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摄取并分解宿主细胞中高达75%的血红蛋白，这种细胞器被称为食物液泡。血红蛋白的分解代谢是正常寄生虫发育所必需的，参与这一过程的多肽酶是抗疟疾药物开发的极有希望的靶点。虽然人们已经很好地研究了将血红蛋白降解为寡肽的最初步骤，但氨基酸的生成过程仍然不清楚。我们鉴定了两种液泡型氨基肽酶：恶性疟原虫氨基肽酶N(PfA-M1)和氨基肽酶P(PfAPP)，推测血红蛋白在食物液泡腔中被广泛降解为氨基酸。这个项目的目标是从生化和细胞生物学的角度了解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。在目标1中，我们研究了这两种在其他真核生物中在中性或碱性pH值下比在酸性pH下更好的催化剂，这两种氨基肽酶是否适应了在酸性环境中有效地发挥作用。PFA-M1是从珠蛋白多肽中产生氨基酸的潜在关键角色，它的底物特异性将在酸性pH下进行分析，以确定其对液泡肽分解代谢的贡献程度。对AIM 2中PFA-M1原子结构的表征将为解释在AIM 1中观察到的底物特异性提供分子基础。此外，对关键底物结合位点的突变分析将有助于阐明这种酶的专一性的分子基础，并有助于设计有效的、特异的抑制剂。在目标3中，将探索PfAPP对食物液泡和胞浆的双重靶向的机制，重点是交替转录或翻译起始是最有可能的候选。疟原虫特异的PfAPP N-末端延伸包含指定双重靶向的序列元件，将被选择性地诱变以揭示靶向和分选信息的存在。从这些研究中获得的洞察力将被用来进一步剖析PfAPP在空泡和胞质中的作用。与公共卫生相关：疟疾每年造成100-200万人死亡。这个项目考察了两种名为氨基肽酶的酶的作用，这两种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裂缝，这些裂缝可以被用来开发新的抗疟疾药物。

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.