LYMPHOCYTE PROTEASE INHIBITORS AS PROBES OF CYTOLYSIS

淋巴细胞释放含有丝氨酸蛋白酶和穿孔素的颗粒 膜孔形成蛋白对病毒感染和肿瘤的杀伤作用 细胞。分离的含有穿孔素的颗粒是细胞溶解的，但需要 活性的蛋白水解酶和钙来调节裂解。在中国有很多的蛋白酶 颗粒，包括糜酶、类胰酶和裂解酶的“酶” 蛋氨酸残基，都与溶血功能有关，但都未知 天然底物只在假设中是蛋白酶裂解的 它们的天然底物只在有钙的情况下存在，而且 天然底物可包括穿孔素原、穿孔素促进蛋白、 和/或穿孔素抑制剂。新的特定底物和抑制剂是 需要解决这些新发现的淋巴细胞的功能 蛋白酶。在这笔赠款中，两个实验室之间的合作 生物有机化学家J.C.鲍尔斯和免疫学家D·胡迪格包括 四个具体目标：(1)合成大鼠特异性多肽底物， 利用计算机模拟小鼠和人类淋巴细胞蛋白水解酶的 确定蛋白质序列以预测合适的底物并使用 测定杀戮过程中释放的个别蛋白酶的底物；(2) 设计和合成更具反应性和特异性的多肽 氯甲基酮、多肽膦和基于机理的异香豆素 淋巴细胞蛋白水解酶的抑制剂及其应用 细胞介导的杀伤机制；(3)合成生物素和 基于放射性机制的丝氨酸蛋白酶抑制剂阻断大鼠RNK-2 16颗粒介导的裂解，并用它们检测在 杀灭；以及(4)确定RNK-16需要哪些蛋白酶 利用从生物素化回收的蛋白酶颗粒介导的裂解， 酰化而不是烷化抑制剂以重建裂解功能 抑制颗粒。丝氨酸蛋白酶对淋巴细胞颗粒溶解的控制 蛋白水解酶生物学的一个新领域，它对特定的 靶向和激活穿孔素蛋白作为肿瘤的生物疗法。 灭活淋巴细胞介导的裂解也具有实用价值 移植排斥反应和限制自身免疫性疾病，特别是在 患者产生了完全分化的细胞毒性淋巴细胞。

Lymphocytes release granules containing serine proteases and perforin membrane pore-forming proteins when they kill virally infected and tumor cells. Isolated perforin-containing granules are cytolytic but require active proteases and calcium to mediate lysis. There are many proteases in the granules, including chymases, tryptases and "Met-ases" which cleave at methionine residues, all implicated in lytic function but all with unknown natural substrates only in the hypothesis is that the proteases cleave their natural substrates only in the presence of calcium and that the natural substrates may include pro-perforins, perforin-promoting proteins, and/or perforin inhibitors. New, specific substrates and inhibitors are needed to address the functions of these newly discovered lymphocyte proteases. In this grant, collaboration between the laboratories of the bioorganic chemist J.C. Powers and the immunologist D. Hudig encompasses four specific aims: (1) to synthesize specific peptide substrates for rat, mouse, and human lymphocyte proteases using computer modeling of cDNA- determined protein sequences to predict appropriate substrates and use the substrates to measure individual proteases released during killing; (2) to design and synthesize more reactive and specific peptide chloromethylketone, peptide phosphonate, and mechanism-based isocoumarin inhibitors for the lymphocyte proteases and to use the inhibitors to probe the mechanism of cell-mediated killing; (3) to synthesize biotinylated and radioactive mechanism-based serine protease inhibitors that block rat RNK- 16 granule-mediated lysis and use them to detect proteases released during killing; and (4) to determine which proteases are required for RNK-16 granule-mediated lysis by using proteases recovered from biotinylated, acylating but not alkylating inhibitors to reconstitute lytic function to inhibited granules. Serine protease control of lymphocyte granule lysis is a new area of protease biology that has implications for specifically targeting and activating perforin proteins as biotherapies for tumors. Inactivation of lymphocyte-mediated lysis also has practical value to block graft rejection and to limit autoimmune diseases, especially after the patient has generated fully differentiated cytotoxic lymphocytes.