Structure and Function of Eukaryotic Phosphatidylserine Decarboxylase

真核磷脂酰丝氨酸脱羧酶的结构和功能

• 批准号: 8579734
• 负责人: DENNIS R. VOELKER
• 金额: $ 30.43万
• 依托单位: NATIONAL JEWISH HEALTH
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8579734
• 关键词: Active Sites; Address; Arachidonic Acids; Autophagocytosis; Bacteria; Behavior; Binding; Binding Sites; Biochemical; Biological Metamorphosis; C-terminal; Carboxy-Lyases; Cell division; Cell membrane; Cells; Complementary DNA; Coupled; Data; Detergents; Embryo; Enzyme Precursors; Enzymes; Escherichia coli; Eukaryota; Event; Family; Gene Silencing; Genetic; Genetic Transcription; Growth; Human; In Vitro; Individual; Integral Membrane Protein; Leukotrienes; Lipids; Mammalian Cell; Membrane; Membrane Lipids; Mitochondria; Modeling; Molecular; Mus; N-terminal; Organelles; Organism; Parasites; Pathway interactions; Peptide Hydrolases; Phosphatidic Acid; Phosphatidylethanolamine; Phosphatidylglycerols; Phosphatidylinositols; Phosphatidylserines; Phospholipid Metabolism; Phospholipids; Plasmodium knowlesi; Play; Population; Post-Translational Protein Processing; Post-Translational Regulation; Process; Production; Prokaryotic Cells; Property; Prostaglandins; Prosthesis; Protein Isoforms; Proteolysis; Pyruvate; Reaction; Regulation; Relative (related person); Research; Retinaldehyde; Role; Serine Protease; Site; Source; Structure; System; Systems Analysis; Testing; Tissues; Translations; Work; enzyme activity; fungus; insight; interest; lipid metabolism; member; membrane biogenesis; molecular rearrangement; novel; pathogen; polypeptide; public health relevance; visual cycle

DESCRIPTION (provided by applicant): Phosphatidylethanolamine (PE) is an essential lipid in organisms ranging from bacteria to humans and a pivotal enzyme in the production of this phospholipid is phosphatidylserine decarboxylase (PSD). Although the deduced primary structure of eukaryotic PSD has been known for more than a decade, the details about how the activity of this enzyme is regulated have been elusive. In addition, PSD belongs to an unusual family of enzymes that contain a pyruvoyl prosthetic group. Progress in understanding eukaryotic PSD enzymes has been hampered by its integral membrane structure and relative lability in the presence of detergents. Recently, we cloned a cDNA encoding PSD from the parasite Plasmodium knowlesi (PkPSD). The PkPSD exists in both soluble and membrane bound forms. The availability of soluble forms of PkPSD has now enabled new lines of inquiry into the structure and function of this enzyme. Using coupled in vitro transcription/ translation systems we have begun to dissect the early events that regulate the conversion of nascent proenzyme to the mature enzyme, consisting of a small ¿-subunit containing the pyruvoyl prosthetic group, and a large ¿-subunit. We have now devised a system for examining the in vitro processing of the proenzyme to the mature enzyme, and have succeeded in expressing high levels of the proenzyme in bacteria. Utilizing these systems we now plan to conduct experimentation to elucidate the mechanisms of proenzyme processing and post translational regulation of catalytic activity. This work will be undertaken in a research plan containing three Specific Aims. The first Specific Aim will test the hypothesis that the PkPSD proenzyme is initially a serine protease that undergoes a molecular metamorphosis to become a decarboxylase. The protease activity of the proenzyme is proposed to be activated by phosphatidylserine and inhibited by phosphatidylglycerol, phosphatidylinositol and phosphatidic acid. The second Specific Aim will examine the lipid regulation of the PkPSD by testing for the presence of specific phospholipid binding sites. The third Specific Aim will investigate the hypothesis that lipid regulation of PkPSD is mechanistically coupled to proenzyme processing by inducing conformational changes to the enzyme that either activate or inhibit the protease function. From these studies we will obtain a comprehensive view of how the lipid composition of cell membranes allosterically influences the activation of an essential enzyme in phospholipid synthesis. Understanding this aspect of PSD regulation coupled to membrane lipid composition will have important consequences for intervening in phospholipid metabolism of pathogens and mammalian cells with unregulated growth.

描述（由适用提供）：磷脂酰乙醇胺（PE）是从细菌到人类到人类的生物的必不可少的脂质，在该磷脂的产生中是磷脂酰甲酯脱铁蛋白脱羧酶（PSD）。尽管被推导的真核PSD的一级结构已闻名已有十多年了，但有关该酶的活性如何调节的细节是难以捉摸的。此外，PSD属于一个不寻常的酶家族，其中含有曲折的假体。理解真核PSD酶的进展受到其整体膜结构和在确定剂存在下的相对不稳定的阻碍。最近，我们从寄生虫Knewlesi（PKPSD）中克隆了一个编码PSD的cDNA。 PKPSD以可溶性和膜结合形式存在。固体形式的PKPSD的可用性现已实现了对该酶的结构和功能的新调查线。使用耦合的体外转录/翻译系统，我们已经开始剖析早期事件，这些事件调节了新生酶转化为成熟酶的转化，该酶由包含pyruvoyl假体群的小`subumunit组成，以及一个大的 - subunit。现在，我们已经设计了一个系统，用于检查原始酶对成熟酶的体外加工，并成功地表达了细菌中高水平的蛋白酶。利用这些系统，我们计划进行实验，以阐明催化活性的酶处理和翻译后调节的机制。这项工作将在包含三个特定目标的研究计划中进行。第一个具体目的将检验以下假设：PKPSD蛋白最初是一种丝氨酸蛋白酶，它经历了分子变形成为脱羧酶。提议该酶的蛋白酶活性被磷脂酰丝氨酸激活，并被磷脂酰甘油，磷脂酰肌醇和磷脂酸抑制。第二个特定目的将通过测试存在特定的磷脂结合位点来检查PKPSD的脂质调节。第三个具体目的将调查以下假设：PKPSD的脂质调节通过诱导激活或抑制蛋白酶功能的酶的会议变化来机械地耦合到原酶处理。从这些研究中，我们将获得有关细胞膜脂质组成如何在磷脂合成中影响基本酶的激活的全面观点。了解与膜脂质组成结合的PSD调节的这一方面将对干预病原体和哺乳动物细胞的磷脂代谢具有重要的影响。