RESUMO
HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), is a critical regulator of inflammation. However, how HOIP itself is regulated to control inflammatory responses is unclear. Here, we discover that site-specific ubiquitination of K784 within human HOIP promotes tumor necrosis factor (TNF)-induced inflammatory signaling. A HOIP K784R mutant is catalytically active but shows reduced induction of an NF-κB reporter relative to wild-type HOIP. HOIP K784 is evolutionarily conserved, equivalent to HOIP K778 in mice. We generated HoipK778R/K778R knock-in mice, which show no overt developmental phenotypes; however, in response to TNF, HoipK778R/K778R mouse embryonic fibroblasts display mildly suppressed NF-κB activation and increased apoptotic markers. On the other hand, HOIP K778R enhances the TNF-induced formation of TNFR complex II and an interaction between TNFR complex II and LUBAC. Loss of the LUBAC component SHARPIN leads to embryonic lethality in HoipK778R/K778R mice, which is rescued by knockout of TNFR1. We propose that site-specific ubiquitination of HOIP regulates a LUBAC-dependent switch between survival and apoptosis in TNF signaling.
Assuntos
Apoptose/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos , Animais , Feminino , Técnicas de Introdução de Genes , Células HEK293 , Humanos , Masculino , Camundongos , NF-kappa B/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral , Transcriptoma , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/farmacologiaRESUMO
HUWE1 is a universal quality-control E3 ligase that marks diverse client proteins for proteasomal degradation. Although the giant HECT enzyme is an essential component of the ubiquitin-proteasome system closely linked with severe human diseases, its molecular mechanism is little understood. Here, we present the crystal structure of Nematocida HUWE1, revealing how a single E3 enzyme has specificity for a multitude of unrelated substrates. The protein adopts a remarkable snake-like structure, where the C-terminal HECT domain heads an extended alpha-solenoid body that coils in on itself and houses various protein-protein interaction modules. Our integrative structural analysis shows that this ring structure is highly dynamic, enabling the flexible HECT domain to reach protein targets presented by the various acceptor sites. Together, our data demonstrate how HUWE1 is regulated by its unique structure, adapting a promiscuous E3 ligase to selectively target unassembled orphan proteins.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Microsporídios/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas Fúngicas , Insetos , Microsporídios/genética , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genéticaRESUMO
Protein turnover is a tightly controlled process that is crucial for the removal of aberrant polypeptides and for cellular signalling. Whereas ubiquitin marks eukaryotic proteins for proteasomal degradation, a general tagging system for the equivalent bacterial Clp proteases is not known. Here we describe the targeting mechanism of the ClpC-ClpP proteolytic complex from Bacillus subtilis. Quantitative affinity proteomics using a ClpP-trapping mutant show that proteins phosphorylated on arginine residues are selectively targeted to ClpC-ClpP. In vitro reconstitution experiments demonstrate that arginine phosphorylation by the McsB kinase is required and sufficient for the degradation of substrate proteins. The docking site for phosphoarginine is located in the amino-terminal domain of the ClpC ATPase, as resolved at high resolution in a co-crystal structure. Together, our data demonstrate that phosphoarginine functions as a bona fide degradation tag for the ClpC-ClpP protease. This system, which is widely distributed across Gram-positive bacteria, is functionally analogous to the eukaryotic ubiquitin-proteasome system.
Assuntos
Arginina/análogos & derivados , Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Endopeptidase Clp/metabolismo , Proteínas Quinases/metabolismo , Proteólise , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Arginina/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Endopeptidase Clp/química , Endopeptidase Clp/genética , Mutação , Compostos Organofosforados/metabolismo , FosforilaçãoRESUMO
The HOIP ubiquitin E3 ligase generates linear ubiquitin chains by forming a complex with HOIL-1L and SHARPIN in mammals. Here, we provide the first evidence of linear ubiquitination induced by a HOIP orthologue in Drosophila We identify Drosophila CG11321, which we named Linear Ubiquitin E3 ligase (LUBEL), and find that it catalyzes linear ubiquitination in vitro We detect endogenous linear ubiquitin chain-derived peptides by mass spectrometry in Drosophila Schneider 2 cells and adult flies. Furthermore, using CRISPR/Cas9 technology, we establish linear ubiquitination-defective flies by mutating residues essential for the catalytic activity of LUBEL Linear ubiquitination signals accumulate upon heat shock in flies. Interestingly, flies with LUBEL mutations display reduced survival and climbing defects upon heat shock, which is also observed upon specific LUBEL depletion in muscle. Thus, LUBEL is involved in the heat response by controlling linear ubiquitination in flies.
Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Drosophila/fisiologia , Resposta ao Choque Térmico/fisiologia , Proteínas de Ligação a RNA/genética , Animais , Catálise , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , Proteínas de Drosophila/metabolismo , Mutação , NF-kappa B/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Myosin motors are critical for diverse motility functions, ranging from cytokinesis and endocytosis to muscle contraction. The UNC-45 chaperone controls myosin function mediating the folding, assembly, and degradation of the muscle protein. Here, we analyze the molecular mechanism of UNC-45 as a hub in myosin quality control. We show that UNC-45 forms discrete complexes with folded and unfolded myosin, forwarding them to downstream chaperones and E3 ligases. Structural analysis of a minimal chaperone:substrate complex reveals that UNC-45 binds to a conserved FX3HY motif in the myosin motor domain. Disrupting the observed interface by mutagenesis prevents myosin maturation leading to protein aggregation in vivo. We also show that a mutation in the FX3HY motif linked to the Freeman Sheldon Syndrome impairs UNC-45 assisted folding, reducing the level of functional myosin. These findings demonstrate that a faulty myosin quality control is a critical yet unexplored cause of human myopathies.
Assuntos
Motivos de Aminoácidos , Miosinas , Dobramento de Proteína , Humanos , Animais , Miosinas/metabolismo , Miosinas/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Mutação , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Ligação Proteica , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
Inhibitor of apoptosis proteins (IAPs) bind to pro-apoptotic proteases, keeping them inactive and preventing cell death. The atypical ubiquitin ligase BIRC6 is the only essential IAP, additionally functioning as a suppressor of autophagy. We performed a structure-function analysis of BIRC6 in complex with caspase-9, HTRA2, SMAC, and LC3B, which are critical apoptosis and autophagy proteins. Cryo-electron microscopy structures showed that BIRC6 forms a megadalton crescent shape that arcs around a spacious cavity containing receptor sites for client proteins. Multivalent binding of SMAC obstructs client binding, impeding ubiquitination of both autophagy and apoptotic substrates. On the basis of these data, we discuss how the BIRC6/SMAC complex can act as a stress-induced hub to regulate apoptosis and autophagy drivers.
Assuntos
Proteínas Reguladoras de Apoptose , Apoptose , Proteínas Inibidoras de Apoptose , Proteínas Mitocondriais , Humanos , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia , Microscopia Crioeletrônica , Proteínas Inibidoras de Apoptose/química , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Ubiquitinação , Multimerização Proteica , Serina Peptidase 2 de Requerimento de Alta Temperatura A/química , Serina Peptidase 2 de Requerimento de Alta Temperatura A/metabolismoRESUMO
The Linear Ubiquitin Chain Assembly Complex (LUBAC), composed of HOIP, HOIL-1L, and SHARPIN, promotes tumor necrosis factor (TNF)-dependent NF-κB signaling in diverse cell types. HOIL-1L contains an Npl4 Zinc Finger (NZF) domain that specifically recognizes linear ubiquitin chains, but its physiological role in vivo has remained unclear. Here, we demonstrate that the HOIL-1L NZF domain has important regulatory functions in inflammation and immune responses in mice. We generated knockin mice (Hoil-1l T201A;R208A/T201A;R208A ) expressing a HOIL-1L NZF mutant and observed attenuated responses to TNF- and LPS-induced shock, including prolonged survival, stabilized body temperature, reduced cytokine production, and liver damage markers. Cells derived from Hoil-1l T201A;R208A/T201A;R208A mice show reduced TNF-dependent NF-κB activation and incomplete recruitment of HOIL-1L into TNF Receptor (TNFR) Complex I. We further show that HOIL-1L NZF cooperates with SHARPIN to prevent TNFR-dependent skin inflammation. Collectively, our data suggest that linear ubiquitin-chain binding by HOIL-1L regulates immune responses and inflammation in vivo.
RESUMO
The linear ubiquitin chain assembly complex (LUBAC) is the only known ubiquitin ligase for linear/Met1-linked ubiquitin chain formation. One of the LUBAC components, heme-oxidized IRP2 ubiquitin ligase 1 (HOIL-1L), was recently shown to catalyse oxyester bond formation between ubiquitin and some substrates. However, oxyester bond formation in the context of LUBAC has not been directly observed. Here, we present the first 3D reconstruction of human LUBAC obtained by electron microscopy and report its generation of heterotypic ubiquitin chains containing linear linkages with oxyester-linked branches. We found that this event depends on HOIL-1L catalytic activity. By cross-linking mass spectrometry showing proximity between the catalytic RING-in-between-RING (RBR) domains, a coordinated ubiquitin relay mechanism between the HOIL-1-interacting protein (HOIP) and HOIL-1L ligases is suggested. In mouse embryonic fibroblasts, these heterotypic chains were induced by TNF, which is reduced in cells expressing an HOIL-1L catalytic inactive mutant. In conclusion, we demonstrate that LUBAC assembles heterotypic ubiquitin chains by the concerted action of HOIP and HOIL-1L.
Assuntos
Fatores de Transcrição , Ubiquitina-Proteína Ligases , Ubiquitina , Animais , Proteínas de Transporte/metabolismo , Células Cultivadas , Feminino , Fibroblastos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Domínios Proteicos , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Myosin is a motor protein that is essential for a variety of processes ranging from intracellular transport to muscle contraction. Folding and assembly of myosin relies on a specific chaperone, UNC-45. To address its substrate-targeting mechanism, we reconstitute the interplay between Caenorhabditis elegans UNC-45 and muscle myosin MHC-B in insect cells. In addition to providing a cellular chaperone assay, the established system enabled us to produce large amounts of functional muscle myosin, as evidenced by a biochemical and structural characterization, and to directly monitor substrate binding to UNC-45. Data from in vitro and cellular chaperone assays, together with crystal structures of binding-deficient UNC-45 mutants, highlight the importance of utilizing a flexible myosin-binding domain. This so-called UCS domain can adopt discrete conformations to efficiently bind and fold substrate. Moreover, our data uncover the molecular basis of temperature-sensitive UNC-45 mutations underlying one of the most prominent motility defects in C. elegans.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Chaperonas Moleculares/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Linhagem Celular , Cristalização , Técnicas In Vitro , Insetos , Chaperonas Moleculares/genética , Mutação , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Estrutura Terciária de ProteínaRESUMO
Autophagy has an important role in cellular homeostasis by degrading and recycling cytotoxic components. Ubiquitination is known to target cargoes for autophagy; however, key components of this pathway remain elusive. Here we performed an RNAi screen to uncover ubiquitin modifiers that are required for starvation-induced macroautophagy in mammalian cells. Our screen uncovered BRUCE/Apollon/Birc6, an IAP protein, as a new autophagy regulator. Depletion of BRUCE leads to defective fusion of autophagosomes and lysosomes. Mechanistically, BRUCE selectively interacts with two ATG8 members GABARAP and GABARAPL1, as well as with Syntaxin 17, which are all critical regulators of autophagosome-lysosome fusion. In addition, BRUCE colocalizes with LAMP2. Interestingly, a non-catalytic N-terminal BRUCE fragment that is sufficient to bind GABARAP/GABARAPL1 and Syntaxin 17, and to colocalize with LAMP2, rescues autolysosome formation in Bruce -/- cells. Thus, BRUCE promotes autolysosome formation independently of its ubiquitin-conjugating activity and is a regulator of both macroautophagy and apoptosis.
Assuntos
Autofagossomos/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Lisossomos/metabolismo , Fusão de Membrana , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Apoptose/genética , Proteínas Reguladoras de Apoptose , Autofagia/genética , Sistemas CRISPR-Cas , Linhagem Celular , Células Cultivadas , Células HEK293 , Humanos , Proteínas Inibidoras de Apoptose/genética , Lisossomos/genética , Camundongos Knockout , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Ligação Proteica , Interferência de RNARESUMO
Peptide-based fluoromethyl ketones have been considered for many years to be highly specific caspase inhibitors distinctly blocking the progress of apoptosis in a variety of systems. Here we demonstrate that these compounds can significantly reduce rhinovirus multiplication in cell culture. In their methylated forms they block eIF4GI cleavage in vivo and in vitro and inhibit the activity of picornaviral 2A proteinases.
Assuntos
Antivirais/metabolismo , Inibidores de Caspase , Endopeptidases/metabolismo , Infecções por Picornaviridae/tratamento farmacológico , Rhinovirus/enzimologia , Antivirais/uso terapêutico , Apoptose , Fator de Iniciação Eucariótico 4G , Células HeLa , Humanos , Fragmentos de Peptídeos/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , RNA Viral/metabolismo , Rhinovirus/genética , Fatores de TempoRESUMO
Ageing results from complex genetically and epigenetically programmed processes that are elicited in part by noxious or stressful events that cause programmed cell death. Here, we report that administration of spermidine, a natural polyamine whose intracellular concentration declines during human ageing, markedly extended the lifespan of yeast, flies and worms, and human immune cells. In addition, spermidine administration potently inhibited oxidative stress in ageing mice. In ageing yeast, spermidine treatment triggered epigenetic deacetylation of histone H3 through inhibition of histone acetyltransferases (HAT), suppressing oxidative stress and necrosis. Conversely, depletion of endogenous polyamines led to hyperacetylation, generation of reactive oxygen species, early necrotic death and decreased lifespan. The altered acetylation status of the chromatin led to significant upregulation of various autophagy-related transcripts, triggering autophagy in yeast, flies, worms and human cells. Finally, we found that enhanced autophagy is crucial for polyamine-induced suppression of necrosis and enhanced longevity.
Assuntos
Autofagia/efeitos dos fármacos , Longevidade/efeitos dos fármacos , Espermidina/farmacologia , Acetilação , Adulto , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/imunologia , Caenorhabditis elegans/fisiologia , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/imunologia , Drosophila melanogaster/fisiologia , Feminino , Células HeLa , Histonas/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Necrose , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/imunologia , Saccharomyces cerevisiae/fisiologiaRESUMO
The replication of many viruses is absolutely dependent on proteolytic cleavage. Infected cells also use this biological mechanism to induce programmed cell death in response to viral infection. Specific inhibitors for both viral and cellular proteases are therefore of vital importance. We have recently shown that the general caspase inhibitor zVAD.fmk inhibits not only caspases, but also the 2Apro of human rhinoviruses (HRVs) (L. Deszcz, J. Seipelt, E. Vassilieva, A. Roetzer, and E. Kuechler, FEBS Lett. 560:51-55, 2004). Here, we describe a derivative of zVAD.fmk that inhibits HRV2 2Apro but that has no effect on caspase 9. This gain in specificity was achieved by replacing the aspartic acid of zVAD.fmk with methionine to generate zVAM.fmk. Methionine was chosen because an oligopeptide with methionine at the P1 position was a much better substrate than an oligopeptide with an alanine residue, which is found at the P1 position of the wild-type HRV2 2Apro cleavage site. zVAM.fmk inhibits the replication of HRV type 2 (HRV2), HRV14, and HRV16. In contrast to zVAD.fmk, however, zVAM.fmk did not inhibit apoptosis induced by puromycin in HeLa cells. zVAM.fmk inhibited in vitro the intermolecular cleavage of eukaryotic initiation factor 4GI (eIF4GI) by HRV2 2Apro at nanomolar concentrations. However, much higher concentrations of zVAM.fmk were required to inhibit HRV14 2Apro cleavage of eIF4GI. In contrast, intramolecular self-processing of HRV14 2Apro was much more susceptible to inhibition by zVAM.fmk than that of HRV2 2Apro, suggesting that zVAM.fmk inhibits HRV2 and HRV14 replication by targeting different reactions of the same proteinase.
Assuntos
Clorometilcetonas de Aminoácidos/farmacologia , Antivirais/farmacologia , Cisteína Endopeptidases/metabolismo , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Inibidores de Caspase , Caspases/metabolismo , Células HeLa , Humanos , Dados de Sequência Molecular , Picornaviridae/efeitos dos fármacos , Picornaviridae/fisiologia , Replicação ViralRESUMO
HeLa and 16HBE14o(-) bronchial epithelium cells infected with human rhinovirus serotype 14 (HRV14) were found to exhibit typical apoptotic morphological alterations, such as cell contraction and nuclear condensation. These events coincided with high-molecular-weight DNA fragmentation, activation of caspase-9 and caspase-3 and poly(ADP-ribose) polymerase cleavage. Caspase activation was preceded by cytochrome c translocation from the mitochondria to the cytoplasm, indicating that apoptosis caused by HRV14 infection was triggered predominantly via the mitochondrial pathway. Apoptosis did not affect HRV14 replication per se, but it facilitated the release of newly formed virus from cells. As apoptosis was fully induced at the time of maximal accumulation of progeny HRV14, it is postulated that apoptosis contributed to the destabilization of the cell and facilitated viral progeny release.
Assuntos
Apoptose , Células Epiteliais/virologia , Rhinovirus/crescimento & desenvolvimento , Caspase 3 , Caspase 9 , Caspases/análise , Núcleo Celular/patologia , Células Cultivadas , Citocromos c/análise , Citoplasma/química , Fragmentação do DNA , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Humanos , Poli(ADP-Ribose) Polimerases/análiseRESUMO
Dendritic cells (DC) are professional APCs with an unmatched ability to interact with and activate T cells. There is accumulating evidence that DC not only efficiently stimulate T cell activation but also regulate T cell responses. However, little is known about cell surface structures on DC involved in the regulation of T cell responses. We demonstrate that human rhinoviruses (HRV) can efficiently inhibit the accessory function of DC through induction of inhibitory cell surface receptors. We observed that treatment of DC with HRV14 (R-DC), a member of the major group HRV family, diminished their T cell stimulatory capacity and induced a promiscuous and deep anergic state in cocultured T cells despite high levels of MHC molecules as well as costimulatory molecules, e.g., B7-1 (CD80) and B7-2 (CD86), and independent of inhibitory soluble factors such as IL-10. In contrast, expression of inhibitory B7-H1 molecules was up-regulated and R-DC de novo expressed sialoadhesin (Sn). Most importantly, blocking of B7-H1 and Sn on R-DC with specific mAbs against both receptors reverted the inhibitory phenotype. Thus, inhibitory signals delivered from R-DC to T cells via B7-H1 and Sn were critical for the induction of anergy. These observations suggest that an altered accessory molecule repertoire on DC upon interaction with HRV down-modulates adaptive immune responses during the viral infection.