RESUMO
Protein tyrosine phosphatase nonreceptor type 22 (PTPN22) is encoded by a major autoimmunity gene and is a known inhibitor of T cell receptor (TCR) signaling and drug target for cancer immunotherapy. However, little is known about PTPN22 posttranslational regulation. Here, we characterize a phosphorylation site at Ser325 situated C terminal to the catalytic domain of PTPN22 and its roles in altering protein function. In human T cells, Ser325 is phosphorylated by glycogen synthase kinase-3 (GSK3) following TCR stimulation, which promotes its TCR-inhibitory activity. Signaling through the major TCR-dependent pathway under PTPN22 control was enhanced by CRISPR/Cas9-mediated suppression of Ser325 phosphorylation and inhibited by mimicking it via glutamic acid substitution. Global phospho-mass spectrometry showed Ser325 phosphorylation state alters downstream transcriptional activity through enrichment of Swi3p, Rsc8p, and Moira domain binding proteins, and next-generation sequencing revealed it differentially regulates the expression of chemokines and T cell activation pathways. Moreover, in vitro kinetic data suggest the modulation of activity depends on a cellular context. Finally, we begin to address the structural and mechanistic basis for the influence of Ser325 phosphorylation on the protein's properties by deuterium exchange mass spectrometry and NMR spectroscopy. In conclusion, this study explores the function of a novel phosphorylation site of PTPN22 that is involved in complex regulation of TCR signaling and provides details that might inform the future development of allosteric modulators of PTPN22.
Assuntos
Proteína Tirosina Fosfatase não Receptora Tipo 22 , Receptores de Antígenos de Linfócitos T , Transdução de Sinais , Humanos , Fosforilação , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 22/genética , Proteína Tirosina Fosfatase não Receptora Tipo 22/metabolismo , Mutação com Ganho de Função , Linfócitos T/metabolismo , Linfócitos T/imunologia , Células Jurkat , Células HEK293RESUMO
T-cell protein tyrosine phosphatase (TC-PTP) is a negative regulator of T-cell receptor and oncogenic receptor tyrosine kinase signaling and implicated in cancer and autoimmune disease. TC-PTP activity is modulated by an intrinsically disordered C-terminal region (IDR) and suppressed in cells under basal conditions. In vitro structural studies have shown that the dynamic reorganization of IDR around the catalytic domain, driven by electrostatic interactions, can lead to TC-PTP activity inhibition; however, the process has not been studied in cells. Here, by assessing a mutant (378KRKRPR383 mutated into 378EAAAPE383, called TC45E/A) with impaired tail-PTP domain interaction, we obtained evidence that the downmodulation of TC-PTP enzymatic activity by the IDR occurs in cells. However, we found that the regulation of TC-PTP by the IDR is only recapitulated in vitro when crowding polymers that mimic the intracellular environment are present in kinetic assays using a physiological phosphopeptide. Our FRET-based assays in vitro and in cells confirmed that the effect of the mutant correlates with an impairment of the intramolecular inhibitory remodeling of TC-PTP by the IDR. This work presents an early example of the allosteric regulation of a protein tyrosine phosphatase being controlled by the cellular environment and provides a framework for future studies and targeting of TC-PTP function.
Assuntos
Proteína Tirosina Fosfatase não Receptora Tipo 2 , Transdução de Sinais , Proteína Tirosina Fosfatase não Receptora Tipo 2/metabolismo , Regulação Alostérica , Transdução de Sinais/fisiologia , FosforilaçãoRESUMO
Receptor-type protein tyrosine phosphatase α (RPTPα) is an important positive regulator of SRC kinase activation and a known promoter of cancer growth, fibrosis, and arthritis. The domain structure of RPTPs comprises an extracellular region, a transmembrane helix, and two tandem intracellular catalytic domains referred to as D1 and D2. The D2 domain of RPTPs is believed to mostly play a regulatory function; however, no regulatory model has been established for RPTPα-D2 or other RPTP-D2 domains. Here, we solved the 1.8 Å resolution crystal structure of the cytoplasmic region of RPTPα, encompassing D1 and D2, trapped in a conformation that revealed a possible mechanism through which D2 can allosterically inhibit D1 activity. Using a D2-truncation RPTPα variant and mutational analysis of the D1/D2 interfaces, we show that D2 inhibits RPTPα phosphatase activity and identified a 405PFTP408 motif in D1 that mediates the inhibitory effect of D2. Expression of the gain-of-function F406A/T407A RPTPα variant in HEK293T cells enhanced SRC activation, supporting the relevance of our proposed D2-mediated regulation mechanism in cell signaling. There is emerging interest in the development of allosteric inhibitors of RPTPs but a scarcity of validated allosteric sites for RPTPs. The results of our study not only shed light on the regulatory role of RPTP-D2 domains, but also provide a potentially useful tool for the discovery of chemical probes targeting RPTPα and other RPTPs.
Assuntos
Membrana Celular/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/química , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteínas Tirosina Fosfatases/química , Homologia de SequênciaRESUMO
Protein tyrosine phosphatases (PTPs) play major roles in cancer and are emerging as therapeutic targets. Recent reports suggest low-molecular weight PTP (LMPTP)-encoded by the ACP1 gene-is overexpressed in prostate tumors. We found ACP1 up-regulated in human prostate tumors and ACP1 expression inversely correlated with overall survival. Using CRISPR-Cas9-generated LMPTP knockout C4-2B and MyC-CaP cells, we identified LMPTP as a critical promoter of prostate cancer (PCa) growth and bone metastasis. Through metabolomics, we found that LMPTP promotes PCa cell glutathione synthesis by dephosphorylating glutathione synthetase on inhibitory Tyr270. PCa cells lacking LMPTP showed reduced glutathione, enhanced activation of eukaryotic initiation factor 2-mediated stress response, and enhanced reactive oxygen species after exposure to taxane drugs. LMPTP inhibition slowed primary and bone metastatic prostate tumor growth in mice. These findings reveal a role for LMPTP as a critical promoter of PCa growth and metastasis and validate LMPTP inhibition as a therapeutic strategy for treating PCa through sensitization to oxidative stress.
Assuntos
Neoplasias da Próstata , Masculino , Humanos , Camundongos , Animais , Peso Molecular , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Tirosina , Proteínas Tirosina Fosfatases/metabolismoRESUMO
Receptor-type protein phosphatase α (RPTPα) promotes fibroblast-dependent arthritis and fibrosis, in part, by enhancing the activation of the kinase SRC. Synovial fibroblasts lining joint tissue mediate inflammation and tissue damage, and their infiltration into adjacent tissues promotes disease progression. RPTPα includes an ectodomain and two intracellular catalytic domains (D1 and D2) and, in cancer cells, undergoes inhibitory homodimerization, which is dependent on a D1 wedge motif. Through single-molecule localization and labeled molecule interaction microscopy of migrating synovial fibroblasts, we investigated the role of RPTPα dimerization in the activation of SRC, the migration of synovial fibroblasts, and joint damage in a mouse model of arthritis. RPTPα clustered with other RPTPα and with SRC molecules in the context of actin-rich structures. A known dimerization-impairing mutation in the wedge motif (P210L/P211L) and the deletion of the D2 domain reduced RPTPα-RPTPα clustering; however, it also unexpectedly reduced RPTPα-SRC association. The same mutations also reduced recruitment of RPTPα to actin-rich structures and inhibited SRC activation and cellular migration. An antibody against the RPTPα ectodomain that prevented the clustering of RPTPα also inhibited RPTPα-SRC association and SRC activation and attenuated fibroblast migration and joint damage in arthritic mice. A catalytically inactivating RPTPα-C469S mutation protected mice from arthritis and reduced SRC activation in synovial fibroblasts. We conclude that RPTPα clustering retains it to actin-rich structures to promote SRC-mediated fibroblast migration and can be modulated through the extracellular domain.
Assuntos
Actinas , Artrite , Animais , Camundongos , Análise por Conglomerados , Fibroblastos/metabolismo , Fosfoproteínas Fosfatases , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/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
Systemic sclerosis (SSc) is a fibrotic autoimmune disease characterized by pathogenic activation of fibroblasts enhanced by local oxidative stress. The tyrosine phosphatase PTP4A1 was identified as a critical promoter of TGF-ß signaling in SSc. Oxidative stress is known to functionally inactivate tyrosine phosphatases. Here, we assessed whether oxidation of PTP4A1 modulates its profibrotic action and found that PTP4A1 forms a complex with the kinase SRC in scleroderma fibroblasts, but surprisingly, oxidative stress enhanced rather than reduced PTP4A1's association with SRC and its profibrotic action. Through structural assessment of the oxo-PTP4A1-SRC complex, we unraveled an unexpected mechanism whereby oxidation of a tyrosine phosphatase promotes its function through modification of its protein complex. Considering the importance of oxidative stress in the pathogenesis of SSc and fibrosis, our findings suggest routes for leveraging PTP4A1 oxidation as a potential strategy for developing antifibrotic agents.
Assuntos
Escleroderma Sistêmico , Fibroblastos/metabolismo , Fibrose , Humanos , Estresse Oxidativo , Escleroderma Sistêmico/patologia , Tirosina/metabolismoRESUMO
Obesity-associated insulin resistance plays a central role in the pathogenesis of type 2 diabetes. A promising approach to decrease insulin resistance in obesity is to inhibit the protein tyrosine phosphatases that negatively regulate insulin receptor signaling. The low-molecular-weight protein tyrosine phosphatase (LMPTP) acts as a critical promoter of insulin resistance in obesity by inhibiting phosphorylation of the liver insulin receptor activation motif. Here, we report development of a novel purine-based chemical series of LMPTP inhibitors. These compounds inhibit LMPTP with an uncompetitive mechanism and are highly selective for LMPTP over other protein tyrosine phosphatases. We also report the generation of a highly orally bioavailable purine-based analogue that reverses obesity-induced diabetes in mice.
Assuntos
Inibidores Enzimáticos/química , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Purinas/química , Administração Oral , Animais , Sítios de Ligação , Cristalografia por Raios X , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/etiologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Meia-Vida , Humanos , Resistência à Insulina , Cinética , Simulação de Dinâmica Molecular , Obesidade/complicações , Obesidade/patologia , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Purinas/metabolismo , Purinas/farmacologia , Purinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Anthrax toxin consists of the proteins protective antigen (PA), lethal factor (LF) and oedema factor (EF). The first step of toxin entry into host cells is the recognition by PA of a receptor on the surface of the target cell. Subsequent cleavage of receptor-bound PA enables EF and LF to bind and form a heptameric PA63 pre-pore, which triggers endocytosis. Upon acidification of the endosome, PA63 forms a pore that inserts into the membrane and translocates EF and LF into the cytosol. Two closely related host cell receptors, TEM8 and CMG2, have been identified. Both bind to PA with high affinity and are capable of mediating toxicity. Here, we report the crystal structure of the PA-CMG2 complex at 2.5 A resolution. The structure reveals an extensive receptor-pathogen interaction surface mimicking the non-pathogenic recognition of the extracellular matrix by integrins. The binding surface is closely conserved in the two receptors and across species, but is quite different in the integrin domains, explaining the specificity of the interaction. CMG2 engages two domains of PA, and modelling of the receptor-bound PA63 heptamer suggests that the receptor acts as a pH-sensitive brace to ensure accurate and timely membrane insertion. The structure provides new leads for the discovery of anthrax anti-toxins, and should aid the design of cancer therapeutics.
Assuntos
Antígenos de Bactérias/química , Antígenos de Bactérias/metabolismo , Bacillus anthracis/química , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Receptores de Peptídeos/química , Receptores de Peptídeos/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Sequência Conservada , Cristalização , Cristalografia por Raios X , Humanos , Concentração de Íons de Hidrogênio , Integrinas/química , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
Nanotechnology-introduced materials have promising applications as nanocarriers for drugs, peptides, proteins and nucleic acids. Several studies showed that the geometry (shape and size) and chemical properties of nanoparticles affect the kinetics and pathways of cellular uptake and their intracellular trafficking and signaling. Accurate physico-chemical characterization of nanoparticles customarily precedes their use in cell biology and in vivo experiments. However, a fact that is easily overlooked is that nanomaterials decorated with organic matter or resuspended in aqueous buffers can be theoretically contaminated by fungal and bacterial microorganisms. While investigating the effects of extensively characterized PEGylated carbon nanotubes (PNTs) on T lymphocyte activation, we demonstrated bacterial contamination of PNTs, which correlated with low reproducibility and artifacts in cell signaling assays. Contamination and artifacts were easily eliminated by preparing the materials in sterile conditions. We propose that simple sterile preparation procedures should be adopted and sterility evaluation of nanoparticles should be customarily performed, prior to assessing nanoparticle intracellular internalization, trafficking and their effects on cells and entire organisms.
Assuntos
Portadores de Fármacos , Contaminação de Medicamentos , Nanoconjugados/microbiologia , Nanotubos de Carbono/microbiologia , Artefatos , Endocitose/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Interleucina-2/genética , Interleucina-2/metabolismo , Células Jurkat , Microscopia de Força Atômica , Microscopia Confocal , Nanoconjugados/química , Nanotubos de Carbono/química , Tamanho da Partícula , Polietilenoglicóis , Reprodutibilidade dos Testes , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Receptores Toll-Like/metabolismoRESUMO
The hematopoietic-specific protein tyrosine phosphatase nonreceptor type 22 (PTPN22) is encoded by a major autoimmunity risk gene. PTPN22 inhibits T cell activation by dephosphorylating substrates involved in proximal T cell receptor (TCR) signaling. Here, we found by mass spectrometry that PTPN22 was phosphorylated at Ser751 by PKCα in Jurkat and primary human T cells activated with phorbol ester/ionomycin or antibodies against CD3/CD28. The phosphorylation of PTPN22 at Ser751 prolonged its half-life by inhibiting K48-linked ubiquitination and impairing recruitment of the phosphatase to the plasma membrane, which is necessary to inhibit proximal TCR signaling. Additionally, the phosphorylation of PTPN22 at Ser751 enhanced the interaction of PTPN22 with the carboxyl-terminal Src kinase (CSK), an interaction that is impaired by the PTPN22 R620W variant associated with autoimmune disease. The phosphorylation of Ser751 did not affect the recruitment of PTPN22 R620W to the plasma membrane but protected this mutant from degradation. Together, out data indicate that phosphorylation at Ser751 mediates a reciprocal regulation of PTPN22 stability versus translocation to TCR signaling complexes by CSK-dependent and CSK-independent mechanisms.
Assuntos
Proteína Tirosina Fosfatase não Receptora Tipo 22/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Serina/metabolismo , Transdução de Sinais , Doenças Autoimunes/genética , Doenças Autoimunes/metabolismo , Proteína Tirosina Quinase CSK/metabolismo , Células Cultivadas , Células HEK293 , Humanos , Células Jurkat , Espectrometria de Massas/métodos , Mutação de Sentido Incorreto , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 22/genética , Serina/genética , Linfócitos T/metabolismoRESUMO
Fibroblast-like synoviocytes (FLS) are joint-lining cells that promote rheumatoid arthritis (RA) pathology. Current disease-modifying antirheumatic agents (DMARDs) operate through systemic immunosuppression. FLS-targeted approaches could potentially be combined with DMARDs to improve control of RA without increasing immunosuppression. Here, we assessed the potential of immunoglobulin-like domains 1 and 2 (Ig1&2), a decoy protein that activates the receptor tyrosine phosphatase sigma (PTPRS) on FLS, for RA therapy. We report that PTPRS expression is enriched in synovial lining RA FLS and that Ig1&2 reduces migration of RA but not osteoarthritis FLS. Administration of an Fc-fusion Ig1&2 attenuated arthritis in mice without affecting innate or adaptive immunity. Furthermore, PTPRS was down-regulated in FLS by tumor necrosis factor (TNF) via a phosphatidylinositol 3-kinase-mediated pathway, and TNF inhibition enhanced PTPRS expression in arthritic joints. Combination of ineffective doses of TNF inhibitor and Fc-Ig1&2 reversed arthritis in mice, providing an example of synergy between FLS-targeted and immunosuppressive DMARD therapies.
Assuntos
Antirreumáticos , Artrite Reumatoide , Sinoviócitos , Animais , Antirreumáticos/uso terapêutico , Células Cultivadas , Fibroblastos/metabolismo , Camundongos , Sinoviócitos/metabolismo , Sinoviócitos/patologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Genetic variants at the PTPN2 locus, which encodes the tyrosine phosphatase PTPN2, cause reduced gene expression and are linked to rheumatoid arthritis (RA) and other autoimmune diseases. PTPN2 inhibits signaling through the T cell and cytokine receptors, and loss of PTPN2 promotes T cell expansion and CD4- and CD8-driven autoimmunity. However, it remains unknown whether loss of PTPN2 in FoxP3+ regulatory T cells (Tregs) plays a role in autoimmunity. Here we aimed to model human autoimmune-predisposing PTPN2 variants, the presence of which results in a partial loss of PTPN2 expression, in mouse models of RA. We identified that reduced expression of Ptpn2 enhanced the severity of autoimmune arthritis in the T cell-dependent SKG mouse model and demonstrated that this phenotype was mediated through a Treg-intrinsic mechanism. Mechanistically, we found that through dephosphorylation of STAT3, PTPN2 inhibits IL-6-driven pathogenic loss of FoxP3 after Tregs have acquired RORγt expression, at a stage when chromatin accessibility for STAT3-targeted IL-17-associated transcription factors is maximized. We conclude that PTPN2 promotes FoxP3 stability in mouse RORγt+ Tregs and that loss of function of PTPN2 in Tregs contributes to the association between PTPN2 and autoimmunity.
Assuntos
Artrite Reumatoide/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 2/imunologia , Linfócitos T Reguladores/imunologia , Animais , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Modelos Animais de Doenças , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 2/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologia , Linfócitos T Reguladores/patologiaRESUMO
B-cell receptor-associated protein 31 (Bap31) is an integral membrane protein located in the endoplasmic reticulum (ER) that participates in the transport and quality control of membrane proteins and plays a role in determining cell sensitivity to ER stress and apoptosis. Its cytoplasmic region contains two target sites for caspase cleavage in certain apoptotic pathways. Here, the subcloning, expression, purification and crystallization of the Homo sapiens Bap31 leucine-zipper C-terminal fragment, which spans residues Gly160-Glu246, are reported. An N-terminally His-tagged protein was overexpressed in Escherichia coli and purified by chromatographic methods. X-ray diffraction data were collected in-house to 2.5 A resolution. Crystals belong to space group P6(1)22/P6(5)22, with unit-cell parameters a = b = 70.7, c = 80.6 A. Data analysis indicates the presence of one molecule per asymmetric unit.
Assuntos
Zíper de Leucina , Proteínas de Membrana/química , Cristalização , Cristalografia por Raios X , Humanos , Conformação ProteicaRESUMO
Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of skin and internal organs. Protein tyrosine phosphatases have received little attention in the study of SSc or fibrosis. Here, we show that the tyrosine phosphatase PTP4A1 is highly expressed in fibroblasts from patients with SSc. PTP4A1 and its close homolog PTP4A2 are critical promoters of TGFß signaling in primary dermal fibroblasts and of bleomycin-induced fibrosis in vivo. PTP4A1 promotes TGFß signaling in human fibroblasts through enhancement of ERK activity, which stimulates SMAD3 expression and nuclear translocation. Upstream from ERK, we show that PTP4A1 directly interacts with SRC and inhibits SRC basal activation independently of its phosphatase activity. Unexpectedly, PTP4A2 minimally interacts with SRC and does not promote the SRC-ERK-SMAD3 pathway. Thus, in addition to defining PTP4A1 as a molecule of interest for TGFß-dependent fibrosis, our study provides information regarding the functional specificity of different members of the PTP4A subclass of phosphatases.
Assuntos
Proteínas Imediatamente Precoces/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Tirosina Fosfatases/metabolismo , Escleroderma Sistêmico/enzimologia , Fator de Crescimento Transformador beta/fisiologia , Animais , Linhagem Celular , Derme/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Humanos , Proteínas Imediatamente Precoces/antagonistas & inibidores , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/fisiologia , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Escleroderma Sistêmico/metabolismo , Escleroderma Sistêmico/patologia , Proteína Smad3/metabolismoRESUMO
Anthrax toxin comprises three soluble proteins: protective antigen (PA), lethal factor (LF), and edema factor (EF). PA must be cleaved by host proteases before it oligomerizes and forms a prepore, to which LF and EF bind. After endocytosis of this tripartite complex, the prepore transforms into a narrow transmembrane pore that delivers unfolded LF and EF into the host cytosol. Here, we find that translocation of multiple 90-kD LF molecules is rapid and efficient. To probe the molecular basis of this translocation, we calculated a three-dimensional map of the fully loaded (PA63)7-(LF)3 prepore complex by cryo-electron microscopy (cryo-EM). The map shows three LFs bound in a similar way to one another, via their N-terminal domains, to the surface of the PA heptamer. The model also reveals contacts between the N- and C-terminal domains of adjacent LF molecules. We propose that this molecular arrangement plays an important role in the maintenance of translocation efficiency through the narrow PA pore.
Assuntos
Antígenos de Bactérias/química , Toxinas Bacterianas/química , Transporte Biológico , Escherichia coli , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Relação Estrutura-AtividadeRESUMO
The E3 ubiquitin ligase Siah regulates key cellular events that are central to cancer development and progression. A promising route to Siah inhibition is disrupting its interactions with adaptor proteins. However, typical of protein-protein interactions, traditional unbiased approaches to ligand discovery did not produce viable hits against this target, despite considerable effort and a multitude of approaches. Ultimately, a rational structure-based design strategy was successful for the identification of Siah inhibitors in which peptide binding drives specific covalent bond formation with the target. X-ray crystallography, mass spectrometry, and functional data demonstrate that these peptide mimetics are efficient covalent inhibitors of Siah and antagonize Siah-dependent regulation of Erk and Hif signaling in the cell. The proposed strategy may result useful as a general approach to the design of peptide-based inhibitors of other protein-protein interactions.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/química , Proteínas Nucleares/antagonistas & inibidores , Peptídeos/química , Peptidomiméticos/química , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Peptídeos/farmacologia , Peptidomiméticos/farmacologia , Mapas de Interação de Proteínas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Plants use a highly evolved immune system to exhibit defense response against microbial infections. The plant TIR domain, together with the nucleotide-binding (NB) domain and/or a LRR region, forms a type of molecule, named resistance (R) proteins, that interact with microbial effector proteins and elicit hypersensitive responses against infection. Here, we report the first crystal structure of a plant TIR domain from Arabidopsis thaliana (AtTIR) solved at a resolution of 2.0 A. The structure consists of five beta-strands forming a parallel beta-sheet at the core of the protein. The beta-strands are connected by a series of alpha-helices and the overall fold mimics closely that of other mammalian and bacterial TIR domains. However, the region of the alphaD-helix reveals significant differences when compared with other TIR structures, especially the alphaD3-helix that corresponds to an insertion only present in plant TIR domains. Available mutagenesis data suggest that several conserved and exposed residues in this region are involved in the plant TIR signaling function.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/química , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sequência Conservada , Cristalografia por Raios X/métodos , Escherichia coli/genética , Modelos Moleculares , Dados de Sequência Molecular , Mapeamento de Interação de Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Alinhamento de SequênciaRESUMO
Macrophages detect pathogen infection via the activation of their plasma membrane-bound Toll-like receptor proteins (TLRs). The heterotypic interaction between the Toll/interleukin-1 receptor (TIR) domains of TLRs and adaptor proteins, like Myeloid differentiation primary response gene 88 (MyD88), is the first intracellular step in the signaling pathway of the mammalian innate immune response. The hetero-oligomerization of the TIRs of the receptor and adaptor brings about the activation of the transcription factor NF-kappaB, which regulates the synthesis of pro-inflammatory cytokines. Here, we report the first crystal structure of a bacterial TIR domain solved at 2.5 A resolution. The three-dimensional fold of Paracoccus denitrificans TIR is identical to that observed for the TIR of human TLRs and MyD88 proteins. The structure shows a unique dimerization interface involving the DD-loop and EE-loop residues, whereas leaving the BB-loop highly exposed. Peptide amide hydrogen-deuterium exchange mass spectrometry also reveals that the same region is used for dimerization in solution and in the context of the full-length protein. These results, together with a functional interaction between P. denitrificans TIR and MyD88 visualized in a co-immunoprecipitation assay, further substantiate the model that bacterial TIR proteins adopt structural mimicry of the host active receptor TIR domains to interfere with the signaling of TLRs and their adaptors to decrease the inflammatory response.