Formation and nature of serpin complexes with serine and cysteine proteinases

丝蛋白超家族的蛋白水解酶抑制物在调节细胞内和细胞外的蛋白水解酶方面发挥着重要作用，这些酶参与了凝血、纤溶、炎症、细胞凋亡等关键生理过程。Serpins的独特之处在于它们同时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在动力学稳定的共价复合体中捕获蛋白酶的新机制。而多步蛇毒抑制机制通过与疾病相关的自然突变为蛇毒提供了功能。本项目的长期目标是剖析蛇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，并表征蛇抑制蛋白酶的构象捕获机制中所涉及的分子事件，以及表征丝氨酸和半胱氨酸蛋白酶靶标的复合体动力学稳定的分子基础。从这类研究中获得的知识有望加深我们对蛇如何调控蛋白分解的理解，并阐明蛇突变破坏这一调控的多种方式。在这些研究中，将检验三种关于蛇毒如何作为独特的蛋白蛋白酶抑制剂的假说：i)蛇毒作为自杀底物抑制剂，最初被其靶标蛋白酶识别为正常底物，然后被诱导经历主要的构象变化，该变化在蛋白降解的酰基中间阶段捕获蛋白酶；ii)在稳定的丝氨酸-蛋白酶复合体中捕获蛋白酶是由于丝氨酸诱导的蛋白酶的构象变化扰乱了蛋白酶的催化机制，从而阻止了复合体的脱酰化；Iii)蛇毒通过相同的自杀底物动力学捕获机制抑制半胱氨酸蛋白酶，但由于蛇毒蛋白和蛋白水解酶之间的硫酯键具有更大的反应活性而导致不同的结果。检验假说的三个具体目标是：1)阐明蛇毒抑制丝氨酸蛋白酶的新的多步机制；2)确定捕获机制的性质，并阐明蛋白酶构象变化在诱导TRAP中的作用；3)确定蛇毒抑制半胱氨酸蛋白酶的机制，并评估与丝氨酸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.