Recognition of Orphan Ribosomal Subunit Proteins by the Ubiquitin-Proteasome System

泛素-蛋白酶体系统对孤儿核糖体亚基蛋白的识别

• 批准号: 10221916
• 负责人: Samantha Sedor
• 金额: $ 3.96万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10221916
• 关键词: Anemia; Biochemistry; Biological Assay; Blood; Cell Culture System; Cells; Client; Defect; Development; Enzymes; Erythroblasts; Erythrocytes; Erythroid; Erythroid Cells; Erythropoiesis; Etiology; Event; Exhibits; Face; Goals; Health; Hematological Disease; Hematopoietic Neoplasms; Hematopoietic stem cells; Hemoglobin; Homeostasis; Homologous Gene; Human; Hybrids; In Vitro; Individual; Knock-out; Knowledge; Ligase; Link; Malignant Neoplasms; Mammalian Cell; Mediating; Molecular; Molecular Chaperones; Multiprotein Complexes; Orphan; Pathway interactions; Pattern; Physiological; Predisposition; Process; Production; Protein Biosynthesis; Protein Subunits; Proteins; Proteome; Regulation; Research Personnel; Reticulocytes; Ribosomal RNA; Ribosomes; Risk; Role; Specificity; System; Testing; Ubiquitin; Work; Yeasts; base; erythroid differentiation; experimental study; in vivo; insight; multicatalytic endopeptidase complex; novel; prevent; protein degradation; reconstitution; therapeutic target; ubiquitin-protein ligase

Abstract Immature erythroblasts undergo significant proteome remodeling events prior to their development into functional red blood cells. In the final steps of terminal erythropoiesis, ribosomes are cleared from reticulocytes by the ubiquitin-proteasome system (UPS). During this process, individual unassembled, or orphan, ribosomal subunit proteins (RPs) appear to be recognized by E3 ligases, enzymes capable of ubiquitylating proteins and targeting them for proteasomal degradation. Defects in ribosome homeostasis, including in the clearance process, can cause forms of anemia and are linked to blood cancers. Despite the need for ribosome regulation during development, little is known about the mechanisms underlying ribosome homeostasis in erythroid cells. Recently, researchers identified UBE2O as a hybrid E2/E3 enzyme that ubiquitylates orphan RPs during erythroid ribosome clearance. UBE2O recognizes some specific RPs, but it is not known which of all 80 RPs are UBE2O clients or how they are selected. The ability of orphan RP interactors, such as chaperones, to participate in the ubiquitylation process has also not been explored. Although UBE2O is the only protein known to modify RPs during ribosome clearance, other E3 ligases may be involved, as around 60% of RPs can be degraded effectively in the absence of UBE2O. However, additional ligases have not been identified. A candidate is HUWE1, the mammalian homolog of TOM1, an E3 ligase required for ubiquitylation and degradation of many orphan RPs in yeast. The ability of HUWE1 to directly modify and target orphan mammalian RPs for degradation, including those not recognized by UBE2O, has not been studied in depth. I hypothesize that distinct features of individual RPs are recognized and targeted for proteasomal degradation by unique E3 ligases during erythroid differentiation. In this proposal, I will advance the field by combining in vitro biochemistry and cell-based assays to reveal mechanistic details of mammalian orphan RP recognition, ubiquitylation, and proteasomal degradation. In my first aim, I will characterize the ubiquitylation pattern of all 80 RPs and identify interactors of orphan RPs in reticulocyte lysate. In my second aim, I will reconstitute UBE2O-mediated ubiquitylation of orphan uL14 and assess the contribution of the uL14 interactor NAP1L1 to this process. Additionally, I will identify regions of uL14 recognized by both NAP1L1 and UBE2O and assess the influence of both proteins on uL14 degradation in cell culture systems. In my third aim, I will reconstitute HUWE1-mediated ubiquitylation of orphan uS3, identify regions of uS3 recognized by HUWE1, and determine if HUWE1 targets uS3 for degradation in cells. Studying the mechanisms of client recognition and selection will provide insight into how RPs are targeted for degradation in an effective manner during red blood cell development.

摘要 未成熟的红细胞在发育为 功能正常的红细胞。在终末红细胞生成的最后一步，核糖体从网织红细胞中清除。 通过泛素-蛋白酶体系统(UPS)。在这个过程中，单个未组装的或孤立的核糖体 亚单位蛋白(RP)似乎被E3连接酶识别，这种酶能够泛素化蛋白质和 以它们为靶标进行蛋白酶体降解。核糖体稳态缺陷，包括清除缺陷 在此过程中，可导致各种形式的贫血，并与血癌有关。尽管需要核糖体调节 在发育过程中，人们对红系细胞核糖体稳态的机制知之甚少。 最近，研究人员确认UBE2O是一种E2/E3混合酶，在 红系核糖体清除。UBE2O可以识别一些特定的RP，但不知道所有80个RP中的哪些是 UBE2O客户端或如何选择它们。孤立的RP互动子，如伴侣，参与的能力 在泛素化过程中也没有被探索过。尽管UBE2O是已知的唯一可以修饰的蛋白质 RPS在核糖体清除过程中，可能涉及其他E3连接酶，因为大约60%的RPS可以被降解 有效地在没有UBE2O的情况下。然而，其他的连接酶还没有被确定。候选人是 HUWE1，哺乳动物中TOM1的同源物，E3连接酶，泛素化和许多 酵母中的孤儿RPS。HUWE1直接修饰和靶向孤儿哺乳动物RPS进行降解的能力， 包括那些没有被UBE2O识别的，还没有被深入研究。 我假设个别RPS的不同特征被识别并针对蛋白酶体 红系分化过程中独特的E3连接酶的降解作用。在这项提案中，我将通过以下方式推进这一领域 结合体外生物化学和基于细胞的分析揭示哺乳动物孤儿RP的机制细节 识别、泛素化和蛋白酶体降解。在我的第一个目标中，我将描述泛素化 所有80个RPS的模式，并确定网织红细胞裂解物中孤立RPS的相互作用因子。在我的第二个目标中，我将 重组UBE2O介导的孤儿uL14泛素化并评估uL14相互作用的贡献 NAP1L1到这一过程。此外，我将确定NAP1L1和UBE2O都识别的uL14区域和 评估这两种蛋白质在细胞培养系统中对uL14降解的影响。在我的第三个目标中，我将 重组HUWE1介导的孤儿US3泛素化，鉴定HUWE1识别的US3区域，以及 确定HUWE1是否以US3为目标在细胞中降解。研究客户识别和客户识别的机制 选择将提供关于在红血期间如何有效地针对RP进行降解的洞察 细胞发育。