RUI: Cellular Role of CTP:Phosphocholine Cytidylyltransferase Isoforms

本研究项目的目的是了解酶CTP：磷脂酰转移酶（CCT）如何调节细胞磷脂酰胆碱合成。CCT是cdp -胆碱途径的限速酶，因此受到广泛调节。可溶性CCT易位到细胞膜表面导致酶活化，这是一个可逆的过程，可以通过纯化CCT与合成脂质囊泡的结合在体外模拟。在高等真核生物中存在两种CCT亚型，包括黑腹果蝇和人类。本研究项目旨在确定真核细胞中每个酶同工异构体的作用。使用培养的果蝇Schneider 2细胞进行RNA干扰将被用于选择性地“敲除”每个CCT异构体。第一个研究目标是确定每个黑腹果蝇CCT异构体的细胞位置。第二个目标是确定每个CCT亚型是否对细胞生长至关重要。最后的研究目标涉及阐明每个CCT异构体对磷脂酰胆碱合成的贡献。该研究的完成将提供关于每种CCT亚型对细胞磷脂酰胆碱合成的相对贡献的信息，阐明CCT在真核细胞中的其他作用，并为真核生物功能的多种CCT亚型的必要性提供见解。广泛影响：研究的广泛影响表现在它将对本科生的研究发展和以本科生为主的伊利诺伊州立大学的教育环境产生积极影响。支持本科生物化学专业学生进行全日制暑期研究，可以在关键的早期阶段培养科学事业。独立的实验室研究是对本科生获得的广泛课堂训练的宝贵补充，允许在课堂上学到的科学知识的应用。此外，学生将获得使用专业的现代科学设备的技术培训。其结果将是一个毕业生谁是充分准备开始追求科学研究生学位。

The objective of this research project is to understand how the enzyme CTP:phosphocholine cytidylyltransferase (CCT) regulates cellular phosphatidylcholine synthesis. CCT is the rate limiting enzyme of the CDP-choline pathway and therefore is extensively regulated. Translocation of soluble CCT to the surface of a cellular membrane results in enzyme activation, a reversible process that can be mimicked in vitro through association of purified CCT with synthetic lipid vesicles. There are two CCT isoforms present in higher eukaryotes, including Drosophila melanogaster and humans. This research project is designed to determine the role(s) of each individual enzyme isoform within a eukaryotic cell. RNA interference using cultured Drosophila melanogaster Schneider 2 cells will be employed to selectively "knock out" each CCT isoform. The first research objective is to identify the cellular location of each Drosophila melanogaster CCT isoform. A second objective is to determine whether each CCT isoform is essential for cell growth. The final research objective involves elucidation of the contribution of each CCT isoform to phosphatidylcholine synthesis. Completion of the research will provide information about the relative contribution of each CCT isoform to cellular phosphatidylcholine synthesis, elucidate additional roles of CCT within a eukaryotic cell, and provide insight into the necessity of multiple CCT isoforms for the function of eukaryotes. Broad Impact: A broader impact of the research is manifested in the positive effect it will have on the research development of undergraduate students and the educational environment of Illinois State University, a predominantly undergraduate institution. Support for undergraduate Biochemistry majors to conduct full-time summer research fosters a scientific career at a critical, early stage. Independent laboratory research is a valuable complement to the extensive classroom training undergraduate students obtain, allowing application of the scientific knowledge learned in the classroom. In addition, students will obtain technical training in the use of specialized, modern scientific equipment. The result will be a graduate who is well-prepared to begin pursuit of a graduate degree in science.