A Targeted Proteomic Analysis of the Ubiquitin-Like Modifier Nedd8 and Associated Proteins

Abstract

Nedd8 is a small ubiquitin-like protein that can be conjugated to substrate-proteins in a process known as neddylation. Although neddylation plays a critical regulatory role in cell proliferation and development, the spectrum of Nedd8 substrates and its interaction network remain poorly understood. To explore the neddylation pathway at the proteome level, we have affinity purified Nedd8 modified and associated proteins from HEK293 cells stably expressing GST-Nedd8 and employed LC-MS/ MS for subsequent protein identification. A total of 496 GST-Nedd8 modified and associated proteins have been identified, including all of the eight cullin family members (i.e., Cul-1, -2, -3, -4A, -4B, -5, -7, and Parc) that are involved in the neddylation and ubiquitin-proteasome degradation pathway. In addition, a group of proteins involved in transcription, DNA repair and replication, cell cycle regulation and chromatin organization, and remodeling have been copurified and identified. Apart from protein identification, the neddylation sites of cullins were determined by MS/MS analysis, which agree well with previous mutagenesis studies. Furthermore, MS analyses revealed that Nedd8 K11, K22, K48, and K60 can form chains in vivo, whereas Nedd8 K22 and K48 can be neddylated in vitro. These results present the first molecular evidence for in vitro and in vivo polyneddylation, suggesting that chain formation of ubiquitin and ubiquitin-like proteins may be a general phenomenon for these modifications. Although much remains to be explored for the biological significance of the observations, this work provides critically important information regarding Nedd8 chain assembly and its interaction network. The vast amount of proteomic information obtained here

The covalent modifier Nedd8 is critical for the activation of Smurf1 ubiquitin ligase in tumorigenesis

“…Our findings also demonstrate that both mammalian Smurf1 and yeast Rsp5 require neddylation to activate their ubiquitin ligase activity, suggesting that this mechanism is conserved across species. A previous proteomic analysis of Nedd8-associated proteins identified the HECT E3s UBR5/EDD1 and HUWE1/ARF-BP1 as potential Nedd8-interacting proteins 56 , suggesting that the regulatory role of Nedd8 on the activation of E3s might be broader than previously thought. This finding is consistent with the fact that neddylation is essential for the viability of most organisms and is involved in the development of various diseases, including cancer.…”

Post-translational add-ons mark the path in exosomal protein sorting

“…E1 (NAE), E2 (UbCH12), E3 // IsopepƟdase [40,41] Others UBLs: ATG12, FAT10, MNSFβ, UFM1, URM, UBL5, SUMO4 [34][35][36][37][38] ModificaƟon of Ub: Phospho-Ub and Acetyl-Ub PolyubiquiƟn chains: Branched chains PolyUb mixed chains (modified by SUMO, NEDD8, ISG15) [21][22][23][24][25] Nucleo Ɵdes [190,191] Poly (PARylaƟon) Glu Asp Poly(ADP-ribose)polymerases (PARPs) // Poly (ADP-ribose) glycohydrolase (PARG) [191] * Found in prokaryotes A Almost followed by a proline (1 [39,40]. NEDD8 also forms chains through one of its six Lys [41].…”

“…NEDD8 also forms chains through one of its six Lys [41]. Small ubiquitin-related modifier (SUMO) is an UBL involved in nuclear transport and organization, DNA repair, transcription, chromatin remodelling and ribosome biogenesis.…”

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

“…Examples of both contradictory and concordant evidence lead to debate about the chain-forming ability of Rub1 (75)(76)(77)(78). Ub has seven lysine residues (K6, K11, K27, K29, K33, K48, and K63), and all of them have been shown to be involved in chain formation in vivo (79 -81).…”