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导形式，其需要通过孔形成蛋白穿孔素在细胞内递送颗粒相关的丝氨酸蛋白酶（颗粒酶）。颗粒分泌途径中遗传缺陷的鉴定已经清楚地确定颗粒介导的细胞凋亡对于免疫稳态是至关重要的。通过病毒蛋白获得性破坏细胞毒性效应器功能可能触发淋巴细胞增殖和自身反应性以及降低宿主防御反应性。病毒感染的细胞或肿瘤细胞也可以通过抑制该途径的几乎任何方面来抵抗颗粒介导的细胞凋亡。描述这种知之甚少的现象的多个步骤对于理解许多疾病状态的免疫发病机制变得越来越重要。为了实现理解穿孔素的颗粒酶递送需要最近的步骤的分子表征-围绕这些颗粒组分的结合，内化和早期运输的事件。颗粒酶B在被细胞毒性细胞分泌后仍与颗粒相关蛋白聚糖丝甘肽结合。出乎意料的是，颗粒酶经历了从丝甘肽到靶细胞硫酸化糖胺聚糖的静电交换，然后硫酸化糖胺聚糖促进与未定义受体的结合以实现最佳内化。此外，如果达到足够的内体浓度，颗粒酶似乎表现出膜溶解活性。证实了联合穿孔蛋白-颗粒酶递送假说-穿孔蛋白和颗粒酶两者被预测从丝氨酸甘氨酸经历到细胞表面的静电交换，并且一起有助于内体溶解递送过程。将先前的努力扩展到颗粒酶B细胞表面相互作用的糖生物学，并强调成像技术，特别是直接可视化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从丝甘肽转移到靶细胞表面2.确定穿孔素在颗粒酶传递中的作用-分子分析3.评估细胞毒性细胞介导的凋亡过程中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