Serpine structure in noninhibitory serpin function: Angiotensinogen and TBG

血管紧张素和甲状腺激素结合球蛋白(TbG)是人体血浆十肽血管紧张素的两种重要生理活性物质，在血压调节中起着重要作用。TbG是甲状腺激素甲状腺激素(TM)和三碘甲腺原氨酸(T3)的主要和最高亲和力的结合和运输蛋白。目前还不知道这两种蛋白都具有作为蛋白水解酶抑制因子的能力，但它们都是丝氨酸蛋白超家族的成员。我们认为，每个物种都是一条蛇，这并不是巧合，它们之间存在着共同进化。在较明显的生物功能中，每一种都不是很明显的，而更明显的生物功能和不太明显的辅助特性是共同进化的，这些辅助特性依赖于蛇针折叠的存在。我们已经开发了两个具有挑衅性的新奇假设，将在这个项目中进行测试。关于血管紧张素原，我们认为，血管紧张素原与自身或与嗜酸性粒细胞主要碱性蛋白(ProMBP)通过非共价丝氨酸聚合机制形成更高分子量的复合体，有助于改变肾素与其在血管紧张素原N端的靶切割位点的可及性，从而调节血管紧张素肽的产生。关于TBG，我们认为反应中心环已经被优化，使其能够被蛋白酶进行底物样的切割，从而导致分子内丝氨酸样构象变化，从而削弱了与T3和T4的亲和力，从而为激素释放提供了一种蛋白酶调节机制。在这两种情况下的推论是，影响这些构象变化的蛇针核心突变将是有害的。三个特定的目标将检验这些假设：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末端是否可移动，但通过形成高分子复合体使肾素更难接近。目的2将检查血管紧张素原是否是一种亚稳定的丝氨酸，旨在形成环状片状低聚体，但不能在切割上自环插入，并将检查与高血压相关的变异体M235T和T174M的性质。目的3将表征甲状腺激素在天然和切割状态下与TBG的结合，确定在没有蛋白酶抑制的情况下，在切割时插入环是否对TBG的功能至关重要，并检测TBG寡聚体和潜伏的TBG的性质，以了解它们如何进一步了解自然变异体的功能障碍。

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.