Evolution of Novel Farnesyltransferase Activity

新型法尼基转移酶活性的演变

• 批准号: 7477142
• 负责人: James Hougland
• 金额: $ 2.87万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7477142
• 关键词: Affect; Amino Acid Sequence; Behavior; Binding; Binding Sites; Biological; Biological Assay; Boxing; C-terminal; Catalysis; Cellular Membrane; Condition; Consensus; Data; Enzymes; Evolution; Exhibits; Fellowship; In Vitro; Individual; Investigation; Kinetics; Lead; Length; Libraries; Lipids; Measures; Modeling; Modification; Mutagenesis; Mutate; Mutation; Names; Peptides; Post-Translational Protein Processing; Protein Isoprenylation; Proteins; Regulation; Role; Screening procedure; Site-Directed Mutagenesis; Specificity; Substrate Specificity; Tail; Therapeutic Agents; Translations; Variant; Work; base; design; directed evolution; enzyme activity; enzyme substrate; farnesylation; farnesyltranstransferase; in vivo; inhibitor/antagonist; intracellular protein transport; novel; novel strategies; prenylation; protein farnesyltransferase; protein geranylgeranyltransferase; protein localization location; tool

DESCRIPTION (provided by applicant): Prenylation is an important posttranslational modification for many proteins whereby attachment of a lipid tail leads to protein localization to cellular membranes. The two enzymes responsible for the majority of prenylation, farnesyltransferase (FTase) and geranylgeranyltransferase type I (GGTase-l), have been proposed to recognize a consensus "CaaX" box motif at the C-terminus of target proteins. However, recent work has shown that this model incompletely describes the specificity of these enzymes. Furthermore, the interactions between the protein substrates and FTase responsible for specificity have not been delineated. I will investigate these issues using mutagenesis and directed evolution to generate a library of FTase variants with altered substrate specificities and kinetic behavior. Analysis of these mutated FTases will provide a more comprehensive understanding of the features that govern binding and catalysis within FTase and may aid in the identification of novel substrates and design of new FTase inhibitors as therapeutic agents. In addition, this work will provide important information and novel tools for studying protein prenylation in vivo, allowing examination of the myriad biological roles of prenylated proteins.

描述（由申请人提供）：前酰化是许多蛋白质的重要翻译后修饰，通过脂质尾部的附着导致蛋白质定位到细胞膜。两种酶，法尼基转移酶（FTase）和香叶基转移酶I型（ggtase - 1），负责大部分的戊烯酰化，已被提出在靶蛋白的c端识别一个共识的“CaaX”盒基序。然而，最近的研究表明，该模型不能完全描述这些酶的特异性。此外，蛋白质底物和负责特异性的FTase之间的相互作用尚未被描述。我将使用诱变和定向进化来研究这些问题，以生成具有改变底物特异性和动力学行为的FTase变体库。对这些突变的FTase的分析将提供对控制FTase内结合和催化的特征的更全面的理解，并可能有助于鉴定新的底物和设计新的FTase抑制剂作为治疗药物。此外，这项工作将为研究体内蛋白质戊烯酰化提供重要的信息和新的工具，允许检查戊烯酰化蛋白质的无数生物学作用。