Connecting in vitro glutamine synthetase biophysics with the cellular environment

将体外谷氨酰胺合成酶生物物理学与细胞环境联系起来

• 批准号: 10570167
• 负责人: Eric Raymond Greene
• 金额: $ 6.95万
• 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: 2022-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10570167
• 关键词: Active Sites; Address; Architecture; Binding; Biochemical; Biochemistry; Biological Assay; Biophysics; Catalysis; Cell Proliferation; Cells; Cellular biology; Chimeric Proteins; Cryoelectron Microscopy; Disease; Environment; Enzyme Kinetics; Enzymes; Equilibrium; Fellowship; Fluorescence; Genomics; Glutamate-Ammonia Ligase; Goals; Growth; Hand; Hot Spot; In Vitro; Inherited; Kinetics; Learning; Libraries; Life; Link; Malignant Neoplasms; Massive Parallel Sequencing; Measurement; Measures; Mediating; Metabolic; Metabolic Diseases; Metabolic Pathway; Modeling; Molecular Conformation; Multienzyme Complexes; Mutagenesis; Mutation; Nature; Nutritional; Outcome; Photometry; Play; Procedures; Productivity; Proliferating; Reaction; Regulation; Reporting; Research; Research Personnel; Resolution; Role; Somatic Mutation; Structure; Techniques; Technology; Therapeutic; Thermodynamics; Tissues; Training; Universities; Variant; cancer therapy; career; chemical reaction; data integration; experimental study; fitness; in vitro Assay; in vitro activity; in vivo; inhibitor; innovation; insight; monomer; mutant; mutation screening; next generation; novel; single molecule; three dimensional structure; tumor; variant of interest

Project Summary/Abstract – Connecting in vitro glutamine synthetase biophysics with the cellular environment Enzyme catalysis of vital chemical reactions sustains life. Dysregulation of these essential metabolic reactions contributes to rapid proliferation in the cancer state, devastating inherited metabolic disorders, and is involved in numerous other diseases. Contradictions between in vitro and in vivo measurements of enzyme catalysis hamper our understanding of Nature’s rules governing enzyme regulation. I propose to address these contradictions with an integrated approach using glutamine synthetase (GS), an essential metabolic enzyme, as a model. I hypothesize that integrating multiple metrics of GS function in vivo will yield more accurate functional models of GS and that modes of GS regulation will be uncovered through reconciling any differences made between orthogonal in vivo and in vitro measures of activity and composition. In my first aim, I will use cellular readouts of GS fitness and abundance multiplexed with deep mutational scanning (DMS) to elucidate the sequence determinants of GS specific activity in vivo. Next, in aim 2, I will define the thermodynamic landscape underlying GS activity as a function of oligomeric state using in vitro assays that can be compared to in vivo measurements in aim 1. Finally, I will reveal the fine conformational details that trigger GS mediated catalysis and allosteric control elicited by different oligomeric states and effectors using cryo-EM and novel kinetic assays in my third aim. Furthermore, with cryo-EM, enzyme kinetics, and oligomeric state analysis procedures in hand, those variants of interest identified from aim 1 will be fully characterized to uncover mechanisms of regulation and generate a holistic model of GS function. My in vivo specific activity metric is predicted to yield more precise information on the effect of GS variants allow for inference of allosteric networks underlying GS activity. This in vivo specific activity metric will be supported and validated by in vitro measurements. Any differences between in vitro and in vivo measurements provide the opportunity to be reconciled through additional experimentation, such as expanding in vivo assays to include unique cellular conditions. Establishing GS in vivo and in vitro connections through this approach will allow prediction of cancer somatic mutation effect on GS function. As GS occupies key nodes in vital metabolic pathways required for cell proliferation, specific inhibitors would support existing cancer therapies in GS addicted/associated cancers. Given that a tumor metabolic state is less heterogeneous than its genomic landscape, precise targeting of rouge metabolic enzyme states remains an attractive therapeutic option. Moreover, GS is a representative multimeric metabolic enzyme whose biophysical principles governing function and regulation can be compared and extended to other critical metabolic enzymes. The training I’ll receive to establish the experimental pipeline from the proposed research herein will serve me well to further expand on these regulatory principles in my career as an independent researcher at a research-intensive university.

项目摘要/摘要-将体外谷氨酰胺合成酶生物物理学与细胞 环境 重要化学反应的酶催化作用维持生命。这些基本代谢反应的失调 有助于癌症状态下的快速增殖，毁灭性的遗传性代谢紊乱，并参与 在许多其他疾病中。酶催化体外测定与体内测定的矛盾 阻碍了我们对自然界酶调节规则的理解。我建议解决这些问题 与使用谷氨酰胺合成酶(GS)的综合方法相矛盾，GS是一种重要的代谢酶， 作为一名模特。我假设在活体内整合GS功能的多个度量将产生更准确的结果 通过协调任何差异，将揭示GS的功能模型和GS的监管模式 在体内和体外进行活性和成分的正交化测量。 在我的第一个目标中，我将使用GS适合度和丰度的细胞读数与深度突变相结合 扫描(DMS)以阐明体内GS比活性的序列决定因素。接下来，在《目标2》中，我将 在体外将GS活性下的热力学图景定义为寡聚体状态的函数 可以与目标1中的体内测量进行比较的分析。最后，我将揭示精细构象 不同寡聚态和不同亚基引发的GS介导的催化和变构调控的细节 在我的第三个目标中使用冷冻-EM和新的动力学分析的效应器。此外，使用冷冻-EM，酶动力学， 和寡聚状态分析程序在手，从目标1中确定的那些感兴趣的变体将完全 其特点是揭示调节机制并生成GS功能的整体模型。 我的体内比活度指标预计将提供关于GS变体影响的更准确信息 允许推断GS活性背后的变构网络。这一体内特定活性指标将是 体外测量支持并验证了这一点。体外和体内测量之间的任何差异 提供通过其他实验进行协调的机会，例如扩展体内测试 包括独特的细胞条件。通过这种方法在体内和体外建立GS连接将 允许预测癌症体细胞突变对GS功能的影响。由于GS在至关重要的代谢中占据关键节点 细胞增殖所需的途径，特定的抑制剂将支持GS现有的癌症治疗 成瘾/相关癌症。假设肿瘤的代谢状态比其基因组的异质性要小 此外，精确定位胭脂代谢酶状态仍然是一种有吸引力的治疗选择。 此外，GS是一种具有代表性的多聚体代谢酶，其生物物理原理决定了 功能和调节可以比较和扩展到其他关键的代谢酶。我将接受的训练 从这里建议的研究中收到建立实验管道的建议将很好地帮助我进一步 在我作为一家研究密集型公司的独立研究员的职业生涯中，我对这些监管原则进行了扩展 上大学。