LYMPHOCYTE PROTEASE INHIBITORS AS PROBES OF CYTOLYSIS

淋巴细胞蛋白酶抑制剂作为细胞溶解的探针

• 批准号: 3300736
• 负责人: Dorothy Hudig
• 金额: $ 19.81万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-04-01 至 1993-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3300736
• 关键词: alpha benzopyrone; biotin; cellular immunity; computer simulation; cytolysis; enzyme structure; enzyme substrate; granule; ketones; laboratory rat; lymphocyte; peptidases; peptide chemical synthesis; phosphonate; protease inhibitor

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.

淋巴细胞释放含丝氨酸蛋白酶和穿孔蛋白的颗粒 膜形成孔蛋白杀死病毒感染和肿瘤 细胞。 孤立的含纹红蛋白的颗粒是细胞溶解的，但需要 活性蛋白酶和钙以介导裂解。 有很多蛋白酶 颗粒，包括辣椒酶，胰蛋白酶和“ Met-ass”，它们在 蛋氨酸残基，全部涉及裂解功能，但都属于未知 自然底物仅在假设中是蛋白酶切割 仅在钙的存在下它们的天然底物， 天然底物可能包括pro-proforins，促进性蛋白质蛋白， 和/或穿孔蛋白抑制剂。 新的，特定的底物和抑制剂是 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，合作 生物有机化学家J.C. Powers和免疫学家D. Hudig包括 四个具体目的：（1）合成大鼠的特定肽底物， 小鼠和人类淋巴细胞蛋白酶使用cDNA的计算机建模 确定蛋白质序列以预测适当的底物并使用 测量在杀死过程中释放的单个蛋白酶的底物； （2）至 设计和合成更具反应性和特定肽 氯四甲基酮，磷酸肽和基于机理的异摩蛋白酶 淋巴细胞蛋白酶的抑制剂，并使用抑制剂探测 细胞介导的杀伤机制； （3）合成生物素化和 基于放射性机制的丝氨酸蛋白酶抑制剂，可阻断大鼠RNK- 16颗粒介导的裂解，并使用它们检测在此期间释放的蛋白酶 杀人； （4）确定RNK-16需要哪些蛋白酶 通过使用从生物素化的蛋白酶，颗粒介导的裂解， 酰化但不烷基化抑制剂以将裂解功能重构为 抑制颗粒。 淋巴细胞颗粒裂解的丝氨酸蛋白酶控制是 蛋白酶生物学的新领域，对 靶向和激活穿孔蛋白作为肿瘤的生物疗法。 淋巴细胞介导的裂解的灭活也具有可阻止的实用值 移植拒绝并限制自身免疫性疾病，尤其是在 患者已经产生了完全分化的细胞毒性淋巴细胞。