Role of HSC70 in protein synthesis

项目摘要/摘要 细胞内的细菌通常分泌蛋白质(效应器)，劫持并重新连接细胞路径，以建立其 可复制的利基市场。对这些细菌蛋白功能机制的研究经常导致 识别关键的细胞过程。鉴于蛋白质合成对维持细胞生命和 功能，几个细菌操纵宿主细胞蛋白质的合成。我们最近发现，细胞内的 嗜肺军团菌(Legionella Paopphila(L.P.)分泌真核丝氨酸苏氨酸蛋白激酶(ESTPK) 称为LegK4的效应器，它移位到宿主细胞胞浆中，并使伴侣HSC70磷酸化 T495(PHSC70)。有趣的是，这种单一的磷酸化会导致蛋白质合成的整体受阻。通常， 细菌蛋白质模仿宿主蛋白质的活动来篡夺它们的细胞功能。鉴于HSC70发挥了作用 在细胞中的不同作用，包括调节关键的细胞过程，如蛋白质动态平衡， 我们研究了宿主激酶是否能够磷酸化T495上的HSC70。事实上，我们发现 用甲烷磺酸甲酯处理细胞，这是一种会导致DNA损伤的烷化剂，结果是 PHSC70的积累。在DNA损伤反应(DDR)过程中，全局mRNA翻译被阻止，并且它 众所周知，应激源的类型决定了阻滞的机制。例如，电离辐射和 拓扑异构酶II的抑制导致mTOR信号的抑制，而紫外线暴露则导致 激酶GCN2对真核细胞起始因子2的磷酸化。然而，PHSC70在抑制中的作用 DDR过程中的蛋白质合成从未被研究过。在这里，我们建议研究PHSC70的作用 响应DNA损伤并阐明PHSC70导致蛋白质合成的机制 衰减。 在第一个目标中，我们将使用基于生化和荧光的分析来确定磷酸化是如何 HSC70影响其伴侣功能。在第二个目标中，我们将进行多聚体径流分析来确定 当HSC70被磷酸化时，蛋白质合成的哪一步被阻止。此外，我们建议确定 逃避蛋白质合成的蛋白质被PHSC70介导的蛋白质阻断。最后，在第三个目标中，我们将确定 DNA损伤反应中磷酸化HSC70的激酶及其时空调控 与胁迫相关的信号事件诱导对PHSC70的DNA损伤反应和蛋白质合成抑制。 总体而言，这项提议的目的将有助于阐明多功能伴侣蛋白在调节蛋白质中的作用。 DDR期间的合成抑制。本研究的成果不仅对研究人员具有一定的参考价值 HSC70，也包括那些研究DNA损伤反应和宿主-病原体相互作用的人。

Project Summary/Abstract Intracellular bacteria often secrete proteins (effectors) that hijack and rewire cellular pathways to establish their replicative niche. Studying the mechanisms by which these bacterial proteins function have frequently led to the identification of key cellular processes. Given that protein synthesis is essential to sustain cellular life and function, several bacteria manipulate host cell protein synthesis. We recently discovered that the intracellular bacterium, Legionella pneumophila (L.p.) secretes a eukaryotic-serine-threonine-protein-kinase (eSTPK) effector called LegK4, that translocates into the host cell cytosol and phosphorylates the chaperone HSC70 on T495 (pHSC70). Interestingly, this single phosphorylation causes a global block in protein synthesis. Often, bacterial proteins mimic the activities of host proteins to usurp their cellular function. Given that HSC70 plays diverse roles in the cell that includes the regulation of critical cellular processes such as protein homeostasis, we studied whether a host kinase was capable of phosphorylating HSC70 on T495. Indeed, we discovered that treatment of cells with methyl methanesulfonate, an alkylating agent that causes DNA damage, results in the accumulation of pHSC70. Global mRNA translation is blocked during the DNA damage response (DDR) and it is known that the type of stressor determines the mechanism of the block. For example, ionizing radiation and topoisomerase II inhibition leads to an inhibition of mTOR signaling, while UV exposure leads to the phosphorylation of eukaryotic initiation factor 2 by the kinase GCN2. However, the role of pHSC70 in inhibiting protein synthesis during the DDR has never been studied. Here, we propose to investigate the role of pHSC70 in response to DNA damage and elucidate the mechanisms by which pHSC70 leads to protein synthesis attenuation. In the first aim, we will use biochemical and fluorescence-based assays to determine how phosphorylation of HSC70 affects its chaperone function. In the second aim, we will perform polysome runoff assays to determine which step of protein synthesis is blocked when HSC70 is phosphorylated. In addition, we propose to identify the proteins that escape the protein synthesis block mediated by pHSC70. Finally, in the third aim, we will identify the kinases that phosphorylate HSC70 during the DNA damage response and the spatio-temporal regulation of signaling events that link the stress induced DNA damage response to pHSC70 and protein synthesis inhibition. Overall, the aims of this proposal will help elucidate the role of a multifunctional chaperone in mediating protein synthesis inhibition during DDR. The results of this study will not only be beneficial for the investigators studying HSC70, but also those that study the DNA damage response and host-pathogen interactions.