Regulation of eEF-2K an Energy and Nutrient Sensor

有一个迫切需要破译翻译伸长失调的作用在各种慢性神经系统疾病和许多癌症。推动拟议研究的长期目标是帮助制定针对eEF2K的治疗策略，以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活化和调控的动力学机制；（ii）阐明二价阳离子、pH、ADP和eEF2K调控的结构基础

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.