Biological Regulation Studied In Vitro and In Cellulo with Modified Proteins

用修饰蛋白​​在体外和细胞内研究生物调节

• 批准号: 10164536
• 负责人: Sidney M. Hecht
• 金额: $ 33.76万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10164536
• 关键词: Address; Affinity; Amino Acids; Antiviral Agents; Binding; Biochemical; Biological; Carbohydrates; Cell-Free System; Cells; Crystallization; DNA; DNA-Protein Interaction; Dipeptides; Fingers; Genes; Genetic Transcription; In Vitro; Interferon-beta; Interferons; Metabolic; Modeling; Mus; Nucleic Acids; Phenotype; Phosphorylation; Phosphorylation Site; Positioning Attribute; Preparation; Process; Protein Engineering; Protein Glycosylation; Proteins; RNA, Ribosomal, 23S; Randomized; Regulation; Ribosomes; Roentgen Rays; Serine Phosphorylation Site; Sialic Acids; Site; Specificity; Structure; System; Techniques; analog; design; genetic regulatory protein; glycosylation; in vivo; inorganic phosphate; nanomolar; nucleobase; response; unnatural amino acids

Project Summary/Abstract We have developed a technique for randomizing 23S ribosomal RNA structure, and for selecting modified ribosomes which incorporate into proteins specific types of modified amino acids not ordinarily incorporated by wild-type ribosomes. Species incorporated both in vitro and in vivo have included nucleobase amino acids, dipeptides/dipeptidomimetics, beta-amino acids, phosphorylated amino acids and glycosylated amino acids. The selected ribosomes enable study of two key biochemical regulatory processes, i.e. protein glycosylation and phosphorylation. We will also modify regulatory proteins that interact with nucleic acids, enabling predictable modulation or altered specificity of interactions. We will exemplify new strategies using proteins containing unusual non-proteinogenic amino acids, whose incorporation requires our selected ribosomes. The creation of proteins phosphorylated stoichiometrically at single or multiple positions affords new opportunities. These include the ability to verify natural phosphorylations, and to study their effects. It permits the introduction of (metabolically stable) phosphate groups in vivo, and has enabled new strategies for identifying residues whose phosphorylation modifies function. We showed that phosphorylation of IB- Tyr42 not only relaxes NF-B inhibition, but facilitates the rate of binding to a gene whose expression NF-B regulates. We plan to study two other known phosphorylation sites in IB-, and three in NF-BWhile some sites of serine phosphorylation in NF-B are known, this is not true for Tyr and Thr. Using a new strategy, we have identified four sites of Tyr phosphorylation, and plan to study new Thr and Ser phosphorylation sites. Most mammalian proteins are glycosylated, but understanding/altering carbohydrate functions is challenging. Using a selected ribosome in a cell free system, we prepared murine interferon- (IFN-) containing GlcNAc- Asn at position 29, which can confer antiviral activity. This intermediate acceptor substrate should enable carbohydrate cluster transfer, producing IFN- fully glycosylated at position 29, and expected to have antiviral activity. We have recently introduced the same glycosylated amino acid, and its peracetylated analogue, into a model protein in very good yield in cellulo. This strategy can potentially produce the same intermediate as that prepared in vitro, but in much larger quantities. We wish to prepare fully glycosylated proteins with natural carbohydrate clusters, to simplify these clusters, and to focus on the inclusion of residues such as sialic acid. Cell regulation often involves protein–DNA interactions; low nanomolar affinities and impressive selectivity are typical. Many X-ray crystal structures could guide design changes, but attempts to alter these regulatory processes by changing affinity or specificity have failed. Using Rob proteins having nucleobase amino acids, we modified binding to their micF DNA partners, realizing stronger binding, and enhanced phenotypic cellular responses. We propose to study the Rob–micF DNA interactions further to refine our design techniques. The principles developed will be used to address protein–DNA recognition in a Zn finger system.

项目概要/摘要 我们开发了一种随机化 23S 核糖体 RNA 结构并选择修饰的技术 核糖体掺入蛋白质中通常不掺入的特定类型的修饰酸氨基酸 体外和体内掺入的物种包括核碱基氨基酸， 二肽/二肽模拟物、β-氨基酸、磷酸化氨基酸和糖基化氨基酸。 选定的核糖体能够研究两个关键的生化调节过程，即蛋白质糖基化 我们还将修改与核酸相互作用的调节蛋白，从而使之成为可能。 我们将举例说明使用蛋白质的新策略。 含有不寻常的非蛋白氨基酸，其掺入需要我们选择的核糖体。 在单个或多个位置上化学计量磷酸化的蛋白质的产生提供了新的 这些机会包括验证天然磷酸化并研究其影响的能力。 在体内引入（代谢稳定的）磷酸基团，并启用了新的策略 确定其磷酸化修饰功能的残基 我们表明 IB- Tyr42 的磷酸化。 不仅可以放松 NF-κB 抑制，还可以提高与表达 NF-κB 的基因的结合速率 我们计划研究 IB- 中的另外两个已知磷酸化位点，以及 NF-B 中的三个磷酸化位点。 NF-κB 中的丝氨酸磷酸化位点是已知的，但 Tyr 和 Thr 的情况并非如此。 已确定了四个 Tyr 磷酸化位点，并计划研究新的 Thr 和 Ser 磷酸化位点。 大多数哺乳动物蛋白质都是糖基化的，但理解/改变碳水化合物的功能具有挑战性。 在无细胞系统中使用选定的核糖体，我们制备了含有 GlcNAc- 的鼠干扰素- (IFN-) 29 位的 Asn，可以赋予抗病毒活性。这种中间受体底物应该能够实现。 碳水化合物簇转移，产生29位完全糖基化的IFN-β，预计具有抗病毒作用 我们最近将相同的糖基化氨基酸及其全乙酰化类似物引入到 该策略可以在纤维素中以非常高的产率产生相同的中间体。 体外制备，但数量要大得多，我们希望用天然材料制备完全糖基化的蛋白质。 碳水化合物簇，以简化这些簇，并重点关注唾液酸等残基的包含。 细胞调节通常涉及蛋白质-DNA 相互作用；低纳摩尔亲和力和令人印象深刻的选择性； 许多 X 射线晶体结构可以指导设计变更，但试图改变这些监管。 使用具有核碱基氨基酸的 Rob 蛋白来改变亲和力或特异性的过程失败了。 我们修改了与其 micF DNA 伴侣的结合，实现了更强的结合，并增强了表型细胞 我们建议进一步研究 Rob-micF DNA 相互作用，以完善我们的设计技术。 所开发的原理将用于解决锌指系统中的蛋白质-DNA 识别问题。