Roles of two aminopeptidases in peptide catabolism in the malaria parasite

描述（由申请人提供）：在其在红细胞中的复制周期中，人类疟疾寄生虫恶性疟原虫在称为食物液泡的酸性降解细胞器中摄取并分解宿主细胞中高达75%的血红蛋白。血红蛋白分解代谢是正常寄生虫发育所必需的，参与这一过程的肽酶是抗疟疾药物开发的极有希望的靶点。虽然血红蛋白降解为寡肽的最初步骤已经得到了很好的研究，但氨基酸产生的过程仍然不明确。我们已经鉴定出两种空泡氨肽酶，恶性疟原虫氨肽酶N （PfA-M1）和氨肽酶P (PfAPP)，并提出血红蛋白在食物空泡腔内被广泛降解为氨基酸。该项目的目标是从生物化学和细胞生物学的角度来理解PfA-M1和PfAPP在寄生虫食物液泡中的招募如何增强其降解血红蛋白的能力。在Aim 1中，我们研究了这两种氨基肽酶，它们在其他真核生物中在中性或碱性pH值下比在酸性pH值下是更好的催化剂，是否已经适应在酸性环境中有效地发挥作用。PfA-M1是珠蛋白多肽产生氨基酸的潜在关键角色，其底物特异性将在酸性pH下进行分析，以确定其对空泡肽分解代谢的贡献程度。Aim 2中PfA-M1原子结构的表征将为解释Aim 1中观察到的底物特异性提供分子基础。此外，对一个关键底物结合位点的诱变分析将揭示该酶特异性的分子基础，并有助于设计有效的特异性抑制剂。在Aim 3中，将探索PfAPP双重靶向食物液泡和细胞质的机制，重点关注替代转录或翻译起始作为最有可能的候选。疟原虫特异性PfAPP n端扩展包含指定双重靶向的序列元件，将被选择性诱变以揭示靶向和分类信息的存在。从这些研究中获得的见解将用于进一步剖析PfAPP的空泡和细胞质作用。公共卫生相关性：疟疾每年造成1至200万人死亡。该项目检查了两种酶的作用，称为氨基肽酶，帮助寄生虫消化其宿主红细胞的血红蛋白。通过了解这些酶是如何工作的，我们希望发现寄生虫盔甲上的缝隙，从而开发出新的抗疟疾药物。

DESCRIPTION (provided by applicant): During its replication cycle in red blood cells, the human malaria parasite Plasmodium falciparum ingests and catabolizes up to 75% of the host cell's hemoglobin in an acidic degradative organelle called the food vacuole. Hemoglobin catabolism is required for normal parasite development, and peptidases that participate in this process are highly promising targets for anti-malarial drug development. While the initial steps in the degradation of hemoglobin to oligopeptides have been well studied, the processes by which amino acids are generated have remained ill-defined. We have identified two vacuolar aminopeptidases, P. falciparum aminopeptidase N (PfA-M1) and aminopeptidase P (PfAPP), and propose that hemoglobin is extensively degraded to amino acids in the food vacuole lumen. The goals of this project are to understand, in biochemical and cell biological terms, how the recruitment of PfA-M1 and PfAPP to the parasite's food vacuole has enhanced its ability to degrade hemoglobin. In Aim 1, we examine whether these two aminopeptidases, which in other eukaryotes are much better catalysts at neutral or basic pH values than at acidic pH, have adapted to function efficiently in an acidic environment. The substrate specificity of PfA-M1, a potential key player in the generation of amino acids from globin peptides, will be profiled at acidic pH in order to define the extent of its contribution to vacuolar peptide catabolism. Characterization of the atomic structure of PfA-M1 in Aim 2 will provide a molecular basis for interpreting the substrate specificities observed in Aim 1. In addition, mutagenic analysis of a key substrate binding site will shed light on the molecular basis of specificity in this enzyme and aid in the design of potent, specific inhibitors. In Aim 3, the mechanism underpinning the dual targeting of PfAPP to the food vacuole and cytosol will be explored, with a focus on alternate transcription or translation initiation as the most likely candidates. The Plasmodium-specific PfAPP N-terminal extension, which contains the sequence elements specifying dual targeting, will be selectively mutagenized to reveal the presence of targeting and sorting information. The insight gained from these studies will be used to further dissect the vacuolar and cytosolic roles of PfAPP. PUBLIC HEALTH RELEVANCE: Malaria is responsible for the death of 1-2 million people annually. This project examines the roles of two enzymes called aminopeptidases that help the parasite to digest the hemoglobin of its host red blood cell. By understanding how these enzymes work, we hope to discover chinks in the parasite's armor that could be exploited for the development of new anti-malarial drugs.