Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 4.070
Filtrar
1.
PLoS Pathog ; 16(8): e1008780, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866188

RESUMO

Ubiquitin like protein 5 (UBL5) interacts with other proteins to regulate their function but differs from ubiquitin and other UBLs because it does not form covalent conjugates. Ubiquitin and most UBLs mediate the degradation of target proteins through the 26S proteasome but it is not known if UBL5 can also do that. Here we found that the UBL5s of rice and Nicotiana benthamiana interacted with rice stripe virus (RSV) p3 protein. Silencing of NbUBL5s in N. benthamiana facilitated RSV infection, while UBL5 overexpression conferred resistance to RSV in both N. benthamiana and rice. Further analysis showed that NbUBL5.1 impaired the function of p3 as a suppressor of silencing by degrading it through the 26S proteasome. NbUBL5.1 and OsUBL5 interacted with RPN10 and RPN13, the receptors of ubiquitin in the 26S proteasome. Furthermore, silencing of NbRPN10 or NbRPN13 compromised the degradation of p3 mediated by NbUBL5.1. Together, the results suggest that UBL5 mediates the degradation of RSV p3 protein through the 26S proteasome, a previously unreported plant defense strategy against RSV infection.


Assuntos
Proteínas de Plantas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteínas Repressoras/metabolismo , Tenuivirus/metabolismo , Tabaco/metabolismo , Ubiquitinas/metabolismo , Proteínas Virais/metabolismo , Proteínas de Plantas/genética , Complexo de Endopeptidases do Proteassoma/genética , Proteínas Repressoras/genética , Tenuivirus/genética , Tabaco/genética , Ubiquitinas/genética , Proteínas Virais/genética
2.
Proc Natl Acad Sci U S A ; 117(30): 17510-17512, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32665439

RESUMO

Type I IFN (IFN-I) is thought to be rapidly internalized and degraded following binding to its receptor and initiation of signaling. However, many studies report the persistent effects mediated by IFN-I for days or even weeks, both ex vivo and in vivo. These long-lasting effects are attributed to downstream signaling molecules or induced effectors having a long half-life, particularly in specific cell types. Here, we describe a mechanism explaining the long-term effects of IFN-I. Following receptor binding, IFN-I is siloed into endosomal compartments. These intracellular "IFN silos" persist for days and can be visualized by fluorescence and electron microscopy. However, they are largely dormant functionally, due to IFN-I-induced negative regulators. By contrast, in individuals lacking these negative regulators, such as ISG15 or USP18, this siloed IFN-I can continue to signal from within the endosome. This mechanism may underlie the long-term effects of IFN-I therapy and may contribute to the pathophysiology of type I interferonopathies.


Assuntos
Endossomos/metabolismo , Interferon Tipo I/metabolismo , Animais , Linhagem Celular , Citocinas/genética , Citocinas/metabolismo , Endossomos/ultraestrutura , Humanos , Transporte Proteico , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismo
3.
Proc Natl Acad Sci U S A ; 117(25): 14395-14404, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513696

RESUMO

Retinoic acid-inducible gene I (RIG-I) is up-regulated during granulocytic differentiation of acute promyelocytic leukemia (APL) cells induced by all-trans retinoic acid (ATRA). It has been reported that RIG-I recognizes virus-specific 5'-ppp-double-stranded RNA (dsRNA) and activates the type I interferons signaling pathways in innate immunity. However, the functions of RIG-I in hematopoiesis remain unclear, especially regarding its possible interaction with endogenous RNAs and the associated pathways that could contribute to the cellular differentiation and maturation. Herein, we identified a number of RIG-I-binding endogenous RNAs in APL cells following ATRA treatment, including the tripartite motif-containing protein 25 (TRIM25) messenger RNA (mRNA). TRIM25 encodes the protein known as an E3 ligase for ubiquitin/interferon (IFN)-induced 15-kDa protein (ISG15) that is involved in RIG-I-mediated antiviral signaling. We show that RIG-I could bind TRIM25 mRNA via its helicase domain and C-terminal regulatory domain, enhancing the stability of TRIM25 transcripts. RIG-I could increase the transcriptional expression of TRIM25 by caspase recruitment domain (CARD) domain through an IFN-stimulated response element. In addition, RIG-I activated other key genes in the ISGylation pathway by activating signal transducer and activator of transcription 1 (STAT1), including the modifier ISG15 and several enzymes responsible for the conjugation of ISG15 to protein substrates. RIG-I cooperated with STAT1/2 and interferon regulatory factor 1 (IRF1) to promote the activation of the ISGylation pathway. The integrity of ISGylation in ATRA or RIG-I-induced cell differentiation was essential given that knockdown of TRIM25 or ISG15 resulted in significant inhibition of this process. Our results provide insight into the role of the RIG-I-TRIM25-ISGylation axis in myeloid differentiation.


Assuntos
Diferenciação Celular , Citocinas/metabolismo , Proteína DEAD-box 58/metabolismo , Granulócitos/fisiologia , Fatores de Transcrição/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinas/metabolismo , Linhagem Celular Tumoral , Citocinas/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Estabilidade de RNA , RNA Mensageiro/metabolismo , Fatores de Transcrição/genética , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinas/genética , Regulação para Cima
4.
Cell Rep ; 31(11): 107772, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32553163

RESUMO

ISG15 is a ubiquitin-like modifier that also functions extracellularly, signaling through the LFA-1 integrin to promote interferon (IFN)-γ release from natural killer (NK) and T cells. The signals that lead to the production of extracellular ISG15 and the relationship between its two core functions remain unclear. We show that both epithelial cells and lymphocytes can secrete ISG15, which then signals in either an autocrine or paracrine manner to LFA-1-expressing cells. Microbial pathogens and Toll-like receptor (TLR) agonists result in both IFN-ß-dependent and -independent secretion of ISG15, and residues required for ISG15 secretion are mapped. Intracellular ISGylation inhibits secretion, and viral effector proteins, influenza B NS1, and viral de-ISGylases, including SARS-CoV-2 PLpro, have opposing effects on secretion of ISG15. These results establish extracellular ISG15 as a cytokine-like protein that bridges early innate and IFN-γ-dependent immune responses, and indicate that pathogens have evolved to differentially inhibit the intracellular and extracellular functions of ISG15.


Assuntos
Citocinas/metabolismo , Transdução de Sinais , Ubiquitinas/metabolismo , Animais , Células HEK293 , Humanos , Influenza Humana/imunologia , Influenza Humana/metabolismo , Interferon gama/imunologia , Interferon gama/metabolismo , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Mycobacterium/imunologia , Infecções por Mycobacterium/metabolismo , Padrões Moleculares Associados a Patógenos , Febre Tifoide/imunologia , Febre Tifoide/metabolismo , Proteínas não Estruturais Virais/metabolismo
5.
Virus Res ; 286: 198036, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32492472

RESUMO

Interferon (IFN)-stimulated gene product 15 (ISG15) is a ubiquitin-like protein critical for the control of microbial infections. ISG15 appears to serve a wide variety of functions, which regulate multiple cellular responses contributing to the development of an antiviral state. ISG15 is a versatile molecule directly modulating both host and virus protein function which regulate many signaling pathways, including its own synthesis. Here we review the various roles ISG15 plays in the antiviral immune response, and examine the mechanisms by which viruses attempt to mitigate or exploit ISG15 activity.


Assuntos
Citocinas/metabolismo , Imunidade Inata/imunologia , Ubiquitinas/metabolismo , Viroses/imunologia , Replicação Viral/imunologia , Animais , Citocinas/genética , Humanos , Interferon Tipo I/imunologia , Macrófagos/imunologia , Camundongos , Neutrófilos/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Ubiquitinas/genética , Proteínas Virais/metabolismo , Internalização do Vírus
6.
ACS Infect Dis ; 6(8): 2099-2109, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32428392

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for COVID-19, is a novel human betacoronavirus that is rapidly spreading worldwide. The outbreak currently includes over 3.7 million cases and 260,000 fatalities. As a betacoronavirus, SARS-CoV-2 encodes for a papain-like protease (PLpro) that is likely responsible for cleavage of the coronavirus (CoV) viral polypeptide. The PLpro is also responsible for suppression of host innate immune responses by virtue of its ability to reverse host ubiquitination and ISGylation events. Here, the biochemical activity of SARS-CoV-2 PLpro against ubiquitin (Ub) and interferon-stimulated gene product 15 (ISG15) substrates is evaluated, revealing that the protease has a marked reduction in its ability to process K48 linked Ub substrates compared to its counterpart in SARS-CoV. Additionally, its substrate activity more closely mirrors that of the PLpro from the Middle East respiratory syndrome coronavirus and prefers ISG15s from certain species including humans. Additionally, naphthalene based PLpro inhibitors are shown to be effective at halting SARS-CoV-2 PLpro activity as well as SARS-CoV-2 replication.


Assuntos
Betacoronavirus/enzimologia , Infecções por Coronavirus/virologia , Cisteína Endopeptidases/metabolismo , Citocinas/metabolismo , Enzimas Desubiquitinantes/antagonistas & inibidores , Pneumonia Viral/virologia , Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Chlorocebus aethiops , Cisteína Endopeptidases/química , Citocinas/antagonistas & inibidores , Citocinas/química , Humanos , Naftalenos/farmacologia , Pandemias , Ligação Proteica , Conformação Proteica , Especificidade por Substrato , Ubiquitinas/antagonistas & inibidores , Ubiquitinas/química , Células Vero , Proteínas não Estruturais Virais/química , Replicação Viral/efeitos dos fármacos
7.
Nat Commun ; 11(1): 2682, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32472071

RESUMO

Pancreatic cancer stem cells (PaCSCs) drive pancreatic cancer tumorigenesis, chemoresistance and metastasis. While eliminating this subpopulation of cells would theoretically result in tumor eradication, PaCSCs are extremely plastic and can successfully adapt to targeted therapies. In this study, we demonstrate that PaCSCs increase expression of interferon-stimulated gene 15 (ISG15) and protein ISGylation, which are essential for maintaining their metabolic plasticity. CRISPR-mediated ISG15 genomic editing reduces overall ISGylation, impairing PaCSCs self-renewal and their in vivo tumorigenic capacity. At the molecular level, ISG15 loss results in decreased mitochondrial ISGylation concomitant with increased accumulation of dysfunctional mitochondria, reduced oxidative phosphorylation (OXPHOS) and impaired mitophagy. Importantly, disruption in mitochondrial metabolism affects PaCSC metabolic plasticity, making them susceptible to prolonged inhibition with metformin in vivo. Thus, ISGylation is critical for optimal and efficient OXPHOS by ensuring the recycling of dysfunctional mitochondria, and when absent, a dysregulation in mitophagy occurs that negatively impacts PaCSC stemness.


Assuntos
Carcinoma Ductal Pancreático/patologia , Transformação Celular Neoplásica/genética , Citocinas/metabolismo , Mitofagia/genética , Células-Tronco Neoplásicas/patologia , Neoplasias Pancreáticas/patologia , Ubiquitinas/metabolismo , Linhagem Celular , Plasticidade Celular/fisiologia , Transformação Celular Neoplásica/patologia , Citocinas/genética , Humanos , Metformina/farmacologia , Mitocôndrias/genética , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Neoplasias Pancreáticas/mortalidade , Edição de RNA/genética , Ubiquitinas/genética
8.
Nat Commun ; 11(1): 2343, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32393759

RESUMO

Ubiquitin mediated signaling contributes critically to host cell defenses during pathogen infection. Many pathogens manipulate the ubiquitin system to evade these defenses. Here we characterize a likely effector protein bearing a deubiquitylase (DUB) domain from the obligate intracellular bacterium Orientia tsutsugamushi, the causative agent of scrub typhus. The Ulp1-like DUB prefers ubiquitin substrates over ubiquitin-like proteins and efficiently cleaves polyubiquitin chains of three or more ubiquitins. The co-crystal structure of the DUB (OtDUB) domain with ubiquitin revealed three bound ubiquitins: one engages the S1 site, the second binds an S2 site contributing to chain specificity and the third binds a unique ubiquitin-binding domain (UBD). The UBD modulates OtDUB activity, undergoes a pronounced structural transition upon binding ubiquitin, and binds monoubiquitin with an unprecedented ~5 nM dissociation constant. The characterization and high-resolution structure determination of this enzyme should aid in its development as a drug target to counter Orientia infections.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Orientia tsutsugamushi/enzimologia , Tifo por Ácaros/microbiologia , Ubiquitinas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Interações Hidrofóbicas e Hidrofílicas , Lisina/metabolismo , Ligação Proteica , Domínios Proteicos , Especificidade por Substrato , Termodinâmica
9.
Anim Sci J ; 91(1): e13378, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32329195

RESUMO

Interferon-tau (IFNT) regulates maternal recognition during early pregnancy in ruminants. The liver can serve as a hematopoietic organ, and it has immune functions. This study hypothesized whether mRNA and proteins of interferon-stimulated genes (ISGs) induced by early pregnancy are upregulated in maternal liver. Therefore, we determined the expression of interferon-stimulated gene 15-kDa protein (ISG15), 2',5'-oligoadenylate synthetase 1 (OAS1), myxovirus resistance protein 1 (MX1), interferon-gamma-inducible protein 10 (IP-10), and signal transducer and activator of transcription 1 (STAT1) in maternal livers during early pregnancy in sheep. Ovine livers were sampled on day 16 of the estrous cycle, and days 13, 16, and 25 of pregnancy, and expression of ISGs was detected by quantitative real-time PCR, Western blot, and immunohistochemistry analysis. Our results showed that there were increases in expression of the mRNA and proteins of ISG15, OAS1, IP-10, STAT1, and MX1 during early pregnancy. STAT1 protein was limited to the hepatocytes, and endothelial cells of proper hepatic arteries and hepatic portal veins. In conclusion, the upregulation of ISG15, OAS1, IP-10, STAT1, and MX1 proteins may be implicated in maternal hepatic immune adjustment and other functions during early pregnancy in sheep.


Assuntos
2',5'-Oligoadenilato Sintetase/genética , 2',5'-Oligoadenilato Sintetase/metabolismo , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Citocinas/genética , Citocinas/metabolismo , Expressão Gênica , Fígado/metabolismo , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/metabolismo , Prenhez/metabolismo , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Ovinos/genética , Ovinos/fisiologia , Ubiquitinas/genética , Ubiquitinas/metabolismo , Animais , Ciclo Estral/genética , Ciclo Estral/metabolismo , Feminino , Fígado/imunologia , Gravidez , Prenhez/imunologia , Ovinos/imunologia , Regulação para Cima
10.
Mem Inst Oswaldo Cruz ; 115: e190242, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32130365

RESUMO

BACKGROUND: Ubiquitin (Ub) and Ub-like proteins (Ub-L) are critical regulators of complex cellular processes such as the cell cycle, DNA repair, transcription, chromatin remodeling, signal translation, and protein degradation. Giardia intestinalis possesses an experimentally proven Ub-conjugation system; however, a limited number of enzymes involved in this process were identified using basic local alignment search tool (BLAST). This is due to the limitations of BLAST's ability to identify homologous functional regions when similarity between the sequences dips to < 30%. In addition Ub-Ls and their conjugating enzymes have not been fully elucidated in Giardia. OBJETIVE: To identify the enzymes involved in the Ub and Ub-Ls conjugation processes using intelligent systems based on the hidden Markov models (HMMs). METHODS: We performed an HMM search of functional Pfam domains found in the key enzymes of these pathways in Giardia's proteome. Each open reading frame identified was analysed by sequence homology, domain architecture, and transcription levels. FINDINGS: We identified 118 genes, 106 of which corresponded to the ubiquitination process (Ub, E1, E2, E3, and DUB enzymes). The E3 ligase group was the largest group with 82 members; 71 of which harbored a characteristic RING domain. Four Ub-Ls were identified and the conjugation enzymes for NEDD8 and URM1 were described for first time. The 3D model for Ub-Ls displayed the ß-grasp fold typical. Furthermore, our sequence analysis for the corresponding activating enzymes detected the essential motifs required for conjugation. MAIN CONCLUSIONS: Our findings highlight the complexity of Giardia's Ub-conjugation system, which is drastically different from that previously reported, and provides evidence for the presence of NEDDylation and URMylation enzymes in the genome and transcriptome of G. intestinalis.


Assuntos
Giardia lamblia/metabolismo , Ubiquitina/genética , Ubiquitinação , Ubiquitinas/genética , Giardia lamblia/genética , Modelos Moleculares , Transdução de Sinais , Ubiquitina/metabolismo , Ubiquitinas/metabolismo
11.
Pharmacol Rev ; 72(2): 380-413, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32107274

RESUMO

Ubiquitin (UB) transfer cascades consisting of E1, E2, and E3 enzymes constitute a complex network that regulates a myriad of biologic processes by modifying protein substrates. Deubiquitinating enzymes (DUBs) reverse UB modifications or trim UB chains of diverse linkages. Additionally, many cellular proteins carry UB-binding domains (UBDs) that translate the signals encoded in UB chains to target proteins for degradation by proteasomes or in autophagosomes, as well as affect nonproteolytic outcomes such as kinase activation, DNA repair, and transcriptional regulation. Dysregulation of the UB transfer pathways and malfunctions of DUBs and UBDs play causative roles in the development of many diseases. A greater understanding of the mechanism of UB chain assembly and the signals encoded in UB chains should aid in our understanding of disease pathogenesis and guide the development of novel therapeutics. The recent flourish of protein-engineering approaches such as unnatural amino acid incorporation, protein semisynthesis by expressed protein ligation, and high throughput selection by phage and yeast cell surface display has generated designer proteins as powerful tools to interrogate cell signaling mediated by protein ubiquitination. In this study, we highlight recent achievements of protein engineering on mapping, probing, and manipulating UB transfer in the cell. SIGNIFICANCE STATEMENT: The post-translational modification of proteins with ubiquitin alters the fate and function of proteins in diverse ways. Protein engineering is fundamentally transforming research in this area, providing new mechanistic insights and allowing for the exploration of concepts that can potentially be applied to therapeutic intervention.


Assuntos
Engenharia de Proteínas/métodos , Ubiquitinas/metabolismo , Animais , Enzimas Desubiquitinantes/metabolismo , Humanos , Ubiquitinação
12.
Vet Res ; 51(1): 22, 2020 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-32093773

RESUMO

Interferons (IFNs) induce the expression of interferon-stimulated genes (ISGs) for defense against numerous viral infections, including classical swine fever virus (CSFV). However, the mechanisms underlying the effect of ISGs on CSFV infection are rarely reported. In this study, we demonstrate that IFN-α treatment induces upregulation of ISG15 and thus attenuates CSFV replication. To determine whether ISG15 is critical for controlling CSFV replication, we established porcine alveolar macrophages (PAMs) with stable overexpression or knockdown of ISG15. Overexpression of Flag-ISG15 significantly prevented CSFV replication, whereas loss of ISG15 led to abnormal proliferation of CSFV. Furthermore, upregulated ISG15 promoted beclin-1 (BECN1) ISGylation and dysfunction and subsequently inhibited autophagy, which is indispensable for CSFV replication. In addition, HECT and RLD domain containing E3 ubiquitin protein ligase 5 (HERC5), which functions to catalyze conjugation of ISG15 protein, was confirmed to interact with BECN1. Collectively, these results indicate that IFN-α restricts CSFV replication through ISG15-mediated BECN1 ISGylation and autophagy inhibition, providing insight into the mechanism of CSFV replication control by type I IFN. This mechanism may not be the only antiviral mechanism of ISG15; nonetheless, this study may contribute to the development of CSFV treatment and prevention strategies.


Assuntos
Proteína Beclina-1/metabolismo , Vírus da Febre Suína Clássica/fisiologia , Peste Suína Clássica/imunologia , Citocinas/genética , Macrófagos Alveolares/imunologia , Ubiquitinas/genética , Animais , Autofagia , Citocinas/metabolismo , Suínos , Ubiquitinas/metabolismo , Replicação Viral
13.
J Exp Clin Cancer Res ; 39(1): 30, 2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-32028981

RESUMO

BACKGROUND: BRD7 is a tumor suppressor known to inhibit cell proliferation and cell cycle progression and initiate apoptosis in breast cancer. However, the function and underlying molecular events of BRD7 in tumor invasion and metastasis in breast cancer are not fully understood. METHODS: BRD7 expression was assessed in two stable cell lines MDA231 and MCF7 with BRD7 overexpression and one stable cell line MDA231 with BRD7 interference using qRT-PCR and western blotting. CCK8 assay was used to examine the proliferation ability of MDA231 and MCF7 cells. Scratch wound healing assay was used to evaluate cell migration in MDA231 and MCF7 cells. Both Matrigel and three-dimensional invasion assays were performed to investigate the cell invasion ability after BRD7 overexpression or silencing or YB1 restoration in MDA231 and MCF7 cells. The potential interacting proteins of BRD7 were screened using co-immunoprecipitation combined with mass spectrometry and verified by co-immunoprecipitation in HEK293T cells. Additionally, we confirmed the specific binding region between BRD7 and YB1 in HEK293T cells by constructing a series of deletion mutants of BRD7 and YB1 respectively. Finally, xenograft and metastatic mouse models using MDA231 cells were established to confirm the effect of BRD7 on tumor growth and metastasis. RESULTS: Here, the results of a series of assays in vitro indicated that BRD7 has the ability to inhibit the mobility, migration and invasion of breast cancer cells. In addition, YB1 was identified as a novel interacting protein of BRD7, and BRD7 was found to associate with the C-terminus of YB1 via its N-terminus. BRD7 decreases the expression of YB1 through negatively regulating YB1 phosphorylation at Ser102, thereby promoting its proteasomal degradation. Furthermore, gene set enrichment analysis revealed that epithelial-mesenchymal transition (EMT) is the common change occurring with altered expression of either BRD7 or YB1 and that BRD7 represses mesenchymal genes and activates epithelial genes. Moreover, restoring the expression of YB1 antagonized the inhibitory effect of BRD7 on tumorigenicity, EMT, invasiveness and metastasis through a series of in vitro and in vivo experiments. Additionally, BRD7 expression was negatively correlated with the level of YB1 in breast cancer patients. The combination of low BRD7 and high YB1 expression was significantly associated with poor prognosis, distant metastasis and advanced TNM stage. CONCLUSIONS: Collectively, these findings uncover that BRD7 blocks tumor growth, migration and metastasis by negatively regulating YB1-induced EMT, providing new insights into the mechanism by which BRD7 contributes to the progression and metastasis of breast cancer.


Assuntos
Neoplasias da Mama/genética , Proteínas Cromossômicas não Histona/genética , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Proteína 1 de Ligação a Y-Box/genética , Adulto , Idoso , Animais , Apoptose/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proteínas Cromossômicas não Histona/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Expressão Gênica , Humanos , Camundongos , Pessoa de Meia-Idade , Invasividade Neoplásica , Metástase Neoplásica , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteólise , Ubiquitinas/metabolismo , Proteína 1 de Ligação a Y-Box/metabolismo
14.
J Agric Food Chem ; 68(7): 2054-2062, 2020 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-31995984

RESUMO

Alzheimer's disease (AD) is a common neurodegenerative disease which is partly characterized by the aggregation of hyperphosphorylated Tau proteins forming neurofibrillary tangles that promote AD pathogenesis. In this study, we investigated the effects of tanshinone IIA (Tan IIA) isolated from Salvia miltiorrhiza on Tau degradation in the treatment of AD. The results showed that Tan IIA reduced the Tau expression and attenuated Tau phosphorylation in N2a cells, Tau-overexpressing cells, and 3×Tg-AD mouse primary neuron cells. Moreover, Tan IIA increased polyubiquitinated Tau accumulation and induced proteasomal degradation of the Tau protein. Additionally, Tan IIA became bound to the Tau protein and inhibited the formation of heparin-induced Tau fibrils. In summary, Tan IIA can increase polyubiquitinated Tau accumulation and induce the proteasomal degradation of the Tau protein and the binding of Tan IIA to the Tau protein, inhibiting the formation of Tau fibrils. Tan IIA may be further explored as a potential candidate for AD treatment.


Assuntos
Abietanos/farmacologia , Doença de Alzheimer/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Salvia miltiorrhiza/química , Ubiquitinas/metabolismo , Proteínas tau/química , Proteínas tau/metabolismo , Doença de Alzheimer/genética , Animais , Linhagem Celular , Humanos , Camundongos , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Proteólise/efeitos dos fármacos , Proteínas tau/genética
15.
Biochim Biophys Acta Mol Cell Res ; 1867(4): 118647, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31926942

RESUMO

Cisplatin-based chemotherapies have long been considered as a standard chemotherapy in ovarian cancer. However, cisplatin resistance restricts beneficial therapy for patients with ovarian cancer. The ubiquitin-like protein interferon-stimulated gene 15 (ISG15) encodes a 15-kDa protein, that is implicated in the post-translational modification of diverse proteins. In this work, we found that ISG15 was downregulated in cisplatin resistant tissues and cell lines of ovarian cancer. Functional studies demonstrated that overexpression of wild type (WT) ISG15, but not nonISGylatable (Mut) ISG15 increased cell responses to cisplatin in resistant ovarian cancer cells. Furthermore, we found that WT ISG15 decreased ABCC2 expression at the protein level. Importantly, overexpression of ABCC2 blocked sensitizing effect of ISG15 on cisplatin. In addition, we identified that hnRNPA2B1 was recruited to 5'UTR of ABCC2 mRNA and promoted its translation, which was blocked by ISG15. We further demonstrated that hnRNPA2B1 could be ISGylated, and ISGylation blocked its recruitment to ABCC2 mRNA, thereby suppressed translation of ABCC2. Altogether, our data support targeting ISG15 might be a potential therapeutic strategy for patients with cisplatin-resistant ovarian cancer.


Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Citocinas/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Neoplasias Ovarianas/genética , Biossíntese de Proteínas , Ubiquitinas/metabolismo , Regiões 5' não Traduzidas , Adulto , Idoso , Linhagem Celular Tumoral , Citocinas/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Pessoa de Meia-Idade , Proteínas Associadas à Resistência a Múltiplos Medicamentos/biossíntese , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Neoplasias Ovarianas/metabolismo , RNA Mensageiro/metabolismo , Ubiquitinas/genética
16.
Proc Natl Acad Sci U S A ; 117(5): 2473-2483, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31941714

RESUMO

Neddylation is a ubiquitination-like pathway that controls cell survival and proliferation by covalently conjugating NEDD8 to lysines in specific substrate proteins. However, the physiological role of neddylation in mammalian metabolism remains elusive, and no mitochondrial targets have been identified. Here, we report that mouse models with liver-specific deficiency of NEDD8 or ubiquitin-like modifier activating enzyme 3 (UBA3), the catalytic subunit of the NEDD8-activating enzyme, exhibit neonatal death with spontaneous fatty liver as well as hepatic cellular senescence. In particular, liver-specific UBA3 deficiency leads to systemic abnormalities similar to glutaric aciduria type II (GA-II), a rare autosomal recessive inherited fatty acid oxidation disorder resulting from defects in mitochondrial electron transfer flavoproteins (ETFs: ETFA and ETFB) or the corresponding ubiquinone oxidoreductase. Neddylation inhibition by various strategies results in decreased protein levels of ETFs in neonatal livers and embryonic hepatocytes. Hepatic neddylation also enhances ETF expression in adult mice and prevents fasting-induced steatosis and mortality. Interestingly, neddylation is active in hepatic mitochondria. ETFs are neddylation substrates, and neddylation stabilizes ETFs by inhibiting their ubiquitination and degradation. Moreover, certain mutations of ETFs found in GA-II patients hinder the neddylation of these substrates. Taken together, our results reveal substrates for neddylation and add insight into GA-II.


Assuntos
Flavoproteínas Transferidoras de Elétrons/metabolismo , Ácidos Graxos/metabolismo , Fígado/metabolismo , Deficiência Múltipla de Acil Coenzima A Desidrogenase/metabolismo , Animais , Flavoproteínas Transferidoras de Elétrons/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Deficiência Múltipla de Acil Coenzima A Desidrogenase/genética , Proteína NEDD8/genética , Proteína NEDD8/metabolismo , Oxirredução , Ubiquitinação , Ubiquitinas/genética , Ubiquitinas/metabolismo
17.
Theriogenology ; 142: 251-259, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31711690

RESUMO

Our objective was to elucidate differences in endometrial mRNA expressions of interferon-stimulated genes (ISG15, CTSL1, RSAD2, SLC2A1, CXCL10, and SLC27A6), peroxisome proliferator activated receptors (PPARA, PPARD, and PPARG), retinoic acid receptors (RXRA, RXRB, and RXRG), and mucin 1 (MUC1) in repeat breeder cows, with or without subclinical endometritis (RB + SE and RB, respectively) and normal cows on day 16 post-ovulation (n = 4 cows per group). The CXCL10 and SLC27A6 mRNA abundances were greater for normal cows compared to RB and RB + SE cows (P < 0.05 and P < 0.01 respectively) whereas ISG15 and SLC2A1 mRNA abundances were greater for normal cows compared to RB + SE (P < 0.05). The SLC27A6 mRNA abundances were greater for RB versus RB + SE (P < 0.01). Similarly, PPARD, PPARG, RXRA and RXRG mRNA abundances were greater for normal cows compared to RB and RB + SE (P < 0.01 and P < 0.05, respectively). Abundances of PPARD, PPARG, RXRA and RXRG mRNA were greater for RB versus RB + SE (P < 0.05) and MUC1 was lower in abundance in normal cows compared to RB or RB + SE (P < 0.05). Key predicted molecular functions were binding, signal transducer and transporter; key biological processes were cellular, localization and metabolic; key cellular components were cell part, membrane and organelle components; and key protein classes were nucleic acid binding, receptor, and transcription factors. Gene networking analysis highlighted interactions and pathways involving PAPRs, RXRs, and MUC1, notably among PPARD, PPARG, and MUC1. In conclusion, endometrial mRNA expressions of ISGs (CXCL10 and SLC27A6), PPAR isomers (PPARD and PPARG), and RXRs (RXRA and RXRG) were in lower abundances, whereas MUC1 expression was more abundant in RB or RB + SE compared to normal cows on day 16. In addition, ISG15 and SLC2A1 genes were less abundant in RB + SE versus RB or normal cows. Altered expression of these uterine genes and associated potential impairment in embryo elongation and implantation may promote embryonic loss in repeat breeder cows. Furthermore, interactions among PPARD, PPARG and MUC1 may be therapeutically exploitable.


Assuntos
Bovinos/genética , Endometrite/genética , Endométrio/metabolismo , Ovulação/genética , Transcriptoma , Animais , Cruzamento , Bovinos/metabolismo , Doenças dos Bovinos/genética , Doenças dos Bovinos/metabolismo , Doenças dos Bovinos/patologia , Citocinas/genética , Citocinas/metabolismo , Endometrite/metabolismo , Endometrite/patologia , Endometrite/veterinária , Endométrio/patologia , Feminino , Perfilação da Expressão Gênica , Mucina-1/genética , Mucina-1/metabolismo , Ovulação/metabolismo , Paridade/fisiologia , Receptores Ativados por Proliferador de Peroxissomo/genética , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Gravidez , Receptores X Retinoide/genética , Receptores X Retinoide/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismo
18.
ACS Chem Biol ; 14(12): 2538-2545, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31794190

RESUMO

FAT10 is a ubiquitin-like protein suggested to target proteins for proteasomal degradation. It is highly upregulated upon pro-inflammatory cytokines, namely, TNFα, IFNγ, and IL6, and was found to be highly expressed in various epithelial cancers. Evidence suggests that FAT10 is involved in cancer development and may have a pro-tumorigenic role. However, its biological role is still unclear, as well as its biochemical and cellular regulation. To identify pathways underlying FAT10 expression in the context of pro-inflammatory stimulation, which characterizes the cancerous environment, we implemented a phenotypic transcriptional reporter screen with a library of annotated compounds. We identified AZ960, a potent JAK2 inhibitor, which significantly downregulates FAT10 under pro-inflammatory cytokines induction, in an NFκB-independent manner. We validated JAK2 as a major regulator of FAT10 expression via knockdown, and we suggest that the transcriptional effects are mediated through pSTAT1/3/5. Overall, we have elucidated a pathway regulating FAT10 transcription and discovered a tool compound to chemically downregulate FAT10 expression, and to further study its biology.


Assuntos
Janus Quinase 2/metabolismo , Ubiquitinas/metabolismo , Células A549 , Aminopiridinas/farmacologia , Células HEK293 , Humanos , Janus Quinase 2/antagonistas & inibidores , Fenótipo , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia
19.
mBio ; 10(6)2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31796536

RESUMO

Conjugation of small ubiquitin-like modifiers (SUMOs) to substrate proteins is a posttranslational protein modification that affects a diverse range of physiological processes. Global inhibition of SUMO conjugation in mice results in embryonic lethality, reflecting the importance of the SUMO pathways for embryonic development. Here, we demonstrated that SUMO1 overexpression was not well tolerated in murine embryonic carcinoma and embryonic stem (ES) cells and that only a few clones were recovered after transduction with vectors delivering SUMO1 expression constructs. Differentiated NIH/3T3 cells overexpress SUMO1 without deleterious effects and maintain high levels of both conjugated and free forms of SUMO1. The few embryonic cells surviving after forced overexpression retained all their SUMO1 in the form of a few high-molecular-weight conjugates and maintained undetectable levels of free SUMO1. The absence of free SUMO in embryonic cells was seen specifically upon overexpression of SUMO1, but not SUMO2. Moreover, blocking SUMO1 conjugation to endogenous substrates by C-terminal mutations of SUMO1 or by overexpression of a SUMO1 substrate "sponge" or by overexpression of the deSUMOylating enzyme SUMO-specific peptidase 1 (SENP1) dramatically restored free SUMO1 overexpression. The data suggest that overexpression of SUMO1 protein leading to an excess accumulation of critical SUMO1-conjugated substrates is not tolerated in embryonic cells. Surviving embryonic cells exhibit SUMO1 conjugation to allowed substrates but a complete absence of free SUMO1.IMPORTANCE Embryonic stem (ES) cells exhibit unusual transcriptional, proteomic, and signal response profiles, reflecting their unusual needs for rapid differentiation and replication. The work reported here demonstrated that mouse embryonic cell lines did not tolerate the overexpression of SUMO1, the small ubiquitin-like modifier protein that is covalently attached to many substrates to alter their intracellular localization and functionality. Forced SUMO1 overexpression is toxic to ES cells, and surviving cell populations adapt by dramatically reducing the levels of free SUMO1. Such a response is not seen in differentiated cells or with SUMO2 or with nonconjugatable SUMO1 mutants or in the presence of a SUMO1 "sponge" substrate that accepts the modification. The findings suggest that excess SUMO1 modification of specific substrates is not tolerated by embryonic cells and highlight a distinctive need for these cells to control the levels of SUMO1 available for conjugation.


Assuntos
Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteína SUMO-1/metabolismo , Animais , Linhagem Celular , Cisteína Endopeptidases/genética , Endopeptidases/genética , Células HEK293 , Humanos , Camundongos , Mutação , Células NIH 3T3 , Processamento de Proteína Pós-Traducional/genética , Proteômica/métodos , Sumoilação/genética , Ubiquitinas/metabolismo
20.
PLoS One ; 14(12): e0226415, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31869347

RESUMO

Tick-borne nairoviruses (order Bunyavirales) encode an ovarian tumor domain protease (OTU) that suppresses the innate immune response by reversing the post-translational modification of proteins by ubiquitin (Ub) and interferon-stimulated gene product 15 (ISG15). Ub is highly conserved across eukaryotes, whereas ISG15 is only present in vertebrates and shows substantial sequence diversity. Prior attempts to address the effect of ISG15 diversity on viral protein-ISG15 interactions have focused on only a single species' ISG15 or a limited selection of nairovirus OTUs. To gain a more complete perspective of OTU-ISG15 interactions, we biochemically assessed the relative activities of 14 diverse nairovirus OTUs for 12 species' ISG15 and found that ISG15 activity is predominantly restricted to particular nairovirus lineages reflecting, in general, known virus-host associations. To uncover the underlying molecular factors driving OTUs affinity for ISG15, X-ray crystal structures of Kupe virus and Ganjam virus OTUs bound to sheep ISG15 were solved and compared to complexes of Crimean-Congo hemorrhagic fever virus and Erve virus OTUs bound to human and mouse ISG15, respectively. Through mutational and structural analysis seven residues in ISG15 were identified that predominantly influence ISG15 species specificity among nairovirus OTUs. Additionally, OTU residues were identified that influence ISG15 preference, suggesting the potential for viral OTUs to adapt to different host ISG15s. These findings provide a foundation to further develop research methods to trace nairovirus-host relationships and delineate the full impact of ISG15 diversity on nairovirus infection.


Assuntos
Citocinas/metabolismo , Especificidade de Hospedeiro/genética , Nairovirus/enzimologia , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Domínios e Motivos de Interação entre Proteínas/genética , Ubiquitinas/metabolismo , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Cristalografia por Raios X , Células HEK293 , Interações Hospedeiro-Patógeno/genética , Humanos , Camundongos , Modelos Moleculares , Nairovirus/classificação , Nairovirus/genética , Peptídeo Hidrolases/química , Filogenia , Ligação Proteica/genética , Homologia de Sequência , Ovinos , Especificidade da Espécie , Ubiquitina/metabolismo , Proteínas Virais/química , Proteínas Virais/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA