Changes in the ratio of free NEDD8 to ubiquitin triggers NEDDylation by ubiquitin enzymes

Hjerpe, Roland; Thomas, Y.; Chen, Jesse; Zemla, Aleksandra; Curran, Siobhan; Shpiro, Natalia; Dick, Lawrence R.; Kurz, Thimo

doi:10.1042/bj20111671

Cited by 92 publications

(102 citation statements)

References 39 publications

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“…A study by the late Cecile Pickart and co-workers suggests that the E1 of Ub activates Rub1 in vitro, though with an efficiency about 100 times less than the efficiency of activation of Ub, and vice versa (19). Similarly, another report shows that lowering the Ub level by a mere 3.6-fold in U2OS cells triggers the conjugation of Rub1 to many substrates using the E1 enzyme of the Ub pathway (106). That report further states that U2OS cells contain free forms of Ub and Nedd8 in a roughly equimolar amount, so a 3.6-fold decrease in the free Ub concentration will increase the ratio of [Nedd8]:[Ub] from 1:1 to 3.6:1, and this is sufficient to trigger Ub E1-mediated neddylation in these cells.…”

Section: Discussionmentioning

confidence: 99%

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

Singh

Zerath

Kleifeld

et al. 2012

Molecular & Cellular Proteomics

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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...

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Section: Discussionmentioning

confidence: 99%

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

Singh

Zerath

Kleifeld

et al. 2012

Molecular & Cellular Proteomics

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“…The cullin family of proteins constitutes the major substrates of NEDDylation, accompanied with other recently identified substrates including TGF-b type II receptor, histone H4, p53, p73, ribosomal proteins and L11 Zuo et al 2013;Abida et al 2007;Watson et al 2006;Xirodimas 2008;Xirodimas et al 2004Xirodimas et al , 2008Sundqvist et al 2009). The caveat here is that identification of non-cullin NEDDylation substrates all relied on over-expressed NEDD8 and recent studies have suggested that NEDD8 may serve as a surrogate for ubiquitin when its cellular levels are up-regulated by over-expression (Hjerpe et al 2012;Leidecker et al 2012). Conjugation of NEDD8 to the C-terminal area will initiate a profound structural change of the cullin-RING complex and facilitate the recruitment of E2 and SRs and thus, promote Ub conjugation to substrate proteins (Duda et al 2008).…”

Section: Regulatory Role Of Nedd8 Conjugation In Crl Complexesmentioning

confidence: 99%

Targeting cullin-RING ligases for cancer treatment: rationales, advances and therapeutic implications

Shu-ju

2015

Cytotechnology

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New therapeutic intervention strategies for the treatment of human malignancies are always desired. Approval of bortezomib as a front-line treatment for multiple myeloma highlighted the significance of ubiquitin-proteasome system (UPS) as a promising therapeutic target. However, due to the broad impact of proteasome inhibition, deleterious side effects have been reported with bortezomib treatment. Cullin RING ligases (CRLs)-mediated ubiquitin conjugation process is responsible for the ubiquitin conjugation of 20 % cellular proteins that are designated for degradation through the UPS, most of them are critical proteins involved in cell cycle progression, signaling transduction and apoptosis. Studies have depicted the upstream NEDDylation pathway that controls the CRL activity by regulating the conjugation of an ubiquitin-like-protein NEDD8 to the cullin protein in the complex. A specific pharmaceutical inhibitor of NEDD8 activating enzyme (NAE; E1) MLN4924 was recently developed and has been promoted to Phase I clinical trials for the treatment of several human malignancies. This article summarizes the most recent understanding about the process of NEDD8 conjugation, its relevance for cancer therapy and molecular mechanisms responsible for the potent anti-tumor activity of MLN4924.

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“…Therefore, it is surprising that, to date, only cullins have been recognized as bona fide NEDD8 modification substrates. For NEDD8, a number of non-cullin NEDD8-modified proteins have previously been identified, mainly in animal systems (Xirodimas, 2008;Mergner and Schwechheimer, 2014;Enchev et al, 2015), but recent observations of nonspecific crosstalk between the ubiquitin and the NEDD8 conjugation machineries suggests that at least some of these neddylation substrates may in fact be ubiquitylation substrates that have become neddylated rather than ubiquitylated as a consequence of NEDD8 overexpression (Hjerpe et al, 2012a(Hjerpe et al, , 2012b. It has thus been questioned whether other biologically relevant non-cullin neddylation substrates exist in eukaryotes (Enchev et al, 2015).…”

Section: Nedd8 (Neural Precursor Cell Expressed De-velopmentally Dowmentioning

confidence: 99%

DENEDDYLASE1 Deconjugates NEDD8 from Non-Cullin Protein Substrates in Arabidopsis thaliana

et al. 2015

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The evolutionarily conserved 8-kD protein NEDD8 (NEURAL PRECURSOR CELL EXPRESSED, DEVELOPMENTALLY DOWN-REGULATED8) belongs to the family of ubiquitin-like modifiers. Like ubiquitin, NEDD8 is conjugated to and deconjugated from target proteins. Many targets and functions of ubiquitylation have been described; by contrast, few targets of NEDD8 have been identified. In plants as well as in non-plant organisms, the cullin subunits of cullin-RING E3 ligases are NEDD8 conjugates with a demonstrated functional role for the NEDD8 modification. The existence of other non-cullin NEDD8 targets has generally been questioned. NEDD8 is translated as a precursor protein and proteolytic processing exposes a C-terminal glycine required for NEDD8 conjugation. In animals and yeast, DENEDDYLASE1 (DEN1) processes NEDD8. Here, we show that mutants of a DEN1 homolog from Arabidopsis thaliana have no detectable defects in NEDD8 processing but do accumulate a broad range of NEDD8 conjugates; this provides direct evidence for the existence of non-cullin NEDD8 conjugates. We further identify AUXIN RESISTANT1 (AXR1), a subunit of the heterodimeric NEDD8 E1 activating enzyme, as a NEDD8-modified protein in den1 mutants and wild type and provide evidence that AXR1 function may be compromised in the absence of DEN1 activity. Thus, in plants, neddylation may serve as a regulatory mechanism for cullin and non-cullin proteins.

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Changes in the ratio of free NEDD8 to ubiquitin triggers NEDDylation by ubiquitin enzymes

Cited by 92 publications

References 39 publications

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

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

Targeting cullin-RING ligases for cancer treatment: rationales, advances and therapeutic implications

DENEDDYLASE1 Deconjugates NEDD8 from Non-Cullin Protein Substrates in Arabidopsis thaliana

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