Molecular Mechanism of Granule-mediated Apoptosis

描述(由申请人提供)：细胞毒性细胞颗粒介导的细胞凋亡是一种独特的细胞信号传递形式，需要通过穿孔蛋白穿孔蛋白在细胞内传递颗粒相关丝氨酸蛋白酶(颗粒酶)。对颗粒分泌途径中遗传缺陷的鉴定清楚地表明，颗粒介导的细胞凋亡对免疫稳态至关重要。病毒蛋白对细胞毒性细胞效应器功能的获得性破坏可能会触发淋巴增殖和自身反应，并降低宿主的防御反应。病毒感染的细胞或肿瘤细胞也可以通过抑制颗粒介导的凋亡途径的几乎任何方面来抵抗这一途径。描述这种鲜为人知的现象的多个步骤，对于理解一些疾病状态的免疫发病机制变得越来越重要。为了了解穿孔素对颗粒酶的传递，需要对最近端的步骤--围绕这些颗粒成分的结合、内化和早期运输的事件--进行分子表征。颗粒酶B在细胞毒细胞分泌后，仍然与颗粒相关的蛋白多糖--丝氨酸结合。出乎意料的是，颗粒酶经历了从丝氨酸到靶细胞硫酸糖胺聚糖的静电交换，然后促进了与未定义受体(S)的结合，以实现最佳的内化。此外，如果获得足够的内体浓度，颗粒酶似乎表现出膜溶解活性。题为连体穿孔素-颗粒酶递送假说-穿孔素和颗粒酶都被预测从丝氨酸甘氨酸静电交换到细胞表面，并共同促进内溶递送过程。将前人的工作扩展到颗粒酶B-细胞表面相互作用的糖生物学，并强调成像技术，特别是直接显示颗粒酶和穿孔素与靶细胞相互作用的新方法，具体目的是：1.研究颗粒酶B从丝氨酸乙酯转移到靶细胞表面2.确定穿孔素在颗粒酶递送中的作用--分子分析3.在细胞毒细胞介导的凋亡过程中评估靶细胞的质膜和内体完整性

DESCRIPTION (provided by applicant): Cytotoxic cell granule-mediated apoptosis is a unique form of cell signaling that entails the intracellular delivery of granule-associated serine proteases (granzymes) by the pore forming protein perforin. The identification of genetic defects in the granule secretion pathway have clearly established that granule- mediated apoptosis is crucial for immune homeostasis. Acquired disruption of cytotoxic cell effector function by viral proteins are likely to trigger lymphoproliferation and autoreactivity as well as diminish host defense responsiveness. A virus-infected or tumor cell may also resist granule mediated apoptosis by inhibiting virtually any aspect of this pathway. Delineating the multiple steps of this poorly understood phenomenon has become increasingly important to comprehend the immunopathogenesis of a number of disease states. To achieve an understanding of granzyme delivery by perforin requires molecular characterization of the most proximal steps - the events that surround the binding, internalization and early trafficking of these granule components. Granzyme B remains bound to the granule-associated proteoglycan, serglycin, after secretion by a cytotoxic cell. Unexpectedly, the granzyme undergoes electrostatic exchange from serglycin to target cell sulfated glycosaminoglycans which then facilitate binding to undefined receptor(s) for optimal internalization. In addition, the granzyme appears to manifest membranolytic activity if a sufficient endosomal concentration is achieved. Entitled the conjoined Perforin-Granzyme delivery hypothesis - both perforin and granzymes are predicted to undergo electrostatic exchange to the cell surface from serglycin and together contribute to endosomolytic delivery process. Extending previous efforts to the glycobiology of granzyme B-cell surface interactions and emphasizing imaging techniques, particularly new methods that directly visualize the interaction of the granzyme and perforin with target cells, the Specific Aims are: 1. Study the transfer of granzyme B from serglycin to the target cell surface 2. Ascertain role of perforin in granzyme delivery - a molecular analysis 3. Assess plasma membrane and endosomal integrity of the target cell during cytotoxic cell-mediated apoptosis