DEN1 Is a Dual Function Protease Capable of Processing the C Terminus of Nedd8 and Deconjugating Hyper-neddylated CUL1

Abstract

Nedd8 activates ubiquitination by increasing the efficiency of polyubiquitin chain assembly through its covalent conjugation to cullin molecules. Here we report the isolation, cloning, and characterization of a novel human Nedd8-specific protease called DEN1. Human DEN1 is encoded by AAH31411.1, a previously uncharacterized protein of 212 amino acids that shares homology with the Ulp1 cysteinyl SUMO deconjugating enzyme family. Recombinant human DEN1, purified from bacteria, selectively binds to Nedd8 and hydrolyzes Cterminal derivatives of Nedd8. Interestingly, DEN1 deconjugates cullin 1 (CUL1)-Nedd8 in a concentration-dependent manner. At a low concentration, DEN1 processes hyper-neddylated CUL1 to yield a mononeddylated form, which presumably contains the Lys-720 CUL1 -Nedd8 linkage. At elevated concentrations, DEN1 is able to complete the removal of Nedd8 from CUL1. These activities distinguish DEN1 from the COP9 signalosome, which is capable of efficiently cleaving the Lys-720 CUL1 -Nedd8 conjugate, but lacks Nedd8 Cterminal hydrolytic activity and poorly processes hyperneddylated CUL1. These results suggest a unique role for DEN1 in regulating the modification of cullins by Nedd8.Nedd8 is a small ubiquitin (Ub) 1 -like protein that plays a critical regulatory role in cell proliferation and development. In fission yeast, Nedd8 is essential for cell viability (1). In animals, Nedd8 is required for development as inactivation of the Nedd8 pathway in either mouse (2) or Drosophila (3) results in embryonic lethality. The critical biological function of Nedd8 is conferred by its biochemical activity as a protein modifier, being covalently attached to nearly all members of the cullin family (4). This modification, neddylation, is reminiscent of the ubiquitination reaction. Neddylation occurs by the formation of an isopeptide-bond linking the ⑀-amino group of a conserved lysine residue typically within the C terminus of a cullin to the carboxyl-end of Nedd8 Gly-76 (5). The enzyme components of the neddylation reaction include a Nedd8-specific E1 activating enzyme comprised of the APP-BP1/Uba3 heterodimer, an E2 conjugating enzyme known as Ubc12 (6), and the ROC1/Rbx1 RING finger protein (7).Using in vitro systems, several studies have shown that Nedd8 activates the ubiquitination of IB␣ (8) or p27 (9), through its conjugation to cullin 1 (CUL1). These reactions are mediated by SCF E3 Ub ligases, in which CUL1 functions as a molecular scaffold (10 -12). Subsequently, it was observed that degradation of HIF-␣ by von Hippel-Lindau tumor suppressor required Nedd8 (13). In this case, Nedd8 was conjugated to CUL2 that assembles the von Hippel-Lindau protein E3 Ub ligase (reviewed in Ref. 14). These studies thus suggest a role for Nedd8 in the assembly of an active cullin-based E3 Ub ligase.We initially reported that conjugation of Nedd8 to CUL1 increases the ability of ROC1-CUL1, a sub-complex within the SCF E3 Ub ligase, to assemble polyubiquitin chains in a reaction catalyzed by the Cdc34 E2 conjugating enzyme (15). S...

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

“…To verify the presence of multiple GST-Nedd8-cullin conjugates, proteins copurified with GST-Nedd8 were treated with DEN1, which is capable of hydrolyzing the Nedd8-cullin isopeptide bond in vitro. 19 The results showed that cleavage by DEN1 resulted in formation of CUL-1, -2, -3, and -4 with sizes of unmodified forms as expected ( Figure 3B). Figure 3C displays the relative abundance of all cullin proteins captured during the purification determined by use of normalized spectral abundance factor (NSAF).…”

Nedd8 on cullin: building an expressway to protein destruction

“…c (Wada et al, 1998;Linghu et al, 2002;Gan-Erdene et al, 2003). d Mendoza et al, 2003;Wu et al, 2003). e .…”

“…e . f Schwechheimer et al, 2001;Cope et al, 2002;Yang et al, 2002;Doronkin et al, 2003;Lykke-Andersen et al, 2003;Pintard et al, 2003a;Wu et al, 2003;Yan et al, 2003). g (Mendoza et al, 2003;Wu et al, 2003) Role of Nedd8 in proteolysis Z-Q Pan et al…”

MLN4924 Is an Efficient Inhibitor of NEDD8 Conjugation in Plants

“…CRLs are evolutionarily conserved E3 ligase complexes that share the CUL and RBX1 subunits and that, depending on the identity of the CUL subunit, associate with different adaptor and degradation substrate receptor modules, such as S-PHASE KINASE-ASSOCIATED PROTEIN1 (SKP1) and F-box proteins to form CUL1-containing SCF-type E3 ligases or DNA DAMAGE-BINDING PROTEIN1 (DDB1) and DDB1-CUL4-ASSOCIATED FACTOR (DCAF) subunits to form CUL4-containing DCAF E3s (Hotton and Callis, 2008;Deshaies and Joazeiro, 2009). NEDD8 is hydrolyzed from target proteins by CUL-deneddylating enzymes including subunit 5 of the CONSTITUTIVELY PHOTORMOPHOGENIC9 SIGNALOSOME Schwechheimer et al, 2001;Cope et al, 2002) or the deneddylase DEN1 Wu et al, 2003). Besides CULs, several other NEDD8-modified proteins have been reported from animal species, and in some cases a functional understanding of the role of their neddylation has been obtained (Rabut and Peter, 2008;Xirodimas, 2008).…”