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

基本信息

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
项目类别：
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

项目摘要

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（应用程序提供）：该项目的总体目标是为膜结合的O-酰基转移酶（MBoats）开发一个分子框架，使用细菌DLTB蛋白作为模型系统。酰化已成为生物学中丰富且具有生物学意义的蛋白质修饰，对治疗具有重要意义。 Mboats是一个从细菌到人保守的酰基转移酶家族，并在代谢物的氧原子或蛋白质的丝氨酸和苏氨酸侧链中添加长链脂肪酸分子。这些多重整合性膜蛋白在重要的代谢作用中起着重要的代谢作用，以及几个人类成员，例如二酰基甘油酰基转移酶1（DGAT1），生长素蛋白O-酰基转移酶（Goat）和HedgeHog酰基转移酶（HHAT）已成为新近代谢性疾病的重要药物靶标。细菌MBOAT蛋白DLTB参与了革兰氏阳性细菌细胞壁的主要成分的生物合成，因此代表了革兰氏阳性细菌发病机理的药物靶标。缺乏有关M船蛋白的分子信息是由于难以以重组形式制备这些蛋白质进行生化和结构分析。在初步数据中，我们克服了这些困难来制备重组DGAT1，山羊和DLTB蛋白，并为它们开发了生化测定。最近，我们准备了一个适合使用X射线晶体学测定生化分析和结构测定的DLTB样品。这将使用DLTB蛋白作为模型系统的独特位置，以在理解MBOAT蛋白的结构和功能方面做出重要突破。该提案的具体目的是（1）使用X射线晶体学确定DLTB Mboat蛋白的原子分辨率结构，并且（2）建立与VITR和VIVO DLTB活性的结构 - 功能相关性，以评估DLTB突变体。总之，这些研究将提供有关DLTB Mboat蛋白结构和功能的重要新见解，以及开发新型探针和抑制剂以打击细菌发病机理的脚手架。这些研究还将对理解更大的Mboat家族的结构功能有影响，并为解决其底物特异性并开发新的M船特异性问题和治疗抑制剂的方式铺平了道路。