PROTEINASE INHIBITORS & CRYSTALLIN FRAGMENTS IN CATARACT

描述：蛋白质的修饰在将正常晶状体转变为白内障晶状体的过程中起着关键作用。内源性酶对晶体蛋白中的多肽键的蛋白水解性断裂可能是最常见和最重要的修饰之一。到目前为止，关于晶体蛋白中这些裂解的信息仅限于它们的酶性质、在酶和底物水平上的调节、对晶体蛋白功能的影响以及晶体蛋白片段的二次修饰。根据初步结果，P.I.假设晶状体蛋白的降解在酶和底物水平上都受到调控，被切割的片段通过Mail Lard反应进行二次修饰，如糖基化，从而直接参与晶状体蛋白的交联，这是晶状体混浊形成的关键。为了验证这一假设，将追求以下三个特定目标：(1)特定目标#1：通过去污剂(如脱氧胆酸钠)对BA31A1-晶体蛋白水解酶的假定酶原型激活，确定膜蛋白酶的自溶激活机制。(2)特定目的#2：确定膜蛋白水解酶和BA31A1-晶体蛋白水解酶在选定晶体蛋白体内蛋白分解中的作用。(3)具体目标#3：通过Maillard反应的糖基化机制，确定降解多肽的一般和特定的9 kDa YD-晶体蛋白片段的交联度，以形成同源(单独的晶体蛋白片段)和异源(晶体蛋白片段和天然晶体蛋白)多聚体。上述研究将解决有关体内调节晶状体蛋白降解的问题，以及内源性蛋白酶在这一过程中的作用。本研究还将回答在晶状体混浊形成过程中，降解的多肽经过二次修饰后是否具有交联性并直接参与蛋白质交联性的问题。由于所有拟议的研究都将在人类晶状体上进行，结果将与人类老年性白内障问题直接相关。

DESCRIPTION: Modification of proteins play a pivotal role in changing a normal lens into a cataractous lens. Proteolytic cleavage of peptide bonds in crystallins by endogenous enzymes is probably one of the most frequent and important modifications. So far, information about these cleavages in crystallins is limited in terms of their enzymatic nature, regulation at the enzyme and substrate levels, effect on crystallin functions and secondary modifications in crystallin fragments. Based on preliminary results, the P.I. has hypothesized that the degradation of crystallins is regulated at both enzyme and substrate levels and the cleaved fragments undergo secondary modification such as glycation via the Mail lard reaction and therefore directly participate in the cross-linking of the lens proteins which is central to the development of lens opacity. To test this hypothesis, the following three specific aims will be pursued: (1) specific Aim #1: Determine the mechanism of autolytic activation of a membrane proteinase from a putative zymogen type activation of the BA31A1-crystallin proteinase by a detergent such as sodium deoxycholate. (2) specific Aim #2: Determine the role of the membrane proteinase and BA31A1-crystallin proteinase in the in vivo proteolysis of selected crystallins. (3) Specific Aim #3: Determine crosslinking of degraded polypeptides in general and specifically of a 9 kDa yD-crystallin fragment to form homologous (crystallin fragments alone) and heterologous (crystallin fragments plus native crystallins) multimers possibly via the glycation mechanism of the Maillard reaction. The above studies will address the questions regarding the in vivo regulation of crystallin degradation and the role of endogenous proteinases in this process. This study will also answer the questions whether the degraded polypeptides, on secondary modifications, acquire cross-linking properties and participate directly in protein cross-linking during the development of lens opacity. Since all of the proposed studies will be carried out in human lenses, the results will be directly relevant to the human senile cataract problem.