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。我们对 O-N-乙酰氨基葡萄糖 (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上。这种类型的修饰在真核细胞中普遍存在，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 酰化在蛋白质结构和功能调节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同质糖基​​化蛋白质。在这个提案中，我们希望通过创造新的遗传密码并使用它们“劫持”细胞的翻译机制来规避这个问题。我们的系统将为哺乳动物细胞开发，因为这将允许在其天然环境中产生感兴趣的蛋白质，并进行必要的翻译后修饰，从而实现更自然的折叠，以及在体内进行功能测定的能力。为了生成新的遗传密码，我们将进化出利用糖基化氨基酸的突变 tRNA 合成酶/抑制子 tRNA 对，并且与哺乳动物细胞正交，这意味着它们不与任何内源 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/抑制子-tRNA 对中进化出这些对。然后，新生成的哺乳动物细胞可以在所需位点合成大量含有 O-GlcNAc 部分的均质目标蛋白。这种独特而强大的技术可用于回答有关许多已知进行 O-GlcNAcNA 酰化的蛋白质（例如 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。对其调节机制的完整了解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。作为对我们系统的测试，我们将研究 c-Myc 的 Thr-58 的 O-GlcNA 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 Thr-58 的 O-GlcNA 酰化和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质 c-Myc 蛋白，然后在体内和体外进行各种生物测定。这些研究将揭示分子水平上糖基化调节蛋白质的机制。公共健康相关性：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。由于许多技术障碍，尚未可能对 O-GlcNAc 以及类似分子对蛋白质功能的影响进行全面研究。然而，这是一个非常重要的研究领域，对于我们理解生命系统的功能和生物医学研究至关重要。作为起点，我想探讨 O-GlcNAc 修饰调节癌基因 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并在哺乳动物细胞中生成新的遗传密码或非天然糖基化氨基酸来规避这些问题。新开发的哺乳动物细胞随后将在体内合成一系列均质的糖基化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，例如c-Myc的核重新定位以及磷酸化和O-GlcNAcNA酰化之间的相互作用以调节c-Myc的泛素化和降解。