Chemical Genetic Tools for the Spatial and Temporal Modulation of O-GlcNAcylation

 描述(申请人提供)：O-连接N-乙酰氨基葡萄糖单糖(O-GlcNAc)对细胞内蛋白质的动态修饰在细胞生理学和疾病进展中发挥关键作用，这些疾病包括II型糖尿病、癌症、神经变性、心力衰竭、高血压和衰老(1，2)。然而，O-GlcNAc在这些疾病的病理生理学中所起作用的分子事件还不清楚。在一定程度上，这是由于缺乏工具来促进O-GlcNAc在体外和体内的特定和时间调节。这项研究的目标是开发简便的化学遗传工具，使O-GlcN酰化在体外和体内的空间、时间和剂量依赖的调节成为可能。为了实现这些目标，我们将解决以下具体目标：具体目标1：实现O-GlcN酰化的可调、可逆的化学遗传调节。拟议的研究将用荧光标签和亲和标签标记添加和移除O-GlcNAc的酶的内源基因座，并引入重组诱导的双正交化学遗传调节剂。总之，这些工具将使内源性O-GlcNAc修饰酶的成像和纯化以及蛋白质O-GlcN酰化的快速和合成调节成为可能。具体目标2：在体内实现O-GlcN酰化的可调组织特异性调节。拟议中的研究将产生小鼠，在小鼠中，添加和移除O-GlcNAc的酶的表达可以以剂量依赖、组织特定和时间的方式进行调节。总之，这项建议中描述的创新工具的开发将克服目前对O-GlcN酰化研究的实验限制，促进侧重于在机制水平上确定蛋白质O-GlcN酰化的生物学作用的研究，并产生临床前小鼠模型，在该模型中，O-GlcNAc在一系列疾病的病理生理学中的作用可以得到快速和具体的测试。

 DESCRIPTION (provided by applicant): The dynamic modification of intracellular proteins by monosaccharides of O-linked N-acetylglucosamine (O- GlcNAc) plays key roles in cellular physiology and the progression of diseases which include type II diabetes, cancer, neurodegeneration, heart failure, hypertension and aging (1, 2). However, the molecular events that underlie the role of O-GlcNAc in the pathophysiology of these diseases are ill defined. In part, this is due to a lack of tools that facilitate the specific and temporal modulation of O-GlcNAc in vitro and in vivo. The goal of this study is to generate facile chemical-genetic tools that enable the spatial, temporal and dose-dependent regulation of O-GlcNAcylation in vitro and in vivo. To achieve these goals we will address the following specific aims: Specific Aim #1: Enabling tunable, reversible chemical-genetic regulation of O-GlcNAcylation. The proposed studies will tag the endogenous loci of the enzymes that add and remove O-GlcNAc with fluorescent- and affinity-tags, and introduce recombination-inducible bi-orthogonal chemical-genetic regulators. Together, these tools will enable imaging and purification of the endogenous O-GlcNAc modifying enzymes and the rapid and synthetic regulation of protein O-GlcNAcylation. Specific Aim #2: Enabling tunable tissue-specific regulation of O-GlcNAcylation in vivo. The proposed studies will generate mice in which the expression of the enzymes that add and remove O- GlcNAc can be regulated in a dose-dependent, tissue specific and temporal manner. Together, the development of the innovative tools described in this proposal will overcome current experimental limitations to O-GlcNAcylation research, facilitate studies focused on determining the biological roles of protein O-GlcNAcylation at a mechanistic level, and generate a pre-clinical murine model in which the role of O-GlcNAc in the pathophysiology of a broad range of diseases can be rapidly and specifically tested.