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: 6410591
• 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: 2000-12-01 至 2001-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6410591
• 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）是两种生理上至关重要的血浆前体十肽血管紧张素i，因此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的结合和转运蛋白。两者都不具有任何作为蛋白酶抑制剂的能力，但两者都是蛇形蛋白超家族的成员。我们认为，这不是一个巧合，每一个都是蛇，有共同进化。较明显的生物功能与较不明显的，每一个都是蛇形蛋白，并有共同进化的较明显的生物功能与较不明显的辅助特性，依赖于蛇形蛋白折叠的存在。我们提出了两个具有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，我们提出血管紧张素原通过非共价丝氨酸聚合机制与自身或与嗜酸性粒细胞颗粒主碱性蛋白（proosinophill granermajor basic protein, proMBP）形成高分子量复合物，有助于改变肾素对其在血管紧张素原n端切割位点的可及性，从而调节血管紧张素肽的产生。关于TBG，我们提出对反应性中心环进行了优化，使其可以被蛋白酶进行底物样的切割，从而导致分子内蛇形蛋白的构象变化，从而削弱了对T3和T4的亲和力，从而提供了一种蛋白酶调节的激素释放机制。这两种情况的推论是，影响这些构象变化的丝状蛋白核心突变将是有害的。三个特定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端是否可移动，但由于形成高分子量复合物而使肾素难以接近。目的2将研究血管紧张素原是否是一种亚稳态蛇形蛋白，其设计目的是形成环片寡聚物，但在切割时不能自我插入环，并将研究与高血压相关的变体M235T和T174M的特性。目的3将描述甲状腺激素在天然状态和断裂状态下与TBG的结合，以确定切割时环插入是否对TBG功能至关重要，但没有蛋白酶抑制，并检查TBG低聚物和潜在TBG的特性，以了解它们如何进一步理解自然变异的功能障碍。