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1.
iScience ; 24(11): 103241, 2021 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-34755089

RESUMO

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.

2.
EMBO J ; 39(24): e103303, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33215740

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/farmacologia
3.
FEBS J ; 285(15): 2746-2761, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29679476

RESUMO

Ubiquitin modification (ubiquitination) of target proteins can vary with respect to chain lengths, linkage type, and chain forms, such as homologous, mixed, and branched ubiquitin chains. Thus, ubiquitination can generate multiple unique surfaces on a target protein substrate. Ubiquitin-binding domains (UBDs) recognize ubiquitinated substrates, by specifically binding to these unique surfaces, modulate the formation of cellular signaling complexes and regulate downstream signaling cascades. Among the eight different homotypic chain types, Met1-linked (also termed linear) chains are the only chains in which linkage occurs on a non-Lys residue of ubiquitin. Linear ubiquitin chains have been implicated in immune responses, cell death and autophagy, and several UBDs - specific for linear ubiquitin chains - have been identified. In this review, we describe the main principles of ubiquitin recognition by UBDs, focusing on linear ubiquitin chains and their roles in biology.


Assuntos
Proteínas/química , Proteínas/metabolismo , Ubiquitina/metabolismo , Animais , Sítios de Ligação , Domínio Catalítico , Displasia Ectodérmica/genética , Displasia Ectodérmica/imunologia , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Mutação , Ligação Proteica , Domínios Proteicos , Proteínas/genética , Ubiquitinação , Dedos de Zinco
5.
Cell Rep ; 15(7): 1481-1492, 2016 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-27160902

RESUMO

The HECT domain E3 ligase HACE1 has been identified as a tumor suppressor in multiple cancers. Here, we report that HACE1 is a central gatekeeper of TNFR1-induced cell fate. Genetic inactivation of HACE1 inhibits TNF-stimulated NF-κB activation and TNFR1-NF-κB-dependent pathogen clearance in vivo. Moreover, TNF-induced apoptosis was impaired in hace1 mutant cells and knockout mice in vivo. Mechanistically, HACE1 is essential for the ubiquitylation of the adaptor protein TRAF2 and formation of the apoptotic caspase-8 effector complex. Intriguingly, loss of HACE1 does not impair TNFR1-mediated necroptotic cell fate via RIP1 and RIP3 kinases. Loss of HACE1 predisposes animals to colonic inflammation and carcinogenesis in vivo, which is markedly alleviated by genetic inactivation of RIP3 kinase and TNFR1. Thus, HACE1 controls TNF-elicited cell fate decisions and exerts tumor suppressor and anti-inflammatory activities via a TNFR1-RIP3 kinase-necroptosis pathway.


Assuntos
Linhagem da Célula , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspase 8/metabolismo , Linhagem da Célula/efeitos dos fármacos , Colite/metabolismo , Colite/patologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Sulfato de Dextrana , Embrião de Mamíferos/citologia , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Deleção de Genes , Camundongos Endogâmicos C57BL , Mutação/genética , NF-kappa B/metabolismo , Necrose , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Fator 2 Associado a Receptor de TNF/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitinação/efeitos dos fármacos
6.
Biochem Biophys Res Commun ; 445(2): 304-9, 2014 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-24508257

RESUMO

In the mammalian species studied so far, the L-histidine decarboxylase (HDC) enzyme responsible for histamine biosynthesis has been shown to undergo post-translational processing. The processing is best characterized for the mouse enzyme, where di-asparate DD motifs mediate the production of active ~55 and ~60 kDa isoforms from the ~74 kDa precursor in a caspase-9 dependent manner. The identification of conserved di-aspartate motifs at similar locations in the rat and human HDC protein sequences has led to proposals that these may represent important processing sites in these species also. Here we used transfected Cos7 cells to demonstrate that the rat and human HDC proteins undergo differential processing compared to each other, and found no evidence to suggest that conserved di-aspartate motifs are required absolutely for processing in this cell type. Instead we identified SKD and EEAPD motifs that are important for caspase-6 dependent production of ~54 and ~59 kDa isoforms in the rat and human proteins, respectively. The addition of staurosporine, which is known to pharmacologically activate caspase enzymes, increased processing of the human HDC protein. We propose that caspase-dependent processing is a conserved feature of mammalian HDC enzymes, but that proteolysis may involve different enzymes and occur at diverse sites and sequences.


Assuntos
Caspase 9/metabolismo , Histidina Descarboxilase/genética , Histidina Descarboxilase/metabolismo , Transfecção , Motivos de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Histidina Descarboxilase/química , Humanos , Camundongos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade da Espécie
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