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

用于 O-GlcNAc 酰化时空调节的化学遗传工具

基本信息

批准号：
8984799
负责人：
Natasha Elizabeth Zachara
金额：
$ 28.18万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-07-15 至 2017-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8984799
关键词：
Acetylglucosamine Address Affinity Aging Animal Model Animals Biochemistry Biological Biology Carbohydrate Biochemistry Cell Line Cell physiology Cells Chemicals Chimeric Proteins Collaborations Development Disease Disease Progression Dose Engineering Enzymes Event Excision Functional disorder Gene Transfer Techniques Generations Genetic Engineering Genetic Recombination Goals Grant Heart Heart failure Hypertension Image In Vitro Inflammation Insulin Life Link Malignant Neoplasms Mammalian Cell Mediating Modeling Modification Molecular Monosaccharides Mus Muscle Cells Nerve Degeneration Neurodegenerative Disorders Non-Insulin-Dependent Diabetes Mellitus O-GlcNAc transferase Pathway interactions Physiology Play Post-Translational Protein Processing Process Protein Biochemistry Proteins Publishing Reagent Regulation Research Research Personnel Role Skeletal Muscle System Testing Tissues Transgenic Mice Transgenic Organisms Universities Work base chemical genetics design glucose metabolism in vivo innovation insight medical schools peptide O-linked N-acetylglucosamine-beta-N-acetylglucosaminidase pre-clinical public health relevance recombinase small molecule sugar tool vector

项目摘要

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.

描述（通过应用程序提供）：通过O连锁的N-乙酰基葡萄糖胺（O-GLCNAC）单糖的细胞内蛋白的动态修饰在细胞生理学中起关键作用，以及包括II型糖尿病，糖尿病，癌症，癌症，心脏失败，心脏失败，高压，高压和衰减（1、2）的疾病进展。然而，未定义的分子事件是O-GLCNAC在这些疾病的病理生理中的作用的基础。在某种程度上，这是由于缺乏促进O-GLCNAC体外和体内特定和临时调节的工具。这项研究的目的是生成易于的化学遗传学工具，以实现O-Glcnacylation在体外和体内的空间，临时和剂量依赖性调节。为了实现这些目标，我们将解决以下特定目标：具体目标＃1：实现O-Glcnacylation的可调，可逆的化学遗传调节。拟议的研究将标记酶的内源性局部局部局部，这些酶添加和去除荧光和亲和力标签的O-GLCNAC，并引入重组 - 可诱导的双轴化学遗传遗传调节剂。这些工具将共同实现内源性O-GlCNAC修饰酶的成像和纯化，以及蛋白O-Glcnacylation的快速而合成的调节。特定目标＃2：在体内启用O-Glcnacylation的可调组织特异性调节。拟议的研究将产生小鼠，其中添加和去除O-GLCNAC的酶的表达可以以剂量依赖性，组织特异性和临时方式调节。 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 biologic 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.