RESUMO
In response to many apoptotic stimuli, oligomerization of Bax is essential for mitochondrial outer membrane permeabilization and the ensuing release of cytochrome c. These events are accompanied by mitochondrial fission that appears to require Drp1, a large GTPase of the dynamin superfamily. Loss of Drp1 leads to decreased cytochrome c release by a mechanism that is poorly understood. Here we show that Drp1 stimulates tBid-induced Bax oligomerization and cytochrome c release by promoting tethering and hemifusion of membranes in vitro. This function of Drp1 is independent of its GTPase activity and relies on arginine 247 and the presence of cardiolipin in membranes. In cells, overexpression of Drp1 R247A/E delays Bax oligomerization and cell death. Our findings uncover a function of Drp1 and provide insight into the mechanism of Bax oligomerization.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Mitocondriais/metabolismo , Proteína X Associada a bcl-2/metabolismo , Sequência de Aminoácidos , Animais , Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Cardiolipinas/metabolismo , Sistema Livre de Células , Dinaminas , Células HeLa , Humanos , Lipossomos/metabolismo , Membranas Mitocondriais/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , RatosRESUMO
The death inducing signalling complex (DISC) formed by Fas receptor, FADD (Fas-associated death domain protein) and caspase 8 is a pivotal trigger of apoptosis. The Fas-FADD DISC represents a receptor platform, which once assembled initiates the induction of programmed cell death. A highly oligomeric network of homotypic protein interactions comprised of the death domains of Fas and FADD is at the centre of DISC formation. Thus, characterizing the mechanistic basis for the Fas-FADD interaction is crucial for understanding DISC signalling but has remained unclear largely because of a lack of structural data. We have successfully formed and isolated the human Fas-FADD death domain complex and report the 2.7 A crystal structure. The complex shows a tetrameric arrangement of four FADD death domains bound to four Fas death domains. We show that an opening of the Fas death domain exposes the FADD binding site and simultaneously generates a Fas-Fas bridge. The result is a regulatory Fas-FADD complex bridge governed by weak protein-protein interactions revealing a model where the complex itself functions as a mechanistic switch. This switch prevents accidental DISC assembly, yet allows for highly processive DISC formation and clustering upon a sufficient stimulus. In addition to depicting a previously unknown mode of death domain interactions, these results further uncover a mechanism for receptor signalling solely by oligomerization and clustering events.
Assuntos
Proteína de Domínio de Morte Associada a Fas/química , Proteína de Domínio de Morte Associada a Fas/metabolismo , Agregação de Receptores , Transdução de Sinais , Receptor fas/química , Receptor fas/metabolismo , Cristalografia por Raios X , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/química , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Humanos , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismoRESUMO
Mitochondria are remarkably dynamic organelles that migrate, divide and fuse. Cycles of mitochondrial fission and fusion ensure metabolite and mitochondrial DNA mixing and dictate organelle shape, number and bioenergetic functionality. There is mounting evidence that mitochondrial dysfunction is an early and causal event in neurodegeneration. Mutations in the mitochondrial fusion GTPases mitofusin 2 and optic atrophy 1, neurotoxins and oxidative stress all disrupt the cable-like morphology of functional mitochondria. This results in impaired bioenergetics and mitochondrial migration, and can trigger neurodegeneration. These findings suggest potential new treatment avenues for neurodegenerative diseases.
Assuntos
Mitocôndrias , Proteínas Mitocondriais , Doenças Neurodegenerativas , Animais , Humanos , Fusão de Membrana/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/fisiopatologia , Neurônios/citologia , Neurônios/metabolismo , Estresse Oxidativo , Conformação Proteica , Espécies Reativas de Oxigênio/metabolismoRESUMO
Pathogenesis by Bacillus anthracis requires coordination between two distinct activities: plasmid-encoded virulence factor expression (which protects vegetative cells from immune surveillance during outgrowth and replication) and chromosomally encoded sporulation (required only during the final stages of infection). Sporulation is regulated by at least five sensor histidine kinases that are activated in response to various environmental cues. One of these kinases, BA2291, harbors a sensor domain that has â¼35% sequence identity with two plasmid proteins, pXO1-118 and pXO2-61. Because overexpression of pXO2-61 (or pXO1-118) inhibits sporulation of B. anthracis in a BA2291-dependent manner, and pXO2-61 expression is strongly up-regulated by the major virulence gene regulator, AtxA, it was suggested that their function is to titrate out an environmental signal that would otherwise promote untimely sporulation. To explore this hypothesis, we determined crystal structures of both plasmid-encoded proteins. We found that they adopt a dimeric globin fold but, most unusually, do not bind heme. Instead, they house a hydrophobic tunnel and hydrophilic chamber that are occupied by fatty acid, which engages a conserved arginine and chloride ion via its carboxyl head group. In vivo, these domains may therefore recognize changes in fatty acid synthesis, chloride ion concentration, and/or pH. Structure-based comparisons with BA2291 suggest that it binds ligand and dimerizes in an analogous fashion, consistent with the titration hypothesis. Analysis of newly sequenced bacterial genomes points to the existence of a much broader family of non-heme, globin-based sensor domains, with related but distinct functionalities, that may have evolved from an ancestral heme-linked globin.
Assuntos
Bacillus anthracis/química , Proteínas de Bactérias/química , Dobramento de Proteína , Multimerização Proteica/fisiologia , Transativadores/química , Fatores de Virulência/química , Bacillus anthracis/genética , Bacillus anthracis/metabolismo , Bacillus anthracis/patogenicidade , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Transativadores/genética , Transativadores/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Herpes simplex virus (HSV) glycoprotein B (gB) is an integral part of the multicomponent fusion system required for virus entry and cell-cell fusion. Here we investigated the mechanism of viral neutralization by the monoclonal antibody (MAb) 2c, which specifically recognizes the gB of HSV type 1 (HSV-1) and HSV-2. Binding of MAb 2c to a type-common discontinuous epitope of gB resulted in highly efficient neutralization of HSV at the postbinding/prefusion stage and completely abrogated the viral cell-to-cell spread in vitro. Mapping of the antigenic site recognized by MAb 2c to the recently solved crystal structure of the HSV-1 gB ectodomain revealed that its discontinuous epitope is only partially accessible within the observed multidomain trimer conformation of gB, likely representing its postfusion conformation. To investigate how MAb 2c may interact with gB during membrane fusion, we characterized the properties of monovalent (Fab and scFv) and bivalent [IgG and F(ab')(2)] derivatives of MAb 2c. Our data show that the neutralization capacity of MAb 2c is dependent on cross-linkage of gB trimers. As a result, only bivalent derivatives of MAb 2c exhibited high neutralizing activity in vitro. Notably, bivalent MAb 2c not only was capable of preventing mucocutaneous disease in severely immunodeficient NOD/SCID mice upon vaginal HSV-1 challenge but also protected animals even with neuronal HSV infection. We also report for the first time that an anti-gB specific monoclonal antibody prevents HSV-1-induced encephalitis entirely independently from complement activation, antibody-dependent cellular cytotoxicity, and cellular immunity. This indicates the potential for further development of MAb 2c as an anti-HSV drug.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 2/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/química , Especificidade de Anticorpos , Células COS , Chlorocebus aethiops , Encefalite por Herpes Simples/imunologia , Encefalite por Herpes Simples/prevenção & controle , Mapeamento de Epitopos , Epitopos/imunologia , Feminino , Herpes Genital/imunologia , Herpes Genital/prevenção & controle , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Moleculares , Testes de Neutralização , Mapeamento de Peptídeos , Células Vero , Proteínas do Envelope Viral/químicaRESUMO
Previously, defined naturally occurring isoforms of allergenic proteins were classified as hypoallergens and therefore suggested as an agent for immunotherapy in the future. In this paper, we report for the first time the molecular background of hypoallergenicity by comparing the immunological behavior of hyperallergenic Betula verrucosa major Ag 1a (Bet v 1a) and hypoallergenic Bet v 1d, two isoforms of the major birch pollen allergen Betula verrucosa 1. Despite their cross-reactivity, Bet v 1a and Bet v 1d differ in their capacity to induce protective Ab responses in BALB/c mice. Both isoforms induced similar specific IgE levels, but only Bet v 1d expressed relevant titers of serum IgGs and IgAs. Interestingly, hypoallergenic Bet v 1d activated dendritic cells more efficiently, followed by the production of increased amounts of Th1- as well as Th2-type cytokines. Surprisingly, compared with Bet v 1a, Bet v 1d-immunized mice showed a decreased proliferation of regulatory T cells. Crystallographic studies and dynamic light scattering revealed that Bet v 1d demonstrated a high tendency to form disulfide-linked aggregates due to a serine to cysteine exchange at residue 113. We conclude that aggregation of Bet v 1d triggers the establishment of a protective Ab titer and supports a rationale for Bet v 1d being a promising candidate for specific immunotherapy of birch pollen allergy.
Assuntos
Alérgenos/química , Betula/imunologia , Reações Cruzadas/imunologia , Hipersensibilidade/imunologia , Pólen/imunologia , Multimerização Proteica/imunologia , Alérgenos/imunologia , Animais , Betulaceae , Imunidade Humoral , Isotipos de Imunoglobulinas/análise , Isoanticorpos/imunologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The authors of J. Immunol. 184, 725-735 respond to the article by Rupp (2012), Acta Cryst. F68, 366-376.
Assuntos
Alérgenos/química , Betula/química , Proteínas de Plantas/química , Pólen/química , Má Conduta Científica , Alérgenos/análise , Cristalografia por Raios X , Proteínas de Plantas/análiseRESUMO
PQQ is an exogenous, tricyclic, quino-cofactor for a number of bacterial dehydrogenases. The final step of PQQ formation is catalyzed by PqqC, a cofactorless oxidase. This study focuses on the activation of molecular oxygen in an enzyme active site without metal or cofactor and has identified a specific oxygen binding and activating pocket in PqqC. The active site variants H154N, Y175F,S, and R179S were studied with the goal of defining the site of O(2) binding and activation. Using apo-glucose dehydrogenase to assay for PQQ production, none of the mutants in this "O(2) core" are capable of PQQ/PQQH(2) formation. Spectrophotometric assays give insight into the incomplete reactions being catalyzed by these mutants. Active site variants Y175F, H154N, and R179S form a quinoid intermediate (Figure 1) anaerobically. Y175S is capable of proceeding further from quinoid to quinol, whereas Y175F, H154N, and R179S require O(2) to produce the quinol species. None of the mutations precludes substrate/product binding or oxygen binding. Assays for the oxidation of PQQH(2) to PQQ show that these O(2) core mutants are incapable of catalyzing a rate increase over the reaction in buffer, whereas H154N can catalyze the oxidation of PQQH(2) to PQQ in the presence of H(2)O(2) as an electron acceptor. Taken together, these data indicate that none of the targeted mutants can react fully to form quinone even in the presence of bound O(2). The data indicate a successful separation of oxidative chemistry from O(2) binding. The residues H154, Y175, and R179 are proposed to form a core O(2) binding structure that is essential for efficient O(2) activation.
Assuntos
Proteínas de Bactérias/metabolismo , Cofator PQQ/biossíntese , Anaerobiose , Apraxia Ideomotora , Proteínas de Bactérias/genética , Domínio Catalítico , Clonagem Molecular , Methylobacterium/enzimologia , Modelos Moleculares , Oxigênio/metabolismo , Conformação ProteicaRESUMO
The innate immune system provides an initial line of defense against infection. Nucleotide-binding domain- and leucine-rich repeat-containing protein (NLR or (NOD-like)) receptors play a critical role in the innate immune response by surveying the cytoplasm for traces of intracellular invaders and endogenous stress signals. NLRs themselves are multi-domain proteins. Their N-terminal effector domains (typically a pyrin or caspase activation and recruitment domain) are responsible for driving downstream signaling and initiating the formation of inflammasomes, multi-component complexes necessary for cytokine activation. However, the currently available structures of NLR effector domains have not yet revealed the mechanism of their differential modes of interaction. Here, we report the structure and dynamics of the N-terminal pyrin domain of NLRP7 (NLRP7 PYD) obtained by NMR spectroscopy. The NLRP7 PYD adopts a six-alpha-helix bundle death domain fold. A comparison of conformational and dynamics features of the NLRP7 PYD with other PYDs showed distinct differences for helix alpha3 and loop alpha2-alpha3, which, in NLRP7, is stabilized by a strong hydrophobic cluster. Moreover, the NLRP7 and NLRP1 PYDs have different electrostatic surfaces. This is significant, because death domain signaling is driven by electrostatic contacts and stabilized by hydrophobic interactions. Thus, these results provide new insights into NLRP signaling and provide a first molecular understanding of inflammasome formation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas do Citoesqueleto , Dobramento de Proteína , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cristalografia por Raios X , Humanos , Interações Hidrofóbicas e Hidrofílicas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Pirina , Eletricidade Estática , Relação Estrutura-AtividadeRESUMO
Proteins of the nucleotide-binding domain, leucine-rich repeat (NLR)-containing family recently gained attention as important components of the innate immune system. Although over 20 of these proteins are present in humans, only a few members including the cytosolic pattern recognition receptors NOD1, NOD2, and NLRP3 have been analyzed extensively. These NLRs were shown to be pivotal for mounting innate immune response toward microbial invasion. Here we report on the characterization of human NLRC5 and provide evidence that this NLR has a function in innate immune responses. We found that NLRC5 is a cytosolic protein expressed predominantly in hematopoetic cells. NLRC5 mRNA and protein expression was inducible by the double-stranded RNA analog poly(I.C) and Sendai virus. Overexpression of NLRC5 failed to trigger inflammatory responses such as the NF-kappaB or interferon pathways in HEK293T cells. However, knockdown of endogenous NLRC5 reduced Sendai virus- and poly(I.C)-mediated type I interferon pathway-dependent responses in THP-1 cells and human primary dermal fibroblasts. Taken together, this defines a function for NLRC5 in anti-viral innate immune responses.
Assuntos
Imunidade Inata , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Vírus/imunologia , Células Cultivadas , Células-Tronco Hematopoéticas , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Poli I-C/imunologia , RNA Mensageiro/análise , Vírus Sendai/imunologia , Ativação Transcricional/imunologiaRESUMO
A decline in mitochondrial function is well recognized in neurodegenerative diseases and aging, and is thought to play a causal role in their biology. Unfortunately, the molecular basis underlying this detrimental loss in mitochondrial function remains mysterious. Interestingly, mitochondria undergo frequent fission and fusion. This process is regulated by molecular machinery that has been highly conserved during evolution, including dynamin-related GTPases that manifest opposing effects. A balance between mitochondrial fission and fusion events is required for normal mitochondrial and cellular function. Emerging evidence indicates that mitochondria undergo rapid and extensive fission at an early stage during apoptosis. A clue that these new findings are of significance for the pathogenesis of neurodegenerative disease is provided by the observation that OPA-1, a dynamin-related GTPase regulating mitochondrial fusion, is mutated in humans with dominant optic atrophy, which is characterized by degeneration of retinal ganglion cells and childhood blindness. Loss of function of OPA-1, analogous to deficiency of its yeast homologue, Mgm1p, is expected to lead to mitochondrial fission, loss of mitochondrial DNA, respiratory deficits and an increase in reactive oxygen species. Here we review the molecular mediators controlling mitochondrial fission and fusion, and how death effector molecules may hijack this ancient machinery. A shift in the rate of mitochondrial fission or fusion may provide a new mechanistic explanation for the mitochondrial dysfunction in neurodegenerative diseases and normal aging, and may offer a new target for therapeutic intervention.
Assuntos
Envelhecimento , Apoptose , Mitocôndrias/patologia , Animais , Caenorhabditis elegans , DNA/química , Drosophila , GTP Fosfo-Hidrolases/metabolismo , Humanos , Mitocôndrias/metabolismo , Modelos Biológicos , Modelos Genéticos , Mutação , Doenças Neurodegenerativas/patologia , Estrutura Terciária de ProteínaRESUMO
The molecular bases underlying the pathogenesis of neurodegenerative diseases are gradually being disclosed. One problem that investigators face is distinguishing primary from secondary events. Rare, inherited mutations causing familial forms of these disorders have provided important insights into the molecular networks implicated in disease pathogenesis. Increasing evidence indicates that accumulation of aberrant or misfolded proteins, protofibril formation, ubiquitin-proteasome system dysfunction, excitotoxic insult, oxidative and nitrosative stress, mitochondrial injury, synaptic failure, altered metal homeostasis and failure of axonal and dendritic transport represent unifying events in many slowly progressive neurodegenerative disorders.
Assuntos
Proteínas de Membrana Transportadoras , Degeneração Neural/genética , Doenças Neurodegenerativas/genética , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Humanos , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Degeneração Neural/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Proteínas Oncogênicas/química , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Peptídeos/metabolismo , Proteína Desglicase DJ-1 , Desnaturação Proteica , Dobramento de Proteína , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina , Proteína Amiloide A Sérica/química , Proteína Amiloide A Sérica/genética , Proteína Amiloide A Sérica/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Apoptosis is executed by caspases, which undergo proteolytic activation in response to cell death stimuli. The apoptotic protease-activating factor 1 (Apaf-1) controls caspase activation downstream of mitochondria. During apoptosis, Apaf-1 binds to cytochrome c and in the presence of ATP/dATP forms an apoptosome, leading to the recruitment and activation of the initiator caspase, caspase-9 (ref. 2). The mechanisms underlying Apaf-1 function are largely unknown. Here we report the 2.2-A crystal structure of an ADP-bound, WD40-deleted Apaf-1, which reveals the molecular mechanism by which Apaf-1 exists in an inactive state before ATP binding. The amino-terminal caspase recruitment domain packs against a three-layered alpha/beta fold, a short helical motif and a winged-helix domain, resulting in the burial of the caspase-9-binding interface. The deeply buried ADP molecule serves as an organizing centre to strengthen interactions between these four adjoining domains, thus locking Apaf-1 in an inactive conformation. Apaf-1 binds to and hydrolyses ATP/dATP and their analogues. The binding and hydrolysis of nucleotides seem to drive conformational changes that are essential for the formation of the apoptosome and the activation of caspase-9.
Assuntos
Difosfato de Adenosina/metabolismo , Proteínas/química , Proteínas/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Apoptose , Fator Apoptótico 1 Ativador de Proteases , Sítios de Ligação , Caspase 9 , Caspases/metabolismo , Cristalografia por Raios X , Citocromos c/metabolismo , Ativação Enzimática , Hidrólise , Modelos Biológicos , Modelos Moleculares , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas/genética , Deleção de Sequência/genética , Relação Estrutura-AtividadeRESUMO
Maturation of neuronal synapses is thought to involve mitochondria. Bcl-xL protein inhibits mitochondria-mediated apoptosis but may have other functions in healthy adult neurons in which Bcl-xL is abundant. Here, we report that overexpression of Bcl-xL postsynaptically increases frequency and amplitude of spontaneous miniature synaptic currents in rat hippocampal neurons in culture. Bcl-xL, overexpressed either pre or postsynaptically, increases synapse number, the number and size of synaptic vesicle clusters, and mitochondrial localization to vesicle clusters and synapses, likely accounting for the changes in miniature synaptic currents. Conversely, knockdown of Bcl-xL or inhibiting it with ABT-737 decreases these morphological parameters. The mitochondrial fission protein, dynamin-related protein 1 (Drp1), is a GTPase known to localize to synapses and affect synaptic function and structure. The effects of Bcl-xL appear mediated through Drp1 because overexpression of Drp1 increases synaptic markers, and overexpression of the dominant-negative dnDrp1-K38A decreases them. Furthermore, Bcl-xL coimmunoprecipitates with Drp1 in tissue lysates, and in a recombinant system, Bcl-xL protein stimulates GTPase activity of Drp1. These findings suggest that Bcl-xL positively regulates Drp1 to alter mitochondrial function in a manner that stimulates synapse formation.
Assuntos
Dinaminas/fisiologia , Hipocampo/metabolismo , Sinapses , Proteína bcl-X/fisiologia , Animais , Células Cultivadas , Hipocampo/citologia , Mitocôndrias/metabolismo , Ratos , Transmissão SinápticaRESUMO
Two apical caspases, caspase-8 and -10, are involved in the extrinsic death receptor pathway in humans, but it is mainly caspase-8 in its apoptotic and nonapoptotic functions that has been an intense research focus. In this study we concentrate on caspase-10, its mechanism of activation, and the role of the intersubunit cleavage. Our data obtained through in vitro dimerization assays strongly suggest that caspase-10 follows the proximity-induced dimerization model for apical caspases. Furthermore, we compare the specificity and activity of the wild-type protease with a mutant incapable of autoprocessing by using positional scanning substrate analysis and cleavage of natural protein substrates. These experiments reveal a striking difference between the wild type and the mutant, leading us to hypothesize that the single chain enzyme has restricted activity on most proteins but high activity on the proapoptotic protein Bid, potentially supporting a prodeath role for both cleaved and uncleaved caspase-10.
Assuntos
Caspase 10/biossíntese , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Dimerização , Endopeptidases/metabolismo , Ativação Enzimática , Humanos , Especificidade por SubstratoRESUMO
Pyrroloquinoline quinone [4,5-dihydro-4,5-dioxo-1H-pyrrolo[2,3-f]quinoline-2,7,9-tricarboxylic acid (PQQ)] is a bacterial cofactor in numerous alcohol dehydrogenases including methanol dehydrogenase and glucose dehydrogenase. Its biosynthesis in Klebsiella pneumoniae is facilitated by six genes, pqqABCDEF and proceeds by an unknown pathway. PqqC is one of two metal free oxidases of known structure and catalyzes the last step of PQQ biogenesis which involves a ring closure and an eight-electron oxidation of the substrate [3a-(2-amino-2-carboxyethyl)-4,5-dioxo-4,5,6,7,8,9-hexahydroquinoline-7,9-dicarboxylic acid (AHQQ)]. PqqC has 14 conserved active site residues, which have previously been shown to be in close contact with bound PQQ. Herein, we describe the structures of three PqqC active site variants, H154S, Y175F, and the double mutant R179S/Y175S. The H154S crystal structure shows that, even with PQQ bound, the enzyme is still in the "open" conformation with helices alpha5b and alpha6 unfolded and the active site solvent accessible. The Y175F PQQ complex crystal structure reveals the closed conformation indicating that Y175 is not required for the conformational change. The R179S/Y175S AHQQ complex crystal structure is the most mechanistically informative, indicating an open conformation with a reaction intermediate trapped in the active site. The intermediate seen in R179S/Y175S is tricyclic but nonplanar, implying that it has not undergone oxidation. These studies implicate a stepwise process in which substrate binding leads to the generation of the closed protein conformation, with the latter playing a critical role in O(2) binding and catalysis.
Assuntos
Proteínas de Bactérias/química , Mutação , Oxigênio/química , Cofator PQQ/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Sequência Conservada , Cristalografia por Raios X , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/genética , Oxigênio/metabolismo , Cofator PQQ/metabolismoRESUMO
Recent events have created an urgent need for new therapeutic strategies to treat anthrax. We have applied a mixture-based peptide library approach to rapidly determine the optimal peptide substrate for the anthrax lethal factor (LF), a metalloproteinase with an important role in the pathogenesis of the disease. Using this approach we have identified peptide analogs that inhibit the enzyme in vitro and that protect cultured macrophages from LF-mediated cytolysis. The crystal structures of LF bound to an optimized peptide substrate and to peptide-based inhibitors provide a rationale for the observed selectivity and may be exploited in the design of future generations of LF inhibitors.
Assuntos
Antígenos de Bactérias , Bacillus anthracis/patogenicidade , Toxinas Bacterianas/antagonistas & inibidores , Toxinas Bacterianas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Antraz/tratamento farmacológico , Bacillus anthracis/enzimologia , Bacillus anthracis/genética , Toxinas Bacterianas/genética , Humanos , Técnicas In Vitro , Cinética , Substâncias Macromoleculares , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Biblioteca de Peptídeos , Especificidade por SubstratoRESUMO
The virulent spore-forming bacterium Bacillus anthracis secretes anthrax toxin composed of protective antigen (PA), lethal factor (LF) and edema factor (EF). LF is a Zn-dependent metalloprotease that inactivates key signaling molecules, such as mitogen-activated protein kinase kinases (MAPKK), to ultimately cause cell death. We report here the identification of small molecule (nonpeptidic) inhibitors of LF. Using a two-stage screening assay, we determined the LF inhibitory properties of 19 compounds. Here, we describe six inhibitors on the basis of a pharmacophoric relationship determined using X-ray crystallographic data, molecular docking studies and three-dimensional (3D) database mining from the US National Cancer Institute (NCI) chemical repository. Three of these compounds have K(i) values in the 0.5-5 microM range and show competitive inhibition. These molecular scaffolds may be used to develop therapeutically viable inhibitors of LF.
Assuntos
Antígenos de Bactérias , Bacillus anthracis/patogenicidade , Toxinas Bacterianas/antagonistas & inibidores , Animais , Antraz/tratamento farmacológico , Toxinas Bacterianas/química , Toxinas Bacterianas/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Humanos , Técnicas In Vitro , Cinética , Camundongos , Modelos Moleculares , Estrutura Molecular , Conformação ProteicaRESUMO
Mutations in OPA1, a dynamin-related GTPase involved in mitochondrial fusion, cristae organization and control of apoptosis, have been linked to non-syndromic optic neuropathy transmitted as an autosomal-dominant trait (DOA). We here report on eight patients from six independent families showing that mutations in the OPA1 gene can also be responsible for a syndromic form of DOA associated with sensorineural deafness, ataxia, axonal sensory-motor polyneuropathy, chronic progressive external ophthalmoplegia and mitochondrial myopathy with cytochrome c oxidase negative and Ragged Red Fibres. Most remarkably, we demonstrate that these patients all harboured multiple deletions of mitochondrial DNA (mtDNA) in their skeletal muscle, thus revealing an unrecognized role of the OPA1 protein in mtDNA stability. The five OPA1 mutations associated with these DOA 'plus' phenotypes were all mis-sense point mutations affecting highly conserved amino acid positions and the nuclear genes previously known to induce mtDNA multiple deletions such as POLG1, PEO1 (Twinkle) and SLC25A4 (ANT1) were ruled out. Our results show that certain OPA1 mutations exert a dominant negative effect responsible for multi-systemic disease, closely related to classical mitochondrial cytopathies, by a mechanism involving mtDNA instability.