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

Stx11 和 STXBP2 对健康和疾病中溶解颗粒胞吐作用的作用

• 批准号: 8882583
• 负责人: Claudio Guillermo Giraudo
• 金额: $ 42万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-15 至 2016-07-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8882583
• 关键词: Address; Affect; Affinity; Bacteria; Binding Proteins; Biochemical; Biochemistry; Biological; Biological Assay; Blood Platelets; Cell membrane; Cell physiology; Cell-Mediated Cytolysis; Cells; Clinical; Color; Complex; Cytoplasmic Granules; Cytotoxic T-Lymphocytes; Data; Defect; Development; Diagnosis; Diagnostic; Disease; Environment; Event; Exocytosis; Functional disorder; Future; Gene Mutation; Genes; Germ-Line Mutation; Goals; Health; Immune; Immunologic Deficiency Syndromes; Immunoprecipitation; In Vitro; Inflammatory; Intracellular Membranes; Length; Life; Liposomes; Lytic; Measures; Megakaryocytes; Membrane; Membrane Fusion; Membrane Protein Traffic; Methods; Microscopy; Molecular; Mutate; Mutation; Natural Killer Cells; Normal Cell; Pathologic; Pathway interactions; Patients; Pattern; Phenotype; Physiological; Process; Proteins; Recombinant Proteins; Resolution; Rest; Role; SNAP receptor; Series; Structure; Surface Plasmon Resonance; System; T-Cell Activation; T-Lymphocyte and Natural Killer Cell; Techniques; Testing; Therapeutic; UNC13B gene; base; congenital immunodeficiency; design; disease-causing mutation; insight; interdisciplinary approach; killings; loss of function; mutant; novel; novel strategies; novel therapeutic intervention; perforin; protein protein interaction; protein transport; public health relevance; research study; synaptogenesis; syntaxin; syntaxin 11; syntaxin 4; syntaxin-2

DESCRIPTION (provided by applicant): Primary immune deficiencies with impaired cell-mediated cytotoxicity most commonly manifest as Familial Hemophagocytic Lymphohysticytosis (FHL), a life threatening disorder. FHL subjects harboring germline mutations in the genes encoding Syntaxin 11 (STX11, FHL-4) and Syntaxin Binding Protein 2 (STXBP2, FHL- 5) display abnormal Cytotoxic T-lymphocyte and Natural Killer cell activity owing to impaired lytic granule (LG) exocytosis. This process involves a highly orchestrated series of protein-protein interactions that culminate in fusion of the LG membrane with the plasma membrane, releasing perforin and other LG contents. STX11 and STXBP2 physically interact in as yet poorly characterized ways to orchestrate this fusion event. Due to the complexity of the cellular environment, it has been challenging to define the cellular and molecular defects underlying immunodeficiency in patients with STX11 or STXBP2 mutations. Toward this end, this proposal aims to establish the basic mechanisms through which STX11 and STXBP2 cooperate to effect LG fusion, and to use novel in vitro and ex-vivo fusion assays to understand how these molecular mechanisms are affected by FHL-associated mutations. To address these questions, we will carry out three Specific Aims. First, we will ask whether and how FHL-mutations in STX11 and STXBP2 affect physical interactions with one another and with other SNAREs. We will use a biochemical approach, performing pull-down, liposome co-flotation and surface plasmon resonance experiments to evaluate how mutations affect the structural requirements for protein- protein interactions. To assess the biological significance of our in vitro results, w will perform immunoprecipitation experiments using ex-vivo cells. Second, we will test the effect of FHL-mutations on intracellular membrane trafficking and lytic granule release. We will precisely localize STX11, STXBP2 and other key trafficking protein machinery under resting and activated conditions in ex-vivo cells from normal and FHL subjects using super-resolution STED microscopy. Additionally, we will set up a TIRF-based dual-color secretion assay to accurately evaluate at the single cell level the effects of FHL-mutations on LG release. This assay will also be used to explore novel strategies to rescue the impaired LG release in FHL cells. Third, we will develop novel methods to study the functional effect of FHL-associated mutations on LG secretion with high resolution and without the complexity of the cellular environment. Toward this end, we will perform liposome-liposome fusion assays, specifically designed to assess the molecular function of Stx11 and STXBP2 proteins in LG granule secretion. This unique simplified system, will serve to elucidate the mechanism by which FHL mutations affect membrane fusion. In summary, this project will combine basic biochemistry using recombinant proteins and in-vitro fusion assays with cell-biological experiments in patient-derived cells to provide new insights into the functional consequences of the patients' genetic defects. Results from these studies will also provide insights that can be applied to diagnosis and targeted therapies for FHL.

描述（由申请方提供）：原发性免疫缺陷伴细胞介导的细胞毒性受损，最常见的表现为家族性噬血细胞性巨噬细胞增多症（FHL），这是一种危及生命的疾病。在编码突触融合蛋白11（STX 11，FHL-4）和突触融合蛋白结合蛋白2（STXBP 2，FHL- 5）的基因中携带种系突变的FHL受试者由于受损的溶解颗粒（LG）胞吐作用而显示异常的细胞毒性T淋巴细胞和自然杀伤细胞活性。这一过程涉及一系列高度协调的蛋白质-蛋白质相互作用，最终导致LG膜与质膜融合，释放穿孔素和其他LG内容物。STX 11和STXBP 2在物理上以尚未充分表征的方式相互作用，以协调这种融合事件。由于细胞环境的复杂性，确定STX 11或STXBP 2突变患者免疫缺陷的细胞和分子缺陷一直是一个挑战。为此，该提案旨在建立STX 11和STXBP 2合作实现LG融合的基本机制，并使用新的体外和离体融合试验来了解这些分子机制如何受到FHL相关突变的影响。为了解决这些问题，我们将实现三个具体目标。首先，我们将询问STX 11和STXBP 2中的FHL突变是否以及如何影响彼此之间以及与其他SNARE之间的物理相互作用。我们将使用生物化学方法，进行下拉，脂质体共浮选和表面等离子体共振实验，以评估突变如何影响蛋白质-蛋白质相互作用的结构要求。为了评估我们体外结果的生物学意义，我们将使用离体细胞进行免疫沉淀实验。其次，我们将测试FHL突变对细胞内膜运输和溶解颗粒释放的影响。我们将使用超分辨率STED显微镜在正常和FHL受试者的离体细胞中精确定位STX 11，STXBP 2和其他关键的运输蛋白机械。此外，我们将建立一个基于TIRF的双色分泌测定，以准确地评估在单细胞水平上的FHL突变对LG释放的影响。该试验也将用于探索新的策略，以挽救受损的LG释放FHL细胞。第三，我们将开发新的方法来研究FHL相关突变对LG分泌的功能性影响，具有高分辨率，并且没有细胞环境的复杂性。为此，我们将进行脂质体-脂质体融合试验，专门设计用于评估Stx 11和STXBP 2蛋白在LG颗粒分泌中的分子功能。这种独特的简化系统，将有助于阐明FHL突变影响膜融合的机制。总之，该项目将联合收割机基础生物化学与重组蛋白和体外融合试验相结合，并在患者来源的细胞中进行细胞生物学实验，以提供对患者遗传缺陷的功能后果的新见解。这些研究的结果也将提供可应用于FHL诊断和靶向治疗的见解。