RESUMO
The RNA exosome is an essential 3' to 5' exoribonuclease complex that mediates degradation, processing and quality control of virtually all eukaryotic RNAs. The nucleolar RNA exosome, consisting of a nine-subunit core and a distributive 3' to 5' exonuclease EXOSC10, plays a critical role in processing and degrading nucleolar RNAs, including pre-rRNA. However, how the RNA exosome is regulated in the nucleolus is poorly understood. Here, we report that the nucleolar ubiquitin-specific protease USP36 is a novel regulator of the nucleolar RNA exosome. USP36 binds to the RNA exosome through direct interaction with EXOSC10 in the nucleolus. Interestingly, USP36 does not significantly regulate the levels of EXOSC10 and other tested exosome subunits. Instead, it mediates EXOSC10 SUMOylation at lysine (K) 583. Mutating K583 impaired the binding of EXOSC10 to pre-rRNAs, and the K583R mutant failed to rescue the defects in rRNA processing and cell growth inhibition caused by knockdown of endogenous EXOSC10. Furthermore, EXOSC10 SUMOylation is markedly reduced in cells in response to perturbation of ribosomal biogenesis. Together, these results suggest that USP36 acts as a SUMO ligase to promote EXOSC10 SUMOylation critical for the RNA exosome function in ribosome biogenesis.
Assuntos
Exorribonucleases , Complexo Multienzimático de Ribonucleases do Exossomo , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , RNA/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Humanos , Linhagem CelularRESUMO
SUMOylation plays a crucial role in regulating diverse cellular processes including ribosome biogenesis. Proteomic analyses and experimental evidence showed that a number of nucleolar proteins involved in ribosome biogenesis are modified by SUMO. However, how these proteins are SUMOylated in cells is less understood. Here, we report that USP36, a nucleolar deubiquitinating enzyme (DUB), promotes nucleolar SUMOylation. Overexpression of USP36 enhances nucleolar SUMOylation, whereas its knockdown or genetic deletion reduces the levels of SUMOylation. USP36 interacts with SUMO2 and Ubc9 and directly mediates SUMOylation in cells and in vitro. We show that USP36 promotes the SUMOylation of the small nucleolar ribonucleoprotein (snoRNP) components Nop58 and Nhp2 in cells and in vitro and their binding to snoRNAs. It also promotes the SUMOylation of snoRNP components Nop56 and DKC1. Functionally, we show that knockdown of USP36 markedly impairs rRNA processing and translation. Thus, USP36 promotes snoRNP group SUMOylation and is critical for ribosome biogenesis and protein translation.
Assuntos
Ribonucleoproteínas Nucleolares Pequenas , Sumoilação , Proteínas de Ciclo Celular/metabolismo , Enzimas Desubiquitinantes/genética , Células HeLa , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteômica , Ribonucleoproteínas Nucleolares Pequenas/genética , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Ubiquitina Tiolesterase/genéticaRESUMO
The stability and activity of the p53 tumor suppressor protein are tightly regulated by various posttranslational modifications, including SUMOylation. p53 can be modified by both SUMO1 and SUMO2, although how SUMOylation regulates p53 activity is still obscure. Whether p53 activity is directly regulated by deSUMOylation is also unclear. Here, we show that SENP1, a SUMO-specific protease implicated in pro-oncogenic roles, is a p53 deSUMOylating enzyme. SENP1 interacts with p53 and deSUMOylates p53 in cells and in vitro. Knockdown of SENP1 markedly induced p53 transactivation activity. We further show that SENP1 depletion synergizes with DNA damage-inducing agent etoposide to induce p53 activation and the expression of p21, leading to synergistic growth inhibition of cancer cells. Our results reveal that SENP1 is a critical p53 deSUMOylating enzyme and a promising therapeutic target in wild-type p53 containing cancer cells.
Assuntos
Cisteína Endopeptidases/metabolismo , Proteína SUMO-1/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Cisteína Endopeptidases/genética , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Etoposídeo/farmacologia , Humanos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/genética , Proteína SUMO-1/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
OBJECTIVE: To assess the benefits of coronary collateral circulation on myocardial perfusion, viability and function in patients with total occlusion of a single coronary artery using the 99mTc-sestamibi SPECT and 18F-fluorodeoxyglucose PET. METHODS: 164 Consecutive patients were included who underwent coronary angiography results exhibited total occlusion of a single coronary artery and received 99mTc-MIBI SPECT and 18F-FDG PET within 90 days of angiography. Myocardial perfusion and viability in patients with collateral circulation and those without it were compared. Long-term follow-up was performed through a review of patient clinical records. RESULTS: Collateral circulation was present in 56 patients (34%) and absent in 108 patients (66%). The total perfusion defect size in patients with collateral circulation decreased when compared to those without (30% ± 13% to 35% ± 14%, P < .05). The myocardial viability was 22% ± 12% in patients with collateral circulation, and 12% ± 9% in those without (P < .001). The left ventricular ejection fraction was higher, and the end-diastolic and end-systolic left ventricular volumes were lower in patients with collateral circulation (39% ± 11%, 138 ± 66, 89 ± 57) compared to patients without collateral circulation (31% ± 9%, 177 ± 55, 125 ± 48, all P < .001, respectively). Multi-factor logistic regression identified that concerning the variables of sex, age, viable myocardium, collateral circulation, treatment type and others, only treatment type was significantly associated with therapeutic effects (OR 3.872, 95% CI 1.915-7.830, P < .001). CONCLUSION: Collateral circulation can preserve resting myocardial blood perfusion and myocardial viability, and help maintain the function of the left ventricular myocardium. The appropriate treatment strategy will have a substantial impact on the therapeutic outcome.
Assuntos
Circulação Colateral , Circulação Coronária , Oclusão Coronária/fisiopatologia , Fluordesoxiglucose F18 , Compostos Radiofarmacêuticos , Tecnécio Tc 99m Sestamibi , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Idoso , Feminino , Coração/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Sobrevivência de TecidosRESUMO
BACKGROUND: A low appropriate therapy rate indicates that a minority of patients will benefit from their implantable cardioverter defibrillator (ICD). Quantitative measurements from 18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET) may predict ventricular arrhythmia (VA) occurrence after ICD placement. METHODS: We performed a prospective observational study and recruited patients who required ICD placement. Pre-procedure image scans were performed. Patients were followed up for VA occurrence. Associations between image results and VA were analyzed. RESULTS: In 51 patients (33 males, 53.9 ± 17.2 years) analyzed, 17 (33.3%) developed VA. Compared with patients without VA, patients with VA had significantly larger values in scar area (17.7 ± 12.4% vs. 7.0 ± 7.9%), phase standard deviation (51.4° ± 14.0° vs. 34.0° ± 15.0°), bandwidth (172.9° ± 39.8° vs. 128.7° ± 49.9°), sum thickening score (STS, 29.5 ± 11.1 vs. 17.8 ± 13.2), and sum motion score (42.9 ± 11.5 vs. 33.0 ± 19.0). Cox regression analysis and receiver operating characteristic curve analysis showed that scar size, dyssynchrony, and STS were associated with VA occurrence (HR, 4.956, 95% CI 1.70-14.46). CONCLUSION: Larger left ventricular scar burden, increased dyssynchrony, and higher STS quantified by 18F-FDG PET may indicate a higher VA incidence after ICD placement.
Assuntos
Técnicas de Imagem Cardíaca/métodos , Desfibriladores Implantáveis/efeitos adversos , Fluordesoxiglucose F18 , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos , Taquicardia Ventricular/etiologia , Fibrilação Ventricular/etiologia , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Modelos de Riscos Proporcionais , Estudos Prospectivos , Reprodutibilidade dos Testes , Tomografia Computadorizada de Emissão de Fóton Único , Função Ventricular EsquerdaRESUMO
BACKGROUND: This study aimed to compare the accuracy of gated-SPECT (GSPECT) and gated-PET (GPET) in the assessment of left ventricular (LV) end-diastolic volumes (EDVs), end-systolic volumes (ESVs) and LV ejection fractions (LVEFs) among patients with prior myocardial infarction (MI). METHODS: One hundred and sixty-eight consecutive patients with MI who underwent GSPECT and GPET were included. Of them, 76 patients underwent CMR in addition to the two imaging modalities. The measurements of LV volumes and LVEF were performed using Quantitative Gated SPECT (QGS), Emory Cardiac Toolbox (ECTB), and 4D-MSPECT (4DM). RESULTS: The correlation between GPET, GSPECT, and CMR were excellent for LV EDV (r = 0.855 to 0.914), ESV (r = 0.852 to 0.949), and LVEF (r = 0.618 to 0.820), as calculated from QGS, ECTB, and 4DM. In addition, subgroup analysis revealed that EDV, ESV, and LVEF measured by GPET were accurate in patients with different extents of total perfusion defect (TPD), viable myocardium, and perfusion/metabolic mismatch. Furthermore, multivariate regression analysis identified that mismatch score was associated with the difference in EDV (P < 0.05) measurements between GPET and CMR. CONCLUSIONS: In patients with MI, LV volumes and LVEF scores measured by both GSPECT and GPET imaging were comparable to those determined by CMR, but should not be interchangeable in individual patients.
Assuntos
Fluordesoxiglucose F18 , Imagem do Acúmulo Cardíaco de Comporta/métodos , Infarto do Miocárdio/fisiopatologia , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos , Tecnécio Tc 99m Sestamibi , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Função Ventricular Esquerda , Idoso , Volume Cardíaco/fisiologia , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/diagnóstico por imagem , Volume Sistólico/fisiologiaRESUMO
Posttranslational modifications play a crucial role in the proper control of c-Myc protein stability and activity. c-Myc can be modified by small ubiquitin-like modifier (SUMO). However, how SUMOylation regulates c-Myc stability and activity remains to be elucidated. The deSUMOylation enzyme, SENP1, has recently been shown to have a prooncogenic role in cancer; however, mechanistic understanding of this is limited. Here we show that SENP1 is a c-Myc deSUMOylating enzyme. SENP1 interacts with and deSUMOylates c-Myc in cells and in vitro. Overexpression of wild-type SENP1, but not its catalytically inactive C603S mutant, markedly stabilizes c-Myc and increases its levels and activity. Knockdown of SENP1 reduces c-Myc levels, induces cell cycle arrest, and drastically suppresses cell proliferation. We further show that c-Myc can be comodified by both ubiquitination and SUMOylation. SENP1-mediated deSUMOylation reduces c-Myc polyubiquitination, suggesting that SUMOylation promotes c-Myc degradation through the proteasome system. Interestingly, SENP1-mediated deSUMOylation promotes the accumulation of monoubiquitinated c-Myc and its phosphorylation at serine 62 and threonine 58. SENP1 is frequently overexpressed, correlating with the high expression of c-Myc, in breast cancer tissues. Together, these results reveal that SENP1 is a crucial c-Myc deSUMOylating enzyme that positively regulates c-Myc's stability and activity.
Assuntos
Cisteína Endopeptidases/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína SUMO-1/metabolismo , Neoplasias da Mama/metabolismo , Pontos de Checagem do Ciclo Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Feminino , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Sumoilação/fisiologia , Ubiquitinação/fisiologiaRESUMO
FOSL1 is frequently overexpressed in multiple types of human cancers including invasive breast cancers and implicated in cancer invasion and metastasis. However, how FOSL1 is overexpressed in cancers remains to be elucidated. Several microRNAs (miRNAs) have been shown to target FOSL1 and are downregulated in human cancers. Here, we report that miR-130a is a novel FOSL1 targeting miRNA. Using gene expression microarray analysis, we found that FOSL1 is among the most up-regulated genes in cells transfected with miR-130a inhibitors. Transient transfection-immunoblot, RNA-immunoprecipitation, and luciferase reporter assays revealed that miR-130a directly targets FOSL1 mRNA at its 3'-UTR. Overexpression of miR-130a significantly reduced the levels of FOSL1 in invasive breast cancer MDA-MB-231 and Hs578T cell lines and suppresses their migration and invasion. This inhibition can be rescued by ectopic expression of miR-130a-resistant FOSL1. Interestingly, we show that overexpression of miR-130a increased the levels of tight-junction protein ZO-1 while inhibition of miR-130a reduced the levels of ZO-1. We further show that miR-130a expression is significantly reduced in cancer tissues from triple-negative breast cancer (TNBC) patients, correlating significantly with the upregulation of FOSL1 expression, compared to non-TNBC tissues. Together, our results reveal that miR-130a directly targets FOSL1 and suppresses the inhibition of ZO-1, thus inhibiting cancer cell migration and invasion, in TNBCs.
Assuntos
Movimento Celular , Regulação Neoplásica da Expressão Gênica , MicroRNAs/biossíntese , Proteínas Proto-Oncogênicas c-fos/biossíntese , RNA Neoplásico/biossíntese , Neoplasias de Mama Triplo Negativas/metabolismo , Regulação para Cima , Proteína da Zônula de Oclusão-1/biossíntese , Linhagem Celular Tumoral , Feminino , Células HEK293 , Humanos , MicroRNAs/genética , Invasividade Neoplásica , Proteínas Proto-Oncogênicas c-fos/genética , RNA Neoplásico/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Proteína da Zônula de Oclusão-1/genéticaRESUMO
c-Myc protein stability and activity are tightly regulated by the ubiquitin-proteasome system. Aberrant stabilization of c-Myc contributes to many human cancers. c-Myc is ubiquitinated by SCF(Fbw7) (a SKP1-cullin-1-F-box complex that contains the F-box and WD repeat domain-containing 7, Fbw7, as the F-box protein) and several other ubiquitin ligases, whereas it is deubiquitinated and stabilized by ubiquitin-specific protease (USP) 28. The bulk of c-Myc degradation appears to occur in the nucleolus. However, whether c-Myc is regulated by deubiquitination in the nucleolus is not known. Here, we report that the nucleolar deubiquitinating enzyme USP36 is a novel c-Myc deubiquitinase. USP36 interacts with and deubiquitinates c-Myc in cells and in vitro, leading to the stabilization of c-Myc. This USP36 regulation of c-Myc occurs in the nucleolus. Interestingly, USP36 interacts with the nucleolar Fbw7γ but not the nucleoplasmic Fbw7α. However, it abolished c-Myc degradation mediated both by Fbw7γ and by Fbw7α. Consistently, knockdown of USP36 reduces the levels of c-Myc and suppresses cell proliferation. We further show that USP36 itself is a c-Myc target gene, suggesting that USP36 and c-Myc form a positive feedback regulatory loop. High expression levels of USP36 are found in a subset of human breast and lung cancers. Altogether, these results identified USP36 as a crucial and bono fide deubiquitinating enzyme controlling c-Myc's nucleolar degradation pathway.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ubiquitina Tiolesterase/metabolismo , Neoplasias da Mama/metabolismo , Catálise , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Proliferação de Células , Proteínas F-Box/metabolismo , Proteína 7 com Repetições F-Box-WD , Feminino , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Neoplasias Pulmonares/metabolismo , Microscopia de Fluorescência , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Our previous study showed that wedelolactone, a compound isolated from Ecliptae herba, has the potential to enhance osteoblastogenesis. However, the molecular mechanisms by which wedelolactone promoted osteoblastogenesis from bone marrow mesenchymal stem cells (BMSCs) remain largely unknown. In this study, treatment with wedelolactone (2 µg/mL) for 3, 6, and 9 days resulted in an increase in phosphorylation of extracellular signal-regulated kinases (ERKs), c-Jun N-terminal protein kinase (JNK), and p38. Phosphorylation of mitogen-activated protein kinases (MAPKs), ERK and JNK started to increase on day 3 of treatment, and p38 phosphorylation was increased by day 6 of treatment. Expression of bone morphogenetic protein (BMP2) mRNA and phosphorylation of Smad1/5/8 was enhanced after treatment of cells with wedelolactone for 6 and 9 days. The addition of the JNK inhibitor SP600125, ERK inhibitor PD98059, and p38 inhibitor SB203580 suppressed wedelolactone-induced alkaline-phosphatase activity, bone mineralization, and osteoblastogenesis-related marker genes including Runx2, Bglap, and Sp7. Increased expression of BMP2 mRNA and Smad1/5/8 phosphorylation was blocked by SP600125 and PD98059, but not by SB203580. These results suggested that wedelolactone enhanced osteoblastogenesis through induction of JNK- and ERK-mediated BMP2 expression and Smad1/5/8 phosphorylation.
Assuntos
Conservadores da Densidade Óssea/farmacologia , Células da Medula Óssea/efeitos dos fármacos , Cumarínicos/farmacologia , Eclipta/química , Regulação da Expressão Gênica/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Animais , Antracenos/farmacologia , Conservadores da Densidade Óssea/isolamento & purificação , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Diferenciação Celular/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Cumarínicos/isolamento & purificação , Flavonoides/farmacologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Imidazóis/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Osteoblastos/citologia , Osteoblastos/metabolismo , Extratos Vegetais/química , Cultura Primária de Células , Piridinas/farmacologia , Transdução de Sinais , Proteínas Smad/genética , Proteínas Smad/metabolismo , Fator de Transcrição Sp7/genética , Fator de Transcrição Sp7/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The ubiquitin (Ub)-proteasome system plays a pivotal role in the regulation of p53 protein stability and activity. p53 is ubiquitinated and destabilized by MDM2 and several other Ub E3s, whereas it is deubiquitinated and stabilized by Ub-specific protease (USP)7 and USP10. Here we show that the ovarian tumour domain-containing Ub aldehyde-binding protein 1 (Otub1) is a novel p53 regulator. Otub1 directly suppresses MDM2-mediated p53 ubiquitination in cells and in vitro. Overexpression of Otub1 drastically stabilizes and activates p53, leading to apoptosis and marked inhibition of cell proliferation in a p53-dependent manner. These effects are independent of its catalytic activity but require residue Asp88. Mutation of Asp88 to Ala (Otub1(D88A)) abolishes activity of Otub1 to suppress p53 ubiquitination. Further, wild-type Otub1 and its catalytic mutant (Otub1(C91S)), but not Otub1(D88A), bind to the MDM2 cognate E2, UbcH5, and suppress its Ub-conjugating activity in vitro. Overexpression of Otub1(D88A) or ablation of endogenous Otub1 by siRNA markedly impaired p53 stabilization and activation in response to DNA damage. Together, these results reveal a novel function for Otub1 in regulating p53 stability and activity.
Assuntos
Cisteína Endopeptidases/metabolismo , Transcrição Gênica/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Sequência de Bases , Biocatálise , Linhagem Celular Tumoral , Proliferação de Células , Cisteína Endopeptidases/genética , Dano ao DNA , Primers do DNA , Enzimas Desubiquitinantes , Técnicas de Silenciamento de Genes , Humanos , Ligação Proteica , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/fisiologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
UNLABELLED: Assembly-activating protein (AAP) of adeno-associated virus serotype 2 (AAV2) is a nucleolar-localizing protein that plays a critical role in transporting the viral capsid VP3 protein to the nucleolus for assembly. Here, we identify and characterize AAV2 AAP (AAP2) nuclear (NLS) and nucleolar (NoLS) localization signals near the carboxy-terminal region of AAP2 (amino acid positions 144 to 184) (AAP2(144-184)). This region contains five basic-amino-acid-rich (BR) clusters, KSKRSRR (AAP2BR1), RRR (AAP2BR2), RFR (AAP2BR3), RSTSSR (AAP2BR4), and RRIK (AAP2BR5), from the amino terminus to the carboxy terminus. We created 30 AAP2BR mutants by arginine/lysine-to-alanine mutagenesis or deletion of AAP2BRs and 8 and 1 green fluorescent protein (GFP)-AAP2BR and ß-galactosidase-AAP2BR fusion proteins, respectively, and analyzed their intracellular localization in HeLa cells by immunofluorescence microscopy. The results showed that AAP2(144-184) has redundant multipartite NLSs and that any combinations of 4 AAP2BRs, but not 3 or less, can constitute a functional NLS-NoLS; AAP2BR1 and AAP2BR2 play the most influential role for nuclear localization, but either one of the two AAP2BRs is dispensable if all 4 of the other AAP2BRs are present, resulting in 3 different, overlapping NLS motifs; and the NoLS is shared redundantly among the five AAP2BRs and functions in a context-dependent manner. AAP2BR mutations not only resulted in aberrant intracellular localization, but also attenuated AAP2 protein expression to various degrees, and both of these abnormalities have a significant negative impact on capsid production. Thus, this study reveals the organization of the intermingling NLSs and NoLSs in AAP2 and provides insights into their functional roles in capsid assembly. IMPORTANCE: Adeno-associated virus (AAV) has become a popular and successful vector for in vivo gene therapy; however, its biology has yet to be fully understood. In this regard, the recent discovery of the assembly-activating protein (AAP), a nonstructural, nucleolar-localizing AAV protein essential for viral capsid assembly, has provided us a new opportunity to better understand the fundamental processes required for virion formation. Here, we identify clusters of basic amino acids in the carboxy terminus of AAP from AAV serotype 2 (AAV2) that act as nuclear and nucleolar localization signals. We also demonstrate their importance in maintaining AAP expression levels and efficient production of viral capsids. Insights into the functions of AAP can elucidate the requirements and process for AAV capsid assembly, which may lead to improved vector production for use in gene therapy. This study also contributes to the growing body of work on nuclear and nucleolar localization signals.
Assuntos
Nucléolo Celular/virologia , Núcleo Celular/virologia , Dependovirus/fisiologia , Sinais de Localização Nuclear , Infecções por Parvoviridae/virologia , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Dependovirus/química , Dependovirus/genética , Humanos , Dados de Sequência Molecular , Transporte Proteico , Alinhamento de Sequência , Proteínas Virais/química , Proteínas Virais/genética , Montagem de VírusRESUMO
Ovarian tumor domain-containing ubiquitin (Ub) aldehyde binding protein 1 (Otub1) regulates p53 stability and activity via non-canonical inhibition of the MDM2 cognate Ub-conjugating enzyme (E2) UbcH5. However, it is not clear how this activity of Otub1 is regulated in cells. Here we report that Otub1 is monoubiquitinated by UbcH5 in cells and in vitro, primarily at the lysine 59 and 109 residues. This monoubiquitination, in turn, contributes to the activity of Otub1 to suppress UbcH5. The lysine-free Otub1 mutant (Otub1(K0)) fails to be monoubiquitinated and is unable to suppress the Ub-conjugating activity of UbcH5 in vitro and the MDM2-mediated p53 ubiquitination in cells. Consistently, this mutant is unable to stabilize p53, induce apoptosis, and suppress cell proliferation. Overexpression of Otub1(K0) inhibits DNA-damage induced apoptosis. Adding either Lys-59 or Lys-109 back to the Otub1(K0) mutant restores the monoubiquitination of Otub1 and its function to stabilize and activate p53. We further show that UbcH5 preferentially binds to the monoubiquitinated Otub1 via Ub interaction with its backside donor Ub-interacting surface, suggesting that this binding interferes with the self-assembly of Ub-charged UbcH5 (UbcH5â¼Ub) conjugates, which is critical for Ub transfer. Thus, our data reveal novel insights into the Otub1 inhibition of E2 wherein monoubiquitination promotes the interaction of Otub1 with UbcH5 and the function to suppress it.
Assuntos
Neoplasias Ovarianas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação , Sequência de Aminoácidos , Linhagem Celular Tumoral , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Dano ao DNA , Enzimas Desubiquitinantes , Feminino , Humanos , Lisina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-mdm2/metabolismoRESUMO
ABSTRACT: A 54-year-old woman with a history of end-stage renal disease was found to have infiltrative cardiomyopathy by echocardiography. 99mTc-pyrophosphate (99mTc-PYP) scintigraphy was positive with a remarkable myocardial uptake. Gene test found a mutation of AGXT, confirming a final diagnosis of primary hyperoxaluria. Radiotracer uptake was due to high myocardial oxalate deposition. This case illustrates false positivity of the 99mTc-PYP scan caused by hyperoxaluria-associated cardiomyopathy, which raises awareness for other conditions apart from amyloid cardiomyopathy.
RESUMO
Ribosome biogenesis is essential for cell growth, proliferation, and animal development. Its deregulation leads to various human disorders such as ribosomopathies and cancer. Thus, tight regulation of ribosome biogenesis is crucial for normal cell homeostasis. Emerging evidence suggests that posttranslational modifications such as ubiquitination and SUMOylation play a crucial role in regulating ribosome biogenesis. Our recent studies reveal that USP36, a nucleolar deubiquitinating enzyme (DUB), acts also as a SUMO ligase to regulate nucleolar protein group SUMOylation, thereby being essential for ribosome biogenesis. Here, we provide an overview of the current understanding of the SUMOylation regulation of ribosome biogenesis and discuss the role of USP36 in nucleolar SUMOylation.
RESUMO
In this study, we explored the changes in plant community diversity and their relationship with soil factors under shrub encroachment pressure by selecting four marsh areas in Sanjiang Plain with different degrees of shrub cover (a, 0≤a≤100%), including marsh with no shrub encroachment (a=0), light shrub encroachment (0
Assuntos
Biodiversidade
, Solo
, Áreas Alagadas
, China
, Solo/química
, Dinâmica Populacional
, Poaceae/crescimento & desenvolvimento
, Plantas/classificação
, Desenvolvimento Vegetal
RESUMO
Nucleostemin (NS) is a nucleolar GTP-binding protein essential for ribosomal biogenesis, proliferation, and animal embryogenesis. It remains largely unclear how this protein is regulated. While working on its role in suppression of MDM2 and activation of p53, we observed that NS protein (but not mRNA) levels decreased drastically in response to GTP depletion. When trying to further elucidate the molecular mechanism(s) underlying this unusual phenomenon, we found that NS was degraded independently of ubiquitin and MDM2 upon GTP depletion. First, depletion of GTP by treating cells with mycophenolic acid decreased the level of NS without apparently affecting the levels of other nucleolar proteins. Second, mutant NS defective in GTP binding and exported to the nucleoplasm was much less stable than wild-type NS. Although NS was ubiquitinated in cells, its polyubiquitination was independent of Lys-48 or Lys-63 in the ubiquitin molecule. Inactivation of E1 in E1 temperature-sensitive mouse embryonic fibroblast (MEF) cells failed to prevent the proteasomal degradation of NS. The proteasomal turnover of NS was also MDM2-independent, as its half-life in p53/MDM2 double knock-out MEF cells was the same as that in wild-type MEF cells. Moreover, NS ubiquitination was MDM2-independent. Mycophenolic acid or doxorubicin induced NS degradation in various human cancerous cells regardless of the status of MDM2. Hence, these results indicate that NS undergoes a ubiquitin- and MDM2-independent proteasomal degradation when intracellular GTP levels are markedly reduced and also suggest that ubiquitination of NS may be involved in regulation of its function rather than stability.
Assuntos
Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Proteínas de Transporte/genética , Linhagem Celular , Núcleo Celular/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Proteínas de Ligação ao GTP/genética , Guanosina Trifosfato/genética , Humanos , Camundongos , Proteínas Nucleares/genética , Complexo de Endopeptidases do Proteassoma/genética , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas de Ligação a RNA , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinação/fisiologiaRESUMO
The ARF tumor suppressor protein activates p53 in response to oncogenic stress, whereas ribosomal protein L11 induces p53 following ribosomal stress. Both proteins bind to central, albeit non-overlapping, regions of MDM2 and suppress MDM2 activity toward p53. However, it is not known whether the two pathways are functionally connected. Here we show that ARF directly binds to L11 in vitro and in cells, which then forms a complex with MDM2 and p53. L11 collaboratively enhances ARF-induced p53 transcriptional activity and cell cycle arrest. Supporting these results, knocking down L11 reduces ARF-mediated p53 accumulation and alleviates ARF-induced cell cycle arrest. Interestingly, overexpression of ARF increases the levels of ribosome-free L11 and enhances the interaction of L11 with MDM2 and p53. These results demonstrate that ARF activates p53, at least partly by induction of ribosomal stress, which results in L11 suppression of MDM2, and suggest that the ARF-MDM2-p53 and the L11-MDM2-p53 pathways are functionally connected.
Assuntos
Pontos de Checagem do Ciclo Celular/fisiologia , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Ribossômicas/biossíntese , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Inibidor p16 de Quinase Dependente de Ciclina/genética , Regulação da Expressão Gênica/fisiologia , Células HEK293 , Humanos , Ligação Proteica/fisiologia , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Ribossômicas/metabolismo , Transdução de Sinais/fisiologia , Estresse Fisiológico/fisiologia , Transcrição Gênica/fisiologia , Proteína Supressora de Tumor p53/genéticaRESUMO
OBJECTIVES: Gated single-photon emission computed tomography (SPECT) myocardial perfusion imaging is useful in assessing left ventricular (LV) myocardial perfusion and function. This study evaluated the LV functional changes after adenosine vasodilator stress, using gated SPECT. METHODS: The study population consisted of 70 patients who underwent adenosine-mediated stress and rest SPECT. All patients underwent coronary angiography. Semi-quantitative assessment of perfusion was analyzed and produced the summed rest score (SRS), the summed stress score (SSS) and the summed difference score (SDS). The global LV function parameters [ejection fraction (EF), end-diastolic volume (EDV), end-systolic volume (ESV)] and regional LV function [the summed motion score (SMS) and the summed thickening score (STS)] were quantified by gated SPECT. RESULTS: Patients were divided into 2 groups: group 1 comprised 16 patients with worsening of LVEF (LVEFrest-LVEFado ≥5%), and group 2 comprised the other 54 patients. Compared with group 2, patients in group 1 had a significantly higher SSS and SDS (9.1 ± 6.8 vs. 5.6 ± 4.5 and 6.6 ± 3.8 vs. 3.6 ± 4.0, respectively; p < 0.05) and the severity of coronary artery stenosis was more serious (p < 0.05). CONCLUSION: Worsening of LVEF after adenosine-induced vasodilator stress, as shown by (99m)Tc-MIBI gated SPECT, is a valuable nonperfusion marker of significant CAD.
Assuntos
Adenosina , Tomografia Computadorizada por Emissão de Fóton Único de Sincronização Cardíaca , Doença da Artéria Coronariana/fisiopatologia , Vasodilatadores , Função Ventricular Esquerda , Adenosina/farmacologia , Idoso , Angiografia Coronária , Doença da Artéria Coronariana/diagnóstico por imagem , Teste de Esforço , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Isquemia Miocárdica/etiologia , Volume Sistólico , Vasodilatadores/farmacologiaRESUMO
To understand the variations in greenhouse gas fluxes during the process of returning cropland to wetland in the Sanjiang Plain, we selected naturally restored wetlands of 4, 7, 11, 16 and 20 years as research objects to compare with a cultivated site (soybean plantation for 13 years) and an uncultivated marsh dominated by Deyeuxia purpurea and Carex schmidtii. We measured carbon dioxide (CO2) and methane (CH4) fluxes using a static chamber-gas chromatography and explored the main influencing factors. The results showed that there were seasonal variations in growing-season CO2 and CH4 fluxes of the restored wetlands, with the seasonal trends in greenhouse gases becoming gradually similar to that of natural marsh with increasing restoration time. The mean growing-season CO2 fluxes increased during the early stage of restoration, but then decreased during the late stage, which decreased from 893.4 mg·m-2·h-1 to 494.0 mg·m-2·h-1 in the 4-year and 20-year sites, respectively. Mean CH4 fluxes increased with restoration time, ranging from a weak CH4 sink (soybean fields, -0.6 mg·m-2·h-1) to a CH4 source of 87.8 mg·m-2·h-1(20-year restored site). The CH4 fluxes of experimental plots were consistently lower than that of natural marsh (96.4 mg·m-2·h-1). Increases in water level and soil conductivity resulting from restoration were the main driving factors for the decrease in CO2 fluxes. The increases in water level and soil dissolved organic carbon resulting from restoration were the primary drivers for the increase of CH4 fluxes in the restored wetlands. The global warming potentials increased with restoration time, ranging from 27.8 t·CO2-eq·hm-2(soybean fields) to 130.8 t·CO2-eq·hm-2(plot of 20-year restoration), which gradually approached that of natural marsh (156.3 t·CO2-eq·hm-2). The emission of GHGs from restored wetlands in the Sanjiang Plain gradually approached those of natural marsh. Further monitoring is required to identify the maturity of restored wetlands for achieving greenhouse gas emissions equivalent to that of natural marshland.