Activation of the transcription factor NF-kappa B in response to proinflammatory stimuli requires the phosphorylation-triggered and ubiquitin-dependent degradation of the NF-kappa B inhibitor, I kappa B alpha. Here, we show the in vitro reconstitution of the phosphorylation-dependent ubiquitination of I kappa B alpha with purified components. ROC1, a novel SCF-associated protein, is recruited by cullin 1 to form a quatemary SCFHOS-ROC1 holenzyme (with Skp1 and the beta-TRCP homolog HOS). SCFHOS-ROC1 binds IKK beta-phosphorylated I kappa B alpha and catalyzes its ubiquitination in the presence of ubiquitin, E1, and Cdc34. ROC1 plays a unique role in the ubiquitination reaction by heterodimerizing with cullin 1 to catalyze ubiquitin polymerization.

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...

Human CC chemokines macrophage inflammatory protein (MIP)-1␣, MIP-1␤, and RANTES (regulated on activation normal T cell expressed) self-associate to form high-molecular mass aggregates. To explore the biological significance of chemokine aggregation, nonaggregating variants were sought. The phenotypes of 105 hMIP-1␣ variants generated by systematic mutagenesis and expression in yeast were determined. hMIP-1␣ residues Asp 26 and Glu 66 were critical to the self-association process. Substitution at either residue resulted in the formation of essentially homogenous tetramers at 0.5 mg/ml. Substitution of identical or analogous residues in homologous positions in both hMIP-1␤ and RAN-TES demonstrated that they were also critical to aggregation. Our analysis suggests that a single charged residue at either position 26 or 66 is insufficient to support extensive aggregation and that two charged residues must be present. Solution of the three-dimensional NMR structure of hMIP-1␣ has enabled comparison of these residues in hMIP-1␤ and RANTES. Aggregated and disaggregated forms of hMIP-1␣, hMIP-1␤, and RANTES generally have equivalent G-protein-coupled receptormediated biological potencies. We have therefore generated novel reagents to evaluate the role of hMIP-1␣, hMIP-1␤, and RANTES aggregation in vitro and in vivo. The disaggregated chemokines retained their human immunodeficiency virus (HIV) inhibitory activities. Surprisingly, high concentrations of RANTES, but not disaggregated RANTES variants, enhanced infection of cells by both M-and T-tropic HIV isolates/strains. This observation has important implications for potential therapeutic uses of chemokines implying that disaggregated forms may be necessary for safe clinical investigation.

We describe a purified ubiquitination system capable of rapidly catalyzing the covalent linkage of polyubiquitin chains onto a model substrate, phosphorylated IB␣. The initial ubiquitin transfer and subsequent polymerization steps of this reaction require the coordinated action of Cdc34 and the SCF HOS/␤-TRCP -ROC1 E3 ligase complex, comprised of four subunits (Skp1, cullin 1 [CUL1], HOS/␤-TRCP, and ROC1). Deletion analysis reveals that the N terminus of CUL1 is both necessary and sufficient for binding Skp1 but is devoid of ROC1-binding activity and, hence, is inactive in catalyzing ubiquitin ligation. Consistent with this, introduction of the N-terminal CUL1 polypeptide into cells blocks the tumor necrosis factor alpha-induced and SCF-mediated degradation of IB by forming catalytically inactive complexes lacking ROC1. In contrast, the C terminus of CUL1 alone interacts with ROC1 through a region containing the cullin consensus domain, to form a complex fully active in supporting ubiquitin polymerization. These results suggest the mode of action of SCF-ROC1, where CUL1 serves as a dual-function molecule that recruits an F-box protein for substrate targeting through Skp1 at its N terminus, while the C terminus of CUL1 binds ROC1 to assemble a core ubiquitin ligase.

Lysine 48-linked polyubiquitin chains are the principle signal for targeting proteins for degradation by the 26 S proteasome. Here we report that the conjugation of Nedd8 to ROC1-CUL1, a subcomplex of the SCF-ROC1 E3 ubiquitin ligase, selectively stimulates Cdc34-catalyzed lysine 48-linked multiubiquitin chain assembly. We have further demonstrated that separate regions within the human Cdc34 C-terminal tail are responsible for multiubiquitin chain assembly and for physical interactions with the Nedd8-conjugated ROC1-CUL1 to assemble extensive ubiquitin polymers. Structural comparisons between Nedd8 and ubiquitin reveal that six charged residues (Lys 4 , Glu 12 , Glu 14 , Arg 25 , Glu 28 , and Glu 31 ) are uniquely present on the surface of Nedd8. Replacement of each of the six residues with the corresponding amino acid in ubiquitin decreases the ability of Nedd8 to activate the ubiquitin ligase activity of ROC1-CUL1. Moreover, maintenance of the proper charges at amino acid positions 14 and 25 are necessary for retaining wild type levels of activity, whereas introduction of the opposite charges at these positions abolishes the Nedd8 activation function. These results suggest that Nedd8 charged surface residues mediate the activation of ROC1-CUL1 to specifically support Cdc34-catalyzed ubiquitin polymerization.Nedd8 (or its orthologue Rub1) is a small ubiquitin (Ub)-like molecule that modifies all members of the Cullin/Cdc53 protein family (1, 2), resulting in the formation of an isopeptide bond linkage between the ⑀-amino group of a conserved Cullin lysine residue and the C-terminal carboxyl group of Nedd8 glycine 76. The conjugation is an ATP-dependent reaction that requires a Nedd8-specific E1 activating enzyme, composed of the APP-BP1 and Uba3 heterodimer, and Ubc12 as the E2 conjugating enzyme (3). In addition, the Cullin-interacting RING finger protein, ROC1/Rbx1/Hrt1, is required for the reaction as disruption of the RING domain abolishes the modification (4).Two well characterized members of the Cullin family, CUL1 and CUL2, serve as subunits of the two multisubunit E3 Ub ligase complexes, SCF-ROC1 (5-8) and pVHL-elongin C/B-CUL2-ROC1 (9), respectively. It has now been well established that the ROC1-CUL1 subassembly acts as a core ubiquitin ligase, capable of supporting Ub polymerization (5,6,8,10,11). In addition, results from transient transfection experiments have shown that ROC1 and its homologue ROC2 interact with all members of the Cullin protein family and that the resulting ROC-Cullin complex is active in supporting Ub polymerization (6). Thus, while the biological roles of Cullin members including CUL3, CUL4A, CUL4B, and CUL5 remain elusive, it is highly likely that all of the ROC-Cullin based complexes are involved in cellular Ub-dependent proteolysis pathways. It is, therefore, conceivable that Nedd8, through its conjugation to Cullins, functions to regulate the stability of cellular proteins.Accumulating genetic evidence has demonstrated a critical role for Nedd8 in the regulation of cell pro...