Roles of two aminopeptidases in peptide catabolism in the malaria parasite

两种氨肽酶在疟原虫肽分解代谢中的作用

• 批准号: 7739488
• 负责人: Michael Klemba
• 金额: $ 26.33万
• 依托单位: VIRGINIA POLYTECHNIC INST AND ST UNIV
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-12-01 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7739488
• 关键词: Active Sites; Amino Acids; Aminopeptidase; Aminopeptidase P; Anabolism; Antimalarials; Binding; Binding Sites; Biochemical; Biological; Biology; Catabolism; Cell Nucleus; Cell physiology; Cells; Cessation of life; Cytosol; Degradation Pathway; Development; Elements; Environment; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Erythrocytes; Eukaryota; Exopeptidase; Food; Functional disorder; Generations; Genetic Transcription; Globin; Goals; Growth; Hemoglobin; Human; Hydrolysis; Kinetics; Knowledge; Lead; Light; Malaria; Mediating; Molecular; N-terminal; Oligopeptides; Organelles; Organism; Parasites; Peptide Hydrolases; Peptides; Pharmaceutical Preparations; Physiological; Plasmodium; Plasmodium falciparum; Play; Process; Proline; Property; Proteins; Recombinants; Recruitment Activity; Role; Solutions; Sorting - Cell Movement; Specific qualifier value; Specificity; Staging; Structure; Substrate Specificity; Translation Initiation; Vacuole; Water; Work; alanine aminopeptidase; base; catalyst; design; drug development; inhibitor/antagonist; insight; multicatalytic endopeptidase complex; novel; public health relevance; uptake

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.

描述（由申请人提供）：在其在红细胞中的复制周期期间，人疟疾寄生虫恶性疟原虫在称为食物泡的酸性降解细胞器中摄取并分解代谢高达75%的宿主细胞血红蛋白。血红蛋白catenase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非常有前途的靶点。虽然血红蛋白降解为寡肽的初始步骤已经得到了很好的研究，但产生氨基酸的过程仍然不清楚。我们已经鉴定了两种空泡氨肽酶，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中被广泛降解为氨基酸。该项目的目标是了解，在生物化学和细胞生物学方面，PfA-M1和PfAPP的招聘到寄生虫的食物泡如何增强其降解血红蛋白的能力。在目标1中，我们研究了这两种氨肽酶是否已适应在酸性环境中有效发挥作用，这两种氨肽酶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。PfA-M1是从珠蛋白肽生成氨基酸的潜在关键因素，其底物特异性将在酸性pH下进行分析，以确定其对液泡肽催化的贡献程度。目标2中PfA-M1的原子结构表征将为解释目标1中观察到的底物特异性提供分子基础。此外，一个关键的底物结合位点的诱变分析将揭示这种酶的特异性的分子基础，并有助于设计有效的，特异性的抑制剂。在目标3中，将探索PfAPP双重靶向食物泡和胞质溶胶的机制，重点是交替转录或翻译起始作为最可能的候选者。疟原虫特异性PfAPP N-末端延伸，其含有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选信息的存在。从这些研究中获得的见解将用于进一步剖析PfAPP的液泡和胞质作用。公共卫生相关性：疟疾每年造成1-2百万人死亡。该项目研究了两种称为氨肽酶的酶的作用，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，这些裂缝可以用于开发新的抗疟疾药物。