Biochemistry of Intramembrane Proteases From Pathogens

简介（由申请人提供）：寄生虫刚地弓形虫和疟原虫是导致疟疾的病原体，是致命的人类病原体。这些专性细胞内寄生虫必须入侵宿主细胞才能生存，因此了解寄生虫的入侵机制是一个重要的目标。这个机制的核心是菱形蛋白酶，它催化寄生虫黏附蛋白的基本切割，而黏附蛋白是附着在宿主细胞上所必需的。菱形体是完整的膜蛋白，穿过膜七次，我们之前推断它们的功能是新的蛋白酶；它们的跨膜结构域（TMDs）结合在膜双分子层内形成丝氨酸蛋白酶活性位点。值得注意的是，粘附素的裂解发生在它们的tmd内。在通常疏水的膜环境中，这种肽键的水解是酶生物化学的新范式。这种模式对人类健康具有更广泛的重要性，因为各种膜内蛋白酶最近被认为是阿尔茨海默病、高胆固醇血症和致病菌感染的核心参与者。然而，人们对这些不寻常的膜酶的生化功能知之甚少。我们试图破译这些神秘的蛋白酶如何在分子水平上发挥作用，特别强调它们在寄生虫入侵中的作用。具体而言，利用我们最近开发的新的研究菱形的生化方法，我们建议研究以下关键问题：1)与其他膜内蛋白酶相比，菱形底物特异性的物理基础；2)寄生虫膜中菱形蛋白酶的排列和调控；3)菱形蛋白酶的结构排列和功能；4)菱形催化的小分子抑制剂的开发。

DESCRIPTION (provided by applicant): Parasites Toxoplasma gondii and Plasmodium, the agent of malaria, are deadly human pathogens. These obligate intracellular parasites must invade host cells to survive, making understanding the parasite invasion machinery an important goal. At the heart of this machinery are rhomboid proteases, which catalyze the essential cleavage of parasite adhesin proteins that are required for attachment to host cells. Rhomboids are integral membrane proteins that cross the membrane seven times, and we previously deduced that they function as novel proteases; their transmembrane domains (TMDs) associate to form a serine protease active site within the membrane bilayer. Remarkably, cleavage of adhesins occurs within their TMDs. Such hydrolysis of peptide bonds within the normally hydrophobic environment of the membrane is a new paradigm in enzyme biochemistry. This paradigm is of wider importance to human health as various intramembrane proteases have recently been implicated as central players in Alzheimers Disease, hypercholesterolemia, and infection by pathogenic bacteria. However, the biochemical function of these unusual membrane enzymes is poorly understood. We seek to decipher how these enigmatic proteases function at the molecular level, with particular emphasis on their role in parasite invasion. Specifically, capitalizing on new biochemical methods for studying rhomboids that we have recently developed, we propose to investigate the following key issues: 1) physical basis of rhomboid substrate specificity compared to that of other intramembrane proteases, 2) arrangement and regulation of rhomboids in parasite membranes, 3) structural arrangement and function of rhomboid proteases, 4) development of small molecule inhibitors of rhomboid catalysis.