Recognition and Cleavage of Related to Ubiquitin 1 (Rub1) and Rub1-Ubiquitin Chains by Components of the Ubiquitin-Proteasome System

Abstract

Of all ubiquitin-like proteins, Rub1 (Nedd8 in mammals) is the closest kin of ubiquitin. We show via NMR that structurally, Rub1 and ubiquitin are fundamentally similar as well. Despite these profound similarities, the prevalence of Rub1/Nedd8 and of ubiquitin as modifiers of the proteome is starkly different, and their attachments to specific substrates perform different functions. Recently, some proteins, including p53, p73, EGFR, caspase-7, and Parkin, have been shown to be modified by both Rub1/ Nedd8 and ubiquitin within cells. To understand whether and how it might be possible to distinguish among the same target protein modified by Rub1 or ubiquitin or both, we examined whether ubiquitin receptors can differentiate between Rub1 and ubiquitin. Surprisingly, Rub1 interacts with proteasome ubiquitin-shuttle proteins comparably to ubiquitin but binds more weakly to a proteasomal ubiquitin receptor Rpn10. We identified Rub1-ubiquitin heteromers in yeast and Nedd8-Ub heteromers in human cells. We validate that in human cells and in vitro, human Rub1 (Nedd8) forms chains with ubiquitin where it acts as a chain terminator. Interestingly, enzymatically assembled K48-linked Rub1-ubiquitin heterodimers are recognized by various proteasomal ubiquitin shuttles and receptors comparably to K48-linked ubiquitin homodimers. Furthermore, these heterologous chains are cleaved by COP9 signalosome or 26S proteasome. A derubylation function of the proteasome expands the repertoire of its enzymatic activities. In contrast, Rub1 conjugates may be somewhat resilient to the actions of other canonical deubiquitinating enzymes. Taken together, these findings suggest that once Rub1/Nedd8 is channeled into ubiquitin pathways, it is recognized essentially like The selective degradation of many proteins in eukaryotic cells is a highly specific and irreversible process required to perform vital cellular functions such as cell cycle progression (1, 2), differentiation and development (3, 4), and transcriptional control (5). One of the major pathways involved in this selective degradation is the ubiquitin-proteasome pathway. In this pathway, ubiquitin (Ub), a 76-amino-acid protein, is attached to the substrate, and this is followed by the recognition and degradation of the substrate by a protein complex collectively known as proteasome (6 -8).The attachment of Ub (ubiquitination) to a substrate protein is achieved via a cascade of enzymatic reactions (involving E1, E2, and E3 enzymes) that results in the formation of an isopeptide bond between the C-terminal glycine of Ub and a lysine residue of the substrate (9 -11). Sequential repetition of this cascade can result in the attachment of a chain of Ub molecules (polyubiquitin) to the substrate protein. The length and topology of the polyubiquitin (polyUb) tag decide the fate of the substrate protein. For example, we and others have shown that a K48-linked tetraubiquitin (tetraUb) chain that acts as a proteasomal degradation signal forms a "closed" structure (12, 13), whereas a K63-linked Ub...

NEDD8 Ultimate Buster 1 Long (NUB1L) Protein Suppresses Atypical Neddylation and Promotes the Proteasomal Degradation of Misfolded Proteins

“…Supporting this model, the deneddylase CSN associates with the proteasome in cells and is able to process Ub-NEDD8 heterodimers in vitro (40). Furthermore, loss of CSN deneddylation activity leads to the accumulation of neddylated proteins and impairs UPS proteolysis in mouse hearts (14,15).…”

“…A substantial change in the Ub-to-NEDD8 ratio under stress condition has been proposed as a trigger to stimulate atypical neddylation (17,18). Given the similarity of NEDD8 and Ub in amino acid sequence and quaternary structure, it has been postulated that NEDD8 may substitute for Ub and cap the extension of Ub chains, therefore preserving the free Ub pool and avoiding otherwise catastrophic effects on cells (17,40).…”

“…The NEDD8 signals detected in the precipitates from TUBEs, which enrich essentially Lys-48-linked polyubiquitinated proteins under proteasome inhibition condition, suggest that NEDD8 likely incorpo- rates into the Lys-48-linked polyubiquitin chains. NEDD8 and Ub are recognized by Ub shuttle proteins with equivalent affinity in vitro and can form heterodimers in cultured cells (40). This Ub-NEDD8 heterodimer can bind to, and be processed by, the proteasome in vitro (40).…”

“…NEDD8 and Ub are recognized by Ub shuttle proteins with equivalent affinity in vitro and can form heterodimers in cultured cells (40). This Ub-NEDD8 heterodimer can bind to, and be processed by, the proteasome in vitro (40). Moreover, neddylated proteins have been reported to be copurified with the proteasome complex in mammalian cells (41).…”

Synthesis of Post-translational Modifier Protein NEDD8 via Ligation of Peptide Hydrazides

“…研究表明当 NEDD8 共价结合 至 Cullin 蛋白上后, 会使 Cullin 的构象发生改变、活性 增强, 从而能够更加高效地将 Ub 带到底物蛋白上完成 泛素化 [5] . 2012 年 Fushman 等 [6] 发现 NEDD 化的多聚 Ub [8] . 然而使用大肠杆菌体系表达 NEDD8 的效率 较低 [9] , 同时, 为了纯化蛋白需要融入 His-tag 或者 GST 标签, 然后再使用特异性的酶来切除.…”