Serpine structure in noninhibitory serpin function: Angiotensinogen and TBG

非抑制性丝氨酸蛋白酶抑制剂功能中的丝氨酸结构：血管紧张素原和 TBG

• 批准号: 6313246
• 负责人: PHILIP A PATSTON
• 金额: $ 21.47万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-22 至 2004-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6313246
• 关键词: angiotensinogen; biochemical evolution; blood proteins; chemical binding; chemical kinetics; chemical stability; conformation; cysteine endopeptidases; enzyme substrate complex; fluorescent dye /probe; hormone regulation /control mechanism; hypertension; nuclear magnetic resonance spectroscopy; polymerization; protease inhibitor; protein folding; protein protein interaction; protein structure function; proteolysis; renin; serine proteinases; thyroid hormone binding protein; tissue /cell culture

Angiotensin and thyroxine binding globulin (TBG) are two physiologically critical human plasma precursor decapeptide angiotensin I. It is therefore critical for blood pressure regulation. TBG is the principal and highest affinity binding and transport protein for the thyroid hormones thyroxine (TM) and triiodothyronine (T3). Neither is known to have any ability to act as proteinase inhibitor, yet both are members of the serpin superfamily. We consider that it is not a coincidence that each is a serpin that there has been co-evolution. of the more obvious biological function with less obvious that each is a serpin and that there has been co- evolution of the more obvious biological function with less obvious auxiliary properties that rely on the presence of the serpin fold. We have developed two provocative and novel hypotheses that will be tested in this project. Concerning angiotensinogen, we propose that formation of higher molecular weight complexes of angiotensinogen, either with itself through a non-covalent serpin-based polymerization mechanism, or with pro-eosinophil granule major basic protein (proMBP), serves to alter the accessibility of renin to its target cleavage site in the N-terminus of angiotensinogen and thereby regulates the production of angiotensin peptides. Concerning TBG, we propose that the reactive center loop has been optimized to give substrate-like cleavage by proteinase, with consequent intramolecular serpin-like conformational change, that weakens the affinity for T3 and T4 and thereby provides a proteinase- regulated mechanism for hormone release. The corollary in both cases is that mutations in the serpin core that affect these conformational changes will be detrimental. Three specific aims will test these hypotheses: Aim 1 will use spectroscopic and kinetic methods to determine whether the N- terminus of angiotensinogen is mobile, but is made less accessible to renin by formation of high molecular weight complexes. Aim 2 will examine whether angiotensinogen is a metastable serpin designed to form loop-sheet oligomers, but to be incapable of self-loop insertion on cleavage and will examine the properties of variants, M235T and T174M, correlated with hypertension. Aim 3 will characterize the binding of thyroid hormones to TBG in native and cleaved states, to determine whether loop insertion on cleavage, but without proteinase inhibition, is critical for TBG function, and to examine the properties of TBG oligomers and latent TBG to see how they further understanding of the dysfunction of natural variants.

血管紧张素和甲状腺素结合球蛋白（TBG）是人体血浆中两种重要的前体十肽血管紧张素I。因此，它对血压调节至关重要。TBG是甲状腺激素甲状腺素（TM）和三碘甲状腺原氨酸（T3）的主要和最高亲和力结合和转运蛋白。两者都不知道有任何能力作为蛋白酶抑制剂，但都是丝氨酸蛋白酶抑制剂超家族的成员。我们认为，这不是一个巧合，每一个都是一个蛇蛋白，有共同的进化。更明显的生物学功能与不太明显的每一个都是丝氨酸蛋白酶抑制剂，并且存在更明显的生物学功能与依赖于丝氨酸蛋白酶抑制剂折叠存在的不太明显的辅助性质的共同进化。我们已经开发了两个挑衅性和新颖的假设，将在这个项目中进行测试。关于血管紧张素原，我们提出，形成更高分子量的复合物的血管紧张素原，无论是通过非共价丝氨酸蛋白酶为基础的聚合机制，或与亲嗜酸性粒细胞颗粒主要碱性蛋白（proMBP），用于改变血管紧张素原的N-末端的目标切割位点的肾素的可及性，从而调节血管紧张素肽的生产。关于TBG，我们提出，反应中心环已被优化，得到底物样裂解蛋白酶，随之而来的分子内丝氨酸蛋白酶样构象变化，削弱了T3和T4的亲和力，从而提供了一个蛋白酶调节的激素释放机制。在这两种情况下的必然结果是，影响这些构象变化的丝氨酸蛋白酶抑制剂核心的突变将是有害的。三个具体的目标将测试这些假设：目标1将使用光谱和动力学方法来确定血管紧张素原的N-末端是否是移动的，但通过形成高分子量复合物使其不易接近肾素。目的2将检查血管紧张素原是否是一种亚稳定的丝氨酸蛋白酶抑制剂，其设计用于形成环片寡聚体，但不能在切割时自环插入，并将检查与高血压相关的变体M235 T和T174 M的性质。目的3将表征甲状腺激素与TBG在天然和切割状态下的结合，以确定切割时的环插入，但没有蛋白酶抑制，是否对TBG功能至关重要，并检查TBG寡聚体和潜伏TBG的性质，以了解它们如何进一步理解天然变体的功能障碍。