Formation and nature of serpin complexes with serine and cysteine proteinases

丝氨酸蛋白酶抑制剂超家族的蛋白酶抑制剂在调节细胞内和细胞外的蛋白水解酶在凝血、纤溶、炎症、细胞凋亡等关键生理过程中发挥重要作用。丝氨酸蛋白酶抑制剂的区别在于它们抑制丝氨酸和半胱氨酸蛋白酶的能力，以及它们通过主要构象变化将蛋白酶捕获在动力学稳定的共价复合物中的新机制。而多步丝氨酸蛋白酶抑制剂抑制机制通过与疾病相关的天然突变提供丝氨酸蛋白酶抑制剂功能。本项目的长期目标是剖析丝氨酸蛋白酶抑制剂抑制蛋白酶的构象捕获机制中所涉及的分子事件的逐步序列，并表征丝氨酸蛋白酶抑制剂抑制蛋白酶的构象捕获机制中所涉及的分子事件，并表征丝氨酸和半胱氨酸蛋白酶靶标的复合物的动力学稳定的分子基础。从这些研究中获得的知识有望加深我们对丝氨酸蛋白酶抑制剂如何调节蛋白水解的理解，并阐明丝氨酸蛋白酶抑制剂突变破坏这种调节的多种方式。在这些研究中，将测试关于丝氨酸蛋白酶抑制剂如何作为独特的蛋白质蛋白酶抑制剂的三个假设：i）丝氨酸蛋白酶抑制剂作为自杀底物抑制剂，其最初被其靶蛋白酶识别为正常底物，然后被诱导经历主要构象变化，该构象变化将蛋白酶捕获在蛋白质水解的酰基中间阶段; ii）在稳定的丝氨酸蛋白酶抑制剂-蛋白酶复合物中蛋白酶的捕获是由丝氨酸蛋白酶抑制剂在蛋白酶中诱导的构象变化引起的，所述构象变化破坏蛋白酶催化机制，从而防止复合物的脱酰作用; iii）半胱氨酸蛋白酶被丝氨酸蛋白酶抑制剂通过相同的自杀底物动力学捕获机制抑制，但具有不同的结果，这由丝氨酸蛋白酶抑制剂和蛋白酶之间的硫酯键的更大反应性决定。将测试假设的三个具体目标是：1）阐明丝氨酸蛋白酶抑制剂抑制丝氨酸蛋白酶的新的多步骤机制; 2）确定捕获机制的性质并阐明蛋白酶构象变化在诱导捕获中的作用;以及3）确定丝氨酸蛋白酶抑制剂抑制半胱氨酸蛋白酶的机制并评估与丝氨酸蛋白酶抑制机制的任何差异。

Protein proteinase inhibitors of the serpin superfamily play an important role in regulating intracellular and extracellular proteolytic enzymes in blood coagulation, fibrinolysis, inflammation, apoptosis and other key physiological processes. Serpins are distinguished by their ability to inhibit both serine and cysteine proteinases and by their novel mechanisms of trapping proteinases in kinetically stable covalent complexes through major conformational changes. While the multi-step serpin inhibitory mechanism has provided serpins with function through natural mutations associated with disease. The long range goals of this project are to dissect the stepwise sequence of molecular events involved in the conformational trapping mechanism by which serpins inhibit proteinases and to characterize the molecular events involved in the conformational trapping mechanisms by which serpins inhibit proteinases and to characterize the molecular basis of kinetic stabilization of the complexes for both serine and cysteine proteinase targets. The knowledge gained from such studies is expected to deepen our understanding of how serpins regulate proteolysis and to illuminate the multiple ways in which serpin mutations disrupt this regulation. Three hypotheses for how serpins function as unique protein proteinase inhibitors will be tested in these studies: i) serpins function as suicide substrate inhibitors, being initially recognized as normal substrates by their target proteinase, by then being induced to undergo a major conformational change which traps the proteinase at the acyl-intermediate stage of proteolysis; ii) the trapping of proteinases in stable serpin-proteinase complexes results from conformational changes induced in the proteinase by the serpin which disrupt the proteinase catalytic machinery and thereby prevent deacylation of the complex; iii) cysteine proteinases are inhibited by serpins by the same suicide substrate mechanism of kinetic trapping but with different outcomes dictated by the greater reactivity of the thioester linkage between serpin and proteinase. The three specific aims which will test the hypotheses are: 1) to elucidate the novel multi-step mechanism by which serpins inhibit serine proteinases; 2) to determine the nature of the trapping mechanism and elucidate the role of proteinase conformation changes in inducing the trap; and 3) to determine the mechanism by which serpins inhibit cysteine proteinases and assess any differences from serine proteinase inhibition mechanism.