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1.
PLoS Biol ; 17(6): e3000324, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31226106

RESUMO

The molecular mechanism controlling the zygotic genome activation (ZGA) in mammals remains poorly understood. The 2-cell (2C)-like cells spontaneously emerging from cultures of mouse embryonic stem cells (ESCs) share some key transcriptional and epigenetic programs with 2C-stage embryos. By studying the transition of ESCs into 2C-like cells, we identified developmental pluripotency associated 2 and 4 (Dppa2/4) as important regulators controlling zygotic transcriptional program through directly up-regulating the expression of double homeobox (Dux). In addition, we found that DPPA2 protein is sumoylated and its activity is negatively regulated by small ubiquitin-like modifier (Sumo) E3 ligase protein inhibitor of activated STAT 4 (PIAS4). PIAS4 is down-regulated during ZGA process and during transitioning of ESCs into 2C-like cells. Depleting Pias4 or overexpressing Dppa2/4 is sufficient to activate 2C-like transcriptional program, whereas depleting Dppa2/4 or forced expression of Pias4 or Sumo2-Dppa2 inhibits 2C-like transcriptional program. Furthermore, ectopic expression of Pias4 or Sumo2-Dppa2 impairs early mouse embryo development. In summary, our study identifies key molecular rivals consisting of transcription factors and a Sumo2 E3 ligase that regulate zygotic transcriptional program upstream of Dux.


Assuntos
Proteínas Nucleares/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias/metabolismo , Feminino , Genoma , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/fisiologia , Proteínas de Ligação a Poli-ADP-Ribose , Proteínas Inibidoras de STAT Ativados/fisiologia , Proteína SUMO-1/metabolismo , Proteína SUMO-1/fisiologia , Análise de Célula Única , Sumoilação , Fatores de Transcrição/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Zigoto/metabolismo
2.
J Virol ; 93(10)2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30842328

RESUMO

SUMOylation is a posttranslational modification that has crucial roles in diverse cellular biological pathways and in various viral life cycles. In this study, we found that the VP1 protein, the RNA-dependent RNA polymerase of avibirnavirus infectious bursal disease virus (IBDV), regulates virus replication by SUMOylation during infection. Our data demonstrated that the polymerase VP1 is efficiently modified by small ubiquitin-like modifier 1 (SUMO1) in avibirnavirus-infected cell lines. Mutation analysis showed that residues 404I and 406I within SUMO interaction motif 3 of VP1 constitute the critical site for SUMO1 modification. Protein stability assays showed that SUMO1 modification enhanced significantly the stability of polymerase VP1 by inhibiting K48-linked ubiquitination. A reverse genetic approach showed that only IBDV with I404C/T and I406C/F mutations of VP1 could be rescued successfully with decreased replication ability. Our data demonstrated that SUMO1 modification is essential to sustain the stability of polymerase VP1 during IBDV replication and provides a potential target for designing antiviral drugs targeting IBDV.IMPORTANCE SUMOylation is an extensively discussed posttranslational modification in diverse cellular biological pathways. However, there is limited understanding about SUMOylation of viral proteins of IBDV during infection. In the present study, we revealed a SUMO1 modification of VP1 protein, the RNA-dependent RNA polymerase of avibirnavirus infectious bursal disease virus (IBDV). The required site of VP1 SUMOylation comprised residues 404I and 406I of SUMO interaction motif 3, which was essential for maintaining its stability by inhibiting K48-linked ubiquitination. We also showed that IBDV with SUMOylation-deficient VP1 had decreased replication ability. These data demonstrated that the SUMOylation of IBDV VP1 played an important role in maintaining IBDV replication.


Assuntos
Vírus da Doença Infecciosa da Bursa/metabolismo , Proteína SUMO-1/metabolismo , Proteínas Estruturais Virais/metabolismo , Avibirnavirus/metabolismo , Avibirnavirus/patogenicidade , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Vírus da Doença Infecciosa da Bursa/patogenicidade , Vírus da Doença Infecciosa da Bursa/fisiologia , Processamento de Proteína Pós-Traducional , RNA Replicase/genética , Proteína SUMO-1/fisiologia , Sumoilação , Ubiquitinação , Proteínas Virais/metabolismo , Proteínas Estruturais Virais/genética , Replicação Viral/fisiologia
3.
J Biol Chem ; 294(17): 6772-6784, 2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-30824543

RESUMO

Aromatic amino acids buried at a protein's core are often involved in mutual paired interactions. Ab initio energy calculations have highlighted that the conformational orientations and the effects of substitutions are important for stable aromatic interactions among aromatic rings, but studies in the context of a protein's fold and function are elusive. Small ubiquitin-like modifier (SUMO) is a common post-translational modifier that affects diverse cellular processes. Here, we report that a highly conserved aromatic triad of three amino acids, Phe36-Tyr51-Phe64, is a unique SUMO signature that is absent in other ubiquitin-like homologous folds. We found that a specific edge-to-face conformation between the Tyr51-Phe64 pair of interacting aromatics is vital to the fold and stability of SUMO. Moreover, the noncovalent binding of SUMO-interacting motif (SIM) at the SUMO surface was critically dependent on the paired aromatic interactions buried at the core. NMR structural studies revealed that perturbation of the Tyr51-Phe64 conformation disrupts several long-range tertiary contacts in SUMO, leading to a heterogeneous and dynamic protein with attenuated SUMOylation both in vitro and in cells. A subtle perturbation of the edge-to-face conformation by a Tyr to Phe substitution significantly decreased stability, SUMO/SIM affinity, and the rate of SUMOylation. Our results highlight that absolute co-conservation of specific aromatic pairs inside the SUMO protein core has a role in its stability and function.


Assuntos
Proteína SUMO-1/metabolismo , Sequência de Aminoácidos , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Proteína SUMO-1/química , Proteína SUMO-1/fisiologia , Homologia de Sequência de Aminoácidos , Sumoilação , Ubiquitina/metabolismo
5.
Chin Med J (Engl) ; 132(4): 466-473, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30707172

RESUMO

OBJECTIVE: Small ubiquitin-related modifiers (SUMOs) are a group of post-translational modification proteins extensively expressed in eukaryotes. Abnormal SUMOylation can lead to the development of various diseases. This article summarizes the progress on research of the role of SUMOs in various types of kidney diseases to further increase the understanding of the regulatory functions of SUMOylation in the pathogenesis of kidney diseases. DATA SOURCES: This review was based on articles published in the PubMed databases up to January 2018, using the keywords including "SUMOs," "SUMOylation," and "kidney diseases." STUDY SELECTION: Original articles and critical reviews about SUMOs and kidney disease were selected for this review. A total of 50 studies were in English. RESULTS: SUMO participates in the activation of NF-κB inflammatory signaling pathway, playing a central regulatory role in the inflammation and progression of DN, and the secretion of various chemokines in AKI. SUMO involves in the regulation of TG2 and Nrf2 antioxidant stress, affecting renal tubular injury in AKI. SUMO affects the MAPK/ERK pathway, regulating intracellular signal transduction, modulating the transcription and expression of effector molecules in DN. SUMO contributes to the TGF-ß/Smad pathway, leading to fibrosis of the kidney. The conjugate combination of SUMO and p53 regulates cell proliferation and apoptosis, and participates in the regulation of tumorigenesis. In addition, SUMOylation of MITF modulates renal tumors secondary to melanoma, Similarly, SUMOylation of tumor suppressor gene VHL regulates the occurrence of renal cell carcinoma in VHL syndrome. CONCLUSIONS: Tissue injury, inflammatory responses, fibrosis, apoptosis, and tumor proliferation in kidney diseases all involve SUMOs. Further research of the substrate SUMOylation and regulatory mechanisms of SUMO in kidney diseases will improve and develop new treatment measures and strategies targeting kidney diseases.


Assuntos
Nefropatias/etiologia , Proteína SUMO-1/fisiologia , Lesão Renal Aguda/etiologia , Carcinoma de Células Renais/etiologia , Nefropatias Diabéticas/etiologia , Fibrose , Humanos , Rim/patologia , Nefropatias/metabolismo , Neoplasias Renais/etiologia , Sumoilação
6.
PLoS Genet ; 14(9): e1007626, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30180169

RESUMO

Chromosome congression and segregation in C. elegans oocytes depend on a complex of conserved proteins that forms a ring around the center of each bivalent during prometaphase; these complexes are then removed from chromosomes at anaphase onset and disassemble as anaphase proceeds. Here, we uncover mechanisms underlying the dynamic regulation of these ring complexes (RCs), revealing a strategy by which protein complexes can be progressively remodeled during cellular processes. We find that the assembly, maintenance, and stability of RCs is regulated by a balance between SUMO conjugating and deconjugating activity. During prometaphase, the SUMO protease ULP-1 is targeted to the RCs but is counteracted by SUMO E2/E3 enzymes; then in early anaphase the E2/E3 enzymes are removed, enabling ULP-1 to trigger RC disassembly and completion of the meiotic divisions. Moreover, we found that SUMO regulation is essential to properly connect the RCs to the chromosomes and then also to fully release them in anaphase. Altogether, our work demonstrates that dynamic remodeling of SUMO modifications facilitates key meiotic events and highlights how competition between conjugation and deconjugation activity can modulate SUMO homeostasis, protein complex stability, and ultimately, progressive processes such as cell division.


Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/fisiologia , Meiose , Proteína SUMO-1/fisiologia , Sumoilação/fisiologia , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Posicionamento Cromossômico/fisiologia , Segregação de Cromossomos/fisiologia , Modelos Animais , Proteína SUMO-1/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo
7.
Sci Rep ; 8(1): 11502, 2018 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-30065345

RESUMO

Adult stem cells play a critical role in tissue homeostasis and repair. Aging leads to a decline in stem cells' regenerative capacity that contributes significantly to the maintenance of organ and tissue functions. Age-dependent genomic and epigenetic modifications together play a role in the disruption of critical cellular pathways. However, the epigenetic mechanisms responsible for the decline of adult stem cell functions remain to be well established. Here, we investigated age-dependent, genome-wide alterations in the chromatin accessibility of primary human adipose-derived stem cells (ASCs) in comparison to age-matched fibroblasts via ATAC-seq technology. Our results demonstrate that aging ASCs possess globally more stable chromatin accessibility profiles as compared to aging fibroblasts, suggesting that robust regulatory mechanisms maintain adult stem cell chromatin structure against aging. Furthermore, we observed age-dependent subtle changes in promoter nucleosome positioning in selective pathways during aging, concurrent with altered small ubiquitin-related modifier (SUMO) protein expression under stress conditions. Together, our data suggest a significant role for nucleosome positioning in sumoylation pathway regulation in stress response during adult stem cell aging. The differences described here between the chromatin structure of human ASCs and fibroblasts will further elucidate the mechanisms regulating gene expression during aging in both stem cells and differentiated cells.


Assuntos
Envelhecimento/fisiologia , Cromatina/fisiologia , Células-Tronco Mesenquimais/fisiologia , Adipócitos/metabolismo , Adulto , Fatores Etários , Idoso , Diferenciação Celular , Montagem e Desmontagem da Cromatina/fisiologia , Epigênese Genética/genética , Feminino , Genoma , Humanos , Pessoa de Meia-Idade , Cultura Primária de Células , Regiões Promotoras Genéticas/genética , Proteína SUMO-1/metabolismo , Proteína SUMO-1/fisiologia , Células-Tronco/metabolismo , Estresse Fisiológico/fisiologia , Transcriptoma
8.
Lab Invest ; 98(6): 799-813, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29472640

RESUMO

The intestinal epithelium constitutes a crucial defense to the potentially life-threatening effects of gut microbiota. However, due to a complex underlying vasculature, hypoperfusion and resultant tissue ischemia pose a particular risk to function and integrity of the epithelium. The small ubiquitin-like modifier (SUMO) conjugation pathway critically regulates adaptive responses to metabolic stress and is of particular significance in the gut, as inducible knockout of the SUMO-conjugating enzyme Ubc9 results in rapid intestinal epithelial disintegration. Here we analyzed the pattern of individual SUMO isoforms in intestinal epithelium and investigated their roles in intestinal ischemia/reperfusion (I/R) damage. Immunostaining revealed that epithelial SUMO2/3 expression was almost exclusively limited to crypt epithelial nuclei in unchallenged mice. However, intestinal I/R or overexpression of Ubc9 caused a remarkable enhancement of epithelial SUMO2/3 staining along the crypt-villus axis. Unexpectedly, a similar pattern was found in SUMO1 knockout mice. Ubc9 transgenic mice, but also SUMO1 knockout mice were protected from I/R injury as evidenced by better preserved barrier function and blunted inflammatory responses. PCR array analysis of microdissected villus-tip epithelia revealed a specific epithelial contribution to reduced inflammatory responses in Ubc9 transgenic mice, as key chemotactic signaling molecules such as IL17A were significantly downregulated. Together, our data indicate a critical role particularly of the SUMO2/3 isoforms in modulating responses to I/R and provide the first evidence that SUMO1 deletion activates a compensatory process that protects from ischemic damage.


Assuntos
Mucosa Intestinal/irrigação sanguínea , Traumatismo por Reperfusão/prevenção & controle , Proteína SUMO-1/fisiologia , Enzimas de Conjugação de Ubiquitina/fisiologia , Animais , Quimiocinas/análise , Mucosa Intestinal/química , Microdissecção e Captura a Laser , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína SUMO-1/deficiência , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/análise , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/fisiologia , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitinas/análise , Ubiquitinas/fisiologia
9.
Nat Rev Cancer ; 17(3): 184-197, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28134258

RESUMO

Post-translational protein modification by small ubiquitin-like modifier (SUMO), termed sumoylation, is an important mechanism in cellular responses to stress and one that appears to be upregulated in many cancers. Here, we examine the role of sumoylation in tumorigenesis as a possibly necessary safeguard that protects the stability and functionality of otherwise easily misregulated gene expression programmes and signalling pathways of cancer cells.


Assuntos
Neoplasias/etiologia , Proteína SUMO-1/fisiologia , Animais , Hipóxia Celular , Senescência Celular , Dano ao DNA , Resistencia a Medicamentos Antineoplásicos , Regulação da Expressão Gênica , Humanos , NF-kappa B/fisiologia , Transdução de Sinais/fisiologia , Sumoilação
10.
Adv Exp Med Biol ; 1042: 371-393, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29357067

RESUMO

Accurate genome duplication during cell division is essential for life. This process is accomplished by the close collaboration between replication factors and many additional proteins that provide assistant roles. Replication factors establish the replication machineries capable of copying billions of nucleotides, while regulatory proteins help to achieve accuracy and efficiency of replication. Among regulatory proteins, protein modification enzymes can bestow fast and reversible changes to many targets, leading to coordinated effects on replication. Recent studies have begun to elucidate how one type of protein modification, sumoylation, can modify replication proteins and regulate genome duplication through multiple mechanisms. This chapter summarizes these new findings, and how they can integrate with the known regulatory circuitries of replication. As this area of research is still at its infancy, many outstanding questions remain to be explored, and we discuss these issues in light of the new advances.


Assuntos
Replicação do DNA/fisiologia , Proteína SUMO-1/fisiologia , Animais , Humanos , Complexo de Reconhecimento de Origem/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Origem de Replicação/fisiologia , Sumoilação/fisiologia
11.
Bioessays ; 37(10): 1095-105, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26354225

RESUMO

The small ubiquitin-like modifier SUMO regulates many aspects of cellular physiology to maintain cell homeostasis, both under normal conditions and during cell stress. Components of the transcriptional apparatus and chromatin are among the most prominent SUMO substrates. The prevailing view is that SUMO serves to repress transcription. However, as we will discuss in this review, this model needs to be refined, because recent studies have revealed that SUMO can also have profound positive effects on transcription.


Assuntos
Regulação da Expressão Gênica , Proteína SUMO-1/fisiologia , Transcrição Genética , Animais , Humanos , Lisina/metabolismo , Ligação Proteica/genética , Conformação Proteica , Sumoilação/fisiologia
12.
Proteomics ; 15(5-6): 1181-91, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25236368

RESUMO

Small ubiquitin-like modifier (SUMO1-3) conjugation is a posttranslational protein modification whereby SUMOs are conjugated to lysine residues of target proteins. SUMO conjugation can alter the activity, stability, and function of target proteins, and thereby modulate almost all major cellular pathways. Many diseases are associated with SUMO conjugation, including heart failure, arthritis, cancer, degenerative diseases, and brain ischemia/stroke. It is, therefore, of major interest to characterize the SUMO-modified proteome regulated by these disorders. SUMO proteomics analysis is hampered by low levels of SUMOylated proteins. Several strategies have, therefore, been developed to enrich SUMOylated proteins from cell/tissue extracts. These include proteomics analysis on cells expressing epitope-tagged SUMO isoforms, use of monoclonal SUMO antibodies for immunoprecipitation and epitope-specific peptides for elution, and affinity purification with peptides containing SUMO interaction motifs to specifically enrich polySUMOylated proteins. Recently, two mouse models were generated and characterized that express tagged SUMO isoforms, and allow purification of SUMOylated proteins from complex organ extracts. Ultimately, these new analytical tools will help to decipher the SUMO-modified proteome regulated by various human diseases, and thereby, identify new targets for preventive and therapeutic purposes.


Assuntos
Cardiopatias/metabolismo , Neoplasias/metabolismo , Proteoma , Proteômica/métodos , Proteína SUMO-1 , Sumoilação/fisiologia , Animais , Biomarcadores , Humanos , Camundongos , Doenças Neurodegenerativas/metabolismo , Proteoma/análise , Proteoma/metabolismo , Proteoma/fisiologia , Proteína SUMO-1/análise , Proteína SUMO-1/metabolismo , Proteína SUMO-1/fisiologia , Acidente Vascular Cerebral/metabolismo
13.
Mol Reprod Dev ; 81(8): 712-24, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25123474

RESUMO

Small ubiquitin-related modifier-1 (SUMO-1)-dependent modifications of many target proteins are involved in a range of intracellular processes. Previous studies reported the localization of SUMO-1 during oocyte meiosis, and that overexpression of Sentrin/SUMO-specific protease 2 (SENP2), a de-SUMOylation protease, altered SUMO-modified proteins, and caused defects in metaphase-II spindle organization. In this study, we detailed the consequences of SUMO-1-mediated SUMOylation by either inhibition of SUMO-1 or UBC9 with a specific antibody or their depletion by specific siRNA microinjection. Inhibition or depletion of SUMO-1 or UBC9 in germinal vesicle (GV)-stage oocytes decreased the rates of germinal vesicle breakdown and first polar body (PB1) extrusion; caused defective spindle organization and misaligned chromosomes; and led to aneuploidy in matured oocytes. Stage-specific antibody injections suggested that SUMO-1 functions before anaphase I during PB1 extrusion. Further experiments indicated that the localization of γ-tubulin was disordered after SUMO-1 inhibition, and that SUMO-1 depletion disrupted kinetochore-microtubule attachment at metaphase I. Moreover, SUMO-1 inhibition resulted in less-condensed chromosomes, altered localization of REC8 and securin, and reduced BUBR1 accumulation at the centromere. On the other hand, overexpression of SUMO-1 in GV-stage oocytes had no significant effect on oocyte maturation. In conclusion, our results implied that SUMO-1 plays crucial roles during oocyte meiotic maturation, specifically involving spindle assembly and chromosome behavior, by regulating kinetochore-microtubule attachment and the localization of γ-tubulin, BUBR1, REC8, and securin.


Assuntos
Segregação de Cromossomos/fisiologia , Meiose/fisiologia , Oócitos/fisiologia , Proteína SUMO-1/fisiologia , Fuso Acromático/fisiologia , Animais , Proteínas de Ciclo Celular/metabolismo , Camundongos , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Proteína SUMO-1/antagonistas & inibidores , Proteína SUMO-1/metabolismo , Securina/metabolismo , Sumoilação , Tubulina (Proteína)/metabolismo
14.
Insect Biochem Mol Biol ; 51: 71-9, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24880118

RESUMO

SUMOylation is an essential post-translational modification that regulates a variety of cellular processes including cell cycle progression. Although the SUMOylation pathway has been identified and investigated in many eukaryotes, the mechanisms of SUMOylation in regulating the functions of various substrates are still poorly understood. Here, we utilized a model species, the silkworm Bombyx mori that possesses holocentric chromosomes, to exploit the role of the SUMOylation system in cell cycle regulation. We identified all the components that are involved in the SUMOylation pathway in the silkworm genome. Our data revealed a cell cycle-dependent transcription of the SUMOylation genes, localization of the SUMOylation proteins, and abundance of the SUMOylation substrates in cultured silkworm cells. Importantly, the proliferation of the silkworm cells was strikingly inhibited by interference with SUMOylation genes expression, possibly due to an arrest of the SUMOylation-deficient cells at the G2/M phase. Furthermore, disruption of the SUMOylation genes induced the defects of holocentric chromosome congression and segregation during mitosis, which was consistent with high expressions of the SUMOylation genes and high enrichments of global SUMOylation at this stage, suggesting that the SUMOylation system in silkworm is essential for cell cycle regulation, with one particular role in mitosis.


Assuntos
Bombyx/genética , Pontos de Checagem do Ciclo Celular/genética , Processamento de Proteína Pós-Traducional , Transdução de Sinais/genética , Sumoilação/genética , Animais , Linhagem Celular , Cromossomos de Insetos/genética , Genoma de Inseto , Interferência de RNA , Proteína SUMO-1/fisiologia , Sumoilação/fisiologia
15.
FEBS J ; 281(13): 3032-47, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24819975

RESUMO

Small ubiquitin-related modifier-1 (SUMO-1) modification has been implicated in many important cellular processes, including cell cycle progression, apoptosis, cellular proliferation, and development, but its role in all-trans-retinoic acid (ATRA)-induced differentiation processes of cancer cells remains unclear. Here, we report for the first time that ATRA-induced differentiation of leukemia and osteosarcoma is accompanied by a decrease in the level of SUMO-1 protein. Our results also demonstrated that depletion or inhibition of SUMO-1 blocks ATRA-induced differentiation, suggesting that SUMO-1 is critical for the differentiation effect of ATRA. Further studies indicated that SUMO-1-promoted ATRA-induced differentiation might be associated with the stabilization of retinoic acid receptor α (RARα), protecting it from degradation. Moreover, our results suggested that Lys399 is a major site for SUMO-1 conjugation of RARα. We also found that RARα enhanced the transcription of its target genes, which might also contribute to the enhanced differentiating effects of ATRA; however, mutation of Lys399 of RARα inhibits the extents of both SUMO-1 modification and ATRA-induced differentiation. Together, these results indicate that SUMO-1 modification of RARα is a potent mechanism for balancing proliferation and differentiation by controlling the stability of RARα in cancer cells. SUMO-1 modification may thus serve an important role in controlling ATRA-induced cell differentiation in cancers.


Assuntos
Diferenciação Celular , Receptores do Ácido Retinoico/metabolismo , Proteína SUMO-1/fisiologia , Sumoilação , Tretinoína/fisiologia , Animais , Células COS , Proliferação de Células , Chlorocebus aethiops , Células HL-60 , Humanos , Estabilidade Proteica , Receptor alfa de Ácido Retinoico
16.
J Hepatol ; 58(6): 1157-64, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23369793

RESUMO

BACKGROUND & AIMS: Constitutive activation of NF-κB is an important event involved in chronic inflammation in hepatocellular carcinoma (HCC). CPAP, which plays important roles in centrosomal functions, was previously identified as the transcriptional co-activator of NF-κB. However, the molecular mechanism is unclear. The goal of this study was to investigate the role of CPAP in activating the NF-κB pathway in HCC. METHODS: SK-Hep1, HuH7, HepG2, HepG2X, Hep3B, and Hep3BX cells with CPAP overexpression or CPAP siRNA were used to evaluate activation of NF-κB under TNF-α stimulation by reporter assay, RT-PCR, Q-PCR, and Western blot analysis. In vivo SUMO modification of CPAP was demonstrated by an in situ PLA assay. Human HCC tissues were used to perform Q-PCR, Western blot, and IHC. RESULTS: CPAP siRNA abolished the interaction between IKKß and NF-κB, whereas overexpression of CPAP enhanced this interaction and finally led to augmented NF-κB activation by increasing the phosphorylation of NF-κB. CPAP could enter nuclei by associating with NF-κB. Furthermore, CPAP was SUMO-1 modified upon TNF-α stimulus, and this is essential for its NF-κB co-activator activity. SUMO-1-deficient CPAP mutant lost its NF-κB co-activator activity and failed to enter nuclei. Importantly, SUMOylated CPAP could synergistically increase the HBx-induced NF-κB activity. CONCLUSIONS: CPAP is essential for the recruitment of the IKK complex to inactivated NF-κB upon TNF-α treatment. Expression of CPAP was positively correlated with a poor prognosis in HBV-HCC. CPAP has the potential to serve as a therapeutic target for inflammation and inflammation-related diseases.


Assuntos
Carcinoma Hepatocelular/etiologia , Quinase I-kappa B/fisiologia , Neoplasias Hepáticas/etiologia , Proteínas Associadas aos Microtúbulos/fisiologia , NF-kappa B/fisiologia , Transdução de Sinais/fisiologia , Sumoilação , Transativadores/fisiologia , Carcinoma Hepatocelular/metabolismo , Humanos , Proteínas I-kappa B/metabolismo , Neoplasias Hepáticas/metabolismo , Inibidor de NF-kappaB alfa , Fosforilação , Proteína SUMO-1/fisiologia , Fator de Necrose Tumoral alfa/farmacologia
17.
Endocrinology ; 154(2): 698-708, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23270804

RESUMO

Properly functioning adipose tissue is essential for normal insulin sensitivity of the body. When mice are kept on high-fat diet (HFD), adipose tissue expands, adipocytes increase in size and number, and the mice become obese. Many of these changes are mediated by the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ), the activity of which is regulated by multiple posttranslational modifications, including SUMOylation. To address the role of small ubiquitin-like modifier-1 (SUMO-1) in PPARγ function in vivo, particularly in fat cell biology, we subjected Sumo1-knockout mice to HFD. Sumo1-null mice gained less weight and had smaller and fewer adipocytes in their gonadal fat tissue on HFD, but their glucose tolerance was similar to that of wild-type littermates. Adipogenesis was impaired in Sumo1-null cells, and expression of PPARγ target genes was attenuated. In addition, both Sumo1-null cells and Sumo1-null mice responded less efficiently to rosiglitazone, a PPARγ agonist. These findings indicate that SUMO-1 is important also for transcriptional activation by the PPARγ signaling pathway and not only for trans-repressive functions of PPARγ as previously reported.


Assuntos
Adipogenia/fisiologia , Peso Corporal/genética , PPAR gama/fisiologia , Proteína SUMO-1/genética , Tiazolidinedionas/farmacologia , Células 3T3-L1 , Tecido Adiposo Branco/efeitos dos fármacos , Animais , Dieta Hiperlipídica , Feminino , Masculino , Camundongos , Camundongos Knockout , PPAR gama/agonistas , Rosiglitazona , Proteína SUMO-1/fisiologia
18.
Biochem Biophys Res Commun ; 426(3): 310-6, 2012 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-22943854

RESUMO

SUMO conjugation is a reversible posttranslational modification that regulates protein function. SENP1 is one of the six SUMO-specific proteases present in vertebrate cells and its altered expression is observed in several carcinomas. To characterize SENP1 role in genome integrity, we generated Senp1 knockout chicken DT40 cells. SENP1(-/-) cells show normal proliferation, but are sensitive to spindle poisons. This hypersensitivity correlates with increased sister chromatid separation, mitotic slippage, and apoptosis. To test whether the cohesion defect had a causal relationship with the observed mitotic events, we restored the cohesive status of sister chromatids by introducing the TOP2α(+/-) mutation, which leads to increased catenation, or by inhibiting Plk1 and Aurora B kinases that promote cohesin release from chromosomes during prolonged mitotic arrest. Although TOP2α is SUMOylated during mitosis, the TOP2α(+/-) mutation had no obvious effect. By contrast, inhibition of Plk1 or Aurora B rescued the hypersensitivity of SENP1(-/-) cells to colcemid. In conclusion, we identify SENP1 as a novel factor required for mitotic arrest and cohesion maintenance during prolonged mitotic arrest induced by spindle poisons.


Assuntos
Endopeptidases/fisiologia , Instabilidade Genômica , Mitose/fisiologia , Proteína SUMO-1/fisiologia , Fuso Acromático/fisiologia , Animais , Antígenos de Neoplasias/genética , Aurora Quinases , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Galinhas , Proteínas Cromossômicas não Histona/deficiência , Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/genética , Demecolcina/farmacologia , Endopeptidases/genética , Mitose/efeitos dos fármacos , Nocodazol/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Proteína SUMO-1/genética , Fuso Acromático/efeitos dos fármacos , Moduladores de Tubulina/farmacologia , Vimblastina/análogos & derivados , Vimblastina/farmacologia , Vinorelbina
19.
Can J Physiol Pharmacol ; 90(6): 765-70, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22486776

RESUMO

The endocrine pancreas is critically important in the regulation of energy metabolism, with defective insulin secretion from pancreatic islet ß-cells a major contributing factor to the development of type 2 diabetes. Small ubiquitin-like modifier (SUMO) proteins have been demonstrated to covalently modify a wide range of target proteins, mediating a broad range of cellular processes. While the effects of SUMOylation on ß-cell gene transcription have been previously reviewed, recent reports indicate roles for SUMO outside of the nucleus. In this review we shall focus on the reported non-nuclear roles of SUMOylation in the regulation of ß-cells, including SUMOylation as a novel signaling pathway in the acute regulation of insulin secretion.


Assuntos
Núcleo Celular/fisiologia , Células Secretoras de Insulina/fisiologia , Proteína SUMO-1/fisiologia , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Humanos , Insulina/genética , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Transdução de Sinais
20.
Mol Cell ; 46(3): 287-98, 2012 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-22464730

RESUMO

RanBP2/Nup358 is an essential protein with roles in nuclear transport and mitosis, and is one of the few known SUMO E3 ligases. However, why RanBP2 functions in vivo has been unclear: throughout the cell cycle it stably interacts with RanGAP1*SUMO1 and Ubc9, whose binding sites overlap with the E3 ligase region. Here we show that cellular RanBP2 is quantitatively associated with RanGAP1, indicating that complexed rather than free RanBP2 is the relevant E3 ligase. Biochemical reconstitution of the RanBP2/RanGAP1*SUMO1/Ubc9 complex enabled us to characterize its activity on the endogenous substrate Borealin. We find that the complex is a composite E3 ligase rather than an E2-E3 complex, and demonstrate that complex formation induces activation of a catalytic site that shows no activity in free RanBP2. Our findings provide insights into the mechanism of an important E3 ligase, and extend the concept of multisubunit E3 ligases from ubiquitin to the SUMO field.


Assuntos
Proteínas Ativadoras de GTPase/fisiologia , Chaperonas Moleculares/fisiologia , Complexo de Proteínas Formadoras de Poros Nucleares/fisiologia , Proteína SUMO-1/fisiologia , Enzimas de Conjugação de Ubiquitina/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Proteínas de Ciclo Celular/metabolismo , Cristalografia por Raios X , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Repressoras/fisiologia , Proteína SUMO-1/química , Proteína SUMO-1/metabolismo , Sumoilação , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/fisiologia
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