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: 8899596
• 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 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8899596
• 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; Health; Human; In Vitro; Individual; Integral Membrane Protein; Leukotrienes; Lipid A; 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; 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（由申请人提供）： 磷脂酰乙醇胺（PE）是从细菌到人类的生物体中的必需脂质，并且该磷脂生产中的关键酶是磷脂酰丝氨酸脱羧酶（PSD），尽管真核PSD的推导的一级结构是已知的。十多年来，关于这种酶的活性如何调节的细节一直难以捉摸。此外，PSD 属于一种不寻常的现象。最近，我们从寄生虫诺氏疟原虫 (PkPSD) 中克隆了编码 PSD 的 cDNA。 PkPSD 以可溶形式和膜结合形式存在。可溶形式的 PkPSD 的可用性现在使得人们能够对其结构和功能进行新的研究。使用耦合的体外转录/翻译系统，我们已经开始剖析调节新生酶原向成熟酶的转化的早期事件，成熟酶由含有丙酮酰基辅基的小β-亚基和大β-亚基组成。我们现在设计了一种系统，用于检查酶原在体外加工成成熟酶的过程，并已成功地在细菌中表达高水平的酶原，我们现在计划利用这些系统。阐明酶原加工和催化活性翻译后调节的机制的实验将在包含三个具体目标的研究计划中进行。第一个具体目标将测试 PkPSD 酶原最初是一种经过一系列过程的丝氨酸蛋白酶的假设。分子变态成为脱羧酶。酶原的蛋白酶活性被磷脂酰丝氨酸激活并被磷脂酰丝氨酸抑制。第二个具体目标将通过测试特定磷脂结合位点的存在来检查 PkPSD 的脂质调节。第三个具体目标将研究 PkPSD 的脂质调节与酶原加工机械耦合的假设。诱导酶的构象变化，从而激活或抑制蛋白酶功能。从这些研究中，我们将获得全面的结果。了解细胞膜的脂质成分如何变构影响磷脂合成中必需酶的激活，了解与膜脂质成分相结合的 PSD 调节的这一方面将对干预生长不受调节的病原体和哺乳动物细胞的磷脂代谢产生重要影响。