Structure and Function of Eukaryotic Phosphatidylserine Decarboxylase

性状（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一种必需脂质，该磷脂生产中的关键酶是磷脂酰丝氨酸脱羧酶（PSD）。虽然真核PSD的推导的一级结构已经知道了十多年，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，含有一个异戊酰辅基。了解真核PSD酶的进展一直受到其完整的膜结构和相对不稳定性的洗涤剂的存在下。最近，我们克隆了一个cDNA编码PSD的寄生虫诺氏疟原虫（PkPSD）。PkPSD以可溶性和膜结合形式存在。可溶性形式的PkPSD的可用性现在已经使对这种酶的结构和功能的新的研究成为可能。利用体外转录/翻译系统，我们已经开始剖析调节新生酶原转化为成熟酶的早期事件，成熟酶由一个含有异戊酰辅基的小β亚基和一个大亚基组成。我们现在已经设计了一个系统，用于检查在体外加工的酶原成熟的酶，并已成功地在细菌中表达高水平的酶原。利用这些系统，我们现在计划进行实验，以阐明酶前加工和翻译后调节催化活性的机制。这项工作将在一项包含三个具体目标的研究计划中进行。第一个具体目标将测试PkPSD酶原最初是一种丝氨酸蛋白酶，经过分子变态成为脱羧酶的假设。酶原的蛋白酶活性被提议由磷脂酰丝氨酸激活并由磷脂酰甘油、磷脂酰肌醇和磷脂酸抑制。第二个特定目的将通过检测是否存在特异性磷脂结合位点来检查PkPSD的脂质调节。第三个具体目标将调查的假设，脂质调节PkPSD是机械耦合到酶原加工诱导构象变化的酶，激活或抑制蛋白酶功能。从这些研究中，我们将获得一个全面的看法，如何变构的细胞膜的脂质组成影响磷脂合成中的一种必需酶的激活。了解PSD调节与膜脂组成的这一方面将对干预病原体和生长不受调节的哺乳动物细胞的磷脂代谢产生重要影响。

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.