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  • The ubiquitin like modifier NEDD NEURAL PRECURSOR CELL EXPRE

    2020-02-10

    The ubiquitin-like modifier NEDD8 (NEURAL PRECURSOR CELL EXPRESSED, DEVELOPMENTALLY DOWN-REGULATED 8) uses similar enzymatic machineries for NEDD8 conjugation as ubiquitin (Figure 1) [11, 12, 13, 14]. In plants as well as in other eukaryotes, the number of E3 ligases for NEDD8 and the number of known NEDD8-modified proteins are, however, much smaller [15, 16, 17, 18, 19, 20, 21]. In view of the substantial sequence identity (50–60%) between ubiquitin and NEDD8, this raises questions about how targeted NEDD8 conjugation to potentially many different substrates could be achieved and whether NEDD8 can give rise to a similar spectrum of posttranslational modifications as ubiquitin. Genetic studies from Arabidopsis thaliana, often aiming at understanding the mode of action of the hormone auxin, have contributed significantly to the identification of NEDD8 pathway components (e.g. [11,21,22]). Here, I review the recent advances in understanding the biochemical processes underlying NEDD8 conjugation (neddylation) and deconjugation (deneddylation) as well as their interplay with ubiquitylation in the context of plant biology.
    NEDD8 is related to ubiquitin Arabidopsis thaliana has three genes encoding NEDD8, RELATED TO UBIQUITIN1 (RUB1), RUB2/UBQ7 and RUB3 [23]. RUB1 and RUB2 differ only by one amino JP 1302 dihydrochloride and they are synthesized as ubiquitin–NEDD8 fusions [23]. RUB3, in turn, is composed of the NEDD8 moiety only [23]. Several ubiquitin C-terminal hydrolases (UCH) may potentially process ubiquitin–NEDD8 fusions in plants, but the identity of the in vivo relevant proteases is unknown [3,16,24]. The processing of ubiquitin–NEDD8 fusions releases NEDD8 (and ubiquitin) terminating in a di-Gly where the terminal Gly will form the stable isopeptide linkage with the ε-amino group of a substrate Lys (Figure 1) [16,25]. In Arabidopsis, rub1 rub2 mutants have an embryonic lethal phenotype, and likewise neddylation is essential in plants as well as in most eukaryotes with the notable exception of Saccharomyces cerevisiae [26]. RUB/NEDD8 as well as the entire neddylation and deneddylation machinery are supposed to be constitutively active.
    Neddylation The identification of genes responsible for auxin-sensitive root growth paved the way for the elucidation of the biochemistry of NEDD8 conjugation: AUXIN RESISTANT1 (AXR1) or the paralogous AXR1-LIKE (AXL) heterodimerize with E1 C-TERMINAL RELATED1 (ECR1) to form the NEDD8 activating enzyme (NAE) (Figure 1) [11,13,27]. Activated NEDD8 is transferred from the NAE via the NEDD8 conjugating enzyme (NCE) RUB CONJUGATING ENZYME1 (RCE1) and the RING-box protein RBX1 to the Cullin subunit of Cullin-RING E3 ubiquitin ligases (Figure 1, Figure 2) [14,18]. RBX1 serves as the E3 ligase for NEDD8 and as an E3 ligase for subsequent ubiquitylation reactions [28]. NAE function is efficiently blocked, also in plants, by MLN4924, a mechanism-based NAE inhibitor that forms a non-reactive NEDD8-MLN4924 adduct (Figure 1) [25,29]. The substrate specificity of the reaction is promoted by interactions with DEFECTIVE IN CULLIN NEDDYLATION1 (DCN1), that restricts the flexible NCE in a catalytically competent orientation (Figure 1) [30]. For a mammalian DCN1-LIKE protein, it was further proposed that it acts as a substrate sensor that bridges between substrate and Cullin [31]. Arabidopsis has two putative DCN1 orthologues, AT3G12760 and ANTI AUXIN RESISTANT3 (AAR3), but neither of them has been functionally implicated in neddylation in Arabidopsis as yet (Figure 1) [21].
    NEDD8 modification of Cullin-RING E3 ligases The Cullin subunits of Cullin-RING E3 ligases (CRLs) are the best-studied neddylation substrates [4]. All CRLs are a priori subject to regulation by neddylation and deneddylation. CRLs are modular complexes composed of Cullin scaffold proteins that bind RBX1 (RING BOX1) at their C-termini and different substrate receptors at their N-termini (Figure 2a–c) [32]. In plants, CRL complexes with Cullin1 (Cul1), Cul3 and Cul4 have been described and each Cullin has specific substrate receptors: SKP1 (SUPPRESSOR OF KINETOCHORE PROTEIN1)-F-box protein heterodimers in the case of Cul1; BTB/POZ (Bric-à-Brac, Tramtrack and Broad Complex/Pox virus and Zinc finger)-domain proteins in the case of Cul3, and DDB1/DCAF (DAMAGED DNA-BINDING PROTEIN1/DDB1 AND CUL4-ASSOCIATED FACTORS) in the case of Cul4 (Figure 2a–c) [32].