RESUMO
Lipid metabolism is important for the maintenance of physiological homeostasis. Several members of the small ubiquitin-like modifier (SUMO)-specific protease (SENP) family have been reported as the regulators of lipid homeostasis. However, the function of Senp7 in lipid metabolism remains unclear. In this study, we generated both conventional and adipocyte-specific Senp7 KO mice to characterize the role of Senp7 in lipid metabolism homeostasis. Both Senp7-deficient mice displayed reduced white adipose tissue mass and decreased size of adipocytes. By analyzing the lipid droplet morphology, we demonstrated that the lipid droplet size was significantly smaller in Senp7-deficient adipocytes. Mechanistically, Senp7 could deSUMOylate the perilipin family protein Plin4 to promote the lipid droplet localization of Plin4. Our results reveal an important role of Senp7 in the maturation of lipid droplets via Plin4 deSUMOylation.
Assuntos
Tecido Adiposo Branco , Gotículas Lipídicas , Camundongos Knockout , Perilipina-4 , Animais , Camundongos , Adipócitos/metabolismo , Tecido Adiposo Branco/metabolismo , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases/genética , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Perilipina-4/metabolismo , Perilipina-4/genética , SumoilaçãoRESUMO
Hepatocellular carcinoma (HCC) is associated with high metastatic potential and high mortality. Accumulating evidence has demonstrated that speckle-type POZ protein (SPOP) is a key adaptor molecule of ubiquitination. However, the molecular mechanism of SPOP-mediated ubiquitination in HCC metastasis remains obscure. In the present study, our results indicated that SPOP expression was significantly downregulated in HCC and was associated with tumor size, differentiation and metastasis. Cox regression model showed that low SPOP expression was a risk factor related to the prognosis of HCC patients. Loss- and gain-of-function assays demonstrated that SPOP inhibited HCC cell migration and invasion in vitro. Mechanisitically, co-immunoprecipitation and ubiquitination assays revealed that SPOP recognized and bound SENP7 and promoted its degradation via ubiquitin-dependent proteolysis. Analysis of immunohistochemistry showed that vimentin expression was correlated negatively with SPOP and positively with SENP7. These results implied that increased degradation of SENP7 by overexpression of SPOP decreased vimentin levels, which in turn attenuated HCC cell metastasis. Moreover, in vivo assays showed that SPOP overexpression also significantly suppressed liver and lung metastases. In summary, SPOP inhibits HCC cell metastasis via ubiquitin-dependent SENP7 proteolysis and may thus serve as a new opinion for the prevention of HCC metastasis.
Assuntos
Carcinoma Hepatocelular/patologia , Endopeptidases/metabolismo , Neoplasias Hepáticas/patologia , Invasividade Neoplásica/patologia , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Proteína SUMO-1/metabolismo , Ubiquitina/metabolismo , Animais , Carcinoma Hepatocelular/metabolismo , Movimento Celular , Endopeptidases/análise , Feminino , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos Nus , Pessoa de Meia-Idade , Proteínas Nucleares/análise , Proteólise , Proteínas Repressoras/análise , Proteína SUMO-1/análise , Ubiquitina/análise , UbiquitinaçãoRESUMO
Covalent attachment of the Small ubiquitin-like modifier (Sumo) polypeptide to proteins regulates many processes in the eukaryotic cell. In the nervous system, Sumo has been associated with the synapsis and with neurodegenerative diseases. However, its involvement in regulating neuronal differentiation remains largely unknown. Here we show that net Sumo deconjugation is observed during neurogenesis and that Sumo overexpression impairs this process. In an attempt to shed light on the underlying mechanisms, we have analyzed the expression profile of genes coding for components of the sumoylation pathway following induction of neuronal differentiation. Interestingly, we observed strong upregulation of the Senp7 protease at both mRNA and protein levels under differentiation conditions. Sumo proteases, by removing Sumo from targets, are key regulators of sumoylation. Strikingly, loss-of-function analysis demonstrated that Senp7 is required for neuronal differentiation not only in a model cell line, but also in the developing neural tube. Finally, reporter-based analysis of the Senp7 promoter indicated that Senp7 was transiently activated at early stages of neuronal differentiation. Thus, the Sumo protease Senp7 adds to the list of factors involved in vertebrate neurogenesis.
Assuntos
Endopeptidases/metabolismo , Células-Tronco Neurais/enzimologia , Tubo Neural/enzimologia , Neurogênese , Animais , Linhagem Celular Tumoral , Embrião de Galinha , Endopeptidases/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Camundongos , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Regiões Promotoras Genéticas , Interferência de RNA , RNA Mensageiro/metabolismo , Transdução de Sinais , Sumoilação , Fatores de Tempo , Ativação Transcricional , Transfecção , Tretinoína/farmacologiaRESUMO
BACKGROUND: Dysregulation of enhancer transcription occurs in multiple cancers. Enhancer RNAs (eRNAs) are transcribed products from enhancers that play critical roles in transcriptional control. Characterizing the genetic basis of eRNA expression may elucidate the molecular mechanisms underlying cancers. METHODS: Initially, a comprehensive analysis of eRNA quantitative trait loci (eRNAQTLs) was performed in The Cancer Genome Atlas (TCGA), and functional features were characterized using multi-omics data. To establish the first eRNAQTL profiles for colorectal cancer (CRC) in China, epigenomic data were used to define active enhancers, which were subsequently integrated with transcription and genotyping data from 154 paired CRC samples. Finally, large-scale case-control studies (34,585 cases and 69,544 controls) were conducted along with multipronged experiments to investigate the potential mechanisms by which candidate eRNAQTLs affect CRC risk. RESULTS: A total of 300,112 eRNAQTLs were identified across 30 different cancer types, which exert their influence on eRNA transcription by modulating chromatin status, binding affinity to transcription factors and RNA-binding proteins. These eRNAQTLs were found to be significantly enriched in cancer risk loci, explaining a substantial proportion of cancer heritability. Additionally, tumor-specific eRNAQTLs exhibited high responsiveness to the development of cancer. Moreover, the target genes of these eRNAs were associated with dysregulated signaling pathways and immune cell infiltration in cancer, highlighting their potential as therapeutic targets. Furthermore, multiple ethnic population studies have confirmed that an eRNAQTL rs3094296-T variant decreases the risk of CRC in populations from China (OR = 0.91, 95%CI 0.88-0.95, P = 2.92 × 10-7) and Europe (OR = 0.92, 95%CI 0.88-0.95, P = 4.61 × 10-6). Mechanistically, rs3094296 had an allele-specific effect on the transcription of the eRNA ENSR00000155786, which functioned as a transcriptional activator promoting the expression of its target gene SENP7. These two genes synergistically suppressed tumor cell proliferation. Our curated list of variants, genes, and drugs has been made available in CancereRNAQTL ( http://canernaqtl.whu.edu.cn/#/ ) to serve as an informative resource for advancing this field. CONCLUSION: Our findings underscore the significance of eRNAQTLs in transcriptional regulation and disease heritability, pinpointing the potential of eRNA-based therapeutic strategies in cancers.
Assuntos
Elementos Facilitadores Genéticos , Neoplasias , Locos de Características Quantitativas , Humanos , Elementos Facilitadores Genéticos/genética , Neoplasias/genética , Variação Genética/genética , Estudo de Associação Genômica Ampla/métodos , Neoplasias Colorretais/genética , Estudos de Casos e Controles , RNA/genética , China , RNAs IntensificadoresRESUMO
SUMO proteases or deSUMOylases regulate the lifetime of SUMO-conjugated targets in the cell by cleaving off the isopetidic bond between the substrate and the SUMO modifier, thus reversing the conjugation activity of the SUMO E3 ligases. In humans the deSUMOylating activity is mainly conducted by the SENP/ULP protease family, which is constituted of six members sharing a homologous catalytic globular domain. SENP6 and SENP7 are the most divergent members of the family and they show a unique SUMO2/3 isoform preference and a particular activity for dismantling polySUMO2 chains. Here, we present the crystal structure of the catalytic domain of human SENP7 bound to SUMO2, revealing structural key elements for the SUMO2 isoform specificity of SENP7. In particular, we describe the specific contacts between SUMO2 and a unique insertion in SENP7 (named Loop1) that is responsible for the SUMO2 isoform specificity. All the other interface contacts between SENP7 and SUMO2, including the SUMO2 C-terminal tail interaction, are conserved among members of the SENP/ULP family. Our data give insight into an evolutionary adaptation to restrict the deSUMOylating activity in SENP6 and SENP7 for the SUMO2/3 isoforms.
Assuntos
Endopeptidases , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina , Sumoilação , Humanos , Cisteína Endopeptidases/química , Endopeptidases/química , Isoformas de Proteínas/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Especificidade por SubstratoRESUMO
The heterochromatin protein 1 (HP1) family is involved in various functions with maintenance of chromatin structure. During murine somatic cell reprogramming, we find that early depletion of HP1γ reduces the generation of induced pluripotent stem cells, while late depletion enhances the process, with a concomitant change from a centromeric to nucleoplasmic localization and elongation-associated histone H3.3 enrichment. Depletion of heterochromatin anchoring protein SENP7 increased reprogramming efficiency to a similar extent as HP1γ, indicating the importance of HP1γ release from chromatin for pluripotency acquisition. HP1γ interacted with OCT4 and DPPA4 in HP1α and HP1ß knockouts and in H3K9 methylation depleted H3K9M embryonic stem cell (ESC) lines. HP1α and HP1γ complexes in ESCs differed in association with histones, the histone chaperone CAF1 complex, and specific components of chromatin-modifying complexes such as DPY30, implying distinct functional contributions. Taken together, our results reveal the complex contribution of the HP1 proteins to pluripotency.
Assuntos
Reprogramação Celular/genética , Cromatina/genética , Células-Tronco Pluripotentes Induzidas/química , Complexos Multiproteicos/genética , Animais , Cromatina/química , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Endopeptidases/química , Endopeptidases/genética , Exorribonucleases , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Camundongos Knockout , Complexos Multiproteicos/química , Proteínas Nucleares/genética , Fator 3 de Transcrição de Octâmero/química , Fator 3 de Transcrição de Octâmero/genética , Proteínas/química , Proteínas/genética , Proteínas Repressoras , Ribonucleases , Fatores de TranscriçãoRESUMO
SUMO posttranslational modification directs gene transcription and epigenetic programming to support normal cell function. The dynamic nature of SUMO-modification makes it difficult to identify endogenous protein substrates. Isolation of chromatin-bound SUMO targets is exceptionally challenging, as conventional immunoprecipitation assays are inefficient at concentrating this protein population. This chapter describes a protocol that effectively precipitates chromatin-associated fractions of SUMOylated heterochromatin protein 1α in cultured cells. Techniques to enrich endogenous SUMO substrates at the chromatin are also demonstrated and discussed. This approach could be adapted to evaluate chromatin-bound SUMO targets in additional in vivo systems.
Assuntos
Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Endopeptidases/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Ubiquitinas/metabolismo , Cromatina/química , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Endopeptidases/genética , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Células MCF-7 , Ligação Proteica , Mapeamento de Interação de Proteínas , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sumoilação , Ubiquitinas/genéticaRESUMO
AIM: To construct and identify the mammary gland cell-specific conditional knockout of SENP7 by the Cre-loxP system. METHODS: The homozygous SENP7
RESUMO
Epigenetic reprogramming allows cancer cells to bypass normal checkpoints and potentiate aberrant proliferation. Several chromatin regulators are subject to reversible SUMO-modification but little is known about how SUMOylation of chromatin-remodelers modulates the cancer epigenome. Recently, we demonstrated that SUMO-protease SENP7L is upregulated in aggressive BCa and maintains hypoSUMOylated heterochromatin protein 1-α (HP1α). Canonical models define HP1α as a "reader" of repressive H3K9m3 marks that supports constitutive heterochromatin. It is unclear how SUMOylation affects HP1α function in BCa cells. This report shows HP1α SUMO-dynamics are closely regulated in a complex with SENP7L and SUMO-E3 Polycomb-2 (PC2/CBX4). This complex accumulates at H3K9m3 sites, hypoSUMOylates HP1α and PC2, and reduces PC2's SUMO-E3 activity. HyperSUMO conditions cause complex dissociation, SUMOylation of PC2 and HP1α, and recruitment of SUMOylated HP1α to multiple DNA-repair genes including Rad51C. SUMOylated HP1α's enrichment at euchromatin requires chromatin-bound non-coding RNA (ncRNA), reduces Rad51C protein, and increases DNA-breaks in BCa cells. Hence, HP1α SUMOylation and consistently low SENP7L increase efficacy of DNA-damaging chemotherapeutic agents. BCa patients on chemotherapy that express low SENP7L exhibit greater survival rates than patients with high SENP7L. Collectively, these studies suggest that SUMOylated HP1α is a critical epigenetic-regulator of DNA-repair in BCa that could define chemotherapy responsiveness.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Proteínas Cromossômicas não Histona/metabolismo , RNA não Traduzido/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Dano ao DNA , Reparo do DNA , Endopeptidases/genética , Endopeptidases/metabolismo , Células HEK293 , Humanos , Estimativa de Kaplan-Meier , Ligases/genética , Ligases/metabolismo , Células MCF-7 , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Interferência de RNA , RNA não Traduzido/genética , SumoilaçãoRESUMO
c-Myc is the most frequently overexpressed oncogene in tumors, including breast cancer, colon cancer and lung cancer. Post-translational modifications comprising phosphorylation, acetylation and ubiquitylation regulate the activity of c-Myc. Recently, it was shown that c-Myc-driven tumors are strongly dependent on the SUMO pathway. Currently, the relevant SUMO target proteins in this pathway are unknown. Here we show that c-Myc is a target protein for SUMOylation, and that SUMOylated c-Myc is subsequently ubiquitylated and degraded by the proteasome. SUMO chains appeared to be dispensable for this process, polymerization-deficient SUMO mutants supported proteolysis of SUMOylated c-Myc. These results indicate that multiple SUMO monomers conjugated to c-Myc could be sufficient to direct SUMOylated c-Myc to the ubiquitin-proteasome pathway. Knocking down the SUMO-targeted ubiquitin ligase RNF4 enhanced the levels of SUMOylated c-Myc, indicating that RNF4 could recognize a multi-SUMOylated protein as a substrate in addition to poly-SUMOylated proteins. Knocking down the SUMO E3 ligase PIAS1 resulted in reduced c-Myc SUMOylation and increased c-Myc transcriptional activity, indicating that PIAS1 mediates c-Myc SUMOylation. Increased SUMOylation of c-Myc was noted upon knockdown of the SUMO protease SENP7, indicating that it also could regulate a multi-SUMOylated protein in addition to poly-SUMOylated proteins. C-Myc lacks KxE-type SUMOylation consensus motifs. We used mass spectrometry to identify 10 SUMO acceptor lysines: K52, K148, K157, K317, K323, K326, K389, K392, K398 and K430. Intriguingly, mutating all 10 SUMO acceptor lysines did not reduce c-Myc SUMOylation, suggesting that SUMO acceptor lysines in c-Myc act promiscuously. Our results provide novel insight into the complexity of c-Myc post-translational regulation.
Assuntos
Endopeptidases/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas Nucleares/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Células HeLa , Humanos , Lisina/química , Espectrometria de Massas , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Transporte Proteico/genética , Proteólise , Sumoilação , UbiquitinaçãoRESUMO
The SENP proteases regulate the SUMO conjugates in the cell by cleaving SUMO from target proteins. SENP6 and SENP7 are the most divergent members of the SENP/ULP protease family in humans by the presence of insertions in their catalytic domains. Loop1 insertion is determinant for the SUMO2/3 activity and specificity on SENP6 and SENP7. To gain structural insights into the role of Loop1, we have designed a chimeric SENP2 with the insertion of Loop1 into its sequence. The structure of SENP2-Loop1 in complex with SUMO2 was solved at 2.15 Å resolution, and reveals the details of an interface exclusive to SENP6/7 and the formation of unique contacts between both proteins. Interestingly, functional data with SUMO substrates showed an increase of the proteolytic activity in the SENP2-Loop1 chimera for diSUMO2 and polySUMO2 substrates.