PROTEIN PRENYLTRANSFERASES OF PLASMODIUM FALCIPARUM

恶性疟原虫的蛋白质异戊烯基转移酶

• 批准号: 6362370
• 负责人: CHARLES M ALLEN
• 金额: $ 22.21万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-03-01 至 2003-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6362370
• 关键词: Plasmodium falciparum; SDS polyacrylamide gel electrophoresis; affinity chromatography; alkyltransferase; electron microscopy; enzyme activity; enzyme inhibitors; enzyme structure; enzyme substrate; geranyl compound; growth inhibitors; immunoaffinity chromatography; immunoprecipitation; isoprenoid; light microscopy; molecular cloning; northern blottings; nucleic acid probes; polymerase chain reaction; posttranslational modifications; prodrugs; protein structure function; site directed mutagenesis; southern blotting; western blottings

Isoprenylated proteins function in important regulatory roles in various cellular processes. Selective inhibition of protein prenylation has been shown to be useful in the control of cell division and differentiation in targeted cells. Little is known about the role of isoprenylation or types of protein prenyl transferases in Plasmodium falciparum. Our hypothesis is that biochemical characterization of protein prenyltransferases from P. falciparum is important for the elucidation of key regulatory components of the signaling pathways of this organism and that studies of the sensitivity of these enzymes to inhibitors will determine whether these enzymes could serve as potential targets for antimalarials. This proposal aims to purify and characterize two types of P. falciparum protein prenyltransferases: protein farnesyl transferase (PFT) and protein geranylgeranyltransferase (PGGT-I). Additional aims are to test the sensitivity of P. falciparum growth and prenyltransferase activity to substrate related inhibitors and to identify physiological substrates for the P. falciparum PFT. The specific aims are 1) to clone the alpha- and beta-subunits of P. falciparum PFT and overexpress them in E. coli or baculovirus, 2) to purify catalytically active recombinant PFT by peptide-affinity chromatography and characterize its subunit composition, 3) to purify native P. falciparum PFT and PGGT-I enzymes to the highest degree possible and compare them with respect to their subunit composition, 4) to characterize and compare recombinant PFT, native PFT and PGGT-I enzymes with respect to their substrate specificity and kinetic properties, 5) to test prodrug peptidomimetic compounds and other derivatives as inhibitors of cell division and growth of P. falciparum, 6) to test peptidomimetics and other prenyltransferase inhibitors as in vitro inhibitors of the PFTs and PGGT-Is, and 7) to identify physiological substrates for P. falciparum PFT by co-immunprecipitation, by identifying peptide sequences interacting with PFT subunits using a phage phage display peptide library and by expression cloning.

等肾上腺素化蛋白在各种重要的调节作用中起作用 细胞过程。 选择性抑制蛋白质原始化的抑制 被证明可用于控制细胞分裂和 靶细胞的分化。 关于角色知之甚少 疟原虫中蛋白质前转移酶的异丙基化或类型 恶意。 我们的假设是 来自恶性疟原虫的蛋白质前转移酶对 阐明信号通路的关键调节组件 这种生物以及对这些酶对这些酶的敏感性的研究 抑制剂将确定这些酶是否可以作为潜力 抗疟疾的靶标。 该建议旨在净化和 表征两种类型的恶性疟原虫蛋白前转移酶： 蛋白Farnesyl转移酶（PFT）和蛋白质黄烷基转移酶 （PGGT-I）。 其他目的是测试恶性疟原虫的灵敏度 对底物相关抑制剂的生长和前转移酶活性 并确定恶性疟原虫PFT的生理底物。 这 具体目的是1）克隆P。 恶性PFT并在大肠杆菌或杆状病毒中过表达它们，2） 通过肽 - 亲属纯化催化活性重组PFT 色谱和表征其亚基组成，3）净化 最高程度的天然恶性疟原虫PFT和PGGT-I酶 可能并将它们相对于其亚基组成进行比较，4） 为了表征和比较重组PFT，本地PFT和PGGT-I 酶相对于其底物特异性和动力学 属性，5）测试前药肽型化合物和其他 衍生物是细胞分裂的抑制剂和恶性疟原虫的生长， 6）测试肽仪和其他前转移酶抑制剂，如 PFT和PGGT-IS的体外抑制剂，以及7） 通过共免疫的恶性疟原虫PFT的生理底物， 通过识别使用A与PFT亚基相互作用的肽序列 噬菌体噬菌体显示肽库和通过表达克隆。