Role of Stx11 and STXBP2 in lytic granule exocytosis in health and disease

Stx11 和 STXBP2 在健康和疾病中溶解颗粒胞吐作用中的作用

• 批准号: 9309328
• 负责人: Claudio Guillermo Giraudo
• 金额: $ 43.58万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-25 至 2021-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9309328
• 关键词: Affect; Affinity; Alleles; Binding; Binding Proteins; Biochemical; Biological; Biological Assay; Biological Process; Cell Proliferation; Cell membrane; Cell physiology; Cell-Mediated Cytolysis; Cells; Chimeric Proteins; Chronic Childhood Arthritis; Clinical; Complex; Cytoplasmic Granules; Cytotoxic T-Lymphocytes; Data; Defect; Development; Diagnostic; Diagnostic tests; Disease; Dominant-Negative Mutation; Endosomes; Environment; Event; Exocytosis; Genes; Genetic; Germ-Line Mutation; Goals; Health; Hereditary Disease; Image; Immune; Immune System Diseases; Immune response; Impairment; In Vitro; Inflammation; Inflammatory Bowel Diseases; Intracellular Membranes; Janus kinase; Knowledge; Lead; Life; Light; Link; Liposomes; Lysosomes; Lytic; Macrophage Activation; Maps; Measures; Mediating; Membrane; Membrane Fusion; Microscopy; Molecular Mechanisms of Action; Mus; Mutate; Mutation; Natural Killer Cells; Normal Cell; Pathway interactions; Patients; Phenotype; Process; Proteins; Proteomics; Resolution; Role; SNAP23 gene; Sampling; Signal Transduction; Signaling Protein; Surface Plasmon Resonance; Symptoms; Syndrome; TNF gene; Techniques; Testing; Therapeutic; Total Internal Reflection Fluorescent; Transmembrane Transport; Viral; Virus Diseases; base; cell killing; congenital immunodeficiency; controlled release; cytotoxic; cytotoxicity; experimental study; immunological synapse; improved; induced pluripotent stem cell; insight; killings; macrophage; mutant; neoplastic cell; novel; novel diagnostics; protein function; protein protein interaction; syntaxin; syntaxin 11; tool; trafficking; tumor

ABSTRACT Primary immune deficiencies with impaired cell-mediated cytotoxicity commonly manifest as Familial Hemophagocytic Lymphohysticytosis (FHL), a life threatening disorder. FHL is characterized by disproportioned immune response mediated by excessively activated Cytotoxic T-lymphocyte (CTLs), Natural Killer (NK) and macrophages. FHL subjects usually harbor biallelic germline-mutations in genes involved in intracellular membrane trafficking including Syntaxin 11 (STX11, FHL-4) and Syntaxin-Binding Protein 2 (STXBP2, FHL-5). Nonetheless, monoallelic mutations have also been associated in FHL, but their clinical and biological significance remain unclear. Despite evidences linking STX11 and STXBP2 mutations to abnormal CTL and NK cell activity in FHL patients, there is a significant gap in knowledge about the normal function of these proteins and how these mutations impact on cytotoxic activity and ultimately lead to disease. This proposal aims to bridge this gap in knowledge between the genetic findings and the clinical manifestations in FHL-patients by establishing the basic mechanisms through which STX11/STXBP2 cooperate to effect LG exocytosis and by dissecting how they are affected in patient cells. STX11 and STXBP2 physically interact in as yet poorly characterized ways to control the release of lytic granule (LG) content at the immunological synapse. Data from our lab provide evidences that STX11 also interact with different sets of membrane fusion proteins that could mediate distinct trafficking steps. We hypothesize that STX11/STXBP2 control endosome- lysosome trafficking and LG fusion at the plasma membrane through these interactions. We further hypothesize that FHL mutations interfere with these interactions and thus impair cell-mediated cytotoxicity and/or survival signaling, a process that ultimately results in an array of immunological disorders with HLH symptoms. First, we will evaluate how FHL-mutations in STX11 and STXBP2 affect the physical relationship with one another and with other interacting proteins involved in exocytic and endo-lysosomal pathways by using biochemical and Mass Spec approaches in patient cells. We will establish how mutations affect the structural requirements for protein-protein interactions by performing pull-down, liposome co-flotation assays and surface plasmon resonance experiments. Second, we will assess the biological significance of both monollelic and biallelic FHL-mutations by investigating the intracellular trafficking defects in FHL-patient cells using super resolution STED and TIRF microscopy. Moreover, we will establish the molecular mechanisms of action of these mutations by using a unique in-vitro fusion assay develop in our lab. In summary, our studies will contribute more broadly to our understanding of the membrane trafficking steps that control cell killing pathways in CTL and NK cells, help in therapeutic decisions for patients carrying monoallelic mutations, establish common and gene-specific pathways affected in wide range of genetic disorders that share some manifestations of HLH symptoms and aid in the development of new tools for rapid diagnostic.

摘要 伴有细胞介导的细胞毒性受损的原发性免疫缺陷通常表现为家族性 噬血细胞性巨噬细胞增多症（FHL）是一种危及生命的疾病。FHL的特点是 过度活化的细胞毒性T淋巴细胞（CTL）介导的免疫应答，天然 杀伤细胞（NK）和巨噬细胞。FHL受试者通常在涉及以下的基因中具有双等位基因种系突变： 包括突触融合蛋白11（STX 11，FHL-4）和突触融合蛋白结合蛋白2的细胞内膜运输 （STXBP2，FHL-5）。尽管如此，单等位基因突变也与FHL相关，但其临床和 生物学意义尚不清楚。尽管有证据表明STX 11和STXBP 2突变与异常 在FHL患者的CTL和NK细胞活性方面，关于FHL患者的正常功能的知识存在显着差距。 这些蛋白质以及这些突变如何影响细胞毒活性并最终导致疾病。这 该提案旨在弥合遗传发现和临床表现之间的知识差距， 通过建立STX 11/STXBP 2合作影响LG的基本机制，FHL患者 胞吐作用，并通过解剖它们如何在患者细胞中受到影响。STX 11和STXBP 2在体内发生物理相互作用。 迄今为止，在免疫学上控制溶解颗粒（LG）内容物释放的方法还不清楚。 突触来自我们实验室的数据提供了证据，表明STX 11也与不同的膜融合集相互作用。 可以介导不同运输步骤的蛋白质。我们假设STX 11/STXBP 2控制内体- 溶酶体运输和LG融合在质膜通过这些相互作用。我们进一步 假设FHL突变干扰这些相互作用，从而削弱细胞介导细胞毒性 和/或生存信号，这一过程最终导致一系列HLH免疫紊乱 症状首先，我们将评估STX 11和STXBP 2中的FHL突变如何影响身体关系。 与其他参与胞吐和内溶酶体途径的相互作用蛋白质， 在病人细胞中使用生物化学和质谱方法。我们将确定突变如何影响 通过进行下拉、脂质体共浮选测定对蛋白质-蛋白质相互作用的结构要求 和表面等离子体共振实验。其次，我们将评估两者的生物学意义。 通过研究FHL患者细胞中的细胞内运输缺陷， 使用超分辨率STED和TIRF显微镜。此外，我们将建立分子机制， 这些突变的作用，通过使用一个独特的体外融合试验在我们的实验室开发。总之，我们的研究 将有助于我们更广泛地了解控制细胞杀伤的膜运输步骤 CTL和NK细胞中的通路，有助于携带单等位基因突变的患者的治疗决策， 建立共同的和基因特异性的途径，影响广泛的遗传性疾病， HLH症状的临床表现，并有助于开发快速诊断的新工具。