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1.
Sci Rep ; 10(1): 16944, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037310

RESUMO

The Spike protein of the novel coronavirus SARS-CoV2 contains an insertion 680SPRRAR↓SV687 forming a cleavage motif RxxR for furin-like enzymes at the boundary of S1/S2 subunits. Cleavage at S1/S2 is important for efficient viral entry into target cells. The insertion is absent in other CoV-s of the same clade, including SARS-CoV1 that caused the 2003 outbreak. However, an analogous cleavage motif was present at S1/S2 of the Spike protein of the more distant Middle East Respiratory Syndrome coronavirus MERS-CoV. We show that a crucial third arginine at the left middle position, comprising a motif RRxR is required for furin recognition in vitro, while the general motif RxxR in common with MERS-CoV is not sufficient for cleavage. Further, we describe a surprising finding that the two serines at the edges of the insert SPRRAR↓SV can be efficiently phosphorylated by proline-directed and basophilic protein kinases. Both phosphorylations switch off furin's ability to cleave the site. Although phospho-regulation of secreted proteins is still poorly understood, further studies, supported by a recent report of ten in vivo phosphorylated sites in the Spike protein of SARS-CoV2, could potentially uncover important novel regulatory mechanisms for SARS-CoV2.


Assuntos
Betacoronavirus/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Vírus da SARS/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Furina/metabolismo , Fosforilação , Proteólise , Glicoproteína da Espícula de Coronavírus/genética , Internalização do Vírus
2.
Mol Cell ; 79(6): 1008-1023.e4, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32871104

RESUMO

TMPRSS2-ERG gene fusion occurs in approximately 50% of cases of prostate cancer (PCa), and the fusion product is a key driver of prostate oncogenesis. However, how to leverage cellular signaling to ablate TMPRSS2-ERG oncoprotein for PCa treatment remains elusive. Here, we demonstrate that DNA damage induces proteasomal degradation of wild-type ERG and TMPRSS2-ERG oncoprotein through ERG threonine-187 and tyrosine-190 phosphorylation mediated by GSK3ß and WEE1, respectively. The dual phosphorylation triggers ERG recognition and degradation by the E3 ubiquitin ligase FBW7 in a manner independent of a canonical degron. DNA damage-induced TMPRSS2-ERG degradation was abolished by cancer-associated PTEN deletion or GSK3ß inactivation. Blockade of DNA damage-induced TMPRSS2-ERG oncoprotein degradation causes chemotherapy-resistant growth of fusion-positive PCa cells in culture and in mice. Our findings uncover a previously unrecognized TMPRSS2-ERG protein destruction mechanism and demonstrate that intact PTEN and GSK3ß signaling are essential for effective targeting of ERG protein by genotoxic therapeutics in fusion-positive PCa.


Assuntos
Proteínas de Ciclo Celular/genética , Glicogênio Sintase Quinase 3 beta/genética , Proteínas de Fusão Oncogênica/genética , PTEN Fosfo-Hidrolase/genética , Neoplasias da Próstata/genética , Proteínas Tirosina Quinases/genética , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Tratamento Farmacológico , Proteína 7 com Repetições F-Box-WD/genética , Xenoenxertos , Humanos , Masculino , Camundongos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Proteólise/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
3.
PLoS One ; 15(9): e0238089, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32903266

RESUMO

A novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) is the source of a current pandemic (COVID-19) with devastating consequences in public health and economic stability. Using a peptide array to map the antibody response of plasma from healing patients (12) and heathy patients (6), we identified three immunodominant linear epitopes, two of which correspond to key proteolytic sites on the spike protein (S1/S2 and S2') known to be critical for cellular entry. We show biochemical evidence that plasma positive for the epitope adjacent to the S1/S2 cleavage site inhibits furin-mediated proteolysis of spike.


Assuntos
Infecções por Coronavirus/patologia , Epitopos/química , Pneumonia Viral/patologia , Sequência de Aminoácidos , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/virologia , Mapeamento de Epitopos , Epitopos/sangue , Epitopos/imunologia , Furina/metabolismo , Humanos , Pandemias , Ácidos Nucleicos Peptídicos/química , Peptídeos/química , Pneumonia Viral/virologia , Análise Serial de Proteínas , Estrutura Terciária de Proteína , Proteólise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
4.
Nat Commun ; 11(1): 4586, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32934222

RESUMO

Frequent mutation of the tumour suppressor RNF43 is observed in many cancers, particularly colon malignancies. RNF43, an E3 ubiquitin ligase, negatively regulates Wnt signalling by inducing degradation of the Wnt receptor Frizzled. In this study, we discover that RNF43 activity requires phosphorylation at a triplet of conserved serines. This phospho-regulation of RNF43 is required for zebrafish development and growth of mouse intestinal organoids. Cancer-associated mutations that abrogate RNF43 phosphorylation cooperate with active Ras to promote tumorigenesis by abolishing the inhibitory function of RNF43 in Wnt signalling while maintaining its inhibitory function in p53 signalling. Our data suggest that RNF43 mutations cooperate with KRAS mutations to promote multi-step tumorigenesis via the Wnt-Ras-p53 axis in human colon cancers. Lastly, phosphomimetic substitutions of the serine trio restored the tumour suppressive activity of extracellular oncogenic mutants. Therefore, harnessing phospho-regulation of RNF43 might be a potential therapeutic strategy for tumours with RNF43 mutations.


Assuntos
Carcinogênese/metabolismo , Receptores Wnt/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Carcinogênese/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteína Oncogênica p21(ras)/genética , Proteína Oncogênica p21(ras)/metabolismo , Fosforilação , Proteólise , Receptores Wnt/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/genética , Via de Sinalização Wnt
5.
Nat Commun ; 11(1): 4382, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32873802

RESUMO

Fusarium graminearum is a causal agent of Fusarium head blight (FHB) and a deoxynivalenol (DON) producer. In this study, OSP24 is identified as an important virulence factor in systematic characterization of the 50 orphan secreted protein (OSP) genes of F. graminearum. Although dispensable for growth and initial penetration, OSP24 is important for infectious growth in wheat rachis tissues. OSP24 is specifically expressed during pathogenesis and its transient expression suppresses BAX- or INF1-induced cell death. Osp24 is translocated into plant cells and two of its 8 cysteine-residues are required for its function. Wheat SNF1-related kinase TaSnRK1α is identified as an Osp24-interacting protein and shows to be important for FHB resistance in TaSnRK1α-overexpressing or silencing transgenic plants. Osp24 accelerates the degradation of TaSnRK1α by facilitating its association with the ubiquitin-26S proteasome. Interestingly, TaSnRK1α also interacts with TaFROG, an orphan wheat protein induced by DON. TaFROG competes against Osp24 for binding with the same region of TaSnRKα and protects it from degradation. Overexpression of TaFROG stabilizes TaSnRK1α and increases FHB resistance. Taken together, Osp24 functions as a cytoplasmic effector by competing against TaFROG for binding with TaSnRK1α, demonstrating the counteracting roles of orphan proteins of both host and fungal pathogens during their interactions.


Assuntos
Proteínas Fúngicas/metabolismo , Fusarium/patogenicidade , Doenças das Plantas/imunologia , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Triticum/microbiologia , Fatores de Virulência/metabolismo , Resistência à Doença , Fusarium/imunologia , Fusarium/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Plantas Geneticamente Modificadas , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Proteólise , Tricotecenos/metabolismo , Triticum/imunologia
6.
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
7.
Nat Commun ; 11(1): 4625, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32934225

RESUMO

A hallmark of neurodegeneration is defective protein quality control. The E3 ligase Listerin (LTN1/Ltn1) acts in a specialized protein quality control pathway-Ribosome-associated Quality Control (RQC)-by mediating proteolytic targeting of incomplete polypeptides produced by ribosome stalling, and Ltn1 mutation leads to neurodegeneration in mice. Whether neurodegeneration results from defective RQC and whether defective RQC contributes to human disease have remained unknown. Here we show that three independently-generated mouse models with mutations in a different component of the RQC complex, NEMF/Rqc2, develop progressive motor neuron degeneration. Equivalent mutations in yeast Rqc2 selectively interfere with its ability to modify aberrant translation products with C-terminal tails which assist with RQC-mediated protein degradation, suggesting a pathomechanism. Finally, we identify NEMF mutations expected to interfere with function in patients from seven families presenting juvenile neuromuscular disease. These uncover NEMF's role in translational homeostasis in the nervous system and implicate RQC dysfunction in causing neurodegeneration.


Assuntos
Doenças Neuromusculares/metabolismo , Ribossomos/metabolismo , Sequência de Aminoácidos , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Mutação , Doenças Neuromusculares/genética , Doenças Neuromusculares/patologia , Proteólise , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribossomos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência
8.
PLoS Pathog ; 16(9): e1008952, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32960936

RESUMO

Understanding how the protozoan protein degradation pathway is regulated could uncover new parasite biology for drug discovery. We found the COP9 signalosome (CSN) conserved in multiple pathogens such as Leishmania, Trypanosoma, Toxoplasma, and used the severe diarrhea-causing Entamoeba histolytica to study its function in medically significant protozoa. We show that CSN is an essential upstream regulator of parasite protein degradation. Genetic disruption of E. histolytica CSN by two distinct approaches inhibited cell proliferation and viability. Both CSN5 knockdown and dominant negative mutation trapped cullin in a neddylated state, disrupting UPS activity and protein degradation. In addition, zinc ditiocarb (ZnDTC), a main metabolite of the inexpensive FDA-approved globally-available drug disulfiram, was active against parasites acting in a COP9-dependent manner. ZnDTC, given as disulfiram-zinc, had oral efficacy in clearing parasites in vivo. Our findings provide insights into the regulation of parasite protein degradation, and supports the significant therapeutic potential of COP9 inhibition.


Assuntos
Complexo do Signalossomo COP9/metabolismo , Entamoeba histolytica/metabolismo , Proteólise , Animais , Complexo do Signalossomo COP9/genética , Dissulfiram/farmacologia , Ditiocarb/farmacologia , Entamoeba histolytica/genética , Camundongos , Proteínas de Protozoários/genética
9.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 49(4): 514-523, 2020 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-32985166

RESUMO

Neuroligin is a key protein that mediates synaptic development and maturation, and is closely related to neurodevelopmental diseases such as autism. In recent years, researchers have found that neuroligin can be hydrolyzed by various proteases at different stages of development, neuronal activities or pathological states of some neuropsychiatric diseases, thus affecting synaptic activity and participating in the occurrence and development of neurological diseases. The hydrolysates may have different physiological functions from the whole protein, and play different functions in neural activities, such as regulating synaptic plasticity, increasing synaptic strength and number, affecting amyloid-ß polymerization, promoting glioma proliferation and growth, activating related signaling pathways, and so on. In this article, on the basis of elaborating the structure and function of neuroligin as a whole protein, the conditions and products of its hydrolysis are summarized and analyzed, and the functional consequences and physiological significance of its hydrolysis are discussed.


Assuntos
Moléculas de Adesão Celular Neuronais , Proteólise , Moléculas de Adesão Celular Neuronais/metabolismo , Neurônios/fisiologia , Transdução de Sinais/fisiologia , Sinapses/fisiologia
10.
Mol Pharmacol ; 98(3): 267-279, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32817462

RESUMO

Human cytochrome P450 (P450) CYP2B6 undergoes nitric oxide (NO)-dependent proteasomal degradation in response to the NO donor dipropylenetriamine NONOate (DPTA) and biologic NO in HeLa and HuH7 cell lines. CYP2B6 is also downregulated by NO in primary human hepatocytes. We hypothesized that NO or derivative reactive nitrogen species may generate adducts of tyrosine and/or cysteine residues, causing CYP2B6 downregulation, and selected Tyr and Cys residues for mutation based on predicted solvent accessibility. CYP2B6V5-Y317A, -Y380A, and -Y190A mutant proteins expressed in HuH7 cells were less sensitive than wild-type (WT) enzyme to degradation evoked by DPTA, suggesting that these tyrosines are targets for NO-dependent downregulation. The Y317A or Y380A mutants did not show increases in high molecular mass (HMM) species after treatment with DPTA or bortezomib + DPTA, in contrast to the WT enzyme. Carbon monoxide-releasing molecule 2 treatment caused rapid suppression of 2B6 enzyme activity, significant HMM species generation, and ubiquitination of CYP2B6 protein but did not stimulate CYP2B6 degradation. The CYP2B6 inhibitor 4-(4-chlorophenyl)imidazole blocked NO-dependent CYP2B6 degradation, suggesting that NO access to the active site is important. Molecular dynamics simulations predicted that tyrosine nitrations of CYP2B6 would cause significant destabilizing perturbations of secondary structure and remove correlated motions likely required for enzyme function. We propose that cumulative nitrations of Y190, Y317, and Y380 by reactive nitrogen species cause destabilization of CYP2B6, which may act synergistically with heme nitrosylation to target the enzyme for degradation. SIGNIFICANCE STATEMENT: This work provides novel insight into the mechanisms by which nitric oxide, which is produced in hepatocytes in response to inflammation, triggers the ubiquitin-dependent proteasomal degradation of the cytochrome P450 (P450) enzyme CYP2B6. Our data demonstrate that both nitration of specific tyrosine residues and interaction of nitric oxide (NO) with the P450 heme are necessary for NO to trigger ubiquitination and protein degradation.


Assuntos
Citocromo P-450 CYP2B6/química , Citocromo P-450 CYP2B6/metabolismo , Doadores de Óxido Nítrico/farmacologia , Tirosina/química , Linhagem Celular , Citocromo P-450 CYP2B6/genética , Regulação para Baixo , Células HeLa , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Cultura Primária de Células , Proteólise
11.
PLoS One ; 15(8): e0233247, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32857759

RESUMO

Poly(glycine-alanine) (polyGA) is one of the polydipeptides expressed in Frontotemporal Dementia and/or Amyotrophic Lateral Sclerosis 1 caused by C9ORF72 mutations and accumulates as inclusion bodies in the brain of patients. Superficially these inclusions are similar to those formed by polyglutamine (polyQ)-expanded Huntingtin exon 1 (Httex1) in Huntington's disease. Both have been reported to form an amyloid-like structure suggesting they might aggregate via similar mechanisms and therefore recruit the same repertoire of endogenous proteins. When co-expressed in the same cell, polyGA101 and Httex1(Q97) inclusions adopted immiscible phases suggesting different endogenous proteins would be enriched. Proteomic analyses identified 822 proteins in the inclusions. Only 7 were specific to polyGA and 4 specific to Httex1(Q97). Quantitation demonstrated distinct enrichment patterns for the proteins not specific to each inclusion type (up to ~8-fold normalized to total mass). The proteasome, microtubules, TriC chaperones, and translational machinery were enriched in polyGA aggregates, whereas Dnaj chaperones, nuclear envelope and RNA splicing proteins were enriched in Httex1(Q97) aggregates. Both structures revealed a collection of folding and degradation machinery including proteins in the Httex1(Q97) aggregates that are risk factors for other neurodegenerative diseases involving protein aggregation when mutated, which suggests a convergence point in the pathomechanisms of these diseases.


Assuntos
Corpos de Inclusão/metabolismo , Peptídeos/metabolismo , Proteínas/metabolismo , Animais , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Linhagem Celular , Éxons , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Corpos de Inclusão/genética , Corpos de Inclusão/patologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Microscopia Confocal , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Peptídeos/genética , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/patologia , Proteínas/genética , Proteólise , Proteoma/genética , Proteoma/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Risco , Solubilidade
12.
Mol Cell ; 79(6): 963-977.e3, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32735772

RESUMO

Autophagic degradation of the endoplasmic reticulum (ER-phagy) is triggered by ER stress in diverse organisms. However, molecular mechanisms governing ER stress-induced ER-phagy remain insufficiently understood. Here we report that ER stress-induced ER-phagy in the fission yeast Schizosaccharomyces pombe requires Epr1, a soluble Atg8-interacting ER-phagy receptor. Epr1 localizes to the ER through interacting with integral ER membrane proteins VAPs. Bridging an Atg8-VAP association is the main ER-phagy role of Epr1, as it can be bypassed by an artificial Atg8-VAP tether. VAPs contribute to ER-phagy not only by tethering Atg8 to the ER membrane, but also by maintaining the ER-plasma membrane contact. Epr1 is upregulated during ER stress by the unfolded protein response (UPR) regulator Ire1. Loss of Epr1 reduces survival against ER stress. Conversely, increasing Epr1 expression suppresses the ER-phagy defect and ER stress sensitivity of cells lacking Ire1. Our findings expand and deepen the molecular understanding of ER-phagy.


Assuntos
Estresse do Retículo Endoplasmático/genética , Endorribonucleases/genética , Proteínas R-SNARE/genética , Autofagossomos/metabolismo , Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/genética , Retículo Endoplasmático/genética , Regulação Fúngica da Expressão Gênica/genética , Proteólise , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Resposta a Proteínas não Dobradas/genética
13.
Nat Commun ; 11(1): 4311, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855420

RESUMO

Pulmonary disease increases the risk of developing abdominal aortic aneurysms (AAA). However, the mechanism underlying the pathological dialogue between the lungs and aorta is undefined. Here, we find that inflicting acute lung injury (ALI) to mice doubles their incidence of AAA and accelerates macrophage-driven proteolytic damage of the aortic wall. ALI-induced HMGB1 leaks and is captured by arterial macrophages thereby altering their mitochondrial metabolism through RIPK3. RIPK3 promotes mitochondrial fission leading to elevated oxidative stress via DRP1. This triggers MMP12 to lyse arterial matrix, thereby stimulating AAA. Administration of recombinant HMGB1 to WT, but not Ripk3-/- mice, recapitulates ALI-induced proteolytic collapse of arterial architecture. Deletion of RIPK3 in myeloid cells, DRP1 or MMP12 suppression in ALI-inflicted mice repress arterial stress and brake MMP12 release by transmural macrophages thereby maintaining a strengthened arterial framework refractory to AAA. Our results establish an inter-organ circuitry that alerts arterial macrophages to regulate vascular remodeling.


Assuntos
Lesão Pulmonar Aguda/complicações , Aneurisma da Aorta Abdominal/patologia , Proteína HMGB1/metabolismo , Macrófagos/metabolismo , Remodelação Vascular , Lesão Pulmonar Aguda/patologia , Animais , Aorta Abdominal/citologia , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/etiologia , Aneurisma da Aorta Abdominal/prevenção & controle , Células Cultivadas , Modelos Animais de Doenças , Dinaminas/antagonistas & inibidores , Dinaminas/metabolismo , Humanos , Macrófagos/citologia , Metaloproteinase 12 da Matriz/genética , Metaloproteinase 12 da Matriz/metabolismo , Camundongos , Camundongos Knockout , Dinâmica Mitocondrial/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fosforilação , Cultura Primária de Células , Proteólise/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/complicações , Doença Pulmonar Obstrutiva Crônica/patologia , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Estudos Retrospectivos , Regulação para Cima
14.
J Cell Biol ; 219(10)2020 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-32785687

RESUMO

With the rapid global spread of SARS-CoV-2, we have become acutely aware of the inadequacies of our ability to respond to viral epidemics. Although disrupting the viral life cycle is critical for limiting viral spread and disease, it has proven challenging to develop targeted and selective therapeutics. Synthetic lethality offers a promising but largely unexploited strategy against infectious viral disease; as viruses infect cells, they abnormally alter the cell state, unwittingly exposing new vulnerabilities in the infected cell. Therefore, we propose that effective therapies can be developed to selectively target the virally reconfigured host cell networks that accompany altered cellular states to cripple the host cell that has been converted into a virus factory, thus disrupting the viral life cycle.


Assuntos
Antivirais/farmacologia , Interações entre Hospedeiro e Microrganismos/efeitos dos fármacos , Viroses/tratamento farmacológico , Replicação Viral/efeitos dos fármacos , Descoberta de Drogas , Humanos , Fatores Imunológicos/farmacologia , Redes e Vias Metabólicas/efeitos dos fármacos , Mapas de Interação de Proteínas , Proteólise , Vírus de RNA/efeitos dos fármacos , Vírus de RNA/fisiologia , Viroses/genética
15.
J Phys Chem Lett ; 11(16): 6655-6663, 2020 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-32787225

RESUMO

The COVID-19 pandemic is an urgent global health emergency, and the presence of Furin site in the SARS-CoV-2 spike glycoprotein alters virulence and warrants further molecular, structural, and biophysical studies. Here we report the structure of Furin in complex with SARS-CoV-2 spike glycoprotein, demonstrating how Furin binds to the S1/S2 region of spike glycoprotein and eventually cleaves the viral protein using experimental functional studies, molecular dynamics, and docking. The structural studies underline the mechanism and mode of action of Furin, which is a key process in host cell entry and a hallmark of enhanced virulence. Our whole-exome sequencing analysis shows the genetic variants/alleles in Furin were found to alter the binding affinity for viral spike glycoprotein and could vary in infectivity in humans. Unravelling the mechanisms of Furin action, binding dynamics, and the genetic variants opens the growing arena of bona fide antibodies and development of potential therapeutics targeting the blockage of Furin cleavage.


Assuntos
Betacoronavirus/química , Furina/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Virulência/fisiologia , Sequência de Aminoácidos , Animais , Betacoronavirus/patogenicidade , Células CHO , Domínio Catalítico , Cricetulus , Furina/química , Furina/genética , Expressão Gênica/fisiologia , Hexosaminas/metabolismo , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Proteólise , Inibidores de Serino Proteinase/metabolismo , Glicoproteína da Espícula de Coronavírus/química
16.
Nat Commun ; 11(1): 4200, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826910

RESUMO

Chemoproteomics is a key technology to characterize the mode of action of drugs, as it directly identifies the protein targets of bioactive compounds and aids in the development of optimized small-molecule compounds. Current approaches cannot identify the protein targets of a compound and also detect the interaction surfaces between ligands and protein targets without prior labeling or modification. To address this limitation, we here develop LiP-Quant, a drug target deconvolution pipeline based on limited proteolysis coupled with mass spectrometry that works across species, including in human cells. We use machine learning to discern features indicative of drug binding and integrate them into a single score to identify protein targets of small molecules and approximate their binding sites. We demonstrate drug target identification across compound classes, including drugs targeting kinases, phosphatases and membrane proteins. LiP-Quant estimates the half maximal effective concentration of compound binding sites in whole cell lysates, correctly discriminating drug binding to homologous proteins and identifying the so far unknown targets of a fungicide research compound.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Aprendizado de Máquina , Proteoma , Proteômica/métodos , Sítios de Ligação , Botrytis , Sobrevivência Celular , Biologia Computacional/métodos , Descoberta de Drogas/métodos , Células HeLa , Humanos , Ligantes , Espectrometria de Massas , Fosfotransferases/metabolismo , Ligação Proteica , Proteólise , Saccharomyces cerevisiae
17.
Nat Commun ; 11(1): 4268, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32848159

RESUMO

Current efforts in the proteolysis targeting chimera (PROTAC) field mostly focus on choosing an appropriate E3 ligase for the target protein, improving the binding affinities towards the target protein and the E3 ligase, and optimizing the PROTAC linker. However, due to the large molecular weights of PROTACs, their cellular uptake remains an issue. Through comparing how different warhead chemistry, reversible noncovalent (RNC), reversible covalent (RC), and irreversible covalent (IRC) binders, affects the degradation of Bruton's Tyrosine Kinase (BTK), we serendipitously discover that cyano-acrylamide-based reversible covalent chemistry can significantly enhance the intracellular accumulation and target engagement of PROTACs and develop RC-1 as a reversible covalent BTK PROTAC with a high target occupancy as its corresponding kinase inhibitor and effectiveness as a dual functional inhibitor and degrader, a different mechanism-of-action for PROTACs. Importantly, this reversible covalent strategy is generalizable to improve other PROTACs, opening a path to enhance PROTAC efficacy.


Assuntos
Tirosina Quinase da Agamaglobulinemia/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Acrilamidas/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Tirosina Quinase da Agamaglobulinemia/genética , Linhagem Celular , Sobrevivência Celular , Corantes Fluorescentes , Meia-Vida , Humanos , Espaço Intracelular/metabolismo , Ligantes , Simulação de Dinâmica Molecular , Mutação , Fenômenos de Química Orgânica , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteólise
18.
Nat Commun ; 11(1): 3984, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32770009

RESUMO

The epsin family of endocytic adapter proteins are widely expressed, and interact with both proteins and lipids to regulate a variety of cell functions. However, the role of epsins in atherosclerosis is poorly understood. Here, we show that deletion of endothelial epsin proteins reduces inflammation and attenuates atherosclerosis using both cell culture and mouse models of this disease. In atherogenic cholesterol-treated murine aortic endothelial cells, epsins interact with the ubiquitinated endoplasmic reticulum protein inositol 1,4,5-trisphosphate receptor type 1 (IP3R1), which triggers proteasomal degradation of this calcium release channel. Epsins potentiate its degradation via this interaction. Genetic reduction of endothelial IP3R1 accelerates atherosclerosis, whereas deletion of endothelial epsins stabilizes IP3R1 and mitigates inflammation. Reduction of IP3R1 in epsin-deficient mice restores atherosclerotic progression. Taken together, epsin-mediated degradation of IP3R1 represents a previously undiscovered biological role for epsin proteins and may provide new therapeutic targets for the treatment of atherosclerosis and other diseases.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Aterosclerose/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteólise , Proteínas Adaptadoras de Transporte Vesicular/química , Animais , Aorta/metabolismo , Aorta/patologia , Aterosclerose/patologia , Cálcio/metabolismo , Colesterol/metabolismo , Células Endoteliais/metabolismo , Feminino , Deleção de Genes , Células HEK293 , Homeostase , Humanos , Inflamação/patologia , Masculino , Camundongos Knockout , Ligação Proteica , Domínios Proteicos , Ubiquitinação
20.
Mol Cell ; 79(5): 768-781.e7, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32738194

RESUMO

Misfolded proteins in the endoplasmic reticulum (ER) are degraded by ER-associated degradation (ERAD). Although ERAD components involved in degradation of luminal substrates are well characterized, much less is known about quality control of membrane proteins. Here, we analyzed the degradation pathways of two short-lived ER membrane model proteins in mammalian cells. Using a CRISPR-Cas9 genome-wide library screen, we identified an ERAD branch required for quality control of a subset of membrane proteins. Using biochemical and mass spectrometry approaches, we showed that this ERAD branch is defined by an ER membrane complex consisting of the ubiquitin ligase RNF185, the ubiquitin-like domain containing proteins TMUB1/2 and TMEM259/Membralin, a poorly characterized protein. This complex cooperates with cytosolic ubiquitin ligase UBE3C and p97 ATPase in degrading their membrane substrates. Our data reveal that ERAD branches have remarkable specificity for their membrane substrates, suggesting that multiple, perhaps combinatorial, determinants are involved in substrate selection.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Sistema Enzimático do Citocromo P-450/metabolismo , Células HEK293 , Células HeLa , Humanos , Domínios Proteicos , Dobramento de Proteína , Proteólise , Proteínas de Saccharomyces cerevisiae/metabolismo , Esterol 14-Desmetilase/metabolismo
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