UNIQUE ASPECTS OF C1-- INHIBITOR STRUCTURE / FUNCTION

C1-抑制物是丝氨酸蛋白家族的一种血浆蛋白水解酶抑制物。C_1-抑制剂的生理靶点是补体成分C_1激活的C_1r和C_1s亚基，以及接触系统的酶。遗传性或获得性的C1-抑制物缺乏可导致血管水肿，这是一种潜在的致命疾病。虽然C1-Inhibitor与其他蛇类有许多共同之处，但它也有一些独特的特性，这些特性还没有被详细研究。首先，与其他蛇类相比，它抑制蛋白酶的速度很慢。造成这种情况的结构性原因尚不清楚。最初将确认，作为C1的一部分，当其生理形式时，C1s抑制发生得很慢。然后，将利用C1-抑制剂突变体和C1-抑制剂-α1-蛋白酶抑制剂嵌合体来研究其抑制活性的结构基础。C_1-抑制物含有一个高度糖化的110个残基的氨基末端结构域，与其他蛇类没有同源性。这一区域的功能将被研究，特别是测试它对于快速抑制和随后解离C1是必不可少的这一想法。为了测试这种重组的C1-抑制物，将制造缺少部分或全部氨基末端的变异体。此外，还将在氨基末端区域引入单半胱氨酸，该区域可以用荧光团标记，以评估该区域与C1和目标蛋白酶的相互作用。这些课题将在以下三个目标中进行研究：目的1)确定分离时和在C1复合体中时的C1S抑制率；目的2)确定C1-抑制剂抑制活性的结构基础；以及目的3)确定C1-抑制剂氨基末端结构域的功能。这些研究将提供有关C1-抑制物结构-功能关系的重要信息，这对蛋白质的体内功能、蛋白质的治疗用途以及由自然发生的功能障碍变异引起的缺陷状态具有重要意义。

C1-inhibitor is a plasma proteinase inhibitor of the serpin family. The physiological targets for C1-inhibitor are the activated C1r and C1s subunits of complement component C1, and enzymes of the contact system. Deficiency of C1-inhibitor, either hereditary or acquired can cause angioedema, a potentially fatal condition. Although C1-inhibitor has much in common with other serpins, it has some unique characteristics which have not been investigated in detail. Firstly, it inhibits proteinases slowly compared to other serpins. The structural reasons for this are not known. Initially it will be confirmed that C1s inhibition occurs slowly when in its physiological form, as part of C1. Then the structural basis for the inhibitory activity will be studied by use of C1-inhibitor mutants and C1-inhibitor- alpha1-proteinase inhibitor chimeras. C1-inhibitor has a highly glycosylated 110 residue amino-terminal domain which has no homology with other serpins. The function of this region will be investigated, in particular testing the idea that it is essential for rapid inhibition and subsequent dissociation of C1. To test this recombinant C1-inhibitor variants lacking part or all of the amino-terminal will be made. Also single cysteines will be introduced into the amino-terminal domain which can be labeled with fluorophore to assess the interaction of this region with C1 and the target proteinases. These topics will be studied in the following three aims: Aim 1) To determine the rate of C1s inhibition when isolated and when in the C1 complex; Aim 2) To determine the structural basis for the inhibitory activity of C1-inhibitor; and Aim 3) To determine the function of the amino-terminal domain of C1-inhibitor. These studies will provide important information regarding structure-function relationships in C1-inhibitor, which has implications for the in vivo function of the protein, the therapeutic use of the protein, and for the deficiency states caused by naturally occurring dysfunctional variants.