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.

抽象的 核糖体相关质量控制（RQC）是细胞处理长期翻译失速的过程 通过遗传误差，带电的tRNA不足或mRNA故障，这会导致核糖体碰撞和可能 细胞毒性，不完整的翻译产品。核糖体被分成一个免费的40和60s亚基，不完整 新生链（逮捕产物或AP）作为肽基TRNA加合物，阻塞了60年代的出口隧道和p位。在 胞质溶胶已定义了一个复杂的过程，用于细胞如何去除AP，回收60S亚基并降解该细胞。 AP。但是，相应的研究如何管理该过程的内质产生的蛋白质 缺乏网状（ER）膜。胞质和ER-RQC之间的关键区别在于，在ER膜上 AP集成到SEC61转运中，因此与胞质RQC和降解机械分开。如何 细胞管理这种拓扑限制是该提案的重点。 Kopito实验室和其他实验室表明 局部核糖体碰撞导致小型泛素样蛋白UFM1与核糖体蛋白L26结合 （RPL26）在60S亚基（Ufmylation）上。 UFM1基因敲除细胞（UFM1KO）防止ER的蛋白酶体降解 局部但不是胞质的AP，建议在ER-RQC中ufmylation和AP降解之间存在联系。我假设 RPL26的ufmylation ufmylation severs作为标记，以募集蛋白质以解决60s-ap-- Translcoon复合体可以降解AP。在AIM 1和AIM 2中，我将确定 使用两种免费方法的ufmylation：通过融合生物素连接酶与UFM1和亲和力的接近标记 UFM1结合蛋白的纯化。这些实验的设计使我将确定与之相互作用的蛋白质 共轭UFM1，而不是免费的UFM1。初步结果表明，ER局部易位相关蛋白是 富含接近性标记实验，该实验支持ufmylation在ER膜上发生的模型 转运。初步亲和力纯化实验表明，已知的胞质RQC蛋白富含在 紫外线核糖体支持ufmylation在ER-RQC中的作用。在本提案的AIM 3中，我将定义 ufmylation导致AP降解的机制。我将首先评估所确定的ufmylation读取器的作用 在AIM 1和2中，AP稳定性通过Emetine Chase和代谢脉冲/追逐实验。我还将测试ufmylation是否 促进从核糖体或易位释放AP的释放。 ufmylation途径在大多数 真核生物物种，对于后生动物细胞中的造血和神经系统发育至关重要。其中突变 途径与各种各样的人类疾病有关。尽管它很重要，但我们并不完全了解 这条路正在做。在此提案中，我将发现读者的ufmylation，并更好地定义其在ER-RQC中的作用。一个 有必要更好地理解ufmylation的缺陷如何导致人类疾病。