Connecting in vitro glutamine synthetase biophysics with the cellular environment

项目概要/摘要-将体外谷氨酰胺合成酶生物物理学与细胞生物物理学相结合 环境 酶催化重要的化学反应维持生命。这些基本代谢反应的失调 有助于癌症状态的快速扩散，破坏遗传性代谢紊乱，并参与 在许多其他疾病。酶催化作用的体外和体内测量之间的矛盾 阻碍了我们对自然界酶调节规律的理解。我建议解决这些问题， 与使用谷氨酰胺合成酶（GS），一种必需的代谢酶， 做模特我假设，整合体内GS功能的多个指标将产生更准确的结果。 全球服务的功能模式，以及全球服务监管模式将通过协调任何差异来揭示， 在活性和组成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多重化， 扫描（DMS）以阐明GS体内比活性的序列决定因素。接下来，在目标2中，我将 使用体外实验将GS活性的热力学背景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， 在我的第三个目标中，使用cryo-EM和新的动力学测定的效应器。此外，利用冷冻电镜，酶动力学， 和寡聚状态分析程序在手，从目标1中鉴定的那些感兴趣的变体将被完全 其特征在于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更精确的信息GS变体的影响 允许推断GS活性背后的变构网络。该体内比活性度量将是 通过体外测量得到支持和验证。体外和体内测量之间的任何差异 提供了通过额外实验（如扩大体内试验）进行协调的机会 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， 允许预测癌症体细胞突变对GS功能的影响。由于GS占据了重要代谢的关键节点， 细胞增殖所需的通路，特异性抑制剂将支持GS中现有的癌症疗法。 成瘾/相关癌症。鉴于肿瘤代谢状态的异质性低于其基因组 因此，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 功能和调节可以进行比较，并扩展到其他关键的代谢酶。训练我会 从这里提出的研究中获得建立实验管道将有助于我进一步 在我的职业生涯中，我作为一名独立研究员，在一个研究密集型的 大学

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.