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

描述(由申请人提供)：细胞介导的细胞毒性受损的原发免疫缺陷最常见的表现为家族性噬血细胞淋巴囊病(FHL)，这是一种危及生命的疾病。携带Synaxin 11(STX11，FHL-4)和Synaxin结合蛋白2(STXBP2，FHL-5)基因胚系突变的FHL患者，由于溶解颗粒(LG)胞吐功能受损，表现出异常的细胞毒性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成分。STX11和STXBP2以迄今尚不典型的方式相互作用，以协调这一融合事件。由于细胞环境的复杂性，确定STX11或STXBP2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，本提案旨在建立STX11和STXBP2协同作用影响LG融合的基本机制，并使用新的体外和体外融合分析来了解FHL相关突变如何影响这些分子机制。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STX11和STXBP2上的FHL-突变是否以及如何影响彼此之间以及与其他陷阱的物理相互作用。我们将使用生化方法，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。为了评估我们的体外结果的生物学意义，我们将使用体外细胞进行免疫沉淀实验。其次，我们将测试FHL突变对细胞内膜转运和裂解颗粒释放的影响。我们将利用超分辨STED显微镜在正常和FHL受试者的体外细胞中，在静息和激活条件下对STX11、STXBP2和其他关键运输蛋白机制进行精确定位。此外，我们将建立一种基于TIRF的双色分泌试验，在单细胞水平上准确评估FHL突变对LG释放的影响。该检测还将被用于探索挽救FHL细胞中受损的LG释放的新策略。第三，我们将开发新的方法来研究FHL相关突变对LG分泌的功能影响，以高分辨率并且不需要复杂的细胞环境。为此，我们将进行脂质体-脂质体融合分析，专门设计来评估Stx11和STXBP2蛋白在LG颗粒分泌中的分子功能。这一独特的简化系统将有助于阐明FHL突变影响膜融合的机制。总而言之，该项目将把使用重组蛋白的基础生物化学和体外融合分析与患者来源细胞的细胞生物学实验结合起来，为患者遗传缺陷的功能后果提供新的见解。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。

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.