This review summarizes recent advances concerning the Nedd8 regulatory pathway in four areas. One, substantial progress has been made in delineating the role of cullin family proteins, the only known substrates of the Nedd8 modification system. Cullins are molecular scaffolds responsible for assembling the ROC1/Rbx1 RING-based E3 ubiquitin ligases, of which several play a direct role in tumorigenesis. Two, a large body of work has helped elucidate the molecular details underlying the Nedd8 modification reaction, which results in covalent conjugation of a Nedd8 moiety onto a conserved cullin lysine residue. Three, studies using a variety of genetic model systems have established an essential role for Nedd8 in cell cycle control and in embryogenesis by upregulating the activities of cullin-based E3 ligases. In vitro experiments have revealed a direct role for Nedd8 in activating ubiquitination. Construction of a model of the ROC1/ Rbx1-CUL1-Nedd8 structure suggests a mechanism by which the cullin-linked Nedd8 may assist the neighboring ROC1/Rbx1 in landing and positioning the E2 conjugating enzyme for the ubiquitin transfer reaction. Finally, increasing evidence indicates that removal of Nedd8 from its cullin targets, by the action of COP9 Signalosome and possibly other proteases, plays a significant role in the regulation of cullin-mediated proteolysis.

The resident prokaryotic microflora of the mammalian intestine influences diverse homeostatic functions of the gut, including regulation of cellular growth and immune responses; however, it is unknown how commensal prokaryotic organisms mechanistically influence eukaryotic signaling networks. We have shown that bacterial coculture with intestinal epithelial cells modulates ubiquitin-mediated degradation of important signaling intermediates, including b-catenin and the NF-jB inhibitor IjB-a. Ubiquitination of these proteins as well as others is catalyzed by the SCF bTrCP ubiquitin ligase, which itself requires regulated modification of the cullin-1 subunit by the ubiquitin-like protein NEDD8. Here we show that epithelia contacted by enteric commensal bacteria in vitro and in vivo rapidly generate reactive oxygen species (ROS). Bacterially induced ROS causes oxidative inactivation of the catalytic cysteine residue of Ubc12, the NEDD8-conjugating enzyme, resulting in complete but transient loss of cullin-1 neddylation and consequent effects on NF-jB and b-catenin signaling. Our results demonstrate that commensal bacteria directly modulate a critical control point of the ubiquitin-proteasome system, and suggest how enteric commensal bacterial flora influences the regulatory pathways of the mammalian intestinal epithelia.

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...

We recently described the direct effects of thyroid-stimulating hormone (TSH) on bone and suggested that the bone loss in hyperthyroidism, hitherto attributed solely to elevated thyroid hormone levels, could at least in part arise from accompanying decrements in serum TSH. Recent studies on both mice and human subjects provide compelling evidence that thyroid hormones and TSH have the opposite effects on the skeleton. Here, we show that TSH, when injected intermittently into rodents, even at intervals of 2 weeks, displays a powerful antiresorptive action in vivo. By virtue of this action, together with the possible anabolic effects shown earlier, TSH both prevents bone loss and restores the lost bone after ovariectomy. Importantly, the osteoclast inhibitory action of TSH persists ex vivo even after therapy is stopped for 4 weeks. This profound and lasting antiresorptive action of TSH is mimicked in cells that genetically overexpress the constitutively active ligand-independent TSH receptor (TSHR). In contrast, loss of function of a mutant TSHR (Pro 3 Leu at 556) in congenital hypothyroid mice activates osteoclast differentiation, confirming once again our premise that TSHRs have a critical role in regulating bone remodeling.osteoclast ͉ osteoporosis ͉ pituitary ͉ osteoblast ͉ bisphosphonate C linical data available since von Recklinghausen's first description of thyrotoxic bone disease, the strong correlation between fracture risk and serum thyroid-stimulating hormone (TSH), recent evidence that TSH receptor (TSHR) polymorphisms are associated with low bone mass, and evidence that bone loss occurs in patients with subclinical hyperthyroidism with normal and low TSH levels all support a role for low TSH in the pathogenesis of hyperthyroid osteoporosis, hitherto attributed solely to high circulating levels of thyroid hormones (1-4).Evidence supporting this premise comes in parallel from genetically manipulated mice lacking receptors for either TSH or thyroid hormones (5, 6). We demonstrated that TSHR deficiency induces a high-turnover osteoporosis, with elevated bone formation and resorption even in TSHR haploinsufficient mice that have normal thyroid function. We found that in ex vivo cell cultures continually exposed to TSH, both osteoblastic bone formation and osteoclastic bone resorption were suppressed. Although these studies point to a primary function for TSH in skeletal homeostasis, data have reestablished a role for the two thyroid hormone receptor (TR) isoforms, TR␣ and TR␤ (6). The deficiency of TR␣ induces osteosclerosis in adult mice, whereas hyperthyroid TR␤-null mice display osteopenia despite high circulating TSH levels (6). Thus, evidence from human and mouse studies underscore the paradigm that both thyroid hormone excess and low TSH levels contribute to hyperthyroid bone loss (7).Hyperthyroidism can arise from adenomatous or inflammatory diseases of the thyroid or from overzealous replacement with thyroid hormones. Hyperthyroidism is Ϸ10-fold more common in women, with an incidence of 1 in 1,000...

SCF (Skp1⅐CUL1⅐F-box protein⅐ROC1) E3 ubiquitin ligase and Cdc34 E2-conjugating enzyme catalyze polyubiquitination in a precisely regulated fashion. Here, we describe biochemical evidence suggesting an autoinhibitory role played by the human CUL1 ECTD (extreme C-terminal domain; spanning the C-terminal 50 amino acids), a region that is predicted to contact the ROC1 RING finger protein by structural studies. We showed that ECTD did not contribute to CUL1's stable association with ROC1. Remarkably, deletion of ECTD, or missense mutations designed to disrupt the predicted ECTD⅐ROC1 interaction, markedly increased the ability of SCF ␤TrCP2 to promote IB␣ polyubiquitination and polyubiquitin chain assembly by Cdc34 in vitro. Thus, disruption of ECTD yields in vitro effects that parallel SCF activation by Nedd8 conjugation to CUL1. We propose that SCF may be subject to autoinhibitory regulation, in which Nedd8 conjugation acts as a molecular switch to drive the E3 into an active state by diminishing the inhibitory ECTD⅐ROC1 interaction.autoinhibition ͉ E3 ubiquitin ligase ͉ neddylation