MECHANISM OF CYTOTOXIC CELL GRANULE MEDIATED APOPTOSIS

描述（改编自研究者摘要）：免疫系统使用 颗粒介导的细胞凋亡以消除外来细胞。致命一击包括 将颗粒相关的丝氨酸蛋白酶如颗粒酶B（GrB）递送至 通过穿孔素（一种孔形成蛋白）靶向细胞胞质溶胶。穿孔素被假定为 来传递颗粒酶。实验支持 然而，模型仍然难以捉摸。他们已经积累了数据， 穿孔素在细胞内传递颗粒酶。在这 模型，内吞颗粒酶和穿孔素的靶细胞是 对细胞毒性细胞介导的凋亡的易感性所必需的。 随后，GrB被PFN释放到胞质溶胶中， 通过一种新的方法激活刽子手前蛋白酶-3和-7， 两步机制此外，相关工作表明靶细胞是 暴露于与其同源物复合的多个颗粒酶分子 颗粒相关蛋白聚糖，丝甘氨酸。结合，内化， GrB-丝甘肽细胞内运输和蛋白水解特异性可 与用游离阳离子颗粒酶获得的结果有很大不同。 从概念上讲，他们认为颗粒介导的凋亡模拟策略 通过病毒进入有核细胞，PFN将大分子 信号复合物-一系列与丝甘蛋白毒性连接的颗粒酶。的 我们应用的总体目标是了解PFN是否经历内吞作用 以递送GrB并确定GrB-丝甘肽复合物是否显示出 与细胞毒性细胞相同的半胱天冬酶激活潜力，使用新描述的 半胱氨酸天冬氨酸蛋白酶原-3缺陷型MCF-7细胞和表达该细胞的稳定转染子 会员是否具体目标是： 1.显示靶细胞需要正常的内吞功能， PFN/GrB-以及CTL介导的凋亡。2.表征GRB-SG 从YT细胞的颗粒分离的复合物。3.以确定是否孤立 PFN或由细胞毒性细胞分泌的形式诱导靶细胞的凋亡 仅含有囊泡相关的GrB/SG。4.为了研究泡状的 GRB-SG复合物的贩运。5.为了了解GrB/SG的输送是否模拟 由仅分泌GrB的细胞毒性细胞诱导的有序半胱天冬酶活化。 这些信息将为细胞内的细胞分裂现象提供新的见解。 多聚体酶的生物学功能 细胞内蛋白水解复合物。值得注意的是，这里描述的系统将 首次阐明PFN如何提供颗粒蛋白和GrB激活 在整个细胞中的胱天蛋白酶级联启动死亡程序。

DESCRIPTION (Adapted from the Investigator's abstract): The immune system uses granule-mediated apoptosis to eliminate foreign cells. The lethal hit involves delivery of granule associated serine proteases such as granzyme B (GrB) to the target cell cytosol by perforin, a pore forming protein. Perforin is postulated to deliver granzymes by acting as a conduit. Experimental support for this model, however, remains elusive. They have accumulated data supporting the concept that perforin acts intracellularly to deliver the granzymes. In this model, endocytosis of both granzyme and perforin by the target cell are necessary for susceptibility to cytotoxic cell-mediated apoptosis. Subsequently, GrB is released to the cytosol by PFN where the granzyme specifically activates executioner procaspases-3 and -7 through a novel two-step mechanism. Furthermore, related work indicates a target cell is exposed to multiple granzyme molecules complexed to its cognate granule-associated proteoglycan, serglycin. The binding, internalization, intracellular trafficking and proteolytic specificity of GrB-serglycin may differ greatly from the results obtained with the free, cationic granzyme. Conceptually they suggest that granule mediated apoptosis mimics strategies adopted by viruses to enter nucleated cells where PFN delivers a macromolecular signaling complex-an array of granzymes toxically linked to serglycin. The overall goal of our application is to learn whether PFN undergoes endocytosis to deliver GrB and to determine whether GrB-serglycin complexes display the same caspase-activating potential as cytotoxic cells using newly described procaspase-3 deficient MCF-7 cells and a stable transfectant expressing this member. The Specific Aims are: 1. To show a target cell requires normal endocytic function for susceptibility to PFN/GrB- as well as CTL-mediated apoptosis. 2. To characterize GRB-SG complexes isolated from granules of YT cells. 3. To determine whether isolated PFN or the form secreted by a cytotoxic cell induces apoptosis in a target cell containing only vesicle-associated GrB/SG. 4. To study the vesicular trafficking of GRB-SG complexes. 5. To learn whether delivery of GrB/SG mimics the ordered caspase activation induced by a cytotoxic cell secreting only GrB. The information will provide novel insights to the phenomenon of intracellular protein delivery as well as the biologic function of multimeric enzyme complexes in intracellular proteolysis. Notably, the system described here will clarify for the first time how PFN delivers granule proteins and GrB activates the caspase cascade in whole cells to initiate death program.