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Analysis of Metabolic Capabilities of Prokaryotic Cells

原核细胞代谢能力分析

基本信息

批准号：
10574503
负责人：
JORGE C ESCALANTE
金额：
$ 67.97万
依托单位：
UNIVERSITY OF GEORGIA
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-03-01 至 2025-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10574503
关键词：
Acetylation Acetyltransferase Address Anabolism Antimicrobial Resistance Archaea Area Bacteria Biochemistry Biophysics Biotechnology Cell Aging Cell membrane Cells Coenzymes Collaborations Complex Crystallography Deacetylase Diabetes Mellitus Enzymes Genetic Goals Health Human Knowledge Learning Life Ligands Lysine Malignant Neoplasms Membrane Biology Metabolic Metabolic Control Metabolic stress Metabolism Modeling Molecular Molecular Biology Neurodegenerative Disorders Nutrient Obesity Pathogenesis Pathway interactions Performance Physiological Physiological Processes Physiology Post-Translational Protein Processing Principal Investigator Prokaryotic Cells Regulation Research Role Salmonella Salmonella enterica Sirtuins Spectrum Analysis System Testing Transition Elements Vitamin B 12 Work base cobamamide experimental study human pathogen in vivo innovation microbial microorganism model organism pathogen programs protein function response riboside single molecule structural biology tool

项目摘要

Program Director/Principal Investigator (Last, First, Middle): Escalante, Jorge C. PROJECT SUMMARY/ABSTRACT This MIRA proposal brings together two fields of prokaryotic metabolism and physiology that the PI’s group has contributed extensively to. The first is the assembly of the structurally complex coenzyme B12 (CoB12), and the second one is the regulation of protein function by lysine acetylation in response to metabolic stress. Previous work by the PI’s group in these areas has resulted in the discovery of new enzymes and pathways, and has established fundamental physiologic paradigms that apply to cells of all domains of life. We have learned a great deal about how the complex coenzyme B12 is made, and yet, gaps in our knowledge about its assembly remain. Although the remaining gaps are challenging to solve, recent breakthroughs in our group have generated the tools to address these questions and advance our understanding of the physiological integration of CoB12 biosynthesis in microorganisms of societal importance. We will investigate how the lower ligand base of CoB12 is synthesized and activated to its riboside in human pathogens, how vitamin B12 is converted to CoB12 in several Gram-positive pathogens, and why the last steps of the pathway occur at the cell membrane in all CoB12 producers known to date. Most of the proposed work will be performed in Salmonella enterica because of our deep knowledge of CoB12 biosynthesis in this bacterium, and the sophisticated genetic system available to do in vivo work. We will also use Salmonella to establish the function of heterologous, putative CoB12 biosynthetic functions in other bacteria and archaea. We will continue to investigate the role of lysine acetylation in the control of metabolic stress. Lysine acetylation is a posttranslational modification of profound relevance to human health and biotechnology. The impact of this regulatory mechanism on human cell aging and cancer, neurodegenerative diseases, diabetes, obesity, antimicrobial resistance, microbial pathogenesis, and other research areas of societal relevance emphasizes the need to continue advancing this field of research. Fundamental questions about lysine acetylation remain unanswered. The proposed work will investigate new role(s) of prokaryotic sirtuin deacetylases in prokaryotic physiology, and will continue to elucidate the functions and physiological roles of acetyltransferases in Gram-negative and Gram-positive human pathogens. Our findings obtained from experiments performed with prokaryotic model organisms will inform how the system may work in higher forms of life. A powerful, innovative combination of approaches, including transition metal spectroscopy, structural biology (crystallography), biochemistry, molecular biology, in vivo genetics, physiology, single-molecule biophysics, and system-wide analyses will be applied during the performance of the proposed work. We will collaborate with experts in the fields of spectroscopy, crystallography, molecular biophysics, and membrane biology to provide comprehensive, rigorous testing of hypotheses and working models. OMB No. 0925-0001/0002 (Rev. 03/16 Approved Through 10/31/2018) Page Continuation Format Page

项目总监/首席研究员（最后、第一、中间）：Escalante, Jorge C. 项目概要/摘要这项 MIRA 提案汇集了 PI 小组的原核代谢和生理学两个领域做出了广泛的贡献。第一个是结构复杂的辅酶 B12 (CoB12) 的组装，以及第二个是通过赖氨酸乙酰化来调节蛋白质功能以应对代谢应激。 PI 团队之前在这些领域的工作已经发现了新的酶和途径，并建立了适用于生命各个领域细胞的基本生理学范式。我们已经了解了大量关于复杂辅酶 B12 的制备方法，但我们的知识仍存在空白关于其组装仍然存在。尽管剩余的差距很难解决，但最近我们在小组已经生成了解决这些问题并增进我们对 CoB12 生物合成在具有社会重要性的微生物中的生理整合。我们将调查 CoB12 的下配体碱基如何在人类病原体中合成并激活为其核苷，如何维生素 B12 在几种革兰氏阳性病原体中转化为 CoB12，以及为什么该途径的最后步骤迄今为止已知的所有 CoB12 生产者的细胞膜上都会发生这种情况。大部分拟议工作将得到执行由于我们对这种细菌中 CoB12 生物合成的深入了解，以及复杂的遗传系统可用于体内工作。我们还将使用沙门氏菌来建立该功能其他细菌和古细菌中异源的、推定的 CoB12 生物合成功能。我们将继续研究赖氨酸乙酰化在控制代谢应激中的作用。赖氨酸乙酰化是一种与人类健康和生物技术密切相关的翻译后修饰。这这种调节机制对人类细胞衰老和癌症、神经退行性疾病、糖尿病、肥胖、抗菌素耐药性、微生物发病机制和其他与社会相关的研究领域强调需要继续推进这一研究领域。关于赖氨酸的基本问题乙酰化仍然没有答案。拟议的工作将研究原核去乙酰化酶的新作用脱乙酰酶在原核生理学中的作用，并将继续阐明其功能和生理作用革兰氏阴性和革兰氏阳性人类病原体中的乙酰转移酶。我们的研究结果来自用原核模型生物进行的实验将告诉我们该系统如何以更高的形式工作的生活。强大、创新的方法组合，包括过渡金属光谱学、结构生物学（晶体学）、生物化学、分子生物学、体内遗传学、生理学、单分子生物物理学、在开展拟议工作期间将应用全系统分析。我们将合作与光谱学、晶体学、分子生物物理学和膜生物学领域的专家一起提供对假设和工作模型的全面、严格的测试。 OMB 编号 0925-0001/0002（修订版 03/16 已批准至 10/31/2018）页面延续格式页面