Molecular Mechanisms of Translation Regulation by the eEF2K Pathway

eEF2K 通路翻译调控的分子机制

• 批准号: 10705222
• 负责人: Sarah Loerch
• 金额: $ 37.68万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-15 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10705222
• 关键词: Address; Affect; Autoimmune Diseases; Award; Binding; Biochemical; Biophysics; Cardiovascular Diseases; Cells; Cellular biology; Communicable Diseases; Cryoelectron Microscopy; Cues; Development; Disease; Homeostasis; In Situ; Location; Malignant Neoplasms; Messenger RNA; Molecular; Neurologic Dysfunctions; Neurons; Neurotransmitters; Pathway interactions; Peptide Elongation Factor 2; Phase; Phosphorylation; Process; Protein Biosynthesis; Proteins; Regulation; Research; Research Project Grants; Ribosomes; Role; Signal Transduction; Site; Specificity; Structural Models; Synapses; Testing; Therapeutic; Time; Translational Regulation; Translational Repression; Translations; Work; calmodulin-dependent protein kinase III; cell type; human disease; mRNA Decay; mRNA Stability; mRNA Translation; nervous system disorder; novel; particle; programs; structural biology; therapeutic target

Project summary: This research project aims to provide a mechanistic and structural model of general and location- dependent eukaryotic elongation factor 2 kinase (eEF2K) regulation and its downstream effects on the ribosome. eEF2K is at the confluence of multiple upstream pathways whose signals it integrates. eEF2K’s only known target is the eukaryotic elongation factor 2 (eEF2), which it phosphorylates on a single site. This eEF2K/eEF2- axis is the predominant regulator of translation elongation and has a general role in cell homeostasis. It is also an essential cue-dependent regulator of protein synthesis in localized regions of specific cell types, for example, after neurotransmitter exposure in the synapses of neurons. eEF2 promotes the translation of specific mRNAs while generally inhibiting translation. The mechanism of this paradoxical phenomenon is entirely unknown. We recently showed that active eEF2K imposes a general translation shutdown in which phosphorylated eEF2 and the phase-separating protein SERBP1 stably bind to ribosomes and renders them idle. This assembly suggests possible mechanisms of eEF2-phosphorylation with respect to ribosome stability, mRNA decay, and ribosome localization that collectively explain how eEF2-phosphorylation might lead to preferential translation of certain mRNAs. eEF2K is associated with numerous human diseases, including neurological dysfunctions, infectious diseases, cancers, and autoimmune disorders. Therapeutics targeting eEF2K are under development but currently lack insufficient specificity. Thus, a mechanistic understanding of eEF2K-regulation and its downstream effects are needed. Under this award, we will pursue two key directions: 1) determine how eEF2K structurally integrates signals from upstream pathways and affects its downstream regulation of eEF2, and 2) determine how phosphorylated eEF2 and idle ribosomes regulate mRNA translation globally and locally. Here, we will test the novel hypotheses that eEF2-phosphorylation regulates ribosome stability, mRNA stability, and ribosome localization, which collectively confers the preferential translation of a specific mRNA subset. We will use an integrated structural biology approach using single-particle and in situ cryogenic electron microscopy, paired with biophysical, biochemical, and cell biology approaches to address these general and location-specific roles of the eukaryotic elongation factor 2 kinase (eEF2K) pathway. Our proposed research program will open the door to promising therapeutic approaches for the long list of eEF2K-related human diseases and, more broadly, expand our understanding of translation elongation.

项目总结： 本研究项目旨在提供一个通用和定位的机械和结构模型。 依赖真核细胞延伸因子2激酶(EEF2K)的调控及其对核糖体的下游影响。 EEF2K处于多条上游通路的汇合点，它整合了这些信号。EEF2K是唯一已知的 靶点是真核细胞延伸因子2(EEF2)，它在单个位点上磷酸化。这个eEF2K/eEF2- 轴是翻译伸长的主要调节因子，在细胞内稳态中起着普遍的作用。它也是 在特定细胞类型的局部区域中，蛋白质合成的基本信号依赖调节因子，例如， 在神经元突触中暴露神经递质后。EEF2促进特定mRNAs的翻译 同时普遍抑制翻译。这一矛盾现象的机制是完全未知的。我们 最近的研究表明，活性的eEF2K造成了一种普遍的翻译关闭，其中磷酸化的eEF2和 相分离蛋白SERBP1稳定地与核糖体结合，使核糖体闲置。这次集会表明 EEF2磷酸化与核糖体稳定性、mRNA衰变和核糖体相关的可能机制 共同解释eEF2-磷酸化如何导致某些特定的优先翻译 MRNAs。EEF2K与许多人类疾病有关，包括神经功能障碍、传染性 疾病、癌症和自身免疫性疾病。针对eEF2K的治疗药物正在开发中，但 目前缺乏足够的特异性。因此，对eEF2K-调节及其下游的机制有了一个理解 效果是需要的。 在这个奖项下，我们将追求两个主要方向：1)确定eEF2K如何在结构上整合 来自上游通路的信号并影响其对eEF2的下游调节，以及2)决定如何 磷酸化的eEF2和闲置的核糖体调节全球和局部的mRNA翻译。在这里，我们将测试 EEF2磷酸化调控核糖体稳定性、mRNA稳定性和核糖体的新假说 本地化，共同赋予特定的mRNA亚集的优先翻译。我们将使用 使用单粒子和原位低温电子显微镜的综合结构生物学方法，与 生物物理、生化和细胞生物学方法，以解决这些一般的和特定于位置的角色 真核细胞延伸因子2激酶(EEF2K)途径。我们提议的研究计划将为 EEF2K相关人类疾病的一长串有希望的治疗方法，更广泛地说，扩大 我们对翻译延伸性的理解。