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: 9114599
• 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 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9114599
• 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; 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必需的脂质，生产这种磷脂的关键酶是磷脂酰丝氨酸脱羧酶（PSD）。虽然真核生物PSD的初级结构已经被推断了十多年，但关于这种酶的活性如何被调节的细节一直是难以捉摸的。此外，PSD属于一个不寻常的酶家族，它含有丙酮酰假基。真核PSD酶的整体膜结构和在洗涤剂存在下的相对稳定性阻碍了对其理解的进展。最近，我们从诺氏疟原虫（Plasmodium knowlesi, 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。可溶形式的PkPSD的可用性现在使得对这种酶的结构和功能的研究有了新的思路。利用偶联的体外转录/翻译系统，我们已经开始剖析调控新生原酶向成熟酶转化的早期事件，成熟酶由一个含有丙酮酰假基的小-亚基和一个大-亚基组成。我们现在已经设计了一种系统来检查原酶到成熟酶的体外加工，并成功地在细菌中表达了高水平的原酶。利用这些系统，我们现在计划进行实验来阐明酶原加工和翻译后催化活性调节的机制。这项工作将在一个包含三个具体目标的研究计划中进行。第一个特异性目标将测试PkPSD前酶最初是丝氨酸蛋白酶，经过分子变态成为脱羧酶的假设。原酶的蛋白酶活性被磷脂酰丝氨酸激活，被磷脂酰甘油、磷脂酰肌醇和磷脂酸抑制。第二个特异性目标将通过检测特定磷脂结合位点的存在来检查PkPSD的脂质调节。第三个特定目标将研究PkPSD的脂质调节通过诱导酶的构象变化来激活或抑制蛋白酶功能，从而与酶前加工机制耦合的假设。从这些研究中，我们将全面了解细胞膜的脂质组成如何变构影响磷脂合成中必需酶的激活。了解PSD调控与膜脂组成耦合的这一方面将对干预病原体和生长不调节的哺乳动物细胞的磷脂代谢具有重要意义。