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1.
Cell Rep ; 36(13): 109754, 2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34547223

RESUMO

The SARS-CoV-2 papain-like protease (PLpro) is a target for antiviral drug development. It is essential for processing viral polyproteins for replication and functions in host immune evasion by cleaving ubiquitin (Ub) and ubiquitin-like protein (Ubl) conjugates. While highly conserved, SARS-CoV-2 and SARS-CoV PLpro have contrasting Ub/Ubl substrate preferences. Using a combination of structural analyses and functional assays, we identify a molecular sensor within the S1 Ub-binding site of PLpro that serves as a key determinant of substrate specificity. Variations within the S1 sensor specifically alter cleavage of Ub substrates but not of the Ubl interferon-stimulated gene 15 protein (ISG15). Significantly, a variant of concern associated with immune evasion carries a mutation in the S1 sensor that enhances PLpro activity on Ub substrates. Collectively, our data identify the S1 sensor region as a potential hotspot of variability that could alter host antiviral immune responses to newly emerging SARS-CoV-2 lineages.


Assuntos
Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Proteases Semelhantes à Papaína de Coronavírus/ultraestrutura , SARS-CoV-2/genética , Sequência de Aminoácidos/genética , Sítios de Ligação/genética , COVID-19/genética , COVID-19/metabolismo , Proteases Semelhantes à Papaína de Coronavírus/genética , Células HEK293 , Humanos , Papaína/química , Papaína/metabolismo , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Ligação Proteica/genética , SARS-CoV-2/metabolismo , Especificidade por Substrato/genética , Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Proteínas Virais/metabolismo
2.
Int J Mol Sci ; 22(17)2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-34502293

RESUMO

Members of the ubiquitin-like protein family are known for their ability to modify substrates by covalent conjugation. The highly conserved ubiquitin relative UBL5/Hub1, however, is atypical because it lacks a carboxy-terminal di-glycine motif required for conjugation, and the whole E1-E2-E3 enzyme cascade is likely absent. Though the conjugation-mediated role of UBL5/Hub1 is controversial, it undoubtedly functions by interacting non-covalently with its partners. Several interactors of UBL5/Hub1 identified to date have suggested broad stress-responsive functions of the protein, for example, stress-induced control of pre-mRNA splicing, Fanconi anemia pathway of DNA damage repair, and mitochondrial unfolded protein response. While having an atypical mode of function, UBL5/Hub1 is still a stress protein that regulates feedback to various stimuli in a similar manner to other ubiquitin-like proteins. In this review, I discuss recent progress in understanding the functions of UBL5/Hub1 and the fundamental questions which remain to be answered.


Assuntos
Proteína Semelhante a ELAV 2/metabolismo , Regulação da Expressão Gênica , Estresse Fisiológico , Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Proteína Semelhante a ELAV 2/genética , Humanos , Ubiquitinas/genética
3.
Nat Commun ; 12(1): 4794, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34373456

RESUMO

The cellular NLRP3 protein level is crucial for assembly and activation of the NLRP3 inflammasome. Various posttranslational modifications (PTMs), including phosphorylation and ubiquitination, control NLRP3 protein degradation and inflammasome activation; however, the function of small ubiquitin-like modifier (SUMO) modification (called SUMOylation) in controlling NLRP3 stability and subsequent inflammasome activation is unclear. Here, we show that the E3 SUMO ligase tripartite motif-containing protein 28 (TRIM28) is an enhancer of NLRP3 inflammasome activation by facilitating NLRP3 expression. TRIM28 binds NLRP3, promotes SUMO1, SUMO2 and SUMO3 modification of NLRP3, and thereby inhibits NLRP3 ubiquitination and proteasomal degradation. Concordantly, Trim28 deficiency attenuates NLRP3 inflammasome activation both in vitro and in vivo. These data identify a mechanism by which SUMOylation controls the cellular NLRP3 level and inflammasome activation, and reveal correlations and interactions of NLRP3 SUMOylation and ubiquitination during inflammasome activation.


Assuntos
Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Sumoilação/fisiologia , Proteína 28 com Motivo Tripartido/metabolismo , Animais , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Fosforilação , Processamento de Proteína Pós-Traducional , Proteólise , Proteína SUMO-1/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação/genética , Proteína 28 com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Ubiquitinas/metabolismo
4.
Cancer Sci ; 112(10): 4100-4111, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34339558

RESUMO

SHANK-associated RH domain interacting protein (SHARPIN) plays an important role in carcinogenesis, as well as inflammation and immunity. Our study explored the effects and underlying mechanisms of SHARPIN in clear cell renal cell carcinoma (ccRCC). By analyzing The Cancer Genome Atlas database, we found that upregulated SHARPIN in patients with ccRCC led to a poor prognosis. Semiquantitative immunohistochemical analysis of clinical samples was carried out and the results suggested the positive association between SHARPIN and hypoxia-induced factor-2α (HIF-2α). Von Hippel-Lindau protein (pVHL) is a tumor suppressor that contributes to degrading HIF-2α. Mechanically, SHARPIN promoted the ubiquitination and proteasomal degradation of pVHL, resulting in the sustained activation of HIF-2α. The α and ß domains of pVHL and ubiquitin-like domain of SHARPIN are required for the interaction. The knockdown of SHARPIN effectively inhibited acquired sorafenib resistance in ccRCC cell lines and tumor growth in xenograft models. In conclusion, our work reveals a novel posttranslational regulation of SHARPIN on pVHL, indicating that SHARPIN could be a potential target for ccRCC treatment.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinoma de Células Renais/etiologia , Neoplasias Renais/etiologia , Ubiquitinas/fisiologia , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Inativação Gênica , Xenoenxertos , Humanos , Estimativa de Kaplan-Meier , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/patologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Prognóstico , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional , Proteólise , RNA Interferente Pequeno , Distribuição Aleatória , Sorafenibe/farmacologia , Ubiquitinação , Ubiquitinas/genética , Ubiquitinas/metabolismo , Regulação para Cima
5.
Immunol Lett ; 237: 33-41, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34228987

RESUMO

OBJECTIVE: In this study, we focused on the interaction between SARS-CoV-2 and host Type I Interferon (IFN) response, so as to identify whether IFN effects could be influenced by the products of SARS-CoV-2. METHODS: All the structural and non-structural proteins of SARS-CoV-2 were transfected and overexpressed in the bronchial epithelial cell line BEAS-2B respectively, and typical antiviral IFN-stimulated gene (ISG) ISG15 expression was detected by qRT-PCR. RNA-seq based transcriptome analysis was performed between control and Spike (S) protein-overexpressed BEAS-2B cells. The expression of ACE2 and IFN effector JAK-STAT signaling activation were detected in control and S protein-overexpressed BEAS-2B cells by qRT-PCR or/and Western blot respectively. The interaction between S protein with STAT1 and STAT2, and the association between JAK1 with downstream STAT1 and STAT2 were measured in BEAS-2B cells by co-immunoprecipitation (co-IP). RESULTS: S protein could activate IFN effects and downstream ISGs expression. By transcriptome analysis, overexpression of S protein induced a set of genes expression, including series of ISGs and the SARS-CoV-2 receptor ACE2. Mechanistically, S protein enhanced the association between the upstream JAK1 and downstream STAT1 and STAT2, so as to promote STAT1 and STAT2 phosphorylation and ACE2 expression. CONCLUSION: SARS-CoV-2 S protein enhances ACE2 expression via facilitating IFN effects, which may help its infection.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Brônquios/efeitos dos fármacos , COVID-19/virologia , Células Epiteliais/efeitos dos fármacos , Interferon alfa-2/farmacologia , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/metabolismo , Enzima de Conversão de Angiotensina 2/genética , Brônquios/enzimologia , Brônquios/virologia , COVID-19/enzimologia , Citocinas/genética , Citocinas/metabolismo , Células Epiteliais/enzimologia , Células Epiteliais/virologia , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Janus Quinase 1/metabolismo , Fosforilação , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT2/metabolismo , Transdução de Sinais , Glicoproteína da Espícula de Coronavírus/genética , Ubiquitinas/genética , Ubiquitinas/metabolismo , Regulação para Cima
6.
Cell Death Dis ; 12(7): 697, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34257278

RESUMO

The tripartite motif-containing protein 21 (TRIM21) plays important roles in autophagy and innate immunity. Here, we found that HECT and RLD domain containing E3 ubiquitin protein ligase 5 (HERC5), as an interferon-stimulated gene 15 (ISG15) E3 ligase, catalyzes the ISGylation of TRIM21 at the Lys260 and Lys279 residues. Moreover, IFN-ß also induces TRIM21 ISGylation at multiple lysine residues, thereby enhancing its E3 ligase activity for K63-linkage-specific ubiquitination and resulting in increased levels of TRIM21 and p62 K63-linked ubiquitination. The K63-linked ubiquitination of p62 at Lys7 prevents its self-oligomerization and targeting to the autophagosome. Taken together, our study suggests that the ISGylation of TRIM21 plays a vital role in regulating self-oligomerization and localization of p62 in the autophagy induced by IFN-ß.


Assuntos
Autofagossomos/enzimologia , Citocinas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Processamento de Proteína Pós-Traducional , Ribonucleoproteínas/metabolismo , Proteína Sequestossoma-1/metabolismo , Ubiquitinas/metabolismo , Células A549 , Autofagossomos/efeitos dos fármacos , Autofagossomos/genética , Autofagia , Citocinas/genética , Células HEK293 , Humanos , Interferon beta/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lisina , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Ribonucleoproteínas/genética , Proteína Sequestossoma-1/genética , Ubiquitinação , Ubiquitinas/genética
7.
J Phys Chem Lett ; 12(23): 5608-5615, 2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34110168

RESUMO

Papain-like protease (PLpro) from SARS-CoV-2 plays essential roles in the replication cycle of the virus. In particular, it preferentially interacts with and cleaves human interferon-stimulated gene 15 (hISG15) to suppress the innate immune response of the host. We used small-angle X-ray and neutron scattering combined with computational techniques to study the mechanism of interaction of SARS-CoV-2 PLpro with hISG15. We showed that hISG15 undergoes a transition from an extended to a compact state after binding to PLpro, a conformation that has not been previously observed in complexes of SARS-CoV-2 PLpro with ISG15 from other species. Furthermore, computational analysis showed significant conformational flexibility in the ISG15 N-terminal domain, suggesting that it is weakly bound to PLpro and supports a binding mechanism that is dominated by the C-terminal ISG15 domain. This study fundamentally improves our understanding of the SARS-CoV-2 deISGylation complex that will help guide development of COVID-19 therapeutics targeting this complex.


Assuntos
Proteases Semelhantes à Papaína de Coronavírus/química , Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Citocinas/química , Citocinas/metabolismo , Interferons/metabolismo , SARS-CoV-2/metabolismo , Ubiquitinas/química , Ubiquitinas/metabolismo , Proteases Semelhantes à Papaína de Coronavírus/genética , Citocinas/genética , Humanos , Difração de Nêutrons , Conformação Proteica , SARS-CoV-2/enzimologia , SARS-CoV-2/genética , Espalhamento a Baixo Ângulo , Ubiquitinas/genética , Difração de Raios X
8.
Front Immunol ; 12: 658048, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33953720

RESUMO

B cell activation by Tfh cells, i.e., through CD154 engagement of CD40 and IL-21, and survival within GCs are crucial for the T-dependent Ab response. LUBAC, composed of HOIP, SHARPIN, and HOIL-1, catalyzes linear ubiquitination (Linear M1-Ub) to mediate NF-κB activation and cell survival induced by TNF receptor superfamily members, which include CD40. As shown in this study, B cells expressing the Sharpin null mutation cpdm (Sharpincpdm ) could undergo proliferation, CSR, and SHM in response to immunization by a T-dependent Ag, but were defective in survival within GCs, enrichment of a mutation enhancing the BCR affinity, and production of specific Abs. Sharpincpdm B cells stimulated in vitro with CD154 displayed normal proliferation and differentiation, marginally impaired NF-κB activation and survival, but markedly exacerbated death triggered by IL-21. While activating the mitochondria-dependent apoptosis pathway in both Sharpin+/+ and Sharpincpdm B cells, IL-21 induced Sharpincpdm B cells to undergo sustained activation of caspase 9 and caspase 8 of the mitochondria-dependent and independent pathway, respectively, and ultimately caspase 3 in effecting apoptosis. These were associated with loss of the caspase 8 inhibitor cFLIP and reduction in cFLIP Linear M1-Ub, which interferes with cFLIP poly-ubiquitination at Lys48 and degradation. Finally, the viability of Sharpincpdm B cells was rescued by caspase inhibitors but virtually abrogated - together with Linear M1-Ub and cFLIP levels - by a small molecule HOIP inhibitor. Thus, LUBAC controls the cFLIP expression and inhibits the effects of caspase 8 and IL-21-activated caspase 9, thereby suppressing apoptosis of CD40 and IL-21-activated B cells and promoting GC B cell survival.


Assuntos
Apoptose , Linfócitos B/imunologia , Linfócitos B/metabolismo , Antígenos CD40/metabolismo , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Interleucinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Formação de Anticorpos/imunologia , Apoptose/efeitos dos fármacos , Biomarcadores , Caspase 8/metabolismo , Caspase 9 , Comunicação Celular , Sobrevivência Celular , Camundongos , Camundongos Transgênicos , Mutação , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Ubiquitinas/metabolismo
9.
Mol Cell ; 81(11): 2278-2289, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-33984284

RESUMO

Agents that induce DNA damage can cure some cancers. However, the side effects of chemotherapy are severe because of the indiscriminate action of DNA-damaging agents on both healthy and cancerous cells. DNA repair pathway inhibition provides a less toxic and targeted alternative to chemotherapy. A compelling DNA repair target is the Fanconi anemia (FA) E3 ligase core complex due to its critical-and likely singular-role in the efficient removal of specific DNA lesions. FA pathway inactivation has been demonstrated to specifically kill some types of cancer cells without the addition of exogenous DNA damage, including cells that lack BRCA1, BRCA2, ATM, or functionally related genes. In this perspective, we discuss the genetic and biochemical evidence in support of the FA core complex as a compelling drug target for cancer therapy. In particular, we discuss the genetic, biochemical, and structural data that could rapidly advance our capacity to identify and implement the use of FA core complex inhibitors in the clinic.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteína BRCA1/genética , Proteína BRCA2/genética , Reparo do DNA/efeitos dos fármacos , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Anemia de Fanconi/tratamento farmacológico , Ubiquitina-Proteína Ligases/genética , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/deficiência , Proteína BRCA1/deficiência , Proteína BRCA2/deficiência , Dano ao DNA , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/uso terapêutico , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Anemia de Fanconi/patologia , Proteínas de Grupos de Complementação da Anemia de Fanconi/antagonistas & inibidores , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Terapia de Alvo Molecular/métodos , Morfolinas/uso terapêutico , Pironas/uso terapêutico , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Mutações Sintéticas Letais , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinas/antagonistas & inibidores , Ubiquitinas/genética , Ubiquitinas/metabolismo
10.
Biomolecules ; 11(4)2021 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-33806190

RESUMO

The COP9 signalosome (CSN) is a highly conserved eukaryotic multi-subunit enzyme, regulating cullin RING ligase activities and accordingly, substrate ubiquitination and degradation. We showed that the CSN complex of Saccharomyces cerevisiae that is deviated in subunit composition and in sequence homology harbors a highly conserved cullin deneddylase enzymatic core complex. We took advantage of the non-essentiality of the S. cerevisiae CSN-NEDD8/Rub1 axis, together with the enzyme-substrate cross-species activity, to develop a sensitive fluorescence readout assay, suitable for biochemical assessment of cullin deneddylation by CSNs from various origins. We also demonstrated that the yeast catalytic subunit, CSN5/Jab1, is targeted by an inhibitor that was selected for the human orthologue. Treatment of yeast by the inhibitor led to the accumulation of neddylated cullins and the formation of reactive oxygen species. Overall, our data revealed S. cerevisiae as a general platform that can be used for studies of CSN deneddylation and for testing the efficacy of selected CSN inhibitors.


Assuntos
Complexo do Signalossomo COP9/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Complexo do Signalossomo COP9/química , Complexo do Signalossomo COP9/genética , Proteínas Culina/metabolismo , Humanos , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por Substrato , Ubiquitinação , Ubiquitinas/química , Ubiquitinas/genética , Ubiquitinas/metabolismo
11.
Viruses ; 13(3)2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799906

RESUMO

Mayaro virus (MAYV) and chikungunya virus (CHIKV) are known for their arthrotropism, but accumulating evidence shows that CHIKV infections are occasionally associated with serious neurological complications. However, little is known about the capacity of MAYV to invade the central nervous system (CNS). We show that human neural progenitors (hNPCs), pericytes and astrocytes are susceptible to MAYV infection, resulting in the production of infectious viral particles. In primary astrocytes, MAYV, and to a lesser extent CHIKV, elicited a strong antiviral response, as demonstrated by an increased expression of several interferon-stimulated genes, including ISG15, MX1 and OAS2. Infection with either virus led to an enhanced expression of inflammatory chemokines, such as CCL5, CXCL10 and CXCL11, whereas MAYV induced higher levels of IL-6, IL-12 and IL-15 in these cells. Moreover, MAYV was more susceptible than CHIKV to the antiviral effects of both type I and type II interferons. Taken together, this study shows that although MAYV and CHIKV are phylogenetically related, they induce different types of antiviral responses in astrocytes. This work is the first to evaluate the potential neurotropism of MAYV and shows that brain cells and particularly astrocytes and hNPCs are permissive to MAYV, which, consequently, could lead to MAYV-induced neuropathology.


Assuntos
Infecções por Alphavirus/imunologia , Alphavirus/imunologia , Astrócitos/imunologia , Astrócitos/virologia , Encéfalo/imunologia , 2',5'-Oligoadenilato Sintetase/metabolismo , Infecções por Alphavirus/patologia , Animais , Encéfalo/virologia , Linhagem Celular , Quimiocina CCL5/metabolismo , Quimiocina CXCL10/metabolismo , Quimiocina CXCL11/metabolismo , Febre de Chikungunya/imunologia , Vírus Chikungunya/imunologia , Chlorocebus aethiops , Citocinas/metabolismo , Humanos , Interferon Tipo I/imunologia , Interferon gama/imunologia , Proteínas de Resistência a Myxovirus/metabolismo , Células-Tronco Neurais/virologia , Pericitos/virologia , Ubiquitinas/metabolismo , Células Vero
12.
J Cell Mol Med ; 25(9): 4395-4407, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33797839

RESUMO

Drug resistance is often developed during clinical chemotherapy of ovarian cancers. The ubiquitin-like protein interferon-stimulated gene 15 (ISG15) is possibly dependent on tumour context to promote or suppress progression of various tumours. The ubiquitin-like protein interferon-stimulated gene 15 (ISG15) was decreased in cisplatin-resistant ovarian cancer cells. The current study identified that both ectopic wild type and nonISGylatable mutant ISG15 expression inhibited CSC-like phenotypes of cisplatin-resistant ovarian cancer cells. Moreover, ectopic ISG15 expression suppressed tumour formation in nude mice. In addition, ISG15 downregulation promoted CSC-like features of cisplatin-sensitive ovarian cancer cells. Furthermore, low ISG15 expression was associated with poor prognosis in patients with ovarian cancer. Transcriptional repressor Krüppel-like factor 12 (KLF12) downregulated ISG15 in cisplatin-resistant cells. Our data indicated that downregulating ISG15 expression, via weakening effect of KLF12, might be considered as new therapeutic strategy to inhibit CSC phenotypes in the treatment of cisplatin-resistant ovarian cancer.


Assuntos
Cisplatino/farmacologia , Citocinas/metabolismo , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fatores de Transcrição Kruppel-Like/metabolismo , Células-Tronco Neoplásicas/patologia , Neoplasias Ovarianas/patologia , Ubiquitinas/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose , Movimento Celular , Proliferação de Células , Citocinas/genética , Feminino , Humanos , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Células Tumorais Cultivadas , Ubiquitinas/genética , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Int J Mol Sci ; 22(9)2021 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-33921964

RESUMO

Ubiquitin is a small protein that is highly conserved throughout eukaryotes. It operates as a reversible post-translational modifier through a process known as ubiquitination, which involves the addition of one or several ubiquitin moieties to a substrate protein. These modifications mark proteins for proteasome-dependent degradation or alter their localization or activity in a variety of cellular processes. In most eukaryotes, ubiquitin is generated by the proteolytic cleavage of precursor proteins in which it is fused either to itself, constituting a polyubiquitin precursor, or as a single N-terminal moiety to ribosomal proteins, which are practically invariably eL40 and eS31. Herein, we summarize the contribution of the ubiquitin moiety within precursors of ribosomal proteins to ribosome biogenesis and function and discuss the biological relevance of having maintained the explicit fusion to eL40 and eS31 during evolution. There are other ubiquitin-like proteins, which also work as post-translational modifiers, among them the small ubiquitin-like modifier (SUMO). Both ubiquitin and SUMO are able to modify ribosome assembly factors and ribosomal proteins to regulate ribosome biogenesis and function. Strikingly, ubiquitin-like domains are also found within two ribosome assembly factors; hence, the functional role of these proteins will also be highlighted.


Assuntos
Processamento de Proteína Pós-Traducional , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Ubiquitinas/metabolismo , Animais , Humanos
14.
Int J Mol Sci ; 22(9)2021 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-33919255

RESUMO

Proteasomal dysfunction is known to be associated with amyotrophic lateral sclerosis and frontotemporal degeneration (ALS/FTD). Our previous reports have shown that a mutant form of ubiquilin-2 (UBQLN2) linked to ALS/FTD leads to neurodegeneration accompanied by accumulations of the proteasome subunit Rpt1 in transgenic rats, but the precise pathogenic mechanisms of how this mutation impairs the proteasome remains to be elucidated. Here, we reveal that this UBQLN2 mutation in rats disrupted the proteasome integrity prior to neurodegeneration, that it dissociated the 26S proteasome in vitro, and that its depletion did not affect 26S proteasome assembly. During both disease progression and in an age-dependent manner, we found that proteasome subunits were translocated to the nucleus, including both of the 20S core particles (PSMA1 and PSMB7) and the 19S regulatory particles (Rpt1 and Rpn1), suggesting that defective proteasome function may result from the proteasome-subunit mislocalization. Taken together, the present data demonstrate that impaired proteasome assembly is an early event in the pathogenesis of UBQLN2-associated neurodegeneration in mutant UBQLN2 rats.


Assuntos
Esclerose Amiotrófica Lateral/fisiopatologia , Demência Frontotemporal/fisiopatologia , Mutação , Neurônios/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitinas/genética , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Animais , Cisteína Endopeptidases , Modelos Animais de Doenças , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Neurônios/metabolismo , Agregação Patológica de Proteínas , Ratos , Ratos Transgênicos , Ubiquitinas/metabolismo
15.
Nat Struct Mol Biol ; 28(3): 300-309, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33686268

RESUMO

The Fanconi anemia (FA) pathway is essential for the repair of DNA interstrand crosslinks. Central to the pathway is the FA core complex, a ubiquitin ligase of nine subunits that monoubiquitinates the FANCI-FANCD2 (ID) DNA clamp. The 3.1 Å structure of the 1.1-MDa human FA core complex, described here, reveals an asymmetric assembly with two copies of all but the FANCC, FANCE and FANCF subunits. The asymmetry is crucial, as it prevents the binding of a second FANCC-FANCE-FANCF subcomplex that inhibits the recruitment of the UBE2T ubiquitin conjugating enzyme, and instead creates an ID binding site. A single active site then ubiquitinates FANCD2 and FANCI sequentially. We also present the 4.2-Å structures of the human core-UBE2T-ID-DNA complex in three conformations captured during monoubiquitination. They reveal the core-UBE2T complex remodeling the ID-DNA complex, closing the clamp on the DNA before ubiquitination. Monoubiquitination then prevents clamp opening after release from the core.


Assuntos
DNA/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , DNA/química , DNA/ultraestrutura , Proteína do Grupo de Complementação C da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação E da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação F da Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/ultraestrutura , Células HEK293 , Humanos , Modelos Moleculares , Complexos Multienzimáticos/ultraestrutura , Reprodutibilidade dos Testes , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/ultraestrutura , Ubiquitinação , Ubiquitinas/metabolismo
16.
Nat Microbiol ; 6(4): 467-478, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33727702

RESUMO

Activation of the RIG-I-like receptors, retinoic-acid inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5), establishes an antiviral state by upregulating interferon (IFN)-stimulated genes (ISGs). Among these is ISG15, the mechanistic roles of which in innate immunity still remain enigmatic. In the present study, we report that ISG15 conjugation is essential for antiviral IFN responses mediated by the viral RNA sensor MDA5. ISGylation of the caspase activation and recruitment domains of MDA5 promotes its oligomerization and thereby triggers activation of innate immunity against a range of viruses, including coronaviruses, flaviviruses and picornaviruses. The ISG15-dependent activation of MDA5 is antagonized through direct de-ISGylation mediated by the papain-like protease of SARS-CoV-2, a recently emerged coronavirus that has caused the COVID-19 pandemic. Our work demonstrates a crucial role for ISG15 in the MDA5-mediated antiviral response, and also identifies a key immune evasion mechanism of SARS-CoV-2, which may be targeted for the development of new antivirals and vaccines to combat COVID-19.


Assuntos
Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Citocinas/metabolismo , Imunidade Inata , Helicase IFIH1 Induzida por Interferon/antagonistas & inibidores , SARS-CoV-2/enzimologia , SARS-CoV-2/imunologia , Ubiquitinas/metabolismo , Aedes , Animais , Chlorocebus aethiops , Cricetinae , Células HEK293 , Humanos , Helicase IFIH1 Induzida por Interferon/metabolismo , Leucócitos Mononucleares , Camundongos , Células Vero
17.
Zhonghua Yi Xue Za Zhi ; 101(12): 846-850, 2021 Mar 30.
Artigo em Chinês | MEDLINE | ID: mdl-33789365

RESUMO

Objective: To explore the association between rare UBQLN2 variants and amyotrophic lateral sclerosis (ALS) in Chinese population, and the characteristic of phenotypes of their carriers. Methods: A total of 166 ALS patients who visited Department of Neurology of Peking University Third Hospital between January 2018 and July 2020 were recruited. The next-generation sequencing was performed to screen possible pathogenic rare variants of UBQLN2. Meanwhile, control individuals were obtained from 1000 Genome Project (2 504 samples) and an in-house whole-exome sequencing database (1 812 samples), separately. The sequence kernel association test (SKAT) and the SKAT-optimal test (SKAT-O) were used to identify the association between UBQLN2 rare variants and ALS. The clinical characteristics of rare variant carriers were analyzed. Results: A total of 33 familiar ALS and 133 sporadic ALS of Chinese ancestry were enrolled. Of the 166 ALS patients, 12.7% had bulbar-onset, 85.5% had limb-onset, and 5 cases were ALS with frontotemporal dementia (3.0%). The male-to-female ratio was 1.68∶1, with a mean age at symptom onset of (43.8±12.2) years. Three possible pathogenic rare variants of UBQLN2 were detected, including c.128A>G (p.Lys43Arg), c.142G>T (p.Val48Leu) and c.1451T>G (p.Val484Gly), and all of them were novel missense mutations. Compared with 1000 Genome Project, SKAT and SKAT-O showed a P value of 2.49×10-6 and 9.22×10-7, respectively. While compared with the in-house database, SKAT and SKAT-O revealed a P value of 1.42×10-3 and 1.10×10-3, respectively. Patients who carried rare UBQLN2 variants were with a higher rate of bulbar-onset (2/3 vs 19/163, P=0.042). Conclusion: Rare variants of UBQLN2 are associated with ALS in Chinese population, and mutation of UBQLN2 may be relevant to bulbar-onset.


Assuntos
Esclerose Amiotrófica Lateral , Proteínas Adaptadoras de Transdução de Sinal , Esclerose Amiotrófica Lateral/genética , Proteínas Relacionadas à Autofagia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , China , Feminino , Humanos , Masculino , Mutação , Ubiquitinas/genética , Ubiquitinas/metabolismo
18.
Biochim Biophys Acta Mol Basis Dis ; 1867(6): 166102, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33617986

RESUMO

Mitophagy is defective in several neurodegenerative diseases, including Ataxia Telangiectasia (A-T). However, the molecular mechanism underlying defective mitophagy in A-T is unknown. Literature indicates that damaged mitochondria are transported to the perinuclear region prior to their removal via mitophagy. Our previous work has indicated that conjugation of SUMO2 (Small Ubiquitin-like Modifier 2) to mitofusins (Mfns) may be necessary for congression of mitochondria into SUMO2-/ubiquitin-/LC3-positive compact structures resembling mito-aggresomes at the perinuclear region in CCCP-treated HEK293 cells. Here, we demonstrate that Mfns are SUMOylated, and mitochondria are transported to the perinuclear region; however, mitochondria fail to congress into mito-aggresome-like structures in CCCP-treated A-T cells. Defect in mitochondrial congression is causally related to constitutively elevated ISG15 (Interferon-Stimulated Gene 15), an antagonist of the ubiquitin pathway, in A-T cells. Suppression of the ISG15 pathway restores mitochondrial congression, reduce oxidative stress, and level of unhealthy mitochondria, which is suggestive of restoration of mitophagy in A-T cells. ISG15 is also constitutively elevated and mitophagy is defective in Amytrophic Lateral Sclerosis (ALS). The constitutively elevated ISG15 pathway therefore appears to be a common unifying biochemical mechanism underlying defective mitophagy in neurodegenerative disorders thus, implying the broader significance of our findings, and suggest the potential role of ISG15 inhibitors in their treatment.


Assuntos
Ataxia Telangiectasia/patologia , Citocinas/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Mitocôndrias/patologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Mitofagia , Processamento de Proteína Pós-Traducional , Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/metabolismo , Citocinas/genética , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , Células HEK293 , Humanos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/genética , Ubiquitinas/genética
19.
Biochemistry ; 60(8): 584-596, 2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33583181

RESUMO

We report the co-crystal structure of the (catalytic Cys)-to-Ala mutant of the deubiquitinase domain of the Legionella pneumophila effector SdeA (SdeADUB) with its ubiquitin (Ub) product. Most of the intermolecular interactions are preserved in this product-bound structure compared to that of the previously characterized complex of SdeADUB with the suicide inhibitor ubiquitin vinylmethyl ester (Ub-VME), whose structure models the acyl-enzyme thioester intermediate. Nuclear magnetic resonance (NMR) titration studies show a chemical shift perturbation pattern that suggests that the same interactions also exist in solution. Isothermal titration calorimetry and NMR titration data reveal that the affinity of wild-type (WT) SdeADUB for Ub is significantly lower than that of the Cys-to-Ala mutant. This is potentially due to repulsive interaction between the thiolate ion of the catalytic Cys residue in WT SdeADUB and the carboxylate group of the C-terminal Gly76 residue in Ub. In the context of SdeADUB catalysis, this electrostatic repulsion arises after the hydrolysis of the scissile isopeptide bond in the acyl-enzyme intermediate and the consequent formation of the C-terminal carboxylic group in the Ub fragment. We hypothesize that this electrostatic repulsion may expedite the release of the Ub product by SdeADUB. We note that similar repulsive interactions may also occur in other deubiquitinases and hydrolases of ubiquitin-like protein modifiers and may constitute a fairly general mechanism of product release within this family. This is a potentially important feature for a family of enzymes that form extensive protein-protein interactions during enzyme-substrate engagement.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Legionella pneumophila/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Ubiquitinas/metabolismo , Catálise , Cristalografia por Raios X , Hidrólise , Modelos Moleculares , Conformação Proteica , Ubiquitinação
20.
J Biol Chem ; 296: 100450, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33617881

RESUMO

Proteasome-mediated substrate degradation is an essential process that relies on the coordinated actions of ubiquitin (Ub), shuttle proteins containing Ub-like (UBL) domains, and the proteasome. Proteinaceous substrates are tagged with polyUb and shuttle proteins, and these signals are then recognized by the proteasome, which subsequently degrades the substrate. To date, three proteasomal receptors have been identified, as well as multiple shuttle proteins and numerous types of polyUb chains that signal for degradation. While the components of this pathway are well-known, our understanding of their interplay is unclear-especially in the context of Rpn1, the largest proteasomal subunit. Here, using nuclear magnetic resonance (NMR) spectroscopy in combination with competition assays, we show that Rpn1 associates with UBL-containing proteins and polyUb chains, while exhibiting a preference for shuttle protein Rad23. Rpn1 appears to contain multiple Ub/UBL-binding sites, theoretically as many as one for each of its hallmark proteasome/cyclosome repeats. Remarkably, we also find that binding sites on Rpn1 can be shared among Ub and UBL species, while proteasomal receptors Rpn1 and Rpn10 can compete with each other for binding of shuttle protein Dsk2. Taken together, our results rule out the possibility of exclusive recognition sites on Rpn1 for individual Ub/UBL signals and further emphasize the complexity of the redundancy-laden proteasomal degradation pathway.


Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitinas/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Espectroscopia de Ressonância Magnética/métodos , Proteínas de Membrana/metabolismo , Poliubiquitina/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologia , Ligação Proteica , Proteólise , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Ubiquitina/metabolismo
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