New Pathways in Lymphocyte Granule-Mediated Cytotoxicity

描述(申请人提供)：CTL或NK细胞使用两种主要的接触依赖机制来杀伤它们各自的目标：结合肿瘤坏死因子家族的死亡受体，如Fas，或通过胞吐细胞毒性颗粒。颗粒胞吐途径已被确定在清除病毒感染的细胞、保护其他细胞内病原体和监视肿瘤方面起主导作用。排出的颗粒含有一组细胞毒性蛋白，包括穿孔素和一系列丝氨酸蛋白酶，即颗粒酶。颗粒酶A和颗粒酶B(GRB)是最丰富的颗粒酶，GRB是唯一与caspase家族具有底物特异性的颗粒酶。它在天冬氨酸残基之后裂解底物，使其能够模仿启动子caspase来触发靶细胞凋亡。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发挥着重要作用。目前应用的总体目标是阐明GRB用来介导线粒体凋亡级联反应的新的凋亡途径。我们之前的研究已经阐明了Bak是线粒体驻留的成员，也是促凋亡的Bcl2家族成员，对GrB的线粒体反应是必需的。然而，目前尚不清楚胞质中的GRB与线粒体Bak之间的联系。我们的初步数据表明，Mcl-1，一个位于线粒体外膜上的抗凋亡的Bcl-2家族成员，介导了线粒体和胞浆GRB之间的串扰。我们推测caspase级联的线粒体扩增是GRB介导的细胞凋亡的重要组成部分，因此可以作为调节GRB功能的靶点，即增强GRB对转化细胞的活性，抑制其在移植物排斥反应中的活性。为了验证这一假说，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。我们建议将重点放在级联的三个不同阶段：(I)Mcl-1在介导上游信号启动线粒体级联中的作用；(Ii)Bax和Bak在GRB介导的级联中的作用；以及(Iii)XIAP作为GRB抑制剂的作用，被认为是线粒体凋亡环的上游和下游。这些研究有望描述细胞毒性淋巴细胞杀伤靶细胞的未知效应机制，并有助于开发预防移植物排斥反应和克服病毒感染或转化细胞中的凋亡抵抗的策略。

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.