Biochemistry of Intramembrane Proteases From Pathogens

描述（由申请人提供）：寄生虫弓形虫和疟原虫，疟疾的代理人，是致命的人类病原体。这些专性细胞内寄生虫必须侵入宿主细胞才能生存，这使得了解寄生虫入侵机制成为一个重要目标。在这一机制的核心是菱形蛋白酶，它催化寄生虫粘附素蛋白的基本切割，所述寄生虫粘附素蛋白是附着到宿主细胞所需的。菱形是完整的膜蛋白，跨越膜7次，我们以前推断，他们作为新的蛋白酶的功能，其跨膜结构域（TMD）相关联，形成一个丝氨酸蛋白酶活性位点内的膜双层。值得注意的是，粘附素的裂解发生在它们的TMD内。这种在膜的正常疏水环境中的肽键的水解是酶生物化学中的新范例。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、高胆固醇血症和病原菌感染的主要参与者。然而，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研究菱形的新生物化学方法，我们建议研究以下关键问题：1）菱形底物特异性与其他膜内蛋白酶相比的物理基础，2）菱形在寄生虫膜中的排列和调节，3）菱形蛋白酶的结构排列和功能，4）菱形催化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.