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: 7924505
• 负责人: ZHIWEN Jonathan ZHANG
• 金额: $ 26.35万
• 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 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7924505
• 关键词: Acetylglucosamine; Affect; Amino Acids; Amino Acyl-tRNA Synthetases; Antibodies; Apoptosis; Area; Biological; Biological Assay; Biomedical Research; Cell Nucleus; Cell Proliferation; Cells; Complex; Cysteine; Development; Environment; Escherichia coli; Ethers; Eukaryotic Cell; Event; Gene Targeting; Genetic Code; Genetic Transcription; Glycobiology; Glycoproteins; Goals; In Vitro; Lead; Ligase; Light; Link; MYC gene; Malignant Neoplasms; Mammalian Cell; Metabolism; Methodology; Modification; Molecular; Mutate; Mutation; Oncogenes; Organism; Paste substance; Pathway interactions; Phosphorylation; Phosphorylation Site; Positioning Attribute; Post-Translational Protein Processing; Process; Property; Protein Glycosylation; Proteins; Proto-Oncogene Proteins c-myc; 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; directed evolution; genetic selection; glycosylation; in vivo; interest; multicatalytic endopeptidase complex; mutant; novel; overexpression; prevent; protein function; protein structure function; protein transport; public health relevance; 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。我们对O-N-乙酰葡糖胺（O-GlcNAc）修饰特别感兴趣，其中GlcNAc部分通过醚键连接到蛋白质的丝氨酸或苏氨酸残基。这种类型的修饰在真核细胞中是普遍存在的，并且可以与磷酸化结合来调节许多蛋白质的功能。然而，O-GlcNAc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。研究糖基化的主要瓶颈之一是无法产生大量的同质糖基化蛋白质。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”细胞的翻译机制来规避这个问题。我们的系统将被开发用于哺乳动物细胞，因为这将允许感兴趣的蛋白质在其天然环境中产生，具有必要的翻译后修饰，允许更自然的折叠，以及在体内进行功能测定的能力。为了产生新的遗传密码，我们将进化出突变的tRNA合成酶/抑制剂tRNA对，它们利用糖基化氨基酸，并且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-“剪切-粘贴”和“定向进化”从现有的tRNA-合成酶/抑制剂-tRNA对进化这些对。然后，新产生的哺乳动物细胞可以合成大量在所需位点含有O-GlcNAc部分的同源目标蛋白质。这种独特而强大的技术可用于回答关于已知进行O-GlcNAc化的许多蛋白质（如c-Myc蛋白质）的调节模式的剩余问题。c-Myc蛋白是一种重要的癌基因，已被广泛研究。全面了解其调节机制对于开发治疗或预防癌症的新疗法至关重要。已知c-Myc仅在一个位点（Thr-58）被O-GlcNAc部分修饰。作为我们系统的一个测试，我们将研究O-GlcNAc化的Thr-58的蛋白质的稳定性，细胞定位和调节作用的影响，特别是O-GlcNAc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同质c-Myc蛋白，然后在体内和体外进行各种生物学测定。这些研究将在分子水平上阐明糖基化对蛋白质的调控机制。公共卫生相关性：本提案的长期目标是研究O-N-乙酰葡萄糖胺（O-GlcNAc）修饰在生物学途径中的调节作用。O-GlcNAc以及类似分子对蛋白质功能的影响的全面研究由于许多技术障碍而不可能。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。作为一个出发点，我想探索新的方面O-GlcNAc修饰调节癌基因c-Myc的活动，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具有特定修饰的同质蛋白质。我将通过劫持细胞的翻译机器来规避这些问题，并在哺乳动物细胞中产生新的遗传密码或非天然的糖基化氨基酸。然后，新开发的哺乳动物细胞将在体内合成一系列同质糖基化的c-Myc蛋白。这是一种独特而强大的技术，可以回答以前无法回答的问题，例如c-Myc的核重定位以及磷酸化和O-GlcNAc化之间的相互作用，以调节c-Myc的泛素化和降解。