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: 10371143
• 负责人: Sidney M. Hecht
• 金额: $ 51.81万
• 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/10371143
• 关键词: Address; Affinity; Amino Acids; 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.

项目摘要/摘要 我们已经开发了一种用于随机化23S核糖体RNA结构的技术，并选择了修改的技术 核糖体掺入蛋白质特定类型的改性氨基酸中 野生型核糖体。在体外和体内均包含的物种包括核碱基氨基酸， 二肽/二肽仪，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 和磷酸化。我们还将修改与核酸相互作用的调节蛋白 可预测的调制或相互作用的特异性改变。我们将使用蛋白质来体现新策略 含有异常的非蛋白原氨基酸，其就业需要我们选择的核糖体。 在单个或多个位置上磷酸化的蛋白质磷酸化磷酸化提供了新的 机会。这些包括验证自然磷酸化并研究其作用的能力。它允许 在体内引入（代谢稳定的）磷酸组，并为 识别磷酸化修饰功能的响应。我们表明IB-Tyr42的磷酸化 不仅放松NF-B抑制作用，而且促进了与表达NF-B的基因结合速率 调节。我们计划在IB-中研究其他两个已知的磷酸化位点，而在NF-B中有三个 NF-B中丝氨酸磷酸化的位点是已知的，对于Tyr和Thr而言，这是不正确的。使用新策略，我们 已经鉴定了四个Tyr磷酸化位点，并计划研究新的THR磷酸化位点。 大多数哺乳动物蛋白是糖基化的，但是了解/改变碳水化合物功能是具有挑战性的。 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-） 在位置29处的ASN，可以赋予抗病毒活性。此中间受体基板应启用 碳水化合物簇转移，在位置29处产生IFN-完全糖基化，并有望有抗病毒 活动。最近，我们引入了相同的糖基化氨基酸及其核酸氨基酸的类似物 模拟纤维素中非常好的蛋白质。该策略可能会产生与 在体外制备，但数量更大。我们希望用天然准备完全糖基化的蛋白 碳水化合物簇，简化这些簇，并专注于纳尔酸等残留物。 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 是典型的。许多X射线晶体结构都可以指导设计变化，但是试图改变这些调节 通过改变亲和力或特异性的过程失败。使用具有核碱基氨基酸的Rob蛋白， 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 回答。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。这 开发的原理将用于解决Zn手指系统中的蛋白质-DNA识别。