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

摘要 未成熟的成红细胞在发育为成熟的成红细胞之前经历显著的蛋白质组重塑事件。 功能性红细胞在终末红细胞生成的最后步骤中，核糖体从网织红细胞中清除 泛素-蛋白酶体系统（UPS）在这个过程中，单个未组装的，或孤儿，核糖体， 亚基蛋白（RP）似乎被E3连接酶识别，E3连接酶是能够使蛋白质泛素化的酶， 靶向它们进行蛋白酶体降解。核糖体稳态缺陷，包括清除 过程中，可能导致贫血，并与血癌有关。尽管需要核糖体调节 在发育过程中，对红系细胞中核糖体稳态的基本机制知之甚少。 最近，研究人员将UBE 2 O鉴定为一种混合E2/E3酶，其在哺乳动物中使孤儿RP遍在化。 红细胞核糖体清除率。UBE 2 O识别一些特定的RP，但不知道所有80个RP中的哪些是 UBE 2 O客户端或如何选择它们。孤儿RP相互作用者（如伴侣）参与的能力 在泛素化过程中也没有被探索。虽然UBE 2 O是已知的唯一一种蛋白质， 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RP可以被降解 在没有UBE 2 O的情况下有效。然而，尚未鉴定出另外的连接酶。某候选人是获 HUWE 1是TOM 1的哺乳动物同源物，TOM 1是一种E3连接酶，用于泛素化和降解许多 酵母中的孤儿RP。HUWE 1直接修饰和靶向孤儿哺乳动物RP降解的能力， 包括那些未被UBE 2 O识别的，尚未被深入研究。 我推测，个别RP的独特特征被蛋白酶体识别和靶向。 在红细胞分化期间通过独特的E3连接酶降解。在这个建议中，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测定来揭示哺乳动物孤儿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 所有80个RP的模式，并确定网织红细胞裂解物中孤儿RP的相互作用物。在我的第二个目标中，我将 重建UBE 2 O介导的孤儿uL 14泛素化，并评估uL 14相互作用因子的作用 NAP 1 L1在这个过程中。此外，我将鉴定由NAP 1 L1和UBE 2 O识别的uL 14区域， 评估两种蛋白质对细胞培养系统中uL 14降解的影响。在我的第三个目标中，我将 重组HUWE 1介导的孤儿uS 3的泛素化，鉴定HUWE 1识别的uS 3区域， 确定HUWE 1是否靶向uS 3在细胞中降解。研究客户识别机制， 选择将提供对RP如何在红血过程中以有效方式靶向降解的深入了解 细胞发育

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.