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

酰化分泌蛋白在细胞间和生物体信号通路中起重要作用， 多种疾病包括糖尿病和癌症。膜结合O-的蛋白质修饰成员 酰基转移酶（MBOAT）家族通过其作用构成信号传导的关键分子控制点 在修饰分泌蛋白ghrelin、Hedgehog和Wnt中。其中，生长激素释放肽是独特的，因为它调节 摄食行为和能量稳态。由于生长素释放肽需要被GOAT酰化以获得生物活性， 详细了解这个MBOAT家族成员对于了解ghrelin在疾病中的作用至关重要 并靶向生长激素释放肽依赖途径。然而，缺乏关于结构、基底 结合位点和GOAT的催化机制阻碍了对ghrelin信号传导和 小分子工具来研究GOAT在代谢相关疾病中的作用。迫切需要 通过GOAT、Hhat和PORCN定义蛋白质酰化的结构和催化基础， 了解其不同生物学作用的分子基础。本申请的目的是定义 通过膜O-酰基转移酶跨膜蛋白酰化的结构和化学基础。 本文提出的研究将建立一个经过验证和支持的GOAT分子水平结构模型 通过化学和生物化学研究。在初步研究的支持下，我们将在 以下三个具体目的：1）确定人GOAT中酰基供体和ghrelin的结合位点 （hGOAT）; 2）确定hGOAT催化机制，和3）鉴定hGOAT内的抑制剂结合位点。在 第一个目标，通过生物信息学分析与计算相结合产生的hGOAT结构模型， 模型将指导研究，以确定hGOAT内的酰基供体和ghrelin结合位点，最终 目的是定义hGOAT如何完成具有拓扑挑战性的ghrelin跨膜辛酰化。 在第二个目标中，结构引导的诱变和机制探针将揭示位置和 负责胃饥饿素酰化的活性位点的组成。在第三个目标中， 将使用半胱氨酸反应性化学探针和计算对接研究鉴定hGOAT。这 建议是创新的，因为它代表了一个新的和实质性的偏离标准的方法， 研究膜结合酶的结构和机制。我们的工作将建立一个小说 研究结构上难处理的膜蛋白的强有力的和通用的方法。拟议 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，同时提供深入了解MBOAT家族成员的结构和催化策略， 这将促进利用酰化分泌蛋白作为人类疾病的治疗靶标。

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.