Leveraging Adaptive Evolution and High-Throughput Techniques to Dissect the Link Between Biochemical Function and Fitness

利用适应性进化和高通量技术来剖析生化功能与健康之间的联系

• 批准号: 10704076
• 负责人: Margaux Pinney
• 金额: $ 40.38万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-13 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10704076
• 关键词: Active Sites; Address; Affect; Amino Acid Sequence; Antiviral Agents; Appointment; Area; Award; Bar Codes; Basic Science; Benchmarking; Biochemical; Biochemistry; Biological; Biological Assay; Biological Markers; Biology; Biophysics; Bioremediations; Cell Therapy; Cells; Chemicals; Chemistry; Collaborations; Communities; Complex; Data; Data Set; Dedications; Discipline; Doctor of Philosophy; Environment; Enzymes; Evolution; Faculty; Future; Goals; HRK gene; In Vitro; Individual; Industry; Institution; Investigation; Knowledge; Laws; Lead; Learning; Libraries; Life; Link; Maps; Measurement; Measures; Medicine; Mentors; Mentorship; Metabolic Diseases; Methods; Modeling; Molecular; Molecular Evolution; Mutation; Organism; Pathogenesis; Pharmaceutical Preparations; Physics; Positioning Attribute; Postdoctoral Fellow; Predisposition; Property; Proteins; Publications; Regulation; Research; Research Proposals; Specific qualifier value; Specificity; Structure; Techniques; Technology; Temperature; Testing; Therapeutic; Translating; Translational Research; Variant; Virulence; Work; anti-cancer; antimicrobial; biochemical evolution; catalyst; chemical reaction; computing resources; design; enzyme mechanism; experience; faculty mentor; fitness; frontier; genetic variant; graduate student; high throughput technology; human pathogen; improved; in vivo; interest; macromolecule; microfluidic technology; next generation; novel; precision medicine; pressure; protein function; recruit; response; senior faculty; tool; undergraduate student

PROJECT SUMMARY/ABSTRACT Enzymes are the primary functional molecules in cells, providing enormous rate enhancements, specificity and regulation to the diverse chemical reactions that are necessary for life. Enzymes, like all biological macromolecules, are the products of evolution: all enzymes have evolved to operate within the complex environment of the organism/cell in specific environmental niches(s). Thus, an understanding of enzyme function and evolution is fundamental to biology. Enzymes also have tremendous potential in medicine (e.g., as targets for anti-cancer, antimicrobial and antiviral drugs and as therapeutics for metabolic disorders) and in industry (e.g. to make important commodity chemicals and as catalysts for bioremediation). Our central premise is that a quantitative, mechanistic understanding of enzyme function and its relationship to organism fitness is critically needed to precisely manipulate enzymes and to deeply understand biology. To generate this level of understanding, we need: (1) a quantitative, chemical, and physical knowledge of enzyme function, and (2) mechanistic data describing how and when these physical principles contribute to enzyme function within the complex environments where enzymes operate. An enhanced understanding of the relationships between protein sequence, protein function and cellular/organismal fitness will have profound impacts across biology and medicine, from improving our ability to predict how mutations will influence the virulence and drug susceptibility of human pathogens, to enhancing precision medicine by accurately predicting the consequences of allelic variants, to enabling the design of next-generation protein and cellular therapeutics. Achieving this understanding requires new tools and a new conceptual paradigm. Enzymes are highly interconnected, their functions are multifaceted, and their cellular environments are complex. Traditional biochemistry is enormously powerful, allowing for the intensive study of a few individual enzymes in vitro (10s) and providing detailed knowledge of their chemical mechanisms. But identifying the many residues that matter for enzyme function requires investigation of residues beyond the active site at a scale far beyond that of traditional biochemistry. Furthermore, this biochemical information then needs to be translated to organism fitness in vivo in a quantitative manner. Here we will overcome these challenges. We will first use evolutionary sequence information to direct enzyme variant design towards functionally important areas of sequence space. We will adapt high-throughput microfluidic technologies to quantitively measure the biochemical properties (e.g., kcat, Km, Ki, and ∆GFold) of this library of 104 enzyme variants in vitro (Aim 1). Then we will determine how each of these variants affects organismal fitness in vivo using pooled competition and barcode sequencing assays (Aim 2). Finally, we will use this sequence-function-fitness map to test long-standing models in biochemistry and evolution and reveal the biochemical determinants of fitness important for industry and medicine (Aim 3). Such a comprehensive and quantitative mapping of biochemical function to fitness has never been achieved.

项目概要/摘要 酶是细胞中的主要功能分子，提供巨大的速率增强， 像所有生物一样，酶对生命所需的各种化学反应具有特异性和调节性。 大分子是进化的产物：所有酶都已进化为在复合体中运作 特定环境位中的有机体/细胞的环境，从而了解酶的功能。 进化是生物学的基础，酶在医学方面也具有巨大的潜力（例如，作为靶标）。 用于抗癌、抗菌和抗病毒药物以及代谢紊乱的治疗）和工业（例如 制造重要的商品化学品并作为生物修复的催化剂）。 对酶功能及其与生物体适应性的关系的定量、机制理解至关重要 需要精确操纵酶并深入了解生物学。 为了产生这种程度的理解，我们需要：（1）定量、化学和物理知识 酶功能的描述，以及 (2) 描述这些物理原理如何以及何时贡献的机械数据 加深对酶在复杂环境中的作用的理解。 蛋白质序列、蛋白质功能和细胞/有机体适应性之间的关系将产生深远的影响 影响生物学和医学，从提高我们预测突变将如何影响的能力 人类病原体的毒力和药物敏感性，通过准确预测来增强精准医疗 等位基因变异的后果，以实现下一代蛋白质和细胞疗法的设计。 实现这一目标需要新的工具和新的概念范式。 它们相互关联，功能多样，蜂窝环境复杂。 生物化学非常强大，可以在体外对一些单独的酶进行深入研究（10 秒） 并提供其化学机制的详细知识，但确定许多重要的残留物。 对于酶的功能，需要对活性位点以外的残基进行研究，其规模远远超出了 此外，这些生化信息需要转化为有机体。 在这里，我们将首先使用进化来克服这些挑战。 序列信息将酶变体设计指导到序列空间的功能重要区域。 我们将采用高通量微流控技术来定量测量生化特性（例如， kcat、Km、Ki 和 ΔGFold) 的 104 个酶变体体外库（目标 1）。 使用混合竞争和条形码测序分析，这些变异会影响体内的生物适应性 （目标 2）最后，我们将使用这个序列-功能-适应度图来测试生物化学和生物化学领域的长期模型。 进化并揭示对工业和医学很重要的健康的生化决定因素（目标 3）。 从未实现过生化功能与健身的全面定量映射。