Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 19 de 19
Filtrar
1.
Eur J Immunol ; 53(7): e2250056, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37058370

RESUMO

TLRs engage numerous adaptor proteins and signaling molecules, enabling a complex series of post-translational modifications (PTMs) to mount inflammatory responses. TLRs themselves are post-translationally modified following ligand-induced activation, with this being required to relay the full spectrum of proinflammatory signaling responses. Here, we reveal indispensable roles for TLR4 Y672 and Y749 phosphorylation in mounting optimal LPS-inducible inflammatory responses in primary mouse macrophages. LPS promotes phosphorylation at both tyrosine residues, with Y749 phosphorylation being required for maintenance of total TLR4 protein levels and Y672 phosphorylation exerting its pro-inflammatory effects more selectively by initiating ERK1/2 and c-FOS phosphorylation. Our data also support a role for the TLR4-interacting membrane proteins SCIMP and the SYK kinase axis in mediating TLR4 Y672 phosphorylation to permit downstream inflammatory responses in murine macrophages. The corresponding residue in human TLR4 (Y674) is also required for optimal LPS signaling responses. Our study, thus, reveals how a single PTM on one of the most widely studied innate immune receptors orchestrates downstream inflammatory responses.


Assuntos
Citocinas , Lipopolissacarídeos , Humanos , Animais , Camundongos , Fosforilação , Citocinas/metabolismo , Lipopolissacarídeos/farmacologia , Receptor 4 Toll-Like , Tirosina/metabolismo , Tirosina/farmacologia , Macrófagos
2.
J Virol ; 97(11): e0125123, 2023 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-37850747

RESUMO

IMPORTANCE: Dengue virus, an arbovirus, causes an estimated 100 million symptomatic infections annually and is an increasing threat as the mosquito range expands with climate change. Dengue epidemics are a substantial strain on local economies and health infrastructure, and an understanding of what drives severe disease may enable treatments to help reduce hospitalizations. Factors exacerbating dengue disease are debated, but gut-related symptoms are much more frequent in severe than mild cases. Using mouse models of dengue infection, we have shown that inflammation and damage are earlier and more severe in the gut than in other tissues. Additionally, we observed impairment of the gut mucus layer and propose that breakdown of the barrier function exacerbates inflammation and promotes severe dengue disease. This idea is supported by recent data from human patients showing elevated bacteria-derived molecules in dengue patient serum. Therapies aiming to maintain gut integrity may help to abrogate severe dengue disease.


Assuntos
Vírus da Dengue , Dengue Grave , Animais , Humanos , Camundongos , Culicidae , Vírus da Dengue/fisiologia , Inflamação/virologia , Dengue Grave/patologia , Cinética
3.
Mol Cell ; 64(2): 236-250, 2016 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-27746017

RESUMO

Caspase-8 activation can be triggered by death receptor-mediated formation of the death-inducing signaling complex (DISC) and by the inflammasome adaptor ASC. Caspase-8 assembles with FADD at the DISC and with ASC at the inflammasome through its tandem death effector domain (tDED), which is regulated by the tDED-containing cellular inhibitor cFLIP and the viral inhibitor MC159. Here we present the caspase-8 tDED filament structure determined by cryoelectron microscopy. Extensive assembly interfaces not predicted by the previously proposed linear DED chain model were uncovered, and were further confirmed by structure-based mutagenesis in filament formation in vitro and Fas-induced apoptosis and ASC-mediated caspase-8 recruitment in cells. Structurally, the two DEDs in caspase-8 use quasi-equivalent contacts to enable assembly. Using the tDED filament structure as a template, structural analyses reveal the interaction surfaces between FADD and caspase-8 and the distinct mechanisms of regulation by cFLIP and MC159 through comingling and capping, respectively.


Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/química , Caspase 8/química , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/química , Proteína de Domínio de Morte Associada a Fas/química , Proteínas Virais/química , Sequência de Aminoácidos , Apoptose/efeitos dos fármacos , Sítios de Ligação , Proteínas Adaptadoras de Sinalização CARD , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Microscopia Crioeletrônica , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Domínio Efetor de Morte , Proteína de Domínio de Morte Associada a Fas/genética , Proteína de Domínio de Morte Associada a Fas/metabolismo , Expressão Gênica , Humanos , Células Jurkat , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transfecção , Proteínas Virais/genética , Proteínas Virais/metabolismo , Receptor fas/farmacologia
4.
J Enzyme Inhib Med Chem ; 39(1): 2313055, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38416868

RESUMO

Toll-like receptor (TLR) innate immunity signalling protects against pathogens, but excessive or prolonged signalling contributes to a range of inflammatory conditions. Structural information on the TLR cytoplasmic TIR (Toll/interleukin-1 receptor) domains and the downstream adaptor proteins can help us develop inhibitors targeting this pathway. The small molecule o-vanillin has previously been reported as an inhibitor of TLR2 signalling. To study its mechanism of action, we tested its binding to the TIR domain of the TLR adaptor MAL/TIRAP (MALTIR). We show that o-vanillin binds to MALTIR and inhibits its higher-order assembly in vitro. Using NMR approaches, we show that o-vanillin forms a covalent bond with lysine 210 of MAL. We confirm in mouse and human cells that o-vanillin inhibits TLR2 but not TLR4 signalling, independently of MAL, suggesting it may covalently modify TLR2 signalling complexes directly. Reactive aldehyde-containing small molecules such as o-vanillin may target multiple proteins in the cell.


Assuntos
Benzaldeídos , Lisina , Receptor 2 Toll-Like , Humanos , Animais , Camundongos , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Receptores Toll-Like/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Interleucina-1/metabolismo
5.
J Biol Chem ; 298(12): 102666, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36334634

RESUMO

Conventional assays to probe signaling protein interactions and function involve measurement of luciferase reporter expression within the bulk cell population, with lack of control over target-protein expression level. To address this issue, we have developed a rapid and robust flow cytometric assay for analysis of signaling protein function. A fluorescent reporter and fluorescent tagging of the target protein enables simultaneous assessment of protein expression and signaling within individual cells. We have applied our technique to the analysis of variants of the lipopolysaccharide receptor Toll-like receptor 4 (TLR4) and its adapter protein MyD88, using a NF-кB-responsive promoter driving mScarlet-I expression. The assay enables exclusion of nontransfected cells and overexpressing cells that signal spontaneously. Additionally, our assay allows the identification of protein variants that fail to express. We found that the assays were highly sensitive, with cells expressing an appropriate level of GFP-MyD88 showing approximately 200-fold induction of mScarlet-I by lipopolysaccharide, and we can detect subtle protein concentration-dependent effects of mutations. Importantly, the assay is adaptable to various signaling pathways.


Assuntos
Lipopolissacarídeos , Fator 88 de Diferenciação Mieloide , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Transdução de Sinais , Humanos
6.
J Immunol ; 194(1): 455-62, 2015 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-25404358

RESUMO

Inflammasomes are large protein complexes induced by a wide range of microbial, stress, and environmental stimuli that function to induce cell death and inflammatory cytokine processing. Formation of an inflammasome involves dramatic relocalization of the inflammasome adapter protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) into a single speck. We have developed a flow cytometric assay for inflammasome formation, time of flight inflammasome evaluation, which detects the change in ASC distribution within the cell. The transit of ASC into the speck is detected by a decreased width or increased height of the pulse of emitted fluorescence. This assay can be used to quantify native inflammasome formation in subsets of mixed cell populations ex vivo. It can also provide a rapid and sensitive technique for investigating molecular interactions in inflammasome formation, by comparison of wild-type and mutant proteins in inflammasome reconstitution experiments.


Assuntos
Proteínas Reguladoras de Apoptose/imunologia , Citometria de Fluxo/métodos , Inflamassomos/imunologia , Animais , Apoptose/imunologia , Proteínas Reguladoras de Apoptose/genética , Células da Medula Óssea/imunologia , Proteínas Adaptadoras de Sinalização CARD/imunologia , Caspase 1/genética , Linhagem Celular , Células HEK293 , Humanos , Inflamassomos/análise , Mediadores da Inflamação/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Knockout
7.
J Biol Chem ; 290(49): 29217-30, 2015 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-26468282

RESUMO

Inflammasomes mediate inflammatory and cell death responses to pathogens and cellular stress signals via activation of procaspases-1 and -8. During inflammasome assembly, activated receptors of the NLR or PYHIN family recruit the adaptor protein ASC and initiate polymerization of its pyrin domain (PYD) into filaments. We show that ASC filaments in turn nucleate procaspase-8 death effector domain (DED) filaments in vitro and in vivo. Interaction between ASC PYD and procaspase-8 tandem DEDs optimally required both DEDs and represents an unusual heterotypic interaction between domains of the death fold superfamily. Analysis of ASC PYD mutants showed that interaction surfaces that mediate procaspase-8 interaction overlap with those required for ASC self-association and interaction with the PYDs of inflammasome initiators. Our data indicate that multiple types of death fold domain filaments form at inflammasomes and that PYD/DED and homotypic PYD interaction modes are similar. Interestingly, we observed condensation of procaspase-8 filaments containing the catalytic domain, suggesting that procaspase-8 interactions within and/or between filaments may be involved in caspase-8 activation. Procaspase-8 filaments may also be relevant to apoptosis induced by death receptors.


Assuntos
Caspase 8/metabolismo , Proteínas do Citoesqueleto/metabolismo , Inflamassomos/metabolismo , Apoptose , Proteínas Adaptadoras de Sinalização CARD , Caspase 1/metabolismo , Domínio Catalítico , Morte Celular , Células HEK293 , Humanos , Inflamação , Microscopia de Fluorescência , Mutação , Ligação Proteica , Transdução de Sinais
8.
J Biol Chem ; 289(34): 23504-19, 2014 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-25006247

RESUMO

Inflammasomes are macromolecular complexes that mediate inflammatory and cell death responses to pathogens and cellular stress signals. Dysregulated inflammasome activation is associated with autoinflammatory syndromes and several common diseases. During inflammasome assembly, oligomerized cytosolic pattern recognition receptors recruit procaspase-1 and procaspase-8 via the adaptor protein ASC. Inflammasome assembly is mediated by pyrin domains (PYDs) and caspase recruitment domains, which are protein interaction domains of the death fold superfamily. However, the molecular details of their interactions are poorly understood. We have studied the interaction between ASC and pyrin PYDs that mediates ASC recruitment to the pyrin inflammasome, which is implicated in the pathogenesis of familial Mediterranean fever. We demonstrate that both the ASC and pyrin PYDs have multifaceted binding modes, involving three sites on pyrin PYD and two sites on ASC PYD. Molecular docking of pyrin-ASC PYD complexes showed that pyrin PYD can simultaneously interact with up to three ASC PYDs. Furthermore, ASC PYD can self-associate and interact with pyrin, consistent with previous reports that pyrin promotes ASC clustering to form a proinflammatory complex. Finally, the effects of familial Mediterranean fever-associated mutations, R42W and A89T, on structural and functional properties of pyrin PYD were investigated. The R42W mutation had a significant effect on structure and increased stability. Although the R42W mutant exhibited reduced interaction with ASC, it also bound less to the pyrin B-box domain responsible for autoinhibition and hence may be constitutively active. Our data give new insights into the binding modes of PYDs and inflammasome architecture.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Inflamassomos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Proteínas Adaptadoras de Sinalização CARD , Proteínas do Citoesqueleto/genética , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Pirina , Homologia de Sequência de Aminoácidos
9.
J Biol Chem ; 287(50): 41732-43, 2012 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-23066025

RESUMO

A key process underlying an innate immune response to pathogens or cellular stress is activation of members of the NOD-like receptor family, such as NLRP3, to assemble caspase-1-activating inflammasome complexes. Activated caspase-1 processes proinflammatory cytokines into active forms that mediate inflammation. Activation of the NLRP3 inflammasome is also associated with common diseases including cardiovascular disease, diabetes, chronic kidney disease, and Alzheimer disease. However, the molecular details of NLRP3 inflammasome assembly are not established. The adaptor protein ASC plays a key role in inflammasome assembly. It is composed of an N-terminal pyrin domain (PYD) and a C-terminal caspase recruitment domain, which are protein interaction domains of the death fold superfamily. ASC interacts with NLRP3 via a homotypic PYD interaction and recruits procaspase-1 via a homotypic caspase recruitment domain interaction. Here we demonstrate that ASC PYD contains two distinct binding sites important for self-association and interaction with NLRP3 and the modulatory protein POP1. Modeling of the homodimeric ASC PYD complex formed via an asymmetric interaction using both sites resembles a type I interaction found in other death fold domain complexes. This interaction mode also permits assembly of ASC PYDs into filaments. Furthermore, a type I binding mode is likely conserved in interactions with NLRP3 and POP1, because residues critical for interaction of ASC PYD are conserved in these PYDs. We also demonstrate that ASC PYD can simultaneously self-associate and interact with NLRP3, rationalizing the model whereby ASC self-association upon recruitment to NLRP3 promotes clustering and activation of procaspase-1.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/metabolismo , Modelos Biológicos , Multimerização Proteica/fisiologia , Sítios de Ligação/fisiologia , Proteínas Adaptadoras de Sinalização CARD , Proteínas de Transporte/genética , Caspase 1/genética , Caspase 1/metabolismo , Proteínas do Citoesqueleto/genética , Células HEK293 , Humanos , Inflamassomos/genética , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Ligação Proteica , Estrutura Terciária de Proteína/fisiologia
10.
Acta Crystallogr D Biol Crystallogr ; 69(Pt 5): 774-84, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23633586

RESUMO

The caspase recruitment domain (CARD) is present in death-domain superfamily proteins involved in inflammation and apoptosis. BinCARD is named for its ability to interact with Bcl10 and inhibit downstream signalling. Human BinCARD is expressed as two isoforms that encode the same N-terminal CARD region but which differ considerably in their C-termini. Both isoforms are expressed in immune cells, although BinCARD-2 is much more highly expressed. Crystals of the CARD fold common to both had low symmetry (space group P1). Molecular replacement was unsuccessful in this low-symmetry space group and, as the construct contains no methionines, first one and then two residues were engineered to methionine for MAD phasing. The double-methionine variant was produced as a selenomethionine derivative, which was crystallized and the structure was solved using data measured at two wavelengths. The crystal structures of the native and selenomethionine double mutant were refined to high resolution (1.58 and 1.40 Šresolution, respectively), revealing the presence of a cis-peptide bond between Tyr39 and Pro40. Unexpectedly, the native crystal structure revealed that all three cysteines were oxidized. The mitochondrial localization of BinCARD-2 and the susceptibility of its CARD region to redox modification points to the intriguing possibility of a redox-regulatory role.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/química , Proteínas/química , Proteínas/metabolismo , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Cristalografia por Raios X , Cisteína/metabolismo , Células HeLa , Humanos , Mitocôndrias/metabolismo , Modelos Moleculares , Mutação , Oxirredução , Prolina/química , Conformação Proteica , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Proteínas/genética , Selenometionina
11.
Nat Commun ; 12(1): 2578, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33972532

RESUMO

MyD88 and MAL are Toll-like receptor (TLR) adaptors that signal to induce pro-inflammatory cytokine production. We previously observed that the TIR domain of MAL (MALTIR) forms filaments in vitro and induces formation of crystalline higher-order assemblies of the MyD88 TIR domain (MyD88TIR). These crystals are too small for conventional X-ray crystallography, but are ideally suited to structure determination by microcrystal electron diffraction (MicroED) and serial femtosecond crystallography (SFX). Here, we present MicroED and SFX structures of the MyD88TIR assembly, which reveal a two-stranded higher-order assembly arrangement of TIR domains analogous to that seen previously for MALTIR. We demonstrate via mutagenesis that the MyD88TIR assembly interfaces are critical for TLR4 signaling in vivo, and we show that MAL promotes unidirectional assembly of MyD88TIR. Collectively, our studies provide structural and mechanistic insight into TLR signal transduction and allow a direct comparison of the MicroED and SFX techniques.


Assuntos
Cristalografia/métodos , Glicoproteínas de Membrana/química , Fator 88 de Diferenciação Mieloide/química , Receptores de Interleucina-1/química , Receptor 4 Toll-Like/química , Dimerização , Células HEK293 , Humanos , Glicoproteínas de Membrana/genética , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação , Fator 88 de Diferenciação Mieloide/genética , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios Proteicos , Receptores de Interleucina-1/genética , Proteínas Recombinantes , Transdução de Sinais/genética , Receptor 4 Toll-Like/genética
12.
FEBS J ; 275(7): 1427-1449, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18266866

RESUMO

Sorting of membrane proteins into intralumenal endosomal vesicles, multivesicular body (MVB) sorting, is critical for receptor down regulation, antigen presentation and enveloped virus budding. Vps4 is an AAA ATPase that functions in MVB sorting. Although AAA ATPases are oligomeric, mechanisms that govern Vps4 oligomerization and activity remain elusive. Vps4 has an N-terminal microtubule interacting and trafficking domain required for endosome recruitment, an AAA domain containing the ATPase catalytic site and a beta domain, and a C-terminal alpha helix positioned close to the catalytic site in the 3D structure. Previous attempts to identify the role of the C-terminal helix have been unsuccessful. Here, we show that the C-terminal helix is important for Vps4 assembly and ATPase activity in vitro and function in vivo, but not endosome recruitment or interactions with Vta1 or ESCRT-III. Unlike the beta domain, which is also important for Vps4 assembly, the C-terminal helix is not required in vivo for Vps4 homotypic interaction or dominant-negative effects of Vps4-E233Q, carrying a mutation in the ATP hydrolysis site. Vta1 promotes assembly of hybrid complexes comprising Vps4-E233Q and Vps4 lacking an intact C-terminal helix in vitro. Formation of catalytically active hybrid complexes demonstrates an intersubunit catalytic mechanism for Vps4. One end of the C-terminal helix lies in close proximity to the second region of homology (SRH), which is important for assembly and intersubunit catalysis in AAA ATPases. We propose that Vps4 SRH function requires an intact C-terminal helix. Co-evolution of a distinct Vps4 SRH and C-terminal helix in meiotic clade AAA ATPases supports this possibility.


Assuntos
Adenosina Trifosfatases/química , Sequência Conservada , Proteínas de Saccharomyces cerevisiae/química , Homologia de Sequência de Aminoácidos , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/fisiologia , Sequência de Aminoácidos , Catálise , Complexos Endossomais de Distribuição Requeridos para Transporte , Endossomos/metabolismo , Humanos , Meiose/fisiologia , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia
13.
J Mol Biol ; 430(2): 238-247, 2018 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-29100888

RESUMO

Canonical inflammasomes are multiprotein complexes that can activate both caspase-1 and caspase-8. Caspase-1 drives rapid lysis of cells by pyroptosis and maturation of interleukin (IL)-1ß and IL-18. In caspase-1-deficient cells, inflammasome formation still leads to caspase-3 activation and slower apoptotic death, dependent on caspase-8 as an apical caspase. A role for caspase-8 directly upstream of caspase-1 has also been suggested, but here we show that caspase-8-deficient macrophages have no defect in AIM2 inflammasome-mediated caspase-1 activation, pyroptosis, and IL-1ß cleavage. In investigating the inflammasome-induced apoptotic pathway, we previously demonstrated that activated caspase-8 is essential for caspase-3 cleavage and apoptosis in caspase-1-deficient cells. However, here we found that AIM2 inflammasome-initiated caspase-3 cleavage was maintained in Ripk3-/-Casp8-/- macrophages. Gene knockdown showed that caspase-1 was required for the caspase-3 cleavage. Thus inflammasomes activate a network of caspases that can promote both pyroptotic and apoptotic cell death. In cells where rapid pyroptosis is blocked, delayed inflammasome-dependent cell death could still occur due to both caspase-1- and caspase-8-dependent apoptosis. Initiation of redundant cell death pathways is likely to be a strategy for coping with pathogen interference in death processes.


Assuntos
Caspase 1/imunologia , Caspase 3/imunologia , Caspase 8/imunologia , Proteínas de Ligação a DNA/imunologia , Inflamassomos/imunologia , Animais , Apoptose , Caspase 8/genética , Deleção de Genes , Camundongos Endogâmicos C57BL , Piroptose
14.
FEBS J ; 274(8): 1894-907, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17408385

RESUMO

During endocytic transport, specific integral membrane proteins are sorted into intraluminal vesicles that bud from the limiting membrane of the endosome. This process, known as multivesicular body (MVB) sorting, is important for several important biological processes. Moreover, components of the MVB sorting machinery are implicated in virus budding. During MVB sorting, a cargo protein recruits components of the MVB sorting machinery from cytoplasmic pools and these sequentially assemble on the endosome. Disassembly of these proteins and recycling into the cytoplasm is critical for MVB sorting. Vacuolar protein sorting 4 (Vps4) is an AAA (ATPase associated with a variety of cellular activities) ATPase which has been proposed to play a critical role in disassembly of the MVB sorting machinery. However, the mechanism by which it disassembles the complex is not clear. Vps4 contains an N-terminal microtubule interacting and trafficking (MIT) domain, which has previously been shown to be required for recruitment to endosomes, and a single AAA ATPase domain, the activity of which is required for Vps4 function. In this study we have systematically characterized the interaction of Vps4 with other components of the MVB sorting machinery. We demonstrate that Vps4 interacts directly with Vps2 and Bro1. We also show that a subset of Vps4 interactions is regulated by ATP hydrolysis, and one interaction is regulated by ATP binding. Finally, we show that most proteins interact with the Vps4 MIT domain. Our studies indicate that the MIT domain has a dual role in substrate binding and recruitment to endosomes and indicate that Vps4 disassembles the MVB sorting machinery by direct effects on multiple proteins.


Assuntos
Adenosina Trifosfatases/fisiologia , Endossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Adenosina Trifosfatases/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Complexos Endossomais de Distribuição Requeridos para Transporte , Hidrólise , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas de Saccharomyces cerevisiae/química
15.
FEBS J ; 274(16): 4103-25, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17635585

RESUMO

Vrp1p (verprolin, End5p) is the yeast ortholog of human Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP). Vrp1p localizes to the cortical actin cytoskeleton, is necessary for its polarization to sites of growth and is also essential for endocytosis. At elevated temperature, Vrp1p becomes essential for growth. A C-terminal Vrp1p fragment (C-Vrp1p) retains the ability to localize to the cortical actin cytoskeleton and function in actin-cytoskeleton polarization, endocytosis and growth. Here, we demonstrate that two submodules in C-Vrp1p are required for actin-cytoskeleton polarization: a novel C-terminal actin-binding submodule (CABS) that contains a novel G-actin-binding domain, which we call a verprolin homology 2 C-terminal (VH2-C) domain; and a second submodule comprising the Las17p-binding domain (LBD) that binds Las17p (yeast WASP). The LBD localizes C-Vrp1p to membranes and the cortical actin cytoskeleton. Intriguingly, the LBD is sufficient to restore endocytosis and growth at elevated temperature to Vrp1p-deficient cells. The CABS also restores these functions, but only if modified by a lipid anchor to provide membrane association. Our findings highlight the role of Las17p binding for Vrp1p membrane association, suggest general membrane association may be more important than specific targeting to the cortical actin cytoskeleton for Vrp1p function in endocytosis and cell growth, and suggest that Vrp1p binding to individual effectors may alter their physiological activity.


Assuntos
Actinas/metabolismo , Endocitose/fisiologia , Proteínas dos Microfilamentos/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Proteína da Síndrome de Wiskott-Aldrich/fisiologia , Sequência de Aminoácidos , Arginina/genética , Arginina/metabolismo , Sítios de Ligação/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Lisina/genética , Lisina/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microscopia de Fluorescência , Dados de Sequência Molecular , Plasmídeos/genética , Plasmídeos/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Temperatura , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismo
16.
Mol Immunol ; 86: 23-37, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28249680

RESUMO

The innate immune system is the first line of defense against infection and responses are initiated by pattern recognition receptors (PRRs) that detect pathogen-associated molecular patterns (PAMPs). PRRs also detect endogenous danger-associated molecular patterns (DAMPs) that are released by damaged or dying cells. The major PRRs include the Toll-like receptor (TLR) family members, the nucleotide binding and oligomerization domain, leucine-rich repeat containing (NLR) family, the PYHIN (ALR) family, the RIG-1-like receptors (RLRs), C-type lectin receptors (CLRs) and the oligoadenylate synthase (OAS)-like receptors and the related protein cyclic GMP-AMP synthase (cGAS). The different PRRs activate specific signaling pathways to collectively elicit responses including the induction of cytokine expression, processing of pro-inflammatory cytokines and cell-death responses. These responses control a pathogenic infection, initiate tissue repair and stimulate the adaptive immune system. A central theme of many innate immune signaling pathways is the clustering of activated PRRs followed by sequential recruitment and oligomerization of adaptors and downstream effector enzymes, to form higher-order arrangements that amplify the response and provide a scaffold for proximity-induced activation of the effector enzymes. Underlying the formation of these complexes are co-operative assembly mechanisms, whereby association of preceding components increases the affinity for downstream components. This ensures a rapid immune response to a low-level stimulus. Structural and biochemical studies have given key insights into the assembly of these complexes. Here we review the current understanding of assembly of immune signaling complexes, including inflammasomes initiated by NLR and PYHIN receptors, the myddosomes initiated by TLRs, and the MAVS CARD filament initiated by RIG-1. We highlight the co-operative assembly mechanisms during assembly of each of these complexes.


Assuntos
Imunidade Inata , Inflamassomos/imunologia , Proteínas NLR/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Transdução de Sinais/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Domínio de Ativação e Recrutamento de Caspases , Proteína DEAD-box 58/metabolismo , Fungos/fisiologia , Humanos , Proteínas NLR/química , Plantas/imunologia , Receptores Imunológicos , Receptores de Reconhecimento de Padrão/química , Receptores Toll-Like/metabolismo
17.
Nat Struct Mol Biol ; 24(9): 743-751, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28759049

RESUMO

Toll-like receptor (TLR) signaling is a key innate immunity response to pathogens. Recruitment of signaling adapters such as MAL (TIRAP) and MyD88 to the TLRs requires Toll/interleukin-1 receptor (TIR)-domain interactions, which remain structurally elusive. Here we show that MAL TIR domains spontaneously and reversibly form filaments in vitro. They also form cofilaments with TLR4 TIR domains and induce formation of MyD88 assemblies. A 7-Å-resolution cryo-EM structure reveals a stable MAL protofilament consisting of two parallel strands of TIR-domain subunits in a BB-loop-mediated head-to-tail arrangement. Interface residues that are important for the interaction are conserved among different TIR domains. Although large filaments of TLR4, MAL or MyD88 are unlikely to form during cellular signaling, structure-guided mutagenesis, combined with in vivo interaction assays, demonstrated that the MAL interactions defined within the filament represent a template for a conserved mode of TIR-domain interaction involved in both TLR and interleukin-1 receptor signaling.


Assuntos
Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/metabolismo , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/ultraestrutura , Fator 88 de Diferenciação Mieloide/metabolismo , Fator 88 de Diferenciação Mieloide/ultraestrutura , Multimerização Proteica , Receptor 4 Toll-Like/metabolismo , Receptor 4 Toll-Like/ultraestrutura , Linhagem Celular , Microscopia Crioeletrônica , Análise Mutacional de DNA , Humanos , Modelos Moleculares , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/genética , Conformação Proteica , Domínios Proteicos , Transdução de Sinais
18.
FEBS J ; 273(11): 2357-73, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16704411

RESUMO

Endocytic and biosynthetic trafficking pathways to the lysosome/vacuole converge at the prevacuolar endosomal compartment. During transport through this compartment, integral membrane proteins that are destined for delivery to the lysosome/vacuole lumen undergo multivesicular body (MVB) sorting into internal vesicles formed by invagination of the endosomal limiting membrane. Vps4 is an AAA family ATPase which plays a key role in MVB sorting and facilitates transport through endosomes. It possesses an N-terminal microtubule interacting and trafficking domain required for recruitment to endosomes and an AAA domain with an ATPase catalytic site. The recently solved 3D structure revealed a beta domain, which protrudes from the AAA domain, and a final C-terminal alpha-helix. However, the in vivo roles of these domains are not known. In this study, we have identified motifs in these domains that are highly conserved between yeast and human Vps4. We have mutated these motifs and studied the effect on yeast Vps4p function in vivo and in vitro. We show that the beta domain of the budding yeast Vps4p is not required for recruitment to endosomes, but is essential for all Vps4p endocytic functions in vivo. We also show that the beta domain is required for Vps4p homotypic interaction and for full ATPase activity. In addition, it is required for interaction with Vta1p, which works in concert with Vps4p in vivo. Our studies suggest that assembly of a Vps4p oligomeric complex with full ATPase activity that interacts with Vta1p is essential for normal endosome function.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Sequência de Bases , Primers do DNA , Complexos Endossomais de Distribuição Requeridos para Transporte , Genótipo , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fenótipo , Plasmídeos , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética
19.
Curr Protoc Immunol ; 114: 14.40.1-14.40.29, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27479658

RESUMO

Inflammasomes are large protein complexes formed in response to cellular stresses that are platforms for recruitment and activation of caspase 1. Central to most inflammasome functions is the adapter molecule ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain) that links the inflammasome initiator protein to the recruited caspases. ASC is normally diffuse within the cell but within minutes of inflammasome activation relocates to a dense speck in the cytosol. The dramatic redistribution of ASC can be monitored by flow cytometry using parameters of fluorescence peak height and width when immunostained or tagged with a fluorescent protein. This can be used to define cells with active inflammasomes within populations of primary macrophages and monocytes, allowing quantification of responses and flow-sorting of responding cells. Protein structural requirements for ASC speck formation and recruitment of caspases to ASC specks can be assessed by expressing components in HEK293 cells. This provides rapid quantification of responding cell number and correlation with the expression level of inflammasome components within single cells. © 2016 by John Wiley & Sons, Inc.


Assuntos
Separação Celular/métodos , Citometria de Fluxo/métodos , Inflamassomos/metabolismo , Macrófagos/imunologia , Monócitos/imunologia , Animais , Apoptose , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Caspase 1/metabolismo , Células HEK293 , Humanos , Análise de Célula Única
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA