Regulation of eEF-2K an Energy and Nutrient Sensor

能量和营养传感器 eEF-2K 的调节

• 批准号: 10658322
• 负责人: Kevin N Dalby
• 金额: $ 42.75万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-15 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10658322
• 关键词: Affect; Alzheimer' s disease related dementia; Automobile Driving; Binding; Binding Sites; Biochemistry; Biological; Biology; Calmodulin; Cells; Chemicals; Chronic; Complex; Data; Disease; Disparate; Dissociation; Divalent Cations; Docking; Enzyme Kinetics; Funding; Goals; Guanosine Triphosphate Phosphohydrolases; In Vitro; Invaded; Kinetics; Learning; Length; Link; Malignant Neoplasms; Mammalian Cell; Mass Spectrum Analysis; Measurement; Measures; Mediating; Memory; Messenger RNA; Modeling; Molecular Conformation; Neoplasm Metastasis; Nerve Degeneration; Nervous System Trauma; Neurodegenerative Disorders; Neurologic; Nucleotides; Nutrient; Nutrient availability; Output; Parkinson Disease; Pathology; Pathway interactions; Phosphorylation; Phosphotransferases; Post-Translational Protein Processing; Process; Property; Protein Biosynthesis; Protein Kinase; Proteins; Publishing; Regulation; Research; Resolution; Ribosomes; Role; Signal Transduction; Site; Stimulus; Stress; Structure; Synapses; Techniques; Testing; Therapeutic; Translations; Work; X-Ray Crystallography; cell growth regulation; cellular targeting; human disease; immunoregulation; inhibitor; innovation; insight; novel therapeutic intervention; sensor; small molecule; spatiotemporal; tumorigenesis

There is a compelling need to decipher the role of the dysregulation of translation elongation in various chronic neurological conditions and many cancers. The long-term goal driving the proposed research is to help develop therapeutic strategies targeting eEF2K for treating progressive neurodegenerative diseases and malignancies. The overall objectives of this application are to (i) characterize the kinetic mechanism of eEF2K activation and regulation and (ii) elucidate the structural basis for the regulation of eEF2K by divalent cations, pH, ADP, and specific post-translational modifications. The central hypothesis is that calmodulin (CaM) binding activates eEF2K by profoundly altering its conformational dynamics, leading to a state capable of efficient phosphoryl transfer. Multiple regulatory inputs control the attainment of this state. The rationale is that understanding the mechanism of eEF2K regulation is necessary to provide a robust scientific framework for developing novel therapeutic approaches targeting neurodegenerative diseases and cancer. The central hypothesis will be tested through two specific aims: (1) to define the kinetic mechanism of activation of eEF2K by calmodulin and the kinetic basis for the modulation of its activity, (2) to determine the structural and dynamic basis of how specific inputs and post-translational modifications regulate the stability of active states of eEF2K. In the first aim, presteady state kinetics will define the precise allosteric mechanism of eEF2K activation and modulation of its activity. The second aim will determine the modulatory effects of specific post-translational modifications and other regulatory inputs in affecting the CaM sensitivity of the active state using a variety of structural approaches. The findings from both aims will be validated by characterizing eEF2K activation and activity in mammalian cells. In our opinion, the research proposed in this application is innovative because it focuses on understanding the relationships between the structural dynamics and the temporal control of eEF2K in mammalian cells using a unique combination of techniques integrated over multiple length scales from the atomic to the cellular. The proposed research is significant because it is expected to provide new insight into the cellular regulation of protein translation by eEF2K and provide critical advancement in understanding various eEF2K-driven pathologies.

迫切需要破译翻译延长的失调在各种慢性神经疾病和许多癌症中的作用。推动这项拟议研究的长期目标是帮助开发针对eEF2K的治疗策略，用于治疗进行性神经退行性疾病和恶性肿瘤。本申请的总体目标是(I)表征eEF2K的激活和调节的动力学机制，以及(Ii)阐明eEF2K受二价阳离子、pH、ADP和 具体的翻译后修改。核心假设是钙调素(CaM)结合通过深刻改变eEF2K的构象动力学来激活eEF2K，导致一种能够有效地进行磷酸化转移的状态。多个监管投入控制着这种状态的实现。其基本原理是，了解eEF2K的调节机制对于开发针对神经退行性疾病和癌症的新治疗方法提供一个强有力的科学框架是必要的。中心假说将通过两个具体目标进行检验：(1)确定钙调蛋白激活eEF2K的动力学机制和 (2)确定特定的输入和翻译后修饰如何调节eEF2K活性状态的稳定性的结构和动力学基础。在第一个目标中，稳态前动力学将定义eEF2K激活及其活性调节的精确变构机制。第二个目标将使用各种结构方法来确定特定的翻译后修饰和其他调节输入在影响激活状态的CaM敏感性方面的调节作用。这两个AIMS的发现将通过表征哺乳动物细胞中eEF2K的激活和活性来验证。 在我们看来，本申请中提出的研究是创新的，因为它专注于使用从原子到细胞的多个长度尺度集成的独特技术组合来理解哺乳动物细胞中eEF2K的结构动力学和时间控制之间的关系。这项拟议的研究具有重要意义，因为它有望为eEF2K对蛋白质翻译的细胞调控提供新的见解，并在理解eEF2K驱动的各种病理机制方面取得关键进展。

项目成果

ADP enhances the allosteric activation of eukaryotic elongation factor 2 kinase by calmodulin.

• DOI: 10.1073/pnas.2300902120
• 发表时间: 2023-04-25
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Piserchio, Andrea;Long, Kimberly J.;Browning, Luke S.;Bohanon, Amanda L.;Isiorho, Eta A.;Dalby, Kevin N.;Ghose, Ranajeet
• 通讯作者: Ghose, Ranajeet

Structure of the complex between calmodulin and a functional construct of eukaryotic elongation factor 2 kinase bound to an ATP-competitive inhibitor.

• DOI: 10.1016/j.jbc.2023.104813
• 发表时间: 2023-06
• 期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
• 影响因子: 4.8
• 作者: Piserchio, Andrea;Isiorho, Eta A;Dalby, Kevin N;Ghose, Ranajeet
• 通讯作者: Ghose, Ranajeet

Quantification of ERK Kinase Activity in Biological Samples Using Differential Sensing.

使用差分传感定量生物样品中的 ERK 激酶活性。

• DOI: 10.1021/acschembio.9b00580
• 发表时间: 2020
• 期刊: ACS chemical biology
• 影响因子: 4
• 作者: Zamora-Olivares,Diana;Kaoud,TamerS;Zeng,Lingyu;Pridgen,JaceyR;Zhuang,DeborahL;Ekpo,YakndaraE;Nye,JessicaR;Telles,Mitchell;Anslyn,EricV;Dalby,KevinN
• 通讯作者: Dalby,KevinN

Raf protomers: Drug binding preferences in living cells.

Raf 原体：活细胞中的药物结合偏好。

• DOI: 10.1016/j.chembiol.2023.10.002
• 发表时间: 2023
• 期刊: Cell chemical biology
• 影响因子: 8.6
• 作者: Dalby,KevinN

Steady State and Time-Dependent Fluorescent Peptide Assays for Protein Kinases.

蛋白激酶的稳态和时间依赖性荧光肽测定。

• DOI: 10.1002/cpz1.998
• 发表时间: 2024
• 期刊: Current protocols
• 作者: Sammons,RaeM;Devkota,AshwiniK;Kaoud,TamerS;Warthaka,Mangalika;Cho,EunJeong;Dalby,KevinN
• 通讯作者: Dalby,KevinN