Uncovering the Role of UFM1 in the Release of Arrested Peptides from Stalled Ribosomes at the Endoplasmic Reticulum (ER) Membrane

揭示 UFM1 在内质网 (ER) 膜上停滞核糖体释放停滞肽中的作用

• 批准号: 10462233
• 负责人: Justin Tyler Marinko
• 金额: --
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2022-05-02
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10462233
• 关键词: Affect; Affinity; Affinity Chromatography; Animal Model; Binding Proteins; Biological; Biological Process; Biotin; Cardiomyopathies; Cell Fractionation; Cell Line; Cells; Chemicals; Complex; Coupled; Cytosol; DNA Sequence Alteration; Data; Defect; Development; Disease; Emetine; Encapsulated; Endoplasmic Reticulum; Genetic; Goals; Health; Hematopoietic; Human; Inflammatory Bowel Diseases; Knock-out; Label; Lead; Ligase; Link; Maintenance; Malignant Neoplasms; Mass Spectrum Analysis; Membrane; Messenger RNA; Metabolic; Modeling; Modification; Mutation; Neurologic; Pathway interactions; Peptides; Pharmacology; Phenocopy; Phenotype; Physiologic pulse; Play; Process; Proteins; Proteomics; Quality Control; Radial; Reader; Reporter; Research; Ribosomal Proteins; Ribosomes; Role; Route; Site; Streptavidin; System; Testing; Transfer RNA Aminoacylation; Translations; Ubiquitin Like Proteins; Work; adduct; base; cellular targeting; crosslink; cytotoxic; design; developmental disease; endoplasmic reticulum stress; experimental study; glycosylation; human disease; in vivo; multicatalytic endopeptidase complex; novel; peptidyl-tRNA; polypeptide; prevent; proteostasis; recruit; response; restraint; signal sequence receptor

Abstract Ribosome-associated quality control (RQC) is the process by which cells deal with prolonged translational stalls caused by genetic errors, insufficient charged tRNA, or faulty mRNA which results in ribosome collisions and potentially cytotoxic, incomplete, translation products. Ribosomes are split into a free 40S and 60S subunit with an incomplete nascent chain (arrest product or AP) as a peptidyl tRNA adduct which obstructs the exit tunnel and P-site of the 60S. In the cytosol, an intricate process has been defined for how cells remove the AP, recycle the 60S subunit, and degrade the AP. However, corresponding research into how this process is managed for proteins produced at the endoplasmic reticulum (ER) membrane is lacking. The key difference between cytosolic and ER-RQC is that at the ER membrane the AP is integrated into the SEC61 translocon and thus separated from the cytosolic RQC and degradative machinery. How cells manage this topological restraint is the focus of this proposal. The Kopito lab and others have shown that ER localized ribosome collisions result in the conjugation of the small ubiquitin-like protein UFM1 to ribosomal protein L26 (RPL26) on the 60S subunit (UFMylation). UFM1 knockout cells (UFM1KO) prevent proteasomal degradation of ER localized, but not cytosolic, APs suggesting a link between UFMylation and AP degradation in ER-RQC. I hypothesize that UFMylation of RPL26 severs as mark to recruit proteins to stalled ER ribosomes necessary for resolving the 60S-AP- translcoon complex so that the AP may be degraded. In Aim 1 and Aim 2 of this proposal I will identify the readers of UFMylation using two complimentary approaches: proximity labeling via fusion of a biotin ligase to UFM1 and affinity purification of UFM1 binding proteins. These experiments are designed such that I will identify proteins that interact with conjugated UFM1 and not free UFM1. Preliminary results show that ER localized, translocon associated proteins, are enriched in proximity labeling experiments which supports the model that UFMylation occurs at the ER membrane near the translocon. Preliminary affinity purification experiments reveal that known cytosolic RQC proteins are enriched on UFMylated ribosomes supporting the role of UFMylation in ER-RQC. In Aim 3 of this proposal, I will define the mechanism by which UFMylation leads to AP degradation. I will first evaluate the role of UFMylation readers identified in Aims 1 and 2 in AP stability via emetine chase and metabolic pulse/chase experiments. I will also test if UFMylation facilitates release of the AP from the ribosome or translocon. The UFMylation pathway is conserved amongst most eukaryotic species and is essential for hematopoietic and neurological development in metazoan cells. Mutations in this pathway have been linked to a wide variety of human diseases. Despite its importance, we do not fully understand what this pathway is doing. In this proposal, I will uncover the readers of UFMylation and better define its role in ER-RQC. A better understanding is necessary to understand how defects in UFMylation lead to human disease.

摘要 核糖体相关质量控制（RQC）是细胞处理由细胞内的核糖体引起的长时间翻译停滞的过程。 遗传错误，不充足的电荷tRNA，或错误的mRNA，导致核糖体碰撞和潜在的 细胞毒性、不完全、翻译产物。核糖体分裂成游离的40 S和60 S亚基， 新生链（阻滞产物或AP）作为肽基tRNA加合物，其阻碍60 S的出口通道和P位点。在 在胞质溶胶中，已经定义了细胞如何去除AP，回收60 S亚基和降解60 S亚基的复杂过程。 AP.然而，对内质网产生的蛋白质如何控制这一过程的相应研究， 内质网（ER）膜缺乏。胞质和ER-RQC之间的关键区别在于，在ER膜上， AP被整合到SEC 61易位子中，从而与胞质RQC和降解机制分离。如何 单元管理这种拓扑约束是本提议的焦点。Kopito实验室和其他人已经证明， 定位的核糖体碰撞导致小泛素样蛋白UFM 1与核糖体蛋白L26的结合 （RPL 26）在60 S亚基上（UFM化）。UFM 1敲除细胞（UFM 1 KO）阻止ER的蛋白酶体降解 局部的，但不是胞质，AP，表明在ER-RQC中UFM化和AP降解之间的联系。我假设 RPL 26的UFM化作为将蛋白质募集到停滞的ER核糖体的标记，所述停滞的ER核糖体是解析60 S-AP-1所必需的。 使AP可以被降解。在本建议的目标1和目标2中，我将指出 使用两种互补方法的UFM化：通过生物素连接酶与UFM 1融合的邻近标记和亲和标记。 UFM 1结合蛋白的纯化。这些实验的设计是为了让我能识别出与 共轭UFM 1而非游离UFM 1。初步结果表明，ER定位，易位相关蛋白， 富集在邻近标记实验中，其支持UFM化发生在ER膜附近的模型。 易位子初步的亲和纯化实验表明，已知的胞质RQC蛋白富集在 UFM化核糖体支持UFM化在ER-RQC中的作用。在本建议的目标3中，我将界定 UFM化导致AP降解的机制。我将首先评估UFMylation读者的作用， 在目的1和2中，通过代谢追踪和代谢脉冲/追踪实验研究AP稳定性。我还将测试UFM化 促进AP从核糖体或易位子的释放。UFM化途径是保守的， 在真核生物中，它是后生动物细胞造血和神经发育所必需的。在这方面的突变 这一途径与多种人类疾病有关。尽管它很重要，但我们并不完全理解它的意义。 这条路在做什么。在这个建议中，我将揭示UFM化的读者，并更好地定义它在ER-RQC中的作用。一 更好的理解是理解UFM化缺陷如何导致人类疾病所必需的。