RESUMO
MALT1 paracaspase is central for lymphocyte antigen-dependent responses including NF-κB activation. We discovered nanomolar, selective allosteric inhibitors of MALT1 that bind by displacing the side chain of Trp580, locking the protease in an inactive conformation. Interestingly, we had previously identified a patient homozygous for a MALT1 Trp580-to-serine mutation who suffered from combined immunodeficiency. We show that the loss of tryptophan weakened interactions between the paracaspase and C-terminal immunoglobulin MALT1 domains resulting in protein instability, reduced protein levels and functions. Upon binding of allosteric inhibitors of increasing potency, we found proportionate increased stabilization of MALT1-W580S to reach that of wild-type MALT1. With restored levels of stable MALT1 protein, the most potent of the allosteric inhibitors rescued NF-κB and JNK signaling in patient lymphocytes. Following compound washout, MALT1 substrate cleavage was partly recovered. Thus, a molecular corrector rescues an enzyme deficiency by substituting for the mutated residue, inspiring new potential precision therapies to increase mutant enzyme activity in other deficiencies.
Assuntos
Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/antagonistas & inibidores , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , Regulação da Expressão Gênica , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/terapia , Linfócitos/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/genética , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/ultraestrutura , NF-kappa B/metabolismo , Proteínas de Neoplasias , Transdução de SinaisRESUMO
S-Nitrosylation is a redox-based protein post-translational modification in response to nitric oxide signaling and is involved in a wide range of biological processes. Detection and quantification of protein S-nitrosylation have been challenging tasks due to instability and low abundance of the modification. Many studies have used mass spectrometry (MS)-based methods with different thiol-reactive reagents to label and identify proteins with S-nitrosylated cysteine (SNO-Cys). In this study, we developed a novel iodoTMT switch assay (ISA) using an isobaric set of thiol-reactive iodoTMTsixplex reagents to specifically detect and quantify protein S-nitrosylation. Irreversible labeling of SNO-Cys with the iodoTMTsixplex reagents enables immune-affinity detection of S-nitrosylated proteins, enrichment of iodoTMT-labeled peptides by anti-TMT resin, and importantly, unambiguous modification site-mapping and multiplex quantification by liquid chromatography-tandem MS. Additionally, we significantly improved anti-TMT peptide enrichment efficiency by competitive elution. Using ISA, we identified a set of SNO-Cys sites responding to lipopolysaccharide (LPS) stimulation in murine BV-2 microglial cells and revealed effects of S-allyl cysteine from garlic on LPS-induced protein S-nitrosylation in antioxidative signaling and mitochondrial metabolic pathways. ISA proved to be an effective proteomic approach for quantitative analysis of S-nitrosylation in complex samples and will facilitate the elucidation of molecular mechanisms of nitrosative stress in disease.
Assuntos
Iodoacetatos/química , Animais , Linhagem Celular , Lipopolissacarídeos/farmacologia , Camundongos , Anotação de Sequência Molecular , Mapeamento de Peptídeos , Processamento de Proteína Pós-Traducional , Proteômica , Coloração e RotulagemRESUMO
Adaptive immunity is the specialized defence mechanism in vertebrates that evolved to eliminate pathogens. Specialized lymphocytes recognize specific protein epitopes through antigen receptors to mount potent immune responses, many of which are initiated by nuclear factor-kappa B activation and gene transcription. Most, if not all, pathways in adaptive immunity are further regulated by post-translational modification (PTM) of signalling proteins, e.g. phosphorylation, citrullination, ubiquitination and proteolytic processing. The importance of PTMs is reflected by genetic or acquired defects in these pathways that lead to a dysfunctional immune response. Here we discuss the state of the art in targeted proteomics and systems biology approaches to dissect the PTM landscape specifically regarding ubiquitination and proteolysis in B- and T-cell activation. Recent advances have occurred in methods for specific enrichment and targeted quantitation. Together with improved instrument sensitivity, these advances enable the accurate analysis of often rare PTM events that are opaque to conventional proteomics approaches, now rendering in-depth analysis and pathway dissection possible. We discuss published approaches, including as a case study the profiling of the N-terminome of lymphocytes of a rare patient with a genetic defect in the paracaspase protease MALT1, a key regulator protease in antigen-driven signalling, which was manifested by elevated linear ubiquitination.This article is part of the themed issue 'Quantitative mass spectrometry'.
Assuntos
Imunidade Adaptativa , Proteínas/química , Proteínas/metabolismo , Proteólise , Proteômica/métodos , Ubiquitinação , Animais , HumanosRESUMO
Encysted embryos of Artemia franciscana are exceptionally resistant to stress and an important part of this tolerance involves p26, a small heat shock protein which functions as a molecular chaperone. Cloning and sequencing of randomly selected p26 cDNAs produced by RT-PCR with poly(A)(+) mRNA from encysted embryos as template yielded 10 clones encoding identical polypeptides. The noncoding nucleotide sequences extending from the termination codon to the poly(A) tail of each clone were also identical. These data indicated a single p26 gene is expressed during embryo development. However, two-dimensional gel electrophoresis showed that purified p26 consisted of four isoforms, providing evidence for posttranslational modification of the protein, a possibility supported by mass spectrometry and immunoprobing of Western blots. The major isoform observed in two-dimensional gels, termed a, is the primary gene product, whereas isoform c is phosphorylated at serine 50, a residue located in a protein kinase C reactive site. Isoforms b and d were generated posttranslationally, but by unknown processes. The results represent the first description of posttranslationally modified small heat shock proteins in crustaceans and they expand the phylogenetic range of organisms that possess phosphorylated isoforms of these proteins. At least two small heat shock proteins from other organisms contain serine residues equivalent in position to serine 50 of p26, but neither is phosphorylated.
Assuntos
Artemia , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Fosfosserina/metabolismo , Serina/química , Serina/metabolismo , Sequência de Aminoácidos , Animais , Artemia/genética , Artemia/metabolismo , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Eletroforese em Gel Bidimensional , Proteínas de Choque Térmico/genética , Humanos , Dados de Sequência Molecular , Fosforilação , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Alinhamento de Sequência , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Antigen receptor signalling activates the canonical NF-κB pathway via the CARD11/BCL10/MALT1 (CBM) signalosome involving key, yet ill-defined roles for linear ubiquitination. The paracaspase MALT1 cleaves and removes negative checkpoint proteins, amplifying lymphocyte responses in NF-κB activation and in B-cell lymphoma subtypes. To identify new human MALT1 substrates, we compare B cells from the only known living MALT1(mut/mut) patient with healthy MALT1(+/mut) family members using 10-plex Tandem Mass Tag TAILS N-terminal peptide proteomics. We identify HOIL1 of the linear ubiquitin chain assembly complex as a novel MALT1 substrate. We show linear ubiquitination at B-cell receptor microclusters and signalosomes. Late in the NF-κB activation cycle HOIL1 cleavage transiently reduces linear ubiquitination, including of NEMO and RIP1, dampening NF-κB activation and preventing reactivation. By regulating linear ubiquitination, MALT1 is both a positive and negative pleiotropic regulator of the human canonical NF-κB pathway-first promoting activation via the CBM--then triggering HOIL1-dependent negative-feedback termination, preventing reactivation.
Assuntos
Caspases/genética , Síndromes de Imunodeficiência/genética , Linfócitos/imunologia , NF-kappa B/metabolismo , Proteínas de Neoplasias/genética , Ubiquitina-Proteína Ligases/metabolismo , Adolescente , Adulto , Animais , Células Apresentadoras de Antígenos , Linfócitos B/imunologia , Caspases/imunologia , Caspases/metabolismo , Família , Feminino , Imunofluorescência , Proteínas Ativadoras de GTPase/metabolismo , Técnicas de Introdução de Genes , Humanos , Quinase I-kappa B/metabolismo , Immunoblotting , Síndromes de Imunodeficiência/imunologia , Imunoprecipitação , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucócitos Mononucleares , Masculino , Espectrometria de Massas , Camundongos , Microscopia Confocal , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Mutação , NF-kappa B/imunologia , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Tonsila Palatina , Proteômica , Proteínas de Ligação a RNA/metabolismo , Linfócitos T/imunologia , Espectrometria de Massas em Tandem , Fatores de Transcrição , Ubiquitinação/imunologiaRESUMO
The modification of Ser/Thr residues in proteins by addition of single O-linked N-acetylglucosamine (O-GlcNAc) moieties play an important role in cell regulation. However, understanding the cellular mechanisms that regulate O-GlcNAc glycosylation has been challenging due to the difficulty in detection and quantification of this modification. Mass spectrometry-based multiplex quantitative approaches have been successfully employed to measure relative phosphorylation levels using collisionally induced dissociation (CID). However, labile modifications such as O-GlcNAc are lost prior to fragmentation of the peptide backbone in conventional CID, often preventing correct peptide identification, localization of the modified site, and as a result, relative quantification. Compared to CID, Electron Transfer Dissociation (ETD) preserves labile post-translational modifications (PTMs), and allows direct mapping of peptide/protein modifications. This is the first report to assess the utility of combining multiplexed isobaric tandem mass tag (TMT) labeling and ETD for relative quantification of labile PTMs. ETD analysis of both labeled and unlabeled peptides from bovine alpha-crystallins pinpointed at least one O-GlcNAc containing modification site in each of the protein subunits, in addition to a multitude of other PTMs, including glycation, phosphorylation, and acetylation. Moreover, ETD of TMT(6) labeled peptides produced four unique reporter ions that could be used for relative quantification. TMT reporter ion ratios measured by ETD had similar accuracy and precision as those obtained by conventional CID techniques. When applied to glycosylated or otherwise modified peptides, ETD was the only dissociation method which consistently provided confident sequence identification, PTM localization, and quantitative information, all in the same spectrum. This suggests that ETD-based workflows can be complementary to traditional CID approaches when used for simultaneous qualitative and quantitative analysis of modified peptides.
Assuntos
Espectrometria de Massas/métodos , Peptídeos/química , alfa-Cristalinas/química , Sequência de Aminoácidos , Animais , Bovinos , Cromatografia Líquida/métodos , Elétrons , Glicosilação , Modelos Químicos , Dados de Sequência Molecular , Fosforilação , Processamento de Proteína Pós-Traducional , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
A new global protein digestion and selective peptide extraction strategy for the purpose of monitoring differential protein expression, coined as tagless extraction-retentate chromatography, is introduced. Target protein populations are firstly digested under reduced and alkylated conditions, and resultant peptides selectively extracted via covalent attachment to methionine residues by bromoacetyl reactive groups tethered to the surface of glass beads packed in small reaction vessels. After conjugation, reactive beads are stringently washed to remove nonspecifically bound peptides and then later treated with beta-mercaptoethanol to release captured methionine peptides in their nascent state, without complicating affinity tags. Recovered methionine containing peptides are profiled using the surface-enhanced laser desorption/ionization (SELDI) retentate chromatography mass spectrometry (RCMS) method. Selected peptides are further studied employing ProteinChip tandem mass spectrometry (MS/MS) analysis to identify their parent proteins. This approach has been applied to an Escherichia coli lysate model system and has demonstrated facility in reducing global digest complexity, sensitivity to low protein expression levels, and significant quantitative capability. It is envisioned that tagless extraction-RCMS will evolve to be a valuable approach for both basic research and clinical proteomics endeavors.