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 当它们杀死病毒感染和肿瘤时， 细胞 分离的含穿孔素颗粒是溶细胞的，但需要 活性蛋白酶和钙介导裂解。 有许多蛋白酶， 颗粒，包括糜蛋白酶、胰蛋白酶和“Metases”，其在 甲硫氨酸残基，所有涉及裂解功能，但所有未知 天然底物的唯一假设是蛋白酶切割 它们的天然底物只有在钙的存在下， 天然底物可以包括穿孔素原，穿孔素促进蛋白， 和/或穿孔素抑制剂。 新的、特定的底物和抑制剂， 需要解决这些新发现的淋巴细胞的功能 蛋白酶 在这项赠款中， 生物有机化学家J.C.鲍尔斯和免疫学家D. Hudig包含 四个具体目标：（1）合成大鼠特异性肽底物， 小鼠和人类淋巴细胞蛋白酶的cDNA- 确定的蛋白质序列，以预测适当的底物，并使用 底物，以测量在杀伤期间释放的单个蛋白酶;（2） 设计和合成更具反应性和特异性的肽 氯甲基酮、肽膦酸酯和基于机理的异香豆素 淋巴细胞蛋白酶的抑制剂，并使用该抑制剂来探测 细胞介导的杀伤机制;（3）合成生物素化的， 基于放射性机制的丝氨酸蛋白酶抑制剂，阻断大鼠RNK- 16颗粒介导的裂解，并使用它们来检测蛋白酶释放过程中 杀伤;和（4）确定RNK-16需要哪些蛋白酶 通过使用从生物素化的， 酰化而非烷基化抑制剂以重建裂解功能， 抑制颗粒。 丝氨酸蛋白酶控制的淋巴细胞颗粒溶解是 蛋白酶生物学的一个新领域， 靶向和激活穿孔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.