Defining the Molecular Architecture for Transmembrane Acylation by a Membrane Bound O-Acyltransferase

酰化分泌蛋白在细胞间和组织间信号通路中发挥重要作用 多种疾病，包括糖尿病和癌症。膜结合O-的蛋白质修饰元件 酰基转移酶(MBOAT)酶家族是信号转导的关键分子控制点 在修饰分泌蛋白Ghrelin、Hedgehog和Wnt时。其中，Ghrelin是独一无二的，因为它调节 摄食行为和能量平衡。由于Ghrelin需要山羊进行酰化才具有生物活性，因此 对这个MBOAT家族成员的详细了解对于理解Ghrelin在疾病中的作用是至关重要的 并以Ghrelin依赖通路为靶点。然而，关于结构、衬底的信息的缺乏 山羊的结合部位和催化机制阻碍了对Ghrelin信号转导途径的理解 小分子工具研究山羊在代谢相关疾病中的作用。存在着迫切需要 确定山羊、HHAT和PORCN进行蛋白质酰化的结构和催化基础，以便 了解它们不同生物学作用的分子基础。本应用程序的目标是定义 膜O-酰基转移酶跨膜蛋白酰化的结构和化学基础。 本文提出的研究将开发一个得到验证和支持的山羊的分子级结构模型。 通过化学和生物化学研究。在强有力的初步研究的支持下，我们的目标将在 以下三个具体目标：1)确定人山羊体内的酰基供体和Ghrelin结合部位 (HGOAT)；2)确定hGOAT的催化机理；3)确定hGOAT中的抑制剂结合部位。在……里面 第一个目标是通过生物信息学分析和计算相结合的hGOAT结构模型 建模将指导研究确定hGOAT内的酰基供体和ghrelin结合位点，最终 目标是定义hGOAT如何完成具有拓扑学挑战性的Ghrelin跨膜辛酰化反应。 在第二个目标中，结构导向突变和机械探针将揭示位置和 负责Ghrelin酰化反应的活性部位的组成。在第三个目标中，抑制物结合部位在 HGOAT将使用半胱氨酸反应性化学探针和计算对接研究来确定。这 提案是创新的，因为它代表了与标准方法的新的实质性背离 研究膜结合酶的结构和作用机制。我们的工作将建立一部小说 用于研究结构上难以处理的膜蛋白的强大而通用的方法。建议数 这项研究意义重大，因为它将产生MBOAT酶家族蛋白的第一个结构 酰基转移酶在洞察MBOAT家族成员的结构和催化策略的同时， 这将促进酰化分泌蛋白作为人类疾病治疗靶点的开发。

Acylated secreted proteins play essential roles in intercellular and organismal signaling pathways implicated in multiple diseases including diabetes and cancer. Protein-modifying members of the membrane-bound O- acyltransferase (MBOAT) enzyme family constitute key molecular control points for signaling through their roles in modifying the secreted proteins ghrelin, Hedgehog, and Wnt. Of these, ghrelin is unique in that it regulates feeding behavior and energy homeostasis. Since ghrelin requires acylation by GOAT for biological activity, a detailed understanding of this MBOAT family member is imperative to understand the role of ghrelin in disease and to target ghrelin-dependent pathways. However, the dearth of information regarding the structure, substrate binding sites, and catalytic mechanism of GOAT impedes understanding ghrelin signaling and development of small molecule tools to study the role of GOAT in metabolism-related diseases. There exists an urgent need to define the structural and catalytic foundations of protein acylation by GOAT, Hhat, and PORCN in order to understand the molecular basis of their diverse biological roles. The objective in this application is to define the structural and chemical basis for transmembrane protein acylation by a membrane O-acyltransferase. The studies proposed herein will develop a molecular-level structural model of GOAT verified and supported by chemical and biochemical studies. Supported by strong preliminary studies, our objective will be pursued in the following three Specific Aims: 1) Define the acyl donor and ghrelin binding sites within human GOAT (hGOAT); 2) Determine the hGOAT catalytic mechanism, and 3) Identify inhibitor binding sites within hGOAT. In the first Aim, an hGOAT structural model generated by bioinformatic analysis coupled with computational modeling will guide studies to identify the acyl donor and ghrelin binding sites within hGOAT, with the ultimate goal of defining how hGOAT accomplishes the topologically challenging transmembrane octanoylation of ghrelin. In the second Aim, structure-guided mutagenesis and mechanistic probes will reveal the location and composition of the active site responsible for ghrelin acylation. In the third Aim, inhibitor binding sites within hGOAT will be identified using cysteine-reactive chemical probes and computational docking studies. This proposal is innovative because it represents a new and substantive departure from the standard approaches for investigating the structure and mechanism of membrane-bound enzymes. Our work will establish a novel powerful and general approach for investigating structurally intractable membrane proteins. The proposed research is significant because it will generate the first structure of an MBOAT enzyme family protein acyltransferase while providing insight into the structures and catalytic strategies of MBOAT family members, which will advance the exploitation of acylated secreted proteins as therapeutic targets for human diseases.