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

• 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
• 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

 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（由申请人提供）：O-连接的N-乙酰葡糖胺（O-GlcNAc）单糖对细胞内蛋白质的动态修饰在细胞生理学和疾病进展中起关键作用，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的作用的分子事件是不明确的。在某种程度上，这是由于缺乏促进体外和体内O-GlcNAc的特异性和时间调节的工具。本研究的目的是产生简便的化学遗传工具，使空间，时间和剂量依赖性的调节O-GlcNAc酰化在体外和体内。为了实现这些目标，我们将致力于以下具体目标：具体目标#1：实现O-GlcNAcylation的可调、可逆的化学遗传调节。拟议的研究将标记的内源位点的酶，添加和删除O-GlcNAc与荧光和亲和标签，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-GlcNAc化的快速和合成调节成为可能。具体目标#2：实现体内O-GlcNAc酰化的可调组织特异性调节。所提出的研究将产生小鼠，其中添加和去除O-GlcNAc的酶的表达可以以剂量依赖性、组织特异性和时间方式进行调节。总之，本提案中描述的创新工具的开发将克服目前对O-GlcNAc化研究的实验限制，促进专注于确定蛋白质O-GlcNAc化在机制水平上的生物学作用的研究，并生成临床前小鼠模型，其中O-GlcNAc在广泛疾病的病理生理学中的作用可以快速和特异性地进行测试。