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1.
Cell ; 187(11): 2875-2892.e21, 2024 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-38626770

RESUMO

Ubiquitylation regulates most proteins and biological processes in a eukaryotic cell. However, the site-specific occupancy (stoichiometry) and turnover rate of ubiquitylation have not been quantified. Here we present an integrated picture of the global ubiquitylation site occupancy and half-life. Ubiquitylation site occupancy spans over four orders of magnitude, but the median ubiquitylation site occupancy is three orders of magnitude lower than that of phosphorylation. The occupancy, turnover rate, and regulation of sites by proteasome inhibitors are strongly interrelated, and these attributes distinguish sites involved in proteasomal degradation and cellular signaling. Sites in structured protein regions exhibit longer half-lives and stronger upregulation by proteasome inhibitors than sites in unstructured regions. Importantly, we discovered a surveillance mechanism that rapidly and site-indiscriminately deubiquitylates all ubiquitin-specific E1 and E2 enzymes, protecting them against accumulation of bystander ubiquitylation. The work provides a systems-scale, quantitative view of ubiquitylation properties and reveals general principles of ubiquitylation-dependent governance.


Assuntos
Complexo de Endopeptidases do Proteassoma , Ubiquitinação , Humanos , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Proteólise , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Animais , Camundongos , Linhagem Celular
2.
Cell ; 186(9): 1817-1818, 2023 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-37116466

RESUMO

Proper regulation of protein degradation is essential for cell physiology. In the current issue of Cell, Baek et al. elucidated how a large class of ubiquitin ligase, known as CRL, is assembled and disassembled through a key regulator, CAND1.


Assuntos
Fatores de Transcrição , Ubiquitina-Proteína Ligases , Proteínas Culina/metabolismo , Proteólise , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
3.
Annu Rev Biochem ; 91: 295-319, 2022 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-35320687

RESUMO

Methods to direct the degradation of protein targets with proximity-inducing molecules that coopt the cellular degradation machinery are advancing in leaps and bounds, and diverse modalities are emerging. The most used and well-studied approach is to hijack E3 ligases of the ubiquitin-proteasome system. E3 ligases use specific molecular recognition to determine which proteins in the cell are ubiquitinated and degraded. This review focuses on the structural determinants of E3 ligase recruitment of natural substrates and neo-substrates obtained through monovalent molecular glues and bivalent proteolysis-targeting chimeras. We use structures to illustrate the different types of substrate recognition and assess the basis for neo-protein-protein interactions in ternary complex structures. The emerging structural and mechanistic complexity is reflective of the diverse physiological roles of protein ubiquitination. This molecular insight is also guiding the application of structure-based design approaches to the development of new and existing degraders as chemical tools and therapeutics.


Assuntos
Ubiquitina-Proteína Ligases , Ubiquitina , Proteínas/metabolismo , Proteólise , Especificidade por Substrato , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
4.
Cell ; 184(17): 4464-4479.e19, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34384544

RESUMO

Emerging evidence supports that mitochondrial dysfunction contributes to systemic lupus erythematosus (SLE) pathogenesis. Here we show that programmed mitochondrial removal, a hallmark of mammalian erythropoiesis, is defective in SLE. Specifically, we demonstrate that during human erythroid cell maturation, a hypoxia-inducible factor (HIF)-mediated metabolic switch is responsible for the activation of the ubiquitin-proteasome system (UPS), which precedes and is necessary for the autophagic removal of mitochondria. A defect in this pathway leads to accumulation of red blood cells (RBCs) carrying mitochondria (Mito+ RBCs) in SLE patients and in correlation with disease activity. Antibody-mediated internalization of Mito+ RBCs induces type I interferon (IFN) production through activation of cGAS in macrophages. Accordingly, SLE patients carrying both Mito+ RBCs and opsonizing antibodies display the highest levels of blood IFN-stimulated gene (ISG) signatures, a distinctive feature of SLE.


Assuntos
Interferon Tipo I/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Mitocôndrias/metabolismo , Células Mieloides/metabolismo , Adolescente , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Criança , Pré-Escolar , Eritroblastos/metabolismo , Eritroblastos/ultraestrutura , Eritrócitos/metabolismo , Eritropoese , Humanos , Mitofagia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo
5.
Cell ; 184(13): 3410-3425.e17, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34062120

RESUMO

To control viral infection, vertebrates rely on both inducible interferon responses and less well-characterized cell-intrinsic responses composed of "at the ready" antiviral effector proteins. Here, we show that E3 ubiquitin ligase TRIM7 is a cell-intrinsic antiviral effector that restricts multiple human enteroviruses by targeting viral 2BC, a membrane remodeling protein, for ubiquitination and proteasome-dependent degradation. Selective pressure exerted by TRIM7 results in emergence of a TRIM7-resistant coxsackievirus with a single point mutation in the viral 2C ATPase/helicase. In cultured cells, the mutation helps the virus evade TRIM7 but impairs optimal viral replication, and this correlates with a hyperactive and structurally plastic 2C ATPase. Unexpectedly, the TRIM7-resistant virus has a replication advantage in mice and causes lethal pancreatitis. These findings reveal a unique mechanism for targeting enterovirus replication and provide molecular insight into the benefits and trade-offs of viral evolution imposed by a host restriction factor.


Assuntos
Enterovirus/fisiologia , Enterovirus/patogenicidade , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Replicação Viral/fisiologia , Adenosina Trifosfatases/metabolismo , Animais , Linhagem Celular , Feminino , Humanos , Inflamação/patologia , Camundongos Endogâmicos C57BL , Mutação/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteólise , RNA Viral/metabolismo , Ubiquitina/metabolismo , Proteínas Virais/genética
6.
Cell ; 184(16): 4268-4283.e20, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34233163

RESUMO

Ultraviolet (UV) light and incompletely understood genetic and epigenetic variations determine skin color. Here we describe an UV- and microphthalmia-associated transcription factor (MITF)-independent mechanism of skin pigmentation. Targeting the mitochondrial redox-regulating enzyme nicotinamide nucleotide transhydrogenase (NNT) resulted in cellular redox changes that affect tyrosinase degradation. These changes regulate melanosome maturation and, consequently, eumelanin levels and pigmentation. Topical application of small-molecule inhibitors yielded skin darkening in human skin, and mice with decreased NNT function displayed increased pigmentation. Additionally, genetic modification of NNT in zebrafish alters melanocytic pigmentation. Analysis of four diverse human cohorts revealed significant associations of skin color, tanning, and sun protection use with various single-nucleotide polymorphisms within NNT. NNT levels were independent of UVB irradiation and redox modulation. Individuals with postinflammatory hyperpigmentation or lentigines displayed decreased skin NNT levels, suggesting an NNT-driven, redox-dependent pigmentation mechanism that can be targeted with NNT-modifying topical drugs for medical and cosmetic purposes.


Assuntos
Fator de Transcrição Associado à Microftalmia/metabolismo , NADP Trans-Hidrogenases/metabolismo , Pigmentação da Pele/efeitos da radiação , Raios Ultravioleta , Animais , Linhagem Celular , Estudos de Coortes , AMP Cíclico/metabolismo , Dano ao DNA , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Predisposição Genética para Doença , Humanos , Melanócitos/efeitos dos fármacos , Melanócitos/metabolismo , Melanossomas/efeitos dos fármacos , Melanossomas/metabolismo , Melanossomas/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , NADP Trans-Hidrogenases/antagonistas & inibidores , Oxirredução/efeitos dos fármacos , Oxirredução/efeitos da radiação , Polimorfismo de Nucleotídeo Único/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise/efeitos dos fármacos , Proteólise/efeitos da radiação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Pigmentação da Pele/efeitos dos fármacos , Pigmentação da Pele/genética , Ubiquitina/metabolismo , Peixe-Zebra
7.
Nat Rev Mol Cell Biol ; 24(4): 273-287, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36284179

RESUMO

Our understanding of the ubiquitin code has greatly evolved from conventional E1, E2 and E3 enzymes that modify Lys residues on specific substrates with a single type of ubiquitin chain to more complex processes that regulate and mediate ubiquitylation. In this Review, we discuss recently discovered endogenous mechanisms and unprecedented pathways by which pathogens rewrite the ubiquitin code to promote infection. These processes include unconventional ubiquitin modifications involving ester linkages with proteins, lipids and sugars, or ubiquitylation through a phosphoribosyl bridge involving Arg42 of ubiquitin. We also introduce the enzymatic pathways that write and reverse these modifications, such as the papain-like proteases of severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. Furthermore, structural studies have revealed that the ultimate functions of ubiquitin are mediated not simply by straightforward recognition by ubiquitin-binding domains. Instead, elaborate multivalent interactions between ubiquitylated targets or ubiquitin chains and their readers (for example, the proteasome, the MLL1 complex or DOT1L) can elicit conformational changes that regulate protein degradation or transcription. The newly discovered mechanisms provide opportunities for innovative therapeutic interventions for diseases such as cancer and infectious diseases.


Assuntos
COVID-19 , Ubiquitina , Humanos , Ubiquitina/metabolismo , SARS-CoV-2 , Ubiquitinação , Proteínas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo
8.
Nat Rev Mol Cell Biol ; 24(3): 167-185, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36302887

RESUMO

Autophagy is a process that targets various intracellular elements for degradation. Autophagy can be non-selective - associated with the indiscriminate engulfment of cytosolic components - occurring in response to nutrient starvation and is commonly referred to as bulk autophagy. By contrast, selective autophagy degrades specific targets, such as damaged organelles (mitophagy, lysophagy, ER-phagy, ribophagy), aggregated proteins (aggrephagy) or invading bacteria (xenophagy), thereby being importantly involved in cellular quality control. Hence, not surprisingly, aberrant selective autophagy has been associated with various human pathologies, prominently including neurodegeneration and infection. In recent years, considerable progress has been made in understanding mechanisms governing selective cargo engulfment in mammals, including the identification of ubiquitin-dependent selective autophagy receptors such as p62, NBR1, OPTN and NDP52, which can bind cargo and ubiquitin simultaneously to initiate pathways leading to autophagy initiation and membrane recruitment. This progress opens the prospects for enhancing selective autophagy pathways to boost cellular quality control capabilities and alleviate pathology.


Assuntos
Macroautofagia , Proteínas , Animais , Humanos , Proteínas/metabolismo , Autofagia , Ubiquitina/metabolismo , Mamíferos/metabolismo
9.
Cell ; 182(5): 1156-1169.e12, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32795415

RESUMO

Dysregulated microglia are intimately involved in neurodegeneration, including Alzheimer's disease (AD) pathogenesis, but the mechanisms controlling pathogenic microglial gene expression remain poorly understood. The transcription factor CCAAT/enhancer binding protein beta (c/EBPß) regulates pro-inflammatory genes in microglia and is upregulated in AD. We show expression of c/EBPß in microglia is regulated post-translationally by the ubiquitin ligase COP1 (also called RFWD2). In the absence of COP1, c/EBPß accumulates rapidly and drives a potent pro-inflammatory and neurodegeneration-related gene program, evidenced by increased neurotoxicity in microglia-neuronal co-cultures. Antibody blocking studies reveal that neurotoxicity is almost entirely attributable to complement. Remarkably, loss of a single allele of Cebpb prevented the pro-inflammatory phenotype. COP1-deficient microglia markedly accelerated tau-mediated neurodegeneration in a mouse model where activated microglia play a deleterious role. Thus, COP1 is an important suppressor of pathogenic c/EBPß-dependent gene expression programs in microglia.


Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Ligases/metabolismo , Microglia/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/genética , Doença de Alzheimer/metabolismo , Animais , Linhagem Celular , Técnicas de Cocultura/métodos , Feminino , Expressão Gênica/fisiologia , Regulação da Expressão Gênica/fisiologia , Células HEK293 , Humanos , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo
10.
Nat Rev Mol Cell Biol ; 23(5): 350-367, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35079164

RESUMO

Mammalian development demands precision. Millions of molecules must be properly located in temporal order, and their function regulated, to orchestrate important steps in cell cycle progression, apoptosis, migration and differentiation, to shape developing embryos. Ubiquitin and its associated enzymes act as cellular guardians to ensure precise spatio-temporal control of key molecules during each of these important cellular processes. Loss of precision results in numerous examples of embryological disorders or even cancer. This Review discusses the crucial roles of E3 ubiquitin ligases during key steps of early mammalian development and their roles in human disease, and considers how new methods to manipulate and exploit the ubiquitin regulatory machinery - for example, the development of molecular glues and PROTACs - might facilitate clinical therapy.


Assuntos
Neoplasias , Ubiquitina , Animais , Apoptose , Humanos , Mamíferos/metabolismo , Neoplasias/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
11.
Cell ; 176(6): 1490-1501.e12, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30765112

RESUMO

Methylation of histone H3 K79 by Dot1L is a hallmark of actively transcribed genes that depends on monoubiquitination of H2B K120 (H2B-Ub) and is an example of histone modification cross-talk that is conserved from yeast to humans. We report here cryo-EM structures of Dot1L bound to ubiquitinated nucleosome that show how H2B-Ub stimulates Dot1L activity and reveal a role for the histone H4 tail in positioning Dot1L. We find that contacts mediated by Dot1L and the H4 tail induce a conformational change in the globular core of histone H3 that reorients K79 from an inaccessible position, thus enabling this side chain to insert into the active site in a position primed for catalysis. Our study provides a comprehensive mechanism of cross-talk between histone ubiquitination and methylation and reveals structural plasticity in histones that makes it possible for histone-modifying enzymes to access residues within the nucleosome core.


Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Animais , Domínio Catalítico , Cromatina/metabolismo , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/ultraestrutura , Histonas/química , Histonas/genética , Humanos , Metilação , Modelos Moleculares , Nucleossomos/metabolismo , Processamento de Proteína Pós-Traducional , Receptor Cross-Talk , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinação , Xenopus laevis
12.
Cell ; 177(5): 1187-1200.e16, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31006531

RESUMO

The conventional view posits that E3 ligases function primarily through conjugating ubiquitin (Ub) to their substrate molecules. We report here that RIPLET, an essential E3 ligase in antiviral immunity, promotes the antiviral signaling activity of the viral RNA receptor RIG-I through both Ub-dependent and -independent manners. RIPLET uses its dimeric structure and a bivalent binding mode to preferentially recognize and ubiquitinate RIG-I pre-oligomerized on dsRNA. In addition, RIPLET can cross-bridge RIG-I filaments on longer dsRNAs, inducing aggregate-like RIG-I assemblies. The consequent receptor clustering synergizes with the Ub-dependent mechanism to amplify RIG-I-mediated antiviral signaling in an RNA-length dependent manner. These observations show the unexpected role of an E3 ligase as a co-receptor that directly participates in receptor oligomerization and ligand discrimination. It also highlights a previously unrecognized mechanism by which the innate immune system measures foreign nucleic acid length, a common criterion for self versus non-self nucleic acid discrimination.


Assuntos
Imunidade Inata , RNA de Cadeia Dupla/imunologia , Transdução de Sinais/imunologia , Ubiquitina-Proteína Ligases/imunologia , Ubiquitina/imunologia , Células A549 , Animais , Proteína DEAD-box 58/imunologia , Células HEK293 , Humanos , Camundongos , Receptores Imunológicos
13.
Cell ; 177(2): 286-298.e15, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30929903

RESUMO

The 26S proteasome is the principal macromolecular machine responsible for protein degradation in eukaryotes. However, little is known about the detailed kinetics and coordination of the underlying substrate-processing steps of the proteasome, and their correlation with observed conformational states. Here, we used reconstituted 26S proteasomes with unnatural amino-acid-attached fluorophores in a series of FRET- and anisotropy-based assays to probe substrate-proteasome interactions, the individual steps of the processing pathway, and the conformational state of the proteasome itself. We develop a complete kinetic picture of proteasomal degradation, which reveals that the engagement steps prior to substrate commitment are fast relative to subsequent deubiquitination, translocation, and unfolding. Furthermore, we find that non-ideal substrates are rapidly rejected by the proteasome, which thus employs a kinetic proofreading mechanism to ensure degradation fidelity and substrate prioritization.


Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologia , Anisotropia , Sítios de Ligação/fisiologia , Ativação Enzimática , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Processamento de Proteína Pós-Traducional/fisiologia , Proteólise , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato/fisiologia , Ubiquitina/metabolismo
14.
Annu Rev Cell Dev Biol ; 36: 115-139, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33021827

RESUMO

Lipid droplets (LDs) are endoplasmic reticulum-derived organelles that consist of a core of neutral lipids encircled by a phospholipid monolayer decorated with proteins. As hubs of cellular lipid and energy metabolism, LDs are inherently involved in the etiology of prevalent metabolic diseases such as obesity and nonalcoholic fatty liver disease. The functions of LDs are regulated by a unique set of associated proteins, the LD proteome, which includes integral membrane and peripheral proteins. These proteins control key activities of LDs such as triacylglycerol synthesis and breakdown, nutrient sensing and signal integration, and interactions with other organelles. Here we review the mechanisms that regulate the composition of the LD proteome, such as pathways that mediate selective and bulk LD protein degradation and potential connections between LDs and cellular protein quality control.


Assuntos
Gotículas Lipídicas/metabolismo , Proteínas/metabolismo , Animais , Autofagia , Humanos , Proteólise , Proteoma/metabolismo , Ubiquitina/metabolismo
15.
Annu Rev Biochem ; 87: 697-724, 2018 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-29652515

RESUMO

As the endpoint for the ubiquitin-proteasome system, the 26S proteasome is the principal proteolytic machine responsible for regulated protein degradation in eukaryotic cells. The proteasome's cellular functions range from general protein homeostasis and stress response to the control of vital processes such as cell division and signal transduction. To reliably process all the proteins presented to it in the complex cellular environment, the proteasome must combine high promiscuity with exceptional substrate selectivity. Recent structural and biochemical studies have shed new light on the many steps involved in proteasomal substrate processing, including recognition, deubiquitination, and ATP-driven translocation and unfolding. In addition, these studies revealed a complex conformational landscape that ensures proper substrate selection before the proteasome commits to processive degradation. These advances in our understanding of the proteasome's intricate machinery set the stage for future studies on how the proteasome functions as a major regulator of the eukaryotic proteome.


Assuntos
Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , ATPases Associadas a Diversas Atividades Celulares/química , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Enzimas Desubiquitinantes/química , Enzimas Desubiquitinantes/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/metabolismo , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Ubiquitina/química , Ubiquitina/metabolismo
16.
Annu Rev Biochem ; 87: 751-782, 2018 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-29394096

RESUMO

Cells must constantly monitor the integrity of their macromolecular constituents. Proteins are the most versatile class of macromolecules but are sensitive to structural alterations. Misfolded or otherwise aberrant protein structures lead to dysfunction and finally aggregation. Their presence is linked to aging and a plethora of severe human diseases. Thus, misfolded proteins have to be rapidly eliminated. Secretory proteins constitute more than one-third of the eukaryotic proteome. They are imported into the endoplasmic reticulum (ER), where they are folded and modified. A highly elaborated machinery controls their folding, recognizes aberrant folding states, and retrotranslocates permanently misfolded proteins from the ER back to the cytosol. In the cytosol, they are degraded by the highly selective ubiquitin-proteasome system. This process of protein quality control followed by proteasomal elimination of the misfolded protein is termed ER-associated degradation (ERAD), and it depends on an intricate interplay between the ER and the cytosol.


Assuntos
Degradação Associada com o Retículo Endoplasmático , Proteólise , Proteínas de Saccharomyces cerevisiae/metabolismo , Animais , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Modelos Biológicos , Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína , Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Proteína com Valosina/metabolismo
17.
Immunity ; 57(5): 973-986.e7, 2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38697117

RESUMO

The ubiquitin-binding endoribonuclease N4BP1 potently suppresses cytokine production by Toll-like receptors (TLRs) that signal through the adaptor MyD88 but is inactivated via caspase-8-mediated cleavage downstream of death receptors, TLR3, or TLR4. Here, we examined the mechanism whereby N4BP1 limits inflammatory responses. In macrophages, deletion of N4BP1 prolonged activation of inflammatory gene transcription at late time points after TRIF-independent TLR activation. Optimal suppression of inflammatory cytokines by N4BP1 depended on its ability to bind polyubiquitin chains, as macrophages and mice-bearing inactivating mutations in a ubiquitin-binding motif in N4BP1 displayed increased TLR-induced cytokine production. Deletion of the noncanonical IκB kinases (ncIKKs), Tbk1 and Ikke, or their adaptor Tank phenocopied N4bp1 deficiency and enhanced macrophage responses to TLR1/2, TLR7, or TLR9 stimulation. Mechanistically, N4BP1 acted in concert with the ncIKKs to limit the duration of canonical IκB kinase (IKKα/ß) signaling. Thus, N4BP1 and the ncIKKs serve as an important checkpoint against over-exuberant innate immune responses.


Assuntos
Endorribonucleases , Quinase I-kappa B , Inflamação , Macrófagos , Proteínas Serina-Treonina Quinases , Receptores Toll-Like , Animais , Camundongos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Citocinas/metabolismo , Endorribonucleases/metabolismo , Endorribonucleases/genética , Quinase I-kappa B/metabolismo , Quinase I-kappa B/genética , Inflamação/imunologia , Inflamação/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Receptores Toll-Like/metabolismo , Ubiquitina/metabolismo
18.
Cell ; 173(5): 1231-1243.e16, 2018 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-29731171

RESUMO

Ubiquitination constitutes one of the most important signaling mechanisms in eukaryotes. Conventional ubiquitination is catalyzed by the universally conserved E1-E2-E3 three-enzyme cascade in an ATP-dependent manner. The newly identified SidE family effectors of the pathogen Legionella pneumophila ubiquitinate several human proteins by a different mechanism without engaging any of the conventional ubiquitination machinery. We now report the crystal structures of SidE alone and in complex with ubiquitin, NAD, and ADP-ribose, thereby capturing different conformations of SidE before and after ubiquitin and ligand binding. The structures of ubiquitin bound to both mART and PDE domains reveal several unique features of the two reaction steps catalyzed by SidE. Further, the structural and biochemical results demonstrate that SidE family members do not recognize specific structural folds of the substrate proteins. Our studies provide both structural explanations for the functional observations and new insights into the molecular mechanisms of this non-canonical ubiquitination machinery.


Assuntos
Proteínas de Bactérias/química , Legionella pneumophila/metabolismo , Diester Fosfórico Hidrolases/química , Ubiquitina/química , Proteínas de Bactérias/metabolismo , Biocatálise , Cristalografia por Raios X , Dimerização , Diester Fosfórico Hidrolases/metabolismo , Ligação Proteica , Domínios Proteicos , Estrutura Quaternária de Proteína , Ubiquitina/metabolismo , Ubiquitinação
20.
Annu Rev Biochem ; 86: 193-224, 2017 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-28460188

RESUMO

Autophagy and the ubiquitin-proteasome system are the two major quality control pathways responsible for cellular homeostasis. As such, they provide protection against age-associated changes and a plethora of human diseases. Ubiquitination is utilized as a degradation signal by both systems, albeit in different ways, to mark cargoes for proteasomal and lysosomal degradation. Both systems intersect and communicate at multiple points to coordinate their actions in proteostasis and organelle homeostasis. This review summarizes molecular details of how proteasome and autophagy pathways are functionally interconnected in cells and indicates common principles and nodes of communication that can be therapeutically exploited.


Assuntos
Autofagia/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína Sequestossoma-1/metabolismo , Ubiquitina/metabolismo , Células Eucarióticas/citologia , Células Eucarióticas/metabolismo , Regulação da Expressão Gênica , Homeostase , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/química , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Mitofagia/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Fator 2 Relacionado a NF-E2/química , Fator 2 Relacionado a NF-E2/genética , Conformação Proteica , Proteólise , Proteína Sequestossoma-1/química , Proteína Sequestossoma-1/genética , Transdução de Sinais , Ubiquitina/genética , Ubiquitinação
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