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）：本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。酰化已成为生物学中丰富的和具有生物学意义的蛋白质修饰，对治疗具有重要意义。MBOAT是从细菌到人保守的酰基转移酶的一个家族，并且将长链脂肪酸分子添加到代谢物的氧原子或蛋白质的丝氨酸和苏氨酸侧链。这些多位整合膜蛋白发挥重要的代谢作用，并且一些人类成员如二酰基甘油酰基转移酶1（DGAT 1）、ghrelin O-酰基转移酶（GOAT）和刺猬酰基转移酶（HHAT）已经成为代谢疾病和癌症的重要药物靶标。细菌MBOAT蛋白DltB参与革兰氏阳性细菌细胞壁的主要组分的生物合成，因此代表革兰氏阳性细菌发病机制的药物靶标。缺乏MBOAT蛋白的分子信息是由于难以制备重组形式的这些蛋白用于生物化学和结构分析。在初步数据中，我们已经克服了这些困难来制备重组DGAT 1、GOAT和DltB蛋白，并开发了针对它们的生化检测方法。最近，我们已经制备了DltB样品，适用于使用X射线晶体学进行生化分析和结构测定。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。本提案的具体目的是（1）使用X射线晶体学确定DltB MBOAT蛋白的原子分辨率结构，和（2）建立体外和体内DltB活性测定的结构-功能相关性，以评估DltB突变体。总之，这些研究将为DltB MBOAT蛋白的结构和功能提供重要的新见解，并为开发新型探针和抑制剂以对抗细菌致病机制提供支架。这些研究也将对理解更大的MBOAT家族的结构-功能产生影响，并为解决其底物特异性和开发新型MBOAT特异性探针和抑制剂的治疗铺平道路。