Structure and Function of Eukaryotic Phosphatidylserine Decarboxylase

描述(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一种基本脂质，生产这种磷脂的关键酶是磷脂酰丝氨酸脱羧酶(PSD)。虽然推测的真核生物PSD的一级结构已经知道十多年了，但关于这种酶的活性是如何调节的细节一直难以捉摸。此外，PSD属于一个不寻常的酶家族，含有丙酮酰假体基团。由于其完整的膜结构和在洗涤剂存在下的相对不稳定性，阻碍了对真核生物PSD酶的研究进展。最近，我们从诺氏疟原虫(PkPSD)中克隆了编码PSD的基因。PkPSD以可溶形式和膜结合形式存在。PkPSD的可溶性形式的出现现在使人们能够对这种酶的结构和功能进行新的探索。利用体外转录/翻译耦合系统，我们已经开始剖析调节新生前酶向成熟酶转化的早期事件，包括一个含有丙酮酰修复基的小的？亚基和一个大的？亚基。我们现在已经设计了一种检测酶原体外加工成熟酶的系统，并成功地在细菌中表达了高水平的酶原。利用这些系统，我们现在计划进行实验，以阐明酶原加工和催化活性的翻译后调节的机制。这项工作将在一项包含三个具体目标的研究计划中进行。第一个特定目标将测试PkPSD前酶最初是丝氨酸蛋白酶的假设，该酶经历分子变形成为脱羧酶。该酶原的活性被磷脂酰丝氨酸激活，而被磷脂酰甘油、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。第三个特定目标将调查PkPSD的脂质调节是通过诱导激活或抑制蛋白酶功能的酶的构象变化而机械地耦合到酶前加工的假说。通过这些研究，我们将全面了解细胞膜的脂类成分如何变构地影响磷脂合成中的一种关键酶的激活。了解PSD调节与膜脂组成有关的这一方面，将对干预病原体和非调节生长的哺乳动物细胞的磷脂代谢具有重要意义。

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.