Biochemistry of Intramembrane Proteases From Pathogens

描述(申请人提供)：寄生虫弓形虫和疟疾病原体疟原虫是致命的人类病原体。这些专性的细胞内寄生虫必须入侵宿主细胞才能生存，这使得了解寄生虫入侵机制成为一个重要目标。这种机制的核心是菱形蛋白酶，它催化寄生虫附着到宿主细胞所需的粘附素蛋白的基本裂解。菱形蛋白是一种跨膜七次的完整膜蛋白，我们先前推测它们是一种新的蛋白水解酶；它们的跨膜结构域(TMD)结合在一起，在膜双层内形成丝氨酸蛋白酶活性部位。值得注意的是，粘附素的裂解发生在它们的TMD内。在膜的正常疏水环境中，这种多肽键的水解是酶生物化学中的一种新范式。这一模式对人类健康具有更广泛的重要性，因为各种膜内蛋白水解酶最近被认为是阿尔茨海默氏病、高胆固醇血症和病原菌感染的核心角色。然而，人们对这些不寻常的细胞膜酶的生化功能知之甚少。我们试图在分子水平上破译这些神秘的蛋白水解酶是如何发挥作用的，特别强调它们在寄生虫入侵中的作用。具体地说，利用我们最近开发的研究菱形的新的生化方法，我们建议研究以下关键问题：1)菱形底物相对于其他膜内酶的专一性的物理基础，2)菱形在寄生虫膜中的排列和调节，3)菱形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.