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

基本信息

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
项目类别：
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

项目摘要

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连接起来环境重要化学反应的酶催化维持生命。有助于癌症状态的快速增殖，破坏遗传性代谢紊乱，并参与在许多其他疾病中，酶催化的体外和体内测量之间存在矛盾。阻碍了我们对酶调节的自然规则的理解，我建议解决这些问题。与使用谷氨酰胺合成酶（GS）（一种重要的代谢酶）的综合方法相矛盾，作为一个模型，整合体内 GS 功能的多个指标将产生更准确的结果。 GS 的功能模型以及 GS 监管模式将通过协调任何差异来揭示在活性和成分的体内和体外正交测量之间进行。在我的第一个目标中，我将使用 GS 适应性和丰度的细胞读数与深度突变复用接下来，在目标 2 中，我将通过扫描 (DMS) 阐明体内 GS 比活性的序列决定因素。使用体外将 GS 活性的热力学景观定义为寡聚状态的函数可以与目标 1 中的体内测量进行比较的测定。最后，我将揭示精细构象触发 GS 介导的催化和由不同寡聚状态引起的变构控制的细节和在我的第三个目标中，使用冷冻电镜和新颖的动力学分析来实现效应器此外，通过冷冻电镜、酶动力学，和现有的寡聚状态分析程序，从目标 1 中确定的那些感兴趣的变体将完全被旨在揭示调节机制并生成 GS 功能的整体模型。我的体内比活性指标预计将产生有关 GS 变体影响的更精确信息允许推断 GS 活性的变构网络，该体内特定活性度量将是体外测量和体内测量之间的任何差异均得到支持和验证。提供通过额外实验进行协调的机会，例如扩大体内测定通过这种方法建立 GS 体内和体外连接将包括独特的细胞条件。由于 GS 占据重要代谢的关键节点，因此可以预测癌症体细胞突变对 GS 功能的影响。细胞增殖所需的途径，特定抑制剂将支持 GS 中现有的癌症疗法鉴于肿瘤代谢状态的异质性低于其基因组。总体而言，精确靶向胭脂代谢酶状态仍然是一种有吸引力的治疗选择。此外，GS是一种代表性的多聚体代谢酶，其生物物理原理支配着功能和调节可以与其他关键代谢酶进行比较和扩展。从本文提出的研究中建立实验管道将有助于我进一步作为研究密集型机构的独立研究员，我在职业生涯中扩展了这些监管原则大学。