Molecular Mechanisms of Translation Regulation by the eEF2K Pathway

项目概要： 本研究项目旨在提供一个一般和位置的机械和结构模型， 依赖性真核细胞延伸因子2激酶（eEF 2K）调控及其对核糖体的下游作用。 eEF 2K位于多个上游通路的汇合处，其信号被其整合。eEF 2K是唯一已知的 靶是真核延伸因子2（eEF 2），其在单个位点上磷酸化。此eEF 2K/eEF 2- 轴是翻译延伸的主要调节器，并且在细胞内稳态中具有一般作用。也是 在特定细胞类型的局部区域中蛋白质合成的重要线索依赖性调节剂，例如， 在神经元的突触中暴露神经递质之后。eEF 2促进特定mRNA的翻译 同时通常抑制翻译。这种矛盾现象的机制是完全未知的。我们 最近表明，活性eEF 2K强加了一个普遍的翻译关闭，其中磷酸化的eEF 2和 相分离蛋白SERBP 1稳定地与核糖体结合并使它们闲置。本次大会建议 eEF 2磷酸化与核糖体稳定性、mRNA降解和核糖体 共同解释了eEF 2磷酸化如何可能导致某些蛋白的优先翻译。 mRNA。eEF 2K与许多人类疾病相关，包括神经功能障碍、感染性疾病、神经系统疾病 疾病、癌症和自身免疫性疾病。靶向eEF 2K的治疗药物正在开发中， 目前缺乏足够的具体性。因此，对eEF 2K调节及其下游的机制的理解， 需要效果。 在这个奖项下，我们将追求两个关键方向：1）确定eEF 2K如何在结构上集成 信号从上游途径，并影响其下游调控eEF 2，和2）确定如何 磷酸化的eEF 2和空闲的核糖体整体和局部地调节mRNA翻译。在这里，我们将测试 eEF 2磷酸化调节核糖体稳定性、mRNA稳定性和核糖体稳定性的新假说 在一些实施例中，mRNA的翻译是一种定位，其共同赋予特定mRNA子集的优先翻译。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， 生物物理学、生物化学和细胞生物学方法来解决这些一般和特定位置的作用， 真核细胞延伸因子2激酶（eEF 2K）途径。我们提出的研究计划将为 有前途的治疗方法，为一长串的eEF 2K相关的人类疾病，更广泛地说，扩大 我们对翻译延伸的理解。

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.