The prevalence and mechanistic significance of self-association among substrate adaptors for the Cul-Rbx family of ubiquitin ligases remain unclear. We now report that it is as a homodimer that the substrate adaptor Keap1 interacts with Cul3. The resulting complex facilitates ubiquitylation of the Nrf2 transcription factor but only when this substrate possesses within its Neh2 domain a second cryptic Keap1-binding site, the DLG motif, in addition to its previously described ETGE site. Both motifs recognize overlapping surfaces on Keap1, and the seven lysine residues of Nrf2 that act as ubiquitin acceptors lie between them. Based on these data, we propose a "fixed-ends" model for Nrf2 ubiquitylation in which each binding site becomes tethered to a separate subunit of the Keap1 homodimer. This two-site interaction between Keap1 and Nrf2 constrains the mobility of the target lysine residues in the Neh2 domain, increasing their average concentration in the vicinity of the Rbx-bound ubiquitin-conjugating enzyme, and thus the rate at which the transcription factor is ubiquitylated. We show that self-association is a general feature of Cul3 substrate adaptors and propose that the fixed-ends mechanism is commonly utilized to recruit, orientate, and ubiquitylate substrates upon this family of ubiquitin ligases.Ubiquitylation underpins virtually all biological processes as it is the major mechanism regulating the stability of critical effector molecules in eukaryotic cells. It refers to the formation of an isopeptide bond between the C terminus of ubiquitin and the ⑀-NH 2 group of a lysine residue in a target protein. The reaction proceeds in three stages with the final critical step, transfer of activated ubiquitin from an E2 2 ubiquitin-conjugating enzyme to an acceptor lysine, facilitated by E3 ubiquitin ligases (1). Modular complexes based around at least four Cullin-RING box (Cul-Rbx) holoenzymes constitute the largest family of E3 ligases identified to date. These holoenzymes, Cul1-Rbx1, Cul2-Rbx1, Cul3-Rbx1, and Cul5-Rbx2, differ in the nature of the substrate adaptors they bind (2-5). For example, Cul1-Rbx1 recruits protein dimers comprising the S-phase kinase-associated protein 1 (Skp1) bound to the eponymous domain found in over 40 F-box proteins (2). The resulting E3 ligase is termed SCF F-box (Skp1, Cul1, and F-box, with the specific F-box protein identified in supercript). Cul3-Rbx1 recruits Broad complex, Tramtrack, and Bric-a-brac (BTB) proteins to generate a large family of ligases that, by analogy with SCF ligases, are referred to as BC 3 B BTB ubiquitin ligases (6 -9). 3Structural similarities exist among substrate adaptors. For example, Skp1 and BTB proteins all utilize BTB folds to interact with Cul proteins (3). Additionally, the domains utilized to recruit substrates can adopt analogous super-secondary structures; whereas F-box proteins frequently use WD40 domains to recruit substrates and BTB proteins commonly exploit Kelchrepeat domains for this purpose, both domains fold to give sixbladed ␤-propel...

The Nrf2 transcription factor is more rapidly turned over in cells grown under homeostatic conditions than in those experiencing oxidative stress. The variable turnover of Nrf2 is accomplished through the use of at least two degrons and its redox-sensitive interaction with the Kelch-repeat protein Keap1. In homeostatic COS1 cells, the Neh2 degron confers on Nrf2 a half-life of less than 10 min. Analyses of deletion mutants of a Gal4(HA)mNeh2 fusion protein and full-length mNrf2 indicate that full redox-sensitive Neh2 destabilizing activity depends upon two separate sequences within this N-terminal domain. The DIDLID element (amino acids 17-32) is indispensable for Neh2 activity and appears necessary to recruit a ubiquitin ligase to the fusion protein. A second motif within Neh2, the ETGE tetrapeptide (amino acids 79 -82), allows the redox-sensitive recruitment of Nrf2 to Keap1. This interaction, which occurs only in homeostatic cells, enhances the capacity of the Neh2 degron to direct degradation by functioning downstream of ubiquitination mediated by the DIDLID element. By contrast with the situation under homeostatic conditions, the Neh2 degron is neither necessary nor sufficient to account for the characteristic half-life of Nrf2 in oxidatively stressed cells. Instead, the previously uncharacterized, redox-insensitive Neh6 degron (amino acids 329 -379) is essential to ensure that the transcription factor is still appropriately turned over in stressed cells, albeit with an increased half-life of 40 min. A model can now be proposed to explain how the turnover of this protein adapts in response to alterations in cellular redox state.

1. The relative roles of various members of the human sulfotransferase (SULT) enzyme family in the metabolism of apomorphine, a dopamine receptor antagonist used in the treatment of Parkinson's disease and, more recently, erectile dysfunction, were examined. In humans, sulfation is the major route of metabolism of this drug. 2. Using recombinant SULTs expressed in Escherichia coli, R(--)-apomorphine sulfation was studied using the universal barium precipitation assay in the presence of [35S] 3'-phosphoadenosine 5'-phosphosulfate and SULTs 1A1, 1A2, 1A3, 1B1, 1C2, 1E1 and 2A1. It was shown that SULTs 1A1, 1A2, 1A3 and 1E1 all sulfated apomorphine to varying extents. Low activity with SULT1B1 was only seen at the highest concentration (100 microM) and no activity with SULT1C2 or SULT2A1 was observed. 3. Kinetic analysis using purified recombinant SULTs showed that 1A1, 1A3 and 1E1 all had similar Vmax/Km values, although SULT1E1 had a slightly lower Km at around 1 microM compared with approximately 4 microM for the other SULTs. 4. By correlating apomorphine sulfation (at 10 microM) in a bank of 28 liver cytosols with SULT activity towards 10 microM 4-nitrophenol (SULT1A1) and 0.2 microM 17beta-oestradiol (SULT1E1), a strong correlation with SULT1A1 activity was clearly demonstrated, suggesting this enzyme was primarily responsible for hepatic apomorphine sulfation. 5. These findings were confirmed using immuno-inhibition experiments with antibodies against SULT1A and SULT1E1, which showed preferential inhibition of apomorphine sulfation in human liver cytosol by anti-SULT1A. 6. The results strongly implicate SULT1A1 as the major enzyme responsible for hepatic apomorphine metabolism. As SULT1A1 is subject to a common functional polymorphism, sulfation phenotype may be an important determinant of susceptibility to side-effects of apomorphine and/or efficacy of treatment.