Molecular Basis for Activity by Membrane Bound O-Acyltransferases

膜结合 O-酰基转移酶活性的分子基础

• 批准号: 9231362
• 负责人: Ronen Marmorstein
• 金额: $ 20.48万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-01 至 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9231362
• 关键词: Acetylation; Acetyltransferase; Acylation; Acyltransferase; Address; Alanine; Anabolism; Autoradiography; Bacteria; Binding; Biochemical; Biological; Biological Assay; Biological Models; Biological Process; Biology; Cell Wall; Cells; Complement; Crystallization; Data; Development; Diabetes Mellitus; Drug Addiction; Drug Targeting; Electron Microscopy; Enzymes; Erinaceidae; Family; Gene Expression; Goals; Growth; Hand; Human; In Vitro; Integral Membrane Protein; Investigation; Knock-out; Laboratories; Link; Lysine; Malignant Neoplasms; Measures; Mediating; Membrane; Membrane Structure and Function; Metabolic; Metabolic Diseases; Metabolism; Modeling; Molecular; Molecular Sieve Chromatography; Mycoses; Negative Staining; Oxygen; Pathogenesis; Play; Positioning Attribute; Post-Translational Protein Processing; Proteins; Publishing; Radiolabeled; Recombinants; Resolution; Roentgen Rays; Role; Sampling; Serine; Side; Site; Site-Directed Mutagenesis; Solid Neoplasm; Staphylococcus aureus; Structure; Substrate Specificity; Testing; Therapeutic; Threonine; Tunicamycin; Virus Diseases; X-Ray Crystallography; base; combat; diacylglycerol O-acyltransferase; fascinate; ghrelin; human disease; in vivo; inflammatory lung disease; inhibitor/antagonist; insight; leukemia; lipoteichoic acid; long chain fatty acid; man; member; membrane activity; membrane model; microscopic imaging; mutant; novel; particle; protein acyltransferase; public health relevance; scaffold; targeted treatment

 DESCRIPTION (provided by applicant): The overall goal of this project is to develop a molecular framework for the family of membrane bound O-acyltransferases (MBOATs) using the bacterial DltB protein as a model system. Acylation has emerged as an abundant and biologically significant protein modification in biology, with important implications for therapy. The MBOATs are one family of acyltransferases that are conserved from bacteria to man and add a long chain fatty acid molecule to the oxygen atom of a metabolite or a serine and threonine side chain of a protein. These polytopic integral membrane proteins play important metabolic roles and several human members such as diacylglycerol acyltransferase 1 (DGAT1), ghrelin O-acyltransferase (GOAT) and hedgehog acyltransferase (HHAT) have emerged as important drug targets in metabolic diseases and cancer. The bacterial MBOAT protein, DltB, participates in the biosynthesis of a major component of gram-positive bacterial cell wall and therefor represents a drug target for gram-positive bacterial pathogenesis. The lack of molecular information on MBOAT proteins is due to the difficulty in preparing these proteins in recombinant form for biochemical and structural analysis. In preliminary data, we have overcome these difficulties to prepare recombinant DGAT1, GOAT and DltB proteins and have developed biochemical assays for them. Most recently, we have prepared a DltB sample that is suitable for biochemical analysis and structure determination using X-ray crystallography. This places us in a unique position to use the DltB protein as a model system to make important breakthroughs in understanding the structure and function of MBOAT proteins. The specific Aims of this proposal are to (1) Determine the atomic resolution structure of the DltB MBOAT protein using X-ray crystallography, and (2) Establish structure-function correlations with in vitr and in vivo DltB activity assays to evaluate DltB mutants. Together, these studies will provide important new insights into the structure and function of the DltB MBOAT protein and a scaffold for developing novel probes and inhibitors to combat bacterial pathogenesis. These studies will also have implications for understanding the structure-function of the greater MBOAT family and pave the way to address their substrate specificities and to develop novel MBOAT-specific probes and inhibitors for therapy.

 描述（由申请人提供）：该项目的总体目标是使用细菌 DltB 蛋白作为模型系统，开发膜结合 O-酰基转移酶 (MBOAT) 家族的分子框架，酰化已成为一种丰富且具有生物学意义的系统。 MBOAT 是一种从细菌到人类都保守的酰基转移酶家族，它在氧原子上添加了一个长链脂肪酸分子。这些多胞体整合膜蛋白发挥着重要的代谢作用，其中一些人类成员如二酰基甘油酰基转移酶 1 (DGAT1)、生长素释放肽 O-酰基转移酶 (GOAT) 和刺猬酰基转移酶 (HHAT) 具有重要的代谢作用。细菌 MBOAT 蛋白 DltB 参与生物合成，成为代谢疾病和癌症的重要药物靶点。 MBOAT 蛋白是革兰氏阳性细菌细胞壁的主要成分，因此代表革兰氏阳性细菌发病机制的药物靶标。MBOAT 蛋白的分子信息的缺乏是由于难以制备重组形式的这些蛋白以进行生化和结构分析。在初步数据中，我们克服了这些困难，制备了重组 DGAT1、GOAT 和 DltB 蛋白，并开发了它们的生化测定方法。最近，我们制备了适合生化分析和结构测定的 DltB 样品。这使我们处于一个独特的位置，可以使用 DltB 蛋白作为模型系统，在理解 MBOAT 蛋白的结构和功能方面取得重要突破。该提案的具体目标是 (1) 确定原子。使用 X 射线晶体学解析 DltB MBOAT 蛋白的结构，以及 (2) 通过体外和体内 DltB 活性测定建立结构-功能相关性以评估 DltB 突变体。这些研究为了解 DltB MBOAT 蛋白的结构和功能提供了重要的新见解，并为开发对抗细菌发病机制的新型探针和抑制剂提供了重要的新见解，这些研究还将对理解更大的 MBOAT 家族的结构功能产生影响，并为开发新的探针和抑制剂铺平道路。解决其底物特异性并开发用于治疗的新型 MBOAT 特异性探针和抑制剂。