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Poisoning with the organophosphorus nerve agent VX can be life-threatening due to limitations of the standard therapy with atropine and oximes. To date, the underlying pathomechanism of VX affecting the neuromuscular junction has not been fully elucidated structurally. Results of recent studies investigating the effects of VX were obtained from cells of animal origin or immortalized cell lines limiting their translation to humans. To overcome this limitation, motor neurons (MN) of this study were differentiated from in-house feeder- and integration-free-derived human-induced pluripotent stem cells (hiPSC) by application of standardized and antibiotic-free differentiation media with the aim to mimic human embryogenesis as closely as possible. For testing VX sensitivity, MN were initially exposed once to 400 µM, 600 µM, 800 µM, or 1000 µM VX and cultured for 5 days followed by analysis of changes in viability and neurite outgrowth as well as at the gene and protein level using µLC-ESI MS/HR MS, XTT, IncuCyte, qRT-PCR, and Western Blot. For the first time, VX was shown to trigger neuronal cell death and decline in neurite outgrowth in hiPSC-derived MN in a time- and concentration-dependent manner involving the activation of the intrinsic as well as the extrinsic pathway of apoptosis. Consistent with this, MN morphology and neurite network were altered time and concentration-dependently. Thus, MN represent a valuable tool for further investigation of the pathomechanism after VX exposure. These findings might set the course for the development of a promising human neuromuscular test model and patient-specific therapies in the future.
Assuntos
Diferenciação Celular , Sobrevivência Celular , Células-Tronco Pluripotentes Induzidas , Neurônios Motores , Agentes Neurotóxicos , Compostos Organotiofosforados , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Neurônios Motores/efeitos dos fármacos , Compostos Organotiofosforados/toxicidade , Agentes Neurotóxicos/toxicidade , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Crescimento Neuronal/efeitos dos fármacos , Substâncias para a Guerra Química/toxicidade , Relação Dose-Resposta a Droga , Células CultivadasRESUMO
We developed a bioinformatics-led substrate discovery workflow to expand the known substrate repertoire of MALT1. Our approach, termed GO-2-Substrates, integrates protein function information, including GO terms from known substrates, with protein sequences to rank substrate candidates by similarity. We applied GO-2-Substrates to MALT1, a paracaspase and master regulator of NF-κB signalling in adaptive immune responses. With only 12 known substrates, the evolutionarily conserved paracaspase functions and phenotypes of Malt1 -/- mice strongly implicate the existence of undiscovered substrates. We tested the ranked predictions from GO-2-Substrates of new MALT1 human substrates by co-expression of candidates transfected with the oncogenic constitutively active cIAP2-MALT1 fusion protein or CARD11/BCL10/MALT1 active signalosome. We identified seven new MALT1 substrates by the co-transfection screen: TANK, TAB3, CASP10, ZC3H12D, ZC3H12B, CILK1 and ILDR2. Using catalytically inactive cIAP2-MALT1 (Cys464Ala), a MALT1 inhibitor, MLT-748, and noncleavable P1-Arg to Ala mutant versions of each substrate in dual transfections, we validated the seven new substrates in vitro. We confirmed the cleavage of endogenous TANK and the RNase ZC3H12D in B cells by Western blotting and mining TAILS N-terminomics datasets, where we also uncovered evidence for these and 12 other candidate substrates by endogenous MALT1. Thus, protein function information improves substrate predictions. The new substrates and other high-ranked MALT1 candidate substrates should open new biological frontiers for further validation and exploration of the function of MALT1 within and beyond NF-κB regulation.
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Autoimmune lymphoproliferative syndrome is a primary immunodeficiency caused by variants in FAS-mediated apoptosis related genes and is characterized by lymphadenopathy, splenomegaly and autoimmunity. A total of six different variants in CASP10 have been described as potential causative of disease, although two of them have recently been considered polymorphisms. The high allele frequency of these variants in healthy population in addition to the broad clinical spectrum of the disease difficult the interpretation of their pathogenicity. Here, we describe the clinical and analytical findings of three new patients carrying variants in CASP10 and summarize 12 more cases from the literature. Autoimmune cytopenias, adenopathies and increment of TCRαß+CD4-CD8- cells have been the most common findings, being possibly the FAS-mediated apoptosis pathway the pathogenic mechanism of this disease. The clinical impact and the consequences of CASP10 variants are not fully elucidated, therefore the description of new cases will contribute to solve this issue.
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Síndrome Linfoproliferativa Autoimune/enzimologia , Síndrome Linfoproliferativa Autoimune/genética , Caspase 10/genética , Variação Genética , Adolescente , Adulto , Substituição de Aminoácidos , Apoptose/genética , Síndrome Linfoproliferativa Autoimune/diagnóstico , Feminino , Mutação da Fase de Leitura , Humanos , Masculino , Linhagem , Polimorfismo Genético , Polimorfismo de Nucleotídeo Único , Deleção de SequênciaRESUMO
INTRODUCTION: Apoptosis deregulation have been associated to tumorigenesis process and was highlighted as a prominent hallmark of cancer. Several mutations have been reported in several forms of Blood cancer. However, it has never been investigated in familial aggregations of hematological malignancies. METHODS: In this study, we performed a mutational analysis by sequencing the entire coding regions in four key apoptotic genes FAS, FASLG, CASP8 and CASP10 in 92 independent families belonging to French and Tunisian populations and diagnosed with several forms of familial hematological malignancies. RESULTS: We report 15 genetic variations among which 7 were previously reported in several form of cancers and have a potential effect on gene expression. Particularly, the CASP8 variants p.Asp302His and p.Lys337Lys were detected in 15% and 10% of our group of patients respectively and were previously reported in association to breast cancer and to breast cancer susceptibility. DISCUSSION: In this study, we do not report the underlining deleterious mutations in familial hematological malignancies, but we describe some variants with potential risk of developing blood cancer. To gain further insights on the association between apoptosis pathway deregulation and familial hematological malignancies, more apoptotic genes should be investigated.
Assuntos
Apoptose/genética , Caspase 10/genética , Caspase 8/genética , Proteína Ligante Fas/genética , Neoplasias Hematológicas/genética , Receptor fas/genética , Alelos , Estudos Transversais , Análise Mutacional de DNA/métodos , Família , França , Predisposição Genética para Doença , Humanos , Íntrons , Mutação de Sentido Incorreto , Perforina/genética , TunísiaRESUMO
Dilated cardiomyopathy (DCM) is the leading cause of morbidity and mortality in diabetic patients. Long noncoding RNA plasmacytoma variant translocation 1 (PVT1) has been shown to be related to the pathogenesis of DCM. However, the mechanism by which PVT1 regulates DCM pathogenesis is unclear. High glucose level was employed to construct a DCM cell model in vitro. Cell viability was determined via cell counting kit-8 assay. The level of lactate dehydrogenase (LDH) was measured with the corresponding kit. Expression levels of PVT1, miR-23a-3p, and caspase-10 (CASP10) messenger RNA were evaluated with a quantitative real-time polymerase chain reaction. Cell apoptosis was assessed by flow cytometry assay. Protein levels of B-cell lymphoma 2-associated X (Bax), cleaved-caspase-3 (cleaved-casp-3), and CASP10 were examined via western blot analysis. The relationship between PVT1 or CASP10 and miR-23a-3p was verified with dual-luciferase reporter assay. We observed that PVT1 and CASP10 were upregulated while miR-23a-3p was downregulated in high glucose-induced cardiomyocytes. High glucose levels repressed cardiomyocyte activity and induced cardiomyocyte apoptosis, but this influence was antagonized by PVT1 knockdown or miR-23a-3p overexpression. Furthermore, PVT1 acted as a sponge for miR-23a-3p, and miR-23a-3p inhibition counterbalanced the influence of PVT1 silencing on viability and apoptosis of cardiomyocytes under high glucose level treatment. PVT1 could increase CASP10 expression via sponging miR-23a-3p. In conclusion, PVT1 acted as a deleterious lncRNA in DCM. PVT1 facilitated cardiomyocyte death by regulating the miR-23a-3p/CASP10, which offered a new mechanism to comprehend the pathogenesis of DCM.
Assuntos
Caspase 10/metabolismo , Glucose/toxicidade , MicroRNAs/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , RNA Longo não Codificante/metabolismo , Adulto , Apoptose/efeitos dos fármacos , Apoptose/genética , Sequência de Bases , Caspase 10/genética , Morte Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Humanos , MicroRNAs/genética , Miócitos Cardíacos/efeitos dos fármacos , RNA Longo não Codificante/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
We present a Model Quality Assessment Program (MQAP), called MQAPsingle, for ranking and assessing the absolute global quality of single protein models. MQAPsingle is quasi single-model MQAP, a method that combines advantages of both "pure" single-model MQAPs and clustering MQAPs. This approach results in higher accuracy compared to the state-of-the-art single-model MQAPs. Notably, the prediction for a given model is the same regardless if this model is submitted to our server alone or together with other models. Proteins 2016; 84:1021-1028. © 2015 Wiley Periodicals, Inc.
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Caspase 10/química , Biologia Computacional/métodos , Modelos Moleculares , Software , Benchmarking , Humanos , Internet , Conformação ProteicaRESUMO
Autoimmune lymphoproliferative syndrome (ALPS) is a primary immunodeficiency caused by impaired Fas/FasL-mediated apoptosis of lymphocytes and is characterized by chronic nonmalignant or benign lymphoproliferation, autoimmune manifestations and expansion of double negative (DN) T-cells (TCRαß+CD4-CD8-). Most cases of ALPS are associated with germline (ALPS-FAS) or somatic (ALPS-sFAS) heterozygous FAS mutations or a combination of both. Here we report three unrelated patients with ALPS-sFAS. Only one of them showed impaired Fas function in PHA-activated T-cells. In this patient, the genetic analysis of the caspase-10 gene (CASP10) identified a heterozygous germline change in exon 9 (c.1337A>G) causing Y446C substitution in the caspase-10 protein. In addition, this patient had a dysregulated T- and B-cell phenotype; circulating lymphocytes showed expansion of T effector memory CD45RA+ (TEMRA) CD4 T-cells, effector memory CD8 T-cells, CD21(low) B-cells and reduced memory switched B-cells. Additionally, this patient showed altered expression in T-cells of several molecules that change during differentiation from naïve to effector cells (CD27, CD95, CD57 and perforin). Molecular alterations in genes of the Fas pathway are necessary for the development of ALPS and this syndrome could be influenced by the concurrent effect of other mutations hitting different genes involved in Fas or related pathways.
Assuntos
Síndrome Linfoproliferativa Autoimune/genética , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Caspase 10/genética , Receptor fas/genética , Adolescente , Antígenos CD/genética , Antígenos CD/imunologia , Síndrome Linfoproliferativa Autoimune/imunologia , Síndrome Linfoproliferativa Autoimune/patologia , Linfócitos B/patologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/patologia , Caspase 10/imunologia , Éxons , Feminino , Expressão Gênica , Humanos , Memória Imunológica , Doenças Linfáticas/genética , Doenças Linfáticas/imunologia , Doenças Linfáticas/patologia , Ativação Linfocitária/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Mutação , Perforina/genética , Perforina/imunologia , Fito-Hemaglutininas/farmacologia , Cultura Primária de Células , Esplenomegalia/genética , Esplenomegalia/imunologia , Esplenomegalia/patologia , Receptor fas/imunologiaRESUMO
In CASP10, for the first time, contact-assisted structure predictions have been assessed. Sets of pairs of contacting residues from target structures were provided to predictors for a second round of prediction after the initial round in which they were given only sequences. The objective of the experiment was to measure model quality improvement resulting from the added contact information and thereby assess and help develop so-called hybrid prediction methods--methods where some experimentally determined distance constraints are used to augment de novo computational prediction methods. The results of the experiment were, overall, quite promising.
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Biologia Computacional/métodos , Modelos Moleculares , Conformação Proteica , Proteínas/química , Modelos Estatísticos , Alinhamento de SequênciaRESUMO
We develop and test a new pipeline in CASP10 to predict protein structures based on an interplay of I-TASSER and QUARK for both free-modeling (FM) and template-based modeling (TBM) targets. The most noteworthy observation is that sorting through the threading template pool using the QUARK-based ab initio models as probes allows the detection of distant-homology templates which might be ignored by the traditional sequence profile-based threading alignment algorithms. Further template assembly refinement by I-TASSER resulted in successful folding of two medium-sized FM targets with >150 residues. For TBM, the multiple threading alignments from LOMETS are, for the first time, incorporated into the ab initio QUARK simulations, which were further refined by I-TASSER assembly refinement. Compared with the traditional threading assembly refinement procedures, the inclusion of the threading-constrained ab initio folding models can consistently improve the quality of the full-length models as assessed by the GDT-HA and hydrogen-bonding scores. Despite the success, significant challenges still exist in domain boundary prediction and consistent folding of medium-size proteins (especially beta-proteins) for nonhomologous targets. Further developments of sensitive fold-recognition and ab initio folding methods are critical for solving these problems.
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Biologia Computacional/métodos , Modelos Moleculares , Modelos Estatísticos , Conformação Proteica , Proteínas/química , Algoritmos , Simulação por Computador , Alinhamento de SequênciaRESUMO
Template-based modeling (TBM) is a major component of the critical assessment of protein structure prediction (CASP). In CASP10, some 41,740 predicted models submitted by 150 predictor groups were assessed as TBM predictions. The accuracy of protein structure prediction was assessed by geometric comparison with experimental X-ray crystal and NMR structures using a composite score that included both global alignment metrics and distance-matrix-based metrics. These included GDT-HA and GDC-all global alignment scores, and the superimposition-independent LDDT distance-matrix-based score. In addition, a superimposition-independent RPF metric, similar to that described previously for comparing protein models against experimental NMR data, was used for comparing predicted protein structure models against experimental protein structures. To score well on all four of these metrics, models must feature accurate predictions of both backbone and side-chain conformations. Performance rankings were determined independently for server and the combined server plus human-curated predictor groups. Final rankings were made using paired head-to-head Student's t-test analysis of raw metric scores among the top 25 performing groups in each category.
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Biologia Computacional/métodos , Conformação Proteica , Proteínas/química , Algoritmos , Simulação por Computador , Modelos Moleculares , Modelos Estatísticos , Análise de Sequência de ProteínaRESUMO
We present the assessment of predictions for Template-Free Modeling in CASP10 and a report on the first ROLL experiment wherein predictions are collected year round for review at the regular CASP season. Models were first clustered so that duplicated or very similar ones were grouped together and represented by one model in the cluster. The representatives were then compared with targets using GDT_TS, QCS, and three additional superposition-independent score functions newly developed for CASP10. For each target, the top 15 representatives by each score were pooled to form the Top15Union set. All models in this set were visually inspected by four of us independently using the new plugin, EvalScore, which we developed with the UCSF Chimera group. The best models were selected for each target after extensive debate among the four examiners. Groups were ranked by the number of targets (hits) for which a group's model was selected as one of the best models. The Keasar group had most hits in both categories, with four of 19 FM and eight of 36 ROLL targets. The most successful prediction servers were QUARK from Zhang's group for FM category with three hits and Zhang-server for the ROLL category with seven hits. As observed in CASP9, many successful groups were not true "template-free" modelers but used remote templates and/or server models to obtain their winning models. The results of the first ROLL experiment were broadly similar to those of the CASP10 FM exercise.
Assuntos
Biologia Computacional/métodos , Modelos Moleculares , Estrutura Terciária de Proteína , Proteínas/química , Análise por ConglomeradosRESUMO
For the 10th experiment on Critical Assessment of the techniques of protein Structure Prediction (CASP), the prediction target proteins were broken into independent evaluation units (EUs), which were then classified into template-based modeling (TBM) or free modeling (FM) categories. We describe here how the EUs were defined and classified, what issues arose in the process, and how we resolved them. EUs are frequently not the whole target proteins but the constituting structural domains. However, the assessors from CASP7 on combined more than one domain into 1 EU for some targets, which implied that the assessment also included evaluation of the prediction of the relative position and orientation of these domains. In CASP10, we followed and expanded this notion by defining multidomain EUs for a number of targets. These included 3 EUs, each made of two domains of familiar fold but arranged in a novel manner and for which the focus of evaluation was the interdomain arrangement. An EU was classified to the TBM category if a template could be found by sequence similarity searches and to FM if a structural template could not be found by structural similarity searches. The EUs that did not fall cleanly in either of these cases were classified case-by-case, often including consideration of the overall quality and characteristics of the predictions.