Molecular Basis for Activity by Membrane Bound O-Acyltransferases

 描述(由申请人提供)：这个项目的总体目标是利用细菌DltB蛋白作为模型系统来开发膜结合O-酰基转移酶(MBOATs)家族的分子框架。酰化修饰在生物学中是一种丰富的、具有生物学意义的蛋白质修饰，具有重要的治疗意义。MBOAT是一个家族的酰基转移酶，从细菌到人是保守的，它将一个长链脂肪酸分子添加到代谢物或蛋白质的丝氨酸和苏氨酸侧链的氧原子上。这些多聚体完整膜蛋白具有重要的代谢作用，一些人类成员如二酰甘油酰基转移酶1(DGAT1)、Ghrelin O-酰基转移酶(山羊)和刺猬酰基转移酶(HHAT)已成为治疗代谢性疾病和癌症的重要药物靶点。细菌MBOAT蛋白DltB参与革兰氏阳性细菌细胞壁的主要成分的生物合成，因此是革兰氏阳性细菌致病的药物靶点。缺乏关于MBOAT蛋白的分子信息是因为很难将这些蛋白以重组形式制备用于生化和结构分析。在初步数据中，我们克服了这些困难，制备了重组DGAT1、山羊和DltB蛋白，并建立了它们的生化分析方法。最近，我们制备了一种适用于生化分析和X射线结晶学结构测定的DltB样品。这使我们处于独特的地位，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。本建议的具体目的是(1)利用X射线结晶学确定DltB MBOAT蛋白的原子分辨结构，以及(2)建立结构-功能相关性与体内和体内DltB活性测定，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，并为开发新的探针和抑制剂来对抗细菌的发病机制提供一个支架。这些研究还将对理解MBOAT家族的结构和功能产生影响，并为解决其底物特异性和开发用于治疗的新型MBOAT特异性探针和抑制剂铺平道路。

 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.