Structural Determinants of Glycogen Synthase Regulation

描述(由申请人提供)：本提案的总体目标是了解负责合成和延长葡萄糖储存聚合物糖原的酶的结构和功能特性。糖原是葡萄糖的重要储备物，糖原合成的启动和延长机制从酵母到人类都是保守的。我们最近在酵母糖原合成酶方面的结构工作为了解所有真核酶的功能特性提供了重要的新见解。这项建议的长期目标是在这一初步工作的基础上，描绘由糖原合成酶催化的反应的结构背景，并揭示糖原结合和糖原合成酶的葡萄糖-6-磷酸激活之间的结构相互作用。作为这项工作的一部分，我们将启动人类糖原合酶小分子调节剂的筛选，这些小分子调节剂可以操纵酶的活性状态，并在2型糖尿病或抑制糖原合酶的作用下促进葡萄糖的处置，作为治疗糖原储存障碍的潜在方法，如Lafora和Pompe病。结构测定将是我们努力的一个组成部分，因为糖原合成酶及其底物与葡萄糖-6-磷酸或激活剂结合位点的新型激动剂/拮抗剂之间的络合物将提供重要的结构/活性关系，这将指导我们研究的进一步发展。作为我们在目标2中筛选工作的一部分，我们将产生代表均一磷酸化状态的新的酶形式，并将这些形式与在该目标中确定的糖原合成酶的新调节剂进行测试。此外，我们将在底物、底物类似物或葡萄糖-6-磷酸存在和不存在的情况下对这些新型调节剂进行结构测定，这将与我们在目标1中的工作相联系。目标3将确定催化的分子机制和糖基转移的特异性。这一目标的目的之一是了解糖原合成酶产生的催化错误是否最终导致糖原加工过程中的异常。这一目标将利用目标1中开发的结构信息，并将使用酵母Gsy2p和人糖原合成酶的野生型和突变形式，并将作为目标2的一部分，为我们的调节剂发现和开发提供重要的结构/活性信息。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to understand the structural and functional properties of the enzymes responsible for the synthesis and elongation of the glucose storage polymer, glycogen. Glycogen is an important storage reserve of glucose and the mechanism by which the synthesis of glycogen is initiated and elongated is conserved from yeast to humans. Our recent structural work on yeast glycogen synthase has provided important new insight into the functional properties of all eukaryotic enzymes. The long-range goal of this proposal is to build off this initial work to delineate the structural context for the reaction catalyzed by glycogen synthase and uncover the structural interplay between glycogen association and glucose-6- phosphate activation of glycogen synthase. As part of this effort, we will initialize a screen for small molecule modulators of human glycogen synthase that can manipulate the activity state of the enzyme and promote glucose disposal under conditions found in Type 2 Diabetes or inhibit the action of glycogen synthase as a potential approach for treating glycogen storage disorders, such as Lafora and Pompe's Disease. Structure determination will be an integral part or our efforts as complexes between glycogen synthase and its substrates in combination with glucose-6-phosphate or novel agonists/antagonists of the activator binding site will provide important structure/activity relationships that will guide further development of our research. As part of our screening efforts in Aim 2 we will produce novel enzymatic forms representing homogenous phosphorylation states and these will be tested against the novel modulators of glycogen synthase identified in this aim. In addition, we will pursue structure determination of these novel modulators in the presence and absence of substrates, substrate analogs or glucose-6-phosphate which will link to our work in aim 1. Aim 3 will determine the molecular mechanism of catalysis and the specificity of glycosyltransfer. One of the goals of this aim is to understand whether catalytic mistakes produced by glycogen synthase are ultimately responsible for aberrations in glycogen processing. This aim will draw upon the structural information developed in aim 1 and will employ wild-type and mutant forms of both yeast Gsy2p and human glycogen synthase and will feed important structure/activity information into our modulator discovery and development as part of aim 2.