Regulation of structure and function of protein by glycosylation

通过糖基化调节蛋白质的结构和功能

• 批准号: 7528158
• 负责人: ZHIWEN Jonathan ZHANG
• 金额: $ 27.39万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7528158
• 关键词: Acetylglucosamine; Affect; Amino Acids; Amino Acyl-tRNA Synthetases; Antibodies; Apoptosis; Area; Biological; Biological Assay; Biomedical Research; Cell Nucleus; Cell Proliferation; Cells; Class; Complex; Cysteine; Development; Environment; Escherichia coli; Ethers; Ethyl Ether; Eukaryotic Cell; Event; Gene Targeting; Genetic Code; Genetic Transcription; Glycobiology; Glycoproteins; Goals; In Vitro; Lead; Ligase; Light; Link; Malignant Neoplasms; Mammalian Cell; Metabolism; Methodology; Modification; Modification Type; Molecular; Mutate; Mutation; Numbers; Oncogenes; Organism; Paste substance; Pathway interactions; Phosphorylation; Phosphorylation Site; Positioning Attribute; Post-Translational Protein Processing; Process; Property; Protein Glycosylation; Protein Overexpression; Proteins; Proto-Oncogene Proteins c-myc; Public Health; Regulation; Research; Role; Series; Serine; Signal Transduction; Site; Structure; System; Technology; Testing; Threonine; Transfer RNA; Ubiquitin; Ubiquitination; Work; c-myc Genes; cell growth; chemical synthesis; desire; directed evolution; genetic selection; glycosylation; in vivo; interest; intracellular protein transport; multicatalytic endopeptidase complex; mutant; novel; prevent; protein function; protein structure function; protein transport; sugar; transcription factor

DESCRIPTION (provided by applicant): Our long-term goal is to understand the various regulatory roles of glycosylation. We are specifically interested in O-N-acetylglucosamine (O-GlcNAc) modification, where the GlcNAc moiety is attached by an ether linkage to a serine or threonine residue of a protein. This type of modification is ubiquitous in eukaryotic cells and may work in conjunction with phosphorylation to modulate the function of many proteins. However, the exact role of O-GlcNAcylation in the regulation of the structure and function of proteins had yet to be fully determined and exploited. One of the major bottlenecks for studying glycosylation is the inability to generate large amounts of homogenously glycosylated proteins. In this proposal, we would like to circumvent this problem by creating novel genetic codes and using them to `hijack' the translational machinery of the cell. Our system will be developed for mammalian cells, for this would allow the protein of interest to be produced in its native environment with necessary post-translational modifications, allowing for more natural folding, as well as the ability to perform functional assays in vivo. To generate novel genetic codes, we will evolve mutant tRNA-synthetase/suppressor-tRNA pairs that utilize glycosylated amino acids, and are orthogonal to mammalian cells, meaning they do not interact with any endogenous tRNAs or synthetases. We will evolve these pairs from existing tRNA-synthetase/suppressor-tRNA pairs using two approaches - "cut - paste" and "directed evolution". The newly generated mammalian cell can then synthesize large amounts of a homogeneous protein of interest containing an O-GlcNAc moiety at the desired sites. This unique and powerful technology can then be used to answer remaining questions about the mode of regulation of the many proteins that are known to undergo O-GlcNAcylation, such as the c-Myc protein. The c-Myc protein is an important oncogene that has been extensively studied. A complete understanding of its regulatory mechanism will be critical to the development of new therapies to treat or prevent cancer. It is known that c-Myc is modified with O-GlcNAc moiety only at one site (Thr-58). As a test of our system, we will investigate the effect of O-GlcNAcylation of Thr-58 of c-Myc on its protein stability, cellular localization and regulatory roles, especially the interplay between O-GlcNAcylation and phosphorylation at Thr-58. We will produce a series of homogeneous c-Myc proteins with different site-specific specific glycosylations and then performing various biological assays both in vivo and in vitro. These studies will shed light on the mechanism of regulation of proteins by glycosylation at a molecular level. PUBLIC HEALTH RELEVANCE: The long-term goal of this proposal is to study the regulatory role of O-N-acetylglucosamine (O-GlcNAc) modification in biological pathways. Comprehensive studies of the effects of O-GlcNAc as well as similar molecules on protein function have not been possible due to a number of technical obstacles. Yet, it is a highly significant area of research that is critical to our understanding of the functioning of living systems and biomedical research. As a starting point, I would like to probe the novel aspect of O-GlcNAc modification regulating oncogene c-Myc activities and elucidate its mechanism. One of the major bottlenecks for my project and related glycobiology is the inability to generate homogenous proteins bearing specific modifications in mammalian systems. I will circumvent these problems by hijacking the cell's translational machinery and generate new genetic codes or the non-natural glycosylated amino acids in mammalian cells. The newly developed mammalian cell will then synthesize a series of homogeneous glycosylated c-Myc proteins in vivo. This is a unique and powerful technology that can answer questions that could not be answered before such as nucleus relocation of c-Myc and interplay between phosphorylation and O-GlcNAcylation to regulate the ubiquitination and degradation of c-Myc.

描述(由申请人提供)：我们的长期目标是了解糖基化的各种调节作用。我们特别感兴趣的是O-N-乙酰氨基葡萄糖(O-GlcNAc)的修饰，其中GlcNAc部分通过乙醚连接到蛋白质的丝氨酸或苏氨酸残基上。这种类型的修饰在真核细胞中普遍存在，并可能与磷酸化一起调节许多蛋白质的功能。然而，O-GlcN酰化在调节蛋白质结构和功能中的确切作用尚未完全确定和开发。糖基化研究的主要瓶颈之一是不能产生大量均一的糖基化蛋白。在这项提议中，我们希望通过创造新的遗传密码并使用它们来“劫持”细胞的翻译机制来绕过这个问题。我们的系统将为哺乳动物细胞开发，因为这将允许在其自然环境中生产感兴趣的蛋白质，经过必要的翻译后修改，允许更自然的折叠，以及在体内执行功能分析的能力。为了产生新的遗传密码，我们将进化突变的tRNA-合成酶/抑制-tRNA对，这些突变的tRNA-合成酶/抑制-tRNA对利用糖基化的氨基酸，并与哺乳动物细胞正交，这意味着它们不与任何内源性tRNA或合成酶相互作用。我们将使用两种方法从现有的tRNA合成酶/抑制子-tRNA对进化这些对-“剪切-粘贴”和“定向进化”。然后，新产生的哺乳动物细胞可以在所需的位置合成大量含有O-GlcNAc部分的同质蛋白。然后，这项独特而强大的技术可以用来回答关于许多已知经历O-GlcN酰化的蛋白质的调节模式的剩余问题，例如c-Myc蛋白质。C-Myc蛋白是一种被广泛研究的重要癌基因。对其调控机制的全面了解将对开发治疗或预防癌症的新疗法至关重要。已知c-Myc只在一个位点(Thr-58)被O-GlcNAc部分修饰。作为对我们系统的测试，我们将研究c-Myc的Thr-58的O-GlcN酰化对其蛋白质稳定性、细胞定位和调节作用的影响，特别是O-GlcN酰化和Thr-58的磷酸化之间的相互作用。我们将生产一系列具有不同位点特异性糖基化的均一c-Myc蛋白，然后在体内和体外进行各种生物学检测。这些研究将在分子水平上阐明糖基化对蛋白质的调节机制。公共卫生相关性：该提案的长期目标是研究O-N-乙酰氨基葡萄糖(O-GlcNAc)修饰在生物途径中的调节作用。由于一些技术障碍，全面研究O-GlcNAc以及类似分子对蛋白质功能的影响是不可能的。然而，这是一个非常重要的研究领域，对我们理解生命系统的功能和生物医学研究至关重要。作为一个起点，我想探讨O-GlcNAc修饰调控癌基因c-Myc活性的新方面，并阐明其机制。我的项目和相关糖生物学的主要瓶颈之一是无法在哺乳动物系统中产生带有特定修饰的同质蛋白质。我将通过劫持细胞的翻译机制来绕过这些问题，并在哺乳动物细胞中产生新的遗传密码或非天然糖基化氨基酸。然后，新开发的哺乳动物细胞将在体内合成一系列均一的糖基化c-Myc蛋白。这是一项独特而强大的技术，可以回答以前无法回答的问题，如c-Myc的核重新定位以及磷酸化和O-GlcN酰化之间的相互作用，以调节c-Myc的泛素化和降解。