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

摘要 细胞介导的细胞毒作用受损的原发免疫缺陷通常表现为家族性 噬血细胞性淋巴囊肿症(FHL)，一种危及生命的疾病。FHL的特点是 过度激活的细胞毒性T淋巴细胞(CTL)介导的失衡免疫反应，自然 杀伤(NK)和巨噬细胞。FHL患者通常存在双等位基因种系突变，涉及 包括合成素11(STX11，FHL-4)和合成素结合蛋白2在内的细胞膜转运 (STXBP2、FHL-5)。尽管如此，单等位基因突变也与FHL有关，但他们的临床和 生物学意义尚不清楚。尽管有证据表明STX11和STXBP2突变与异常有关 FHL患者的CTL和NK细胞活性，对其正常功能的认识存在明显差距 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这一知识差距 通过建立STX11/STXBP2协同作用影响LG的基本机制 并通过解剖它们在患者细胞中是如何受到影响的。STX11和STXBP2在物理上相互作用 到目前为止，控制溶血颗粒(LG)内容物释放的免疫学方法尚不明确 Synapse。我们实验室的数据提供了证据，STX11还与不同组的膜融合相互作用 可以调节不同的贩运步骤的蛋白质。我们假设STX11/STXBP2控制内体- 通过这些相互作用，溶酶体在质膜上运输和LG融合。我们进一步 假设FHL突变干扰了这些相互作用，从而削弱了细胞介导的细胞毒性 和/或生存信号，这一过程最终导致HLH一系列免疫紊乱 症状。首先，我们将评估STX11和STXBP2中的FHL突变如何影响物理关系 与其他参与胞外和内切溶酶体途径的相互作用蛋白相互作用 在患者细胞中使用生化和质谱学方法。我们将确定突变是如何影响 通过执行下拉式脂质体共浮选分析对蛋白质-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 研究FHL患者细胞内转运缺陷的FHL单基因和双等位基因突变 使用超分辨率STED和TIRF显微镜。此外，我们还将建立分子机制。 我们实验室开发了一种独特的体外融合试验来研究这些突变的作用。总而言之，我们的研究 将有助于我们更广泛地理解控制细胞杀伤的膜运输步骤 CTL和NK细胞中的途径，有助于携带单等位基因突变的患者的治疗决定， 在广泛的遗传疾病中建立共同的和基因特有的途径，这些途径有一些共同点 HLH症状的表现和帮助开发新的快速诊断工具。

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.