activation, implying that the suppressive effect of SUMO-1 on ASK 1 is independent of sumoylation. ASK 1(3M), an ASK 1 mutant in which all three lysines in the psiKXE motif were substituted with alanines, still retained the kinase activity and activated the Jun amino-terminal kinase pathway. However, SUMO-1 failed to interact with ASK 1(3M) and to suppress ASK 1(3M) activation, indicating that the three lysines are important for regulation by SUMO-1. This study shows that SUMO-1 exerts a negative regulatory effect on ASK 1 activation through physical interaction and not through covalent modification.
Project description:To investigate the protein profiling of buffalo oocytes at the germinal vesicle (GV) stage and metaphase II (MII) stage, an iTRAQ-based strategy was applied. A total of 3,763 proteins were identified, which representing the largest buffalo oocytes proteome dataset to date. Among these proteins identified, 173 proteins were differentially expressed in GV oocytes and competent MII oocytes, and 146 proteins were differentially abundant in competent and incompetent matured oocytes. Functional and KEGG pathway analysis revealed that the up-regulated proteins in competent MII oocytes were related to chromosome segregation, microtubule-based process, protein transport, oxidation reduction, ribosome, and oxidative phosphorylation, etc., in comparison with GV and incompetent MII oocytes. This is the first proteomic report on buffalo oocytes from different maturation stages and developmental competent status. These data will provide valuable information for understanding the molecular mechanism underlying buffalo oocyte maturation, and these proteins may potentially act as markers to predict developmental competence of buffalo oocyte during in vitro maturation.
Project description:The 5 S rRNA gene-specific transcription factor IIIA (TFIIIA) interacts with the small ubiquitin-like modifier (SUMO) E3 ligase PIAS2b and with one of its targets, the transcriptional corepressor, XCtBP. PIAS2b is restricted to the cytoplasm of Xenopus oocytes but relocates to the nucleus immediately after fertilization. Following the midblastula transition, PIAS2b and XCtBP are present on oocyte-type, but not somatic-type, 5 S rRNA genes up through the neurula stage, as is a limiting amount of TFIIIA. Histone H3 methylation, coincident with the binding of XCtBP, also occurs exclusively on the oocyte-type genes. Immunohistochemical staining of embryos confirms the occupancy of a subset of the oocyte-type genes by TFIIIA that become positioned at the nuclear periphery shortly after the midblastula transition. Inhibition of SUMOylation activity relieves repression of oocyte-type 5 S rRNA genes and is correlated with a decrease in methylation of H3K9 and H3K27 and disruption of subnuclear localization. These results reveal a novel function for TFIIIA as a negative regulator that recruits histone modification activity through the CtBP repressor complex exclusively to the oocyte-type 5 S rRNA genes, leading to their terminal repression.
Project description:Somatic cells surrounding the oocyte were sampled at the following stages: developmentally incompetent or poorly competent prophase I oocytes (NC1 oocytes), developmentally competent prophase I oocytes (C1 oocytes), and developmentally competent metaphase II oocytes (C2 oocytes). NC1 samples were collected from late vitellogenic females (LV), C1 samples from post-vitellogenic females (PV), and C2 samples from females undergoing meiotic maturation (Germinal Vesicle Breakdown) Global transcriptional profiling was performed using somatic cells collected from rainbow trout ovarian follicles during in vivo oocyte developmental competence acquisition. Somatic cells were collected at 3 stages of oogenesis: NC1 stage follicles (LV, late vitellogenic, prophase I arrested oocytes, meiotically incompetent and developmentally incompetent, n=6), C1 stage follicles (PV, post-vitellogenic, prophase I arrested oocytes, meiotically competent and developmentally competent, n=16). Ovulatory follicles were also collected during oocyte maturation after in vivo induction (metaphase II arrested oocytes, developmentally fully competent, n=6).