New Pathways in Lymphocyte Granule-Mediated Cytotoxicity

描述（由申请人提供）：CTL或NK细胞使用两种主要的接触依赖性机制来杀死其各自的靶标：与TNF家族的死亡受体（如Fas）结合，或通过细胞毒性颗粒的胞吐作用。已确定颗粒胞吐途径在消除病毒感染的细胞、保护免受其他细胞内病原体和肿瘤监视中占主导地位。胞吐的颗粒含有细胞毒性蛋白质的混合物，包括穿孔素和丝氨酸蛋白酶家族，颗粒酶。颗粒酶A和颗粒酶B（GrB）是最丰富的颗粒酶，而GrB是唯一与半胱天冬酶家族共享底物特异性的颗粒酶。它在天冬氨酸残基后切割其底物，使其能够模拟引发靶细胞凋亡的引发剂胱天蛋白酶。尽管GrB能够在多个入口点参与死亡途径，但最近的证据表明线粒体凋亡事件在其功能中起着重要作用。本申请的总体目标是阐明GrB用于介导线粒体凋亡级联的新的凋亡途径。我们以前的研究已经阐明了巴克，线粒体居民和促凋亡Bcl-2家族成员的线粒体响应GrB的要求。然而，目前尚不清楚是什么将GrB在细胞质中的线粒体巴克。我们的初步数据表明，Mcl-1，抗凋亡Bcl-2家族成员，居住在线粒体外膜介导的线粒体和胞质GrB之间的串扰。我们假设，线粒体的caspase级联放大是GrB介导的细胞凋亡的重要组成部分，并因此可能作为一个目标，调节GrB的功能，即增强GrB对转化细胞的活性和抑制其在移植排斥反应的活性。为了验证这一假设，我们建议阐明GrB介导的线粒体凋亡的功能机制和级联性质。我们建议集中在级联的三个可区分的阶段：（i）Mcl-1在介导上游信号启动线粒体级联中的作用;（ii）Bax和巴克在执行GrB介导的级联中的作用;和（iii）XIAP作为GrB抑制剂的作用，假设其在线粒体凋亡环的上游和下游都起作用。拟议的研究预计将表征未知的效应机制所使用的细胞毒性淋巴细胞杀死靶细胞，并有助于发展的战略，以防止移植排斥反应，克服病毒感染或转化细胞的凋亡抗性。

DESCRIPTION (provided by applicant): Two principal contact-dependent mechanisms are used by CTL or NK cells to kill their respective targets: engagement of death receptors of the TNF family, such as Fas, or via exocytosis of cytotoxic granules. The granule exocytosis pathway has been determined to be dominant in elimination of virus-infected cells, protection against other intracellular pathogens, and tumor surveillance. The exocytosed granules contain a cocktail of cytotoxic proteins including perforin and a family of serine proteases, granzymes. Granzyme A and granzyme B (GrB) are the most abundant granzymes, and GrB is the only granzyme to share substrate specificity with the caspase family. It cleaves its substrate after an aspartate residue, allowing it to mimic an initiator caspase in triggering target cell apoptosis. Despite GrB's ability to engage the death pathway at multiple entry points, recent evidence suggests a significant role for mitochondrial apoptotic events in its function. The overall goal of the current application is to elucidate novel apoptotic pathways utilized by GrB to mediate the mitochondrial apoptotic cascade. Our previous studies have elucidated a requirement for Bak, a mitochondrial resident and a proapoptotic Bcl-2 family member for a mitochondrial response toGrB. However, it is not clear what links GrB in the cytosol to mitochondrial Bak. Our preliminary data suggest that Mcl-1, an antiapoptotic Bcl-2 family member that resides on the mitochondrial outer membrane mediates crosstalk between the mitochondria and cytosolic GrB. We hypothesize that mitochondrial amplification of the caspase cascade is a significant component of GrB mediated apoptosis, and as such may serve as a target for regulation of GrB function, i.e. enhancing GrB activity against transformed cells and inhibiting its activity in graft rejection. To test this hypothesis, we propose to elucidate the functional mechanisms and the cascading nature of GrB mediated mitochondrial apoptosis. We propose to focus on three distinguishable phases of the cascade: (i) the role of Mcl-1 in mediating an upstream signal to initiate the mitochondrial cascade; (ii) the roles of Bax and Bak in the execution of the GrB-mediated cascade; and (iii) the role of XIAP as a GrB inhibitor that is hypothesized to function both upstream and downstream of the mitochondrial apoptotic loop. The proposed studies are expected to characterize unknown effector mechanisms used by cytotoxic lymphocytes to kill target cells, and to contribute to the development of strategies to prevent graft rejection and overcome apoptosis resistance in viral infected or transformed cells.