Analysis of Metabolic Capabilities of Prokaryotic Cells

原核细胞代谢能力分析

• 批准号: 10355463
• 负责人: JORGE C ESCALANTE
• 金额: $ 67.95万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10355463
• 关键词: Acetylation; Acetyltransferase; Address; Anabolism; Animal Model; Antimicrobial Resistance; Archaea; Area; Bacteria; Biochemistry; Biophysics; Biotechnology; Cell Aging; Cell membrane; Cells; Coenzymes; Complex; Crystallography; Deacetylase; Diabetes Mellitus; Genetic; Goals; Health; Human; Knowledge; 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; enzyme pathway; experimental study; human pathogen; in vivo; innovation; microbial; microorganism; pathogen; programs; protein function; response; riboside; single molecule; structural biology; tool

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

项目负责人/主要研究者（最后，第一，中间）：Escalante，Jorge C. 项目总结/摘要 MIRA的建议将原核生物代谢和生理学两个领域结合在一起， 做出了广泛的贡献。第一个是结构复杂的辅酶B12（CoB 12）的组装， 第二种是赖氨酸乙酰化对蛋白质功能的调节。 PI小组在这些领域的先前工作已经发现了新的酶和途径， 并建立了适用于所有生命领域的细胞的基本生理学范式。 我们已经了解了很多关于复杂的辅酶B12是如何产生的，然而，我们的知识空白 关于它的集会仍然存在。虽然剩余的差距是具有挑战性的解决，最近的突破，在我们的 小组已经产生了解决这些问题的工具，并促进了我们对 CoB 12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 在人类病原体中，CoB 12的较低配体碱基是如何合成和激活其核苷的， 维生素B12在几种革兰氏阳性病原体中转化为CoB 12，以及为什么该途径的最后一步 在迄今为止已知的所有CoB 12生产者中发生在细胞膜上。大部分拟议的工作将在 由于我们对这种细菌中CoB 12生物合成的深入了解，以及 复杂的遗传系统可以在体内工作。我们还将使用沙门氏菌来建立功能 在其他细菌和古细菌中的异源，推定的CoB 12生物合成功能。 我们将继续研究赖氨酸乙酰化在代谢应激控制中的作用。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。的 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、微生物发病机理和其他社会相关研究领域 强调需要继续推进这一研究领域。赖氨酸的基本问题 乙酰化仍然没有答案。本研究将探讨原核sirtuin的新功能 脱乙酰酶在原核生物生理学，并将继续阐明功能和生理作用， 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 用原核生物模式生物进行的实验将告诉我们该系统如何在更高的形式下工作 生命 一个强大的，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，将进行全系统分析。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， 对假设和工作模型进行全面、严格的测试。 OMB编号0925-0001/0002（2016年3月修订版，批准至2018年10月31日）