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Familial neurodegenerative diseases commonly involve mutations that result in either aberrant proteins or dysfunctional components of the proteolytic machinery that act on aberrant proteins. UBQLN2 is a ubiquitin receptor of the UBL/UBA family that binds the proteasome through its ubiquitin-like domain and is thought to deliver ubiquitinated proteins to proteasomes for degradation. UBQLN2 mutations result in familial amyotrophic lateral sclerosis (ALS)/frontotemporal dementia in humans through an unknown mechanism. Quantitative multiplexed proteomics was used to provide for the first time an unbiased and global analysis of the role of Ubqln2 in controlling the composition of the proteome. We studied several murine models of Ubqln2-linked ALS and also generated Ubqln2 null mutant mice. We identified impacts of Ubqln2 on diverse physiological pathways, most notably serotonergic signaling. Interestingly, we observed an upregulation of proteasome subunits, suggesting a compensatory response to diminished proteasome output. Among the specific proteins whose abundance is linked to UBQLN2 function, the strongest hits were the ubiquitin ligase TRIM32 and two retroelement-derived proteins, PEG10 and CXX1B. Cycloheximide chase studies using induced human neurons and HEK293 cells suggested that PEG10 and TRIM32 are direct clients. Although UBQLN2 directs the degradation of multiple proteins via the proteasome, it surprisingly conferred strong protection from degradation on the Gag-like protein CXX1B, which is expressed from the same family of retroelement genes as PEG10. In summary, this study charts the proteomic landscape of ALS-related Ubqln2 mutants and identifies candidate client proteins that are altered in vivo in disease models and whose degradation is promoted by UBQLN2.

NEDD8 is a ubiquitin‐like protein that activates cullin‐RING E3 ubiquitin ligases (CRLs). Here, we identify a novel role for NEDD8 in regulating the activity of poly(ADP‐ribose) polymerase 1 (PARP‐1) in response to oxidative stress. We show that treatment of cells with H2O2 results in the accumulation of NEDD8 chains, likely by directly inhibiting the deneddylase NEDP1. One chain type, an unanchored NEDD8 trimer, specifically bound to the second zinc finger domain of PARP‐1 and attenuated its activation. In cells in which Nedp1 is deleted, large amounts of tri‐NEDD8 constitutively form, resulting in inhibition of PARP‐1 and protection from PARP‐1‐dependent cell death. Surprisingly, these NEDD8 trimers are additionally acetylated, as shown by mass spectrometry analysis, and their binding to PARP‐1 is reduced by the overexpression of histone de‐acetylases, which rescues PARP‐1 activation. Our data suggest that trimeric, acetylated NEDD8 attenuates PARP‐1 activation after oxidative stress, likely to delay the initiation of PARP‐1‐dependent cell death.

The activity of Cullin-RING ubiquitin E3 ligases (CRL) is regulated by NEDD8 modification. DCN-like proteins promote Cullin neddylation as scaffold-like E3s. One DCNL, DCNL5, is highly expressed in immune tissue. Here, we provide evidence that DCNL5 may be involved in innate immunity, as it is a direct substrate of the kinase IKKα during immune signalling. We find that upon activation of Toll-like receptors, DCNL5 gets rapidly and transiently phosphorylated on a specific N-terminal serine residue (S41). This phosphorylation event is specifically mediated by IKKα and not IKKβ. Our data for the first time provides evidence that DCNL proteins are post-translationally modified in an inducible manner. Our findings also provide the first example of a DCNL member as a kinase substrate in a signalling pathway, indicating that the activity of at least some DCNLs may be regulated.

Deletion of exon 9 from Cullin‐3 (CUL3, residues 403–459: CUL3Δ403–459) causes pseudohypoaldosteronism type IIE (PHA2E), a severe form of familial hyperkalaemia and hypertension (FHHt). CUL3 binds the RING protein RBX1 and various substrate adaptors to form Cullin‐RING‐ubiquitin‐ligase complexes. Bound to KLHL3, CUL3‐RBX1 ubiquitylates WNK kinases, promoting their ubiquitin‐mediated proteasomal degradation. Since WNK kinases activate Na/Cl co‐transporters to promote salt retention, CUL3 regulates blood pressure. Mutations in both KLHL3 and WNK kinases cause PHA2 by disrupting Cullin‐RING‐ligase formation. We report here that the PHA2E mutant, CUL3Δ403–459, is severely compromised in its ability to ubiquitylate WNKs, possibly due to altered structural flexibility. Instead, CUL3Δ403–459 auto‐ubiquitylates and loses interaction with two important Cullin regulators: the COP9‐signalosome and CAND1. A novel knock‐in mouse model of CUL3WT/Δ403–459 closely recapitulates the human PHA2E phenotype. These mice also show changes in the arterial pulse waveform, suggesting a vascular contribution to their hypertension not reported in previous FHHt models. These findings may explain the severity of the FHHt phenotype caused by CUL3 mutations compared to those reported in KLHL3 or WNK kinases., EMBO Molecular Medicine, 7 (10), ISSN:1757-4676, ISSN:1757-4684

Chronic Obstructive Pulmonary Disease (COPD) is a highly prevalent condition characterized by inflammation and progressive obstruction of the airways. At present, there is no treatment that suppresses the chronic inflammation of the disease, and COPD patients often succumb to the condition. Excessive oxidative stress caused by smoke inhalation is a major driving force of the disease. The transcription factor NRF2 is a critical player in the battle against oxidative stress and its function is impaired in COPD. Increasing NRF2 activity may therefore be a viable therapeutic option for COPD treatment. We show that down regulation of KEAP1, a NRF2 inhibitor, protects primary human lung epithelial cells from cigarette-smoke-extract (CSE) induced cell death in an established in vitro model of radical stress. To identify new potential drug targets with a similar effect, we performed a siRNA screen of the ‘druggable’ genome using a NRF2 transcriptional reporter cell line. This screen identified multiple genes that when down regulated increased NRF2 transcriptional activity and provided a survival benefit in the in vitro model. Our results suggest that inhibiting components of the ubiquitin-proteasome system will have the strongest effects on NRF2 transcriptional activity by increasing NRF2 levels. We also find that down regulation of the small GTPase Rab28 or the Estrogen Receptor ESRRA provide a survival benefit. Rab28 knockdown increased NRF2 protein levels, indicating that Rab28 may regulate NRF2 proteolysis. Conversely ESRRA down regulation increased NRF2 transcriptional activity without affecting NRF2 levels, suggesting a proteasome-independent mechanism.

The WNK (with no lysine kinase)–SPAK (SPS1-related proline/alanine-rich kinase)/OSR1 (oxidative stress-responsive kinase 1) signalling pathway plays an important role in controlling mammalian blood pressure by modulating the activity of ion co-transporters in the kidney. Recent studies have identified Gordon's hypertension syndrome patients with mutations in either CUL3 (Cullin-3) or the BTB protein KLHL3 (Kelch-like 3). CUL3 assembles with BTB proteins to form Cullin–RING E3 ubiquitin ligase complexes. To explore how a CUL3–KLHL3 complex might operate, we immunoprecipitated KLHL3 and found that it associated strongly with WNK isoforms and CUL3, but not with other components of the pathway [SPAK/OSR1 or NCC (Na+/Cl− co-transporter)/NKCC1 (Na+/K+/2Cl− co-transporter 1)]. Strikingly, 13 out of the 15 dominant KLHL3 disease mutations analysed inhibited binding to WNK1 or CUL3. The recombinant wild-type CUL3–KLHL3 E3 ligase complex, but not a disease-causing CUL3–KLHL3[R528H] mutant complex, ubiquitylated WNK1 in vitro. Moreover, siRNA (small interfering RNA)-mediated knockdown of CUL3 increased WNK1 protein levels and kinase activity in HeLa cells. We mapped the KLHL3 interaction site in WNK1 to a non-catalytic region (residues 479–667). Interestingly, the equivalent region in WNK4 encompasses residues that are mutated in Gordon's syndrome patients. Strikingly, we found that the Gordon's disease-causing WNK4[E562K] and WNK4[Q565E] mutations, as well as the equivalent mutation in the WNK1[479–667] fragment, abolished the ability to interact with KLHL3. These results suggest that the CUL3–KLHL3 E3 ligase complex regulates blood pressure via its ability to interact with and ubiquitylate WNK isoforms. The findings of the present study also emphasize that the missense mutations in WNK4 that cause Gordon's syndrome strongly inhibit interaction with KLHL3. This could elevate blood pressure by increasing the expression of WNK4 thereby stimulating inappropriate salt retention in the kidney by promoting activation of the NCC/NKCC2 ion co-transporters. The present study reveals how mutations that disrupt the ability of an E3 ligase to interact with and ubiquitylate a critical cellular substrate such as WNK isoforms can trigger a chronic disease such as hypertension.

Hypertension (high blood pressure) is a major public health problem affecting more than a billion people worldwide with complications, including stroke, heart failure and kidney failure. The regulation of blood pressure is multifactorial reflecting genetic susceptibility, in utero environment and external factors such as obesity and salt intake. In keeping with Arthur Guyton’s hypothesis, the kidney plays a key role in blood pressure control and data from clinical studies; physiology and genetics have shown that hypertension is driven a failure of the kidney to excrete excess salt at normal levels of blood pressure. There is a number of rare Mendelian blood pressure syndromes, which have shed light on the molecular mechanisms involved in dysregulated ion transport in the distal kidney. One in particular is Familial hyperkalemic hypertension (FHHt), an autosomal dominant monogenic form of hypertension characterised by high blood pressure, hyperkalemia, hyperchloremic metabolic acidosis, and hypercalciuria. The clinical signs of FHHt are treated by low doses of thiazide diuretic, and it mirrors Gitelman syndrome which features the inverse phenotype of hypotension, hypokalemic metabolic alkalosis, and hypocalciuria. Gitelman syndrome is caused by loss of function mutations in the thiazide-sensitive Na/Cl cotransporter (NCC); however, FHHt patients do not have mutations in the SCL12A3 locus encoding NCC. Instead, mutations have been identified in genes that have revealed a key signalling pathway that regulates NCC and several other key transporters and ion channels in the kidney that are critical for BP regulation. This is the WNK kinase signalling pathway that is the subject of this review.

Familial hyperkalemic hypertension (FHHt) is a rare inherited form of salt-dependent hypertension caused by mutations in proteins that regulate the renal Na(+)-Cl(-) cotransporter NCC Mutations in four genes have been reported to cause FHHt including CUL3 (Cullin3) that encodes a component of a RING E3 ligase. Cullin-3 binds to WNK kinase-bound KLHL3 (the substrate recognition subunit of the ubiquitin ligase complex) to promote ubiquitination and proteasomal degradation of WNK kinases. Deletion of exon 9 from CUL3 (affecting residues 403-459, CUL3(Δ403-459)) causes a severe form of FHHt (PHA2E) that is recapitulated closely in a knock-in mouse model. The loss of functionality of CUL3(Δ403-459) and secondary accumulation of WNK kinases causes substantial NCC activation. This accounts for the hypertension in FHHt but the origin of the hyperkalemia is less clear. Hence, we explored the impact of CUL3(Δ403-459) on expression of the distal secretory K channel, ROMK, both in vitro and in vivo. We found that expressing wild-type but not the CUL3(Δ403-459) mutant form of CUL3 prevented the suppression of ROMK currents by WNK4 expressed in Xenopus oocytes. The mutant CUL3 protein was also unable to affect ROMK-EGFP protein expression at the surface of mouse M-1 cortical collecting duct (CCD) cells. The effects of CUL3 on ROMK expression in both oocytes and M-1 CCD cells was reduced by addition of the neddylation inhibitor, MLN4924. This confirms that neddylation is important for CUL3 activity. Nevertheless, in our knock-in mouse model expressing CUL3(Δ403-459) we could not show any alteration in ROMK expression by either western blotting whole kidney lysates or confocal microscopy of kidney sections. This suggests that the hyperkalemia in our knock-in mouse and human PHA2E subjects with the CUL3(Δ403-459) mutation is not caused by reduced ROMK expression in the distal nephron., British Heart Foundation. Grant Number: PG/13/89/30577, This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.14814/phy2.12850

Ubiquitin and UBL (ubiquitin-like) modifiers are small proteins that covalently modify other proteins to alter their properties or behaviours. Ubiquitin modification (ubiquitylation) targets many substrates, often leading to their proteasomal degradation. NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) is the UBL most closely related to ubiquitin, and its best-studied role is the activation of CRLs (cullin-RING ubiquitin ligases) by its conjugation to a conserved C-terminal lysine residue on cullin proteins. The attachment of UBLs requires three UBL-specific enzymes, termed E1, E2 and E3, which are usually well insulated from parallel UBL pathways. In the present study, we report a new mode of NEDD8 conjugation (NEDDylation) whereby the UBL NEDD8 is linked to proteins by ubiquitin enzymes in vivo. We found that this atypical NEDDylation is independent of classical NEDD8 enzymes, conserved from yeast to mammals, and triggered by an increase in the NEDD8 to ubiquitin ratio. In cells, NEDD8 overexpression leads to this type of NEDDylation by increasing the concentration of NEDD8, whereas proteasome inhibition has the same effect by depleting free ubiquitin. We show that bortezomib, a proteasome inhibitor used in cancer therapy, triggers atypical NEDDylation in tissue culture, which suggests that a similar process may occur in patients receiving this treatment.

In ubiquitin-like protein (UBL) cascades, a thioester-linked E2∼UBL complex typically interacts with an E3 enzyme for UBL transfer to the target. Here we demonstrate a variant mechanism, whereby the E2 Ubc12 functions with two E3s, Hrt1 and Dcn1, for ligation of the UBL Rub1 to Cdc53's WHB subdomain. Hrt1 functions like a conventional RING E3, with its N terminus recruiting Cdc53 and C-terminal RING activating Ubc12∼Rub1. Dcn1's "potentiating neddylation" domain (Dcn1(P)) acts as an additional E3, reducing nonspecific Hrt1-mediated Ubc12∼Rub1 discharge and directing Ubc12's active site to Cdc53. Crystal structures of Dcn1(P)-Cdc53(WHB) and Ubc12 allow modeling of a catalytic complex, supported by mutational data. We propose that Dcn1's interactions with both Cdc53 and Ubc12 would restrict the otherwise flexible Hrt1 RING-bound Ubc12∼Rub1 to a catalytically competent orientation. Our data reveal mechanisms by which two E3s function synergistically to promote UBL transfer from one E2 to a target.

Cullin-based E3 ubiquitin ligases are activated through covalent modification of the cullin subunit by the ubiquitin-like protein Nedd8. Cullin neddylation dissociates the ligase assembly inhibitor Cand1, and promotes E2 recruitment and ubiquitin transfer by inducing a conformational change. Here, we have identified and characterized Lag2 as a likely Saccharomyces cerevisiae orthologue of mammalian Cand1. Similar to Cand1, Lag2 directly interacts with non-neddylated yeast cullin Cdc53 and prevents its neddylation in vivo and in vitro. Binding occurs through a conserved C-terminal beta-hairpin structure that inserts into the Skp1-binding pocket on the cullin, and an N-terminal motif that covers the neddylation lysine. Interestingly, Lag2 is itself neddylated in vivo on a lysine adjacent to this N-terminal-binding site. Overexpression of Lag2 inhibits Cdc53 activity in strains defective for Skp1 or neddylation functions, implying that these activities are important to counteract Lag2 in vivo. Our results favour a model in which binding of substrate-specific adaptors triggers release of Cand1/Lag2, whereas subsequent neddylation of the cullin facilitates the removal and prevents re-association of Lag2/Cand1.

Cullin-based E3 ubiquitin ligases are activated through modification of the cullin subunit with the ubiquitin-like protein Nedd8. Dcn1 regulates cullin neddylation and thus ubiquitin ligase activity. Here we describe the 1.9 A X-ray crystal structure of yeast Dcn1 encompassing an N-terminal ubiquitin-binding (UBA) domain and a C-terminal domain of unique architecture, which we termed PONY domain. A conserved surface on Dcn1 is required for direct binding to cullins and for neddylation. The reciprocal binding site for Dcn1 on Cdc53 is located approximately 18 A from the site of neddylation. Dcn1 does not require cysteine residues for catalytic function, and directly interacts with the Nedd8 E2 Ubc12 on a surface that overlaps with the E1-binding site. We show that Dcn1 is necessary and sufficient for cullin neddylation in a purified recombinant system. Taken together, these data demonstrate that Dcn1 is a scaffold-like E3 ligase for cullin neddylation.

Cullin-RING ligases (CRL) are ubiquitin E3 enzymes that bind substrates through variable substrate receptor proteins and are activated by attachment of the ubiquitin-like protein NEDD8 to the cullin subunit. DCNs are NEDD8 E3 ligases that promote neddylation. Mammalian cells express five DCN-like (DCNL) proteins but little is known about their specific functions or interaction partners. We found that DCNLs form stable stoichiometric complexes with CAND1 and cullins that can only be neddylated in the presence of a substrate adaptor. These CAND–cullin–DCNL complexes might represent ‘reserve’ CRLs that can be rapidly activated when needed. We further found that all DCNLs interact with most cullin subtypes, but that they are probably responsible for the neddylation of different subpopulations of any given cullin. This is consistent with the fact that the subcellular localization of DCNLs in tissue culture cells differs and that they show unique tissue-specific expression patterns in mice. Thus, the specificity between DCNL-type NEDD8 E3 enzymes and their cullin substrates is only apparent in well-defined physiological contexts and related to their subcellular distribution and restricted expression., Highlighted Article: The mammalian DCN-type NEDD8 E3 ligases (DCNL1–5) have overlapping and unique functions that might be related to their subcellular distribution and are mostly governed by their unique N-termini.

The DNA damage response is vigorously activated by DNA double-strand breaks (DSBs). The chief mobilizer of the DSB response is the ATM protein kinase. We discovered that the COP9 signalosome (CSN) is a crucial player in the DSB response and an ATM target. CSN is a protein complex that regulates the activity of cullin ring ubiquitin ligase (CRL) complexes by removing the ubiquitin-like protein, NEDD8, from their cullin scaffold. We find that the CSN is physically recruited to DSB sites in a neddylation-dependent manner, and is required for timely repair of DSBs, affecting the balance between the two major DSB repair pathways—nonhomologous end-joining and homologous recombination repair (HRR). The CSN is essential for the processivity of deep end-resection—the initial step in HRR. Cullin 4a (CUL4A) is recruited to DSB sites in a CSN- and neddylation-dependent manner, suggesting that CSN partners with CRL4 in this pathway. Furthermore, we found that ATM-mediated phosphorylation of CSN subunit 3 on S410 is critical for proper DSB repair, and that loss of this phosphorylation site alone is sufficient to cause a DDR deficiency phenotype in the mouse. This novel branch of the DSB response thus significantly affects genome stability. España Ministerio de Economía y Competitividad SAF2013-43255-P

Accurate and complete DNA replication is fundamental to maintain genome integrity. While the mechanisms and underlying machinery required to duplicate bulk genomic DNA are beginning to emerge, little is known about how cells replicate through damaged areas and special chromosomal regions such as telomeres, centromeres, and highly transcribed loci . Here, we have investigated the role of the yeast cullin Rtt101p in this process. We show that rtt101Delta cells accumulate spontaneous DNA damage and exhibit a G(2)/M delay, even though they are fully proficient to detect and repair chromosome breaks. Viability of rtt101Delta mutants depends on Rrm3p, a DNA helicase involved in displacing proteinaceous complexes at programmed pause sites . Moreover, rtt101Delta cells show hyperrecombination at forks arrested at replication fork barriers (RFBs) of ribosomal DNA. Finally, rtt101Delta mutants are sensitive to fork arrest induced by DNA alkylation, but not by nucleotide depletion. We therefore propose that the cullin Rtt101p promotes fork progression through obstacles such as DNA lesions or tightly bound protein-DNA complexes via a new mechanism involving ubiquitin-conjugation.

The ubiquitin protein conjugation system tags proteins with the small polypeptide ubiquitin. Most poly-ubiquitinated proteins are recognized and degraded by the proteasome, a large multi-subunit protease. Ubiquitin-dependent protein degradation is used as a regulatory tool for many essential processes, the best studied of which is eukaryotic cell cycle progression. More recently, genetic studies in C. elegans have identified multiple roles for the ubiquitin system in early development, where ubiquitin-dependent protein degradation governs such diverse events as passage through meiosis, cytoskeletal regulation and cell fate determination.

SCF-type E3 ubiquitin ligases are multi-protein complexes required for polyubiquitination and subsequent degradation of target proteins by the 26S proteasome. Cullins, together with the RING-finger protein Rbx1, form the catalytic core of the ligase, and recruit the substrate-recognition module. Cycles of covalent modification of cullins by the ubiquitin-like molecule Nedd8 (neddylation) and removal of Nedd8 by the COP9 signalosome (deneddylation) positively regulate E3 ligase activity. Here we report the identification and analysis of a widely conserved protein that is required for cullin neddylation in the nematode Caenorhabditis elegans and the yeast Saccharomyces cerevisiae. C. elegans DCN-1 and S. cerevisiae Dcn1p (defective in cullin neddylation) are characterized by a novel UBA-like ubiquitin-binding domain and a DUF298 domain of unknown function. Consistent with their requirements for neddylation, DCN-1 and Dcn1p directly bind Nedd8 and physically associate with cullins in both species. Moreover, overexpression of Dcn1p in yeast results in the accumulation of Nedd8-modified cullin Cdc53p. Both in vivo and in vitro experiments indicate that Dcn1p does not inhibit deneddylation of Cdc53p by the COP9 signalosome, but greatly increases the kinetics of the neddylation reaction.

Many biological processes, such as development and cell cycle progression are tightly controlled by selective ubiquitin-dependent degradation of key substrates. In this pathway, the E3-ligase recognizes the substrate and targets it for degradation by the 26S proteasome. The SCF (Skp1–Cul1–F-box) and ECS (Elongin C–Cul2–SOCS box) complexes are two well-defined cullin-based E3-ligases1, 2, 3. The cullin subunits serve a scaffolding function and interact through their C terminus with the RING-finger-containing protein Hrt1/Roc1/Rbx1, and through their N terminus with Skp1 or Elongin C, respectively. In Caenorhabditis elegans, the ubiquitin-ligase activity of the CUL-3 complex is required for degradation of the microtubule-severing protein MEI-1/katanin at the meiosis-to-mitosis transition4. However, the molecular composition of this cullin-based E3-ligase is not known. Here we identified the BTB-containing protein MEL-26 as a component required for degradation of MEI-1 in vivo. Importantly, MEL-26 specifically interacts with CUL-3 and MEI-1 in vivo and in vitro, and displays properties of a substrate-specific adaptor. Our results suggest that BTB-containing proteins may generally function as substrate-specific adaptors in Cul3-based E3-ubiquitin ligases.

Background: SCF (Skp1-Cullin-F-box) complexes are a major class of E3 ligases that are required to selectively target substrates for ubiquitin-dependent degradation by the 26S proteasome. Conjugation of the ubiquitin-like protein Nedd8 to the cullin subunit (neddylation) positively regulates activity of SCF complexes, most likely by increasing their affinity for the E2 conjugated to ubiquitin. The Nedd8 conjugation pathway is required in C. elegans embryos for the ubiquitin-mediated degradation of the microtubule-severing protein MEI-1/Katanin at the meiosis-to-mitosis transition. Genetic experiments suggest that this pathway controls the activity of a CUL-3-based E3 ligase. Counteracting the Nedd8 pathway, the COP9/signalosome has been shown to promote deneddylation of the cullin subunit. However, little is known about the role of neddylation and deneddylation for E3 ligase activity in vivo.Results: Here, we identified and characterized the COP9/signalosome in C. elegans and showed that it promotes deneddylation of CUL-3, a critical target of the Nedd8 conjugation pathway. As in other species, the C. elegans signalosome is a macromolecular complex containing at least six subunits that localizes in the nucleus and the cytoplasm. Reducing COP9/signalosome function by RNAi results in a failure to degrade MEI-1, leading to severe defects in microtubule-dependent processes during the first mitotic division. Intriguingly, reducing COP9/signalosome function suppresses a partial defect in the neddylation pathway; this suppression suggests that deneddylation and neddylation antagonize each other.Conclusions: We conclude that both neddylation and deneddylation of CUL-3 is required for MEI-1 degradation and propose that cycles of CUL-3 neddylation and deneddylation are necessary for its ligase activity in vivo.

The Nedd8 ubiquitin-like protein modification pathway regulates cell-cycle progression. Our analysis of Nedd8 requirements during Caenorhabditis elegans embryogenesis indicates that the cytoskeleton is another target. Nedd8 conjugation negatively regulated contractility of the microfilament-rich cell cortex during pronuclear migration and again during cytokinesis. The Nedd8 pathway also was required after meiosis to negatively regulate katanin, a microtubule-severing complex, permitting the assembly of a large mitotic spindle. We propose that Nedd8-modified cullin, as part of an E3 ubiquitin ligase complex, targets katanin for degradation during the transition from meiosis to mitosis.

WNK1 [with no lysine (K)] and WNK4 regulate blood pressure by controlling the activity of ion co-transporters in the kidney. Groundbreaking work has revealed that the ubiquitylation and hence levels of WNK isoforms are controlled by a Cullin-RING E3 ubiquitin ligase complex (CRL3KLHL3) that utilizes CUL3 (Cullin3) and its substrate adaptor, KLHL3 (Kelch-like protein 3). Loss-of-function mutations in either CUL3 or KLHL3 cause the hereditary high blood pressure disease Gordon's syndrome by stabilizing WNK isoforms. KLHL3 binds to a highly conserved degron motif located within the C-terminal non-catalytic domain of WNK isoforms. This interaction is essential for ubiquitylation by CRL3KLHL3 and disease-causing mutations in WNK4 and KLHL3 exert their effects on blood pressure by disrupting this interaction. In the present study, we report on the crystal structure of the KLHL3 Kelch domain in complex with the WNK4 degron motif. This reveals an intricate web of interactions between conserved residues on the surface of the Kelch domain β-propeller and the WNK4 degron motif. Importantly, many of the disease-causing mutations inhibit binding by disrupting critical interface contacts. We also present the structure of the WNK4 degron motif in complex with KLHL2 that has also been reported to bind WNK4. This confirms that KLHL2 interacts with WNK kinases in a similar manner to KLHL3, but strikingly different to how another KLHL protein, KEAP1 (Kelch-like enoyl-CoA hydratase-associated protein 1), binds to its substrate NRF2 (nuclear factor-erythroid 2-related factor 2). The present study provides further insights into how Kelch-like adaptor proteins recognize their substrates and provides a structural basis for how mutations in WNK4 and KLHL3 lead to hypertension., WNK kinases regulate mammalian blood pressure. The level of WNK protein in a cell is regulated by the KLHL3–CUL3 ubiquitin ligase. We define the interaction between KLHL3 and WNK, identifying the WNK degron, and present the crystal structure of the KLHL3–WNK degron complex.

International audience; Cullin-RING ligases (CRLs) are ubiquitin E3 enzymes with variable substrate-adaptor and -receptor subunits. All CRLs are activated by modification of the cullin subunit with the ubiquitin-like protein Nedd8 (neddylation). The protein CAND1 (Cullin-associated-Nedd8-dissociated-1) also promotes CRL activity, even though it only interacts with inactive ligase complexes. The molecular mechanism underlying this behaviour remains largely unclear. Here, we find that yeast SCF (Skp1-Cdc53-F-box) Cullin-RING complexes are remodelled in a CAND1-dependent manner, when cells are switched from growth in fermentable to non-fermentable carbon sources. Mechanistically, CAND1 promotes substrate adaptor release following SCF deneddylation by the COP9 signalosome (CSN). CSN- or CAND1-mutant cells fail to release substrate adaptors. This delays the formation of new complexes during SCF reactivation and results in substrate degradation defects. Our results shed light on how CAND1 regulates CRL activity and demonstrate that the cullin neddylation-deneddylation cycle is not only required to activate CRLs, but also to regulate substrate specificity through dynamic substrate adaptor exchange.

Ubiquitin and ubiquitin-like proteins use unique E1, E2, and E3 enzymes for conjugation to their substrates. We and others have recently reported that increases in the relative concentration of the ubiquitin-like protein NEDD8 over ubiquitin lead to activation of NEDD8 by the ubiquitin E1 enzyme. We now show that this results in erroneous conjugation of NEDD8 to ubiquitin substrates, such as p53, Caspase 7, and Hif1α, demonstrating that overexpression of NEDD8 is not appropriate for identification of substrates of the NEDD8 pathway.

International audience; Cullin proteins are scaffolds for the assembly of multisubunit ubiquitin ligases, which ubiquitylate a large number of proteins involved in widely varying cellular functions. Multiple mechanisms cooperate to regulate cullin activity, including neddylation of their C-terminal domain. Interestingly, we found that the yeast Cul4-type cullin Rtt101 is not only neddylated but also ubiquitylated, and both modifications promote Rtt101 function in vivo. Surprisingly, proper modification of Rtt101 neither correlated with catalytic activity of the RING domain of Hrt1 nor required the Nedd8 ligase Dcn1. Instead, ubiquitylation of Rtt101 was dependent on the ubiquitin-conjugating enzyme Ubc4, while efficient neddylation involves the RING domain protein Tfb3, a subunit of the transcription factor TFIIH. Tfb3 also controls Cul3 neddylation and activity in vivo, and physically interacts with Ubc4 and the Nedd8-conjugating enzyme Ubc12 and the Hrt1/Rtt101 complex. Together, these results suggest that the conserved RING domain protein Tfb3 controls activation of a subset of cullins.

Cullin (Cul)-based E3 ubiquitin ligases are activated through the attachment of Nedd8 to the Cul protein. In yeast, Dcn1 (defective in Cul neddylation 1 protein) functions as a scaffold-like Nedd8 E3-ligase by interacting with its Cul substrates and the Nedd8 E2 Ubc12. Human cells express 5 Dcn1-like (DCNL) proteins each containing a C-terminal potentiating neddylation domain but distinct amino-terminal extensions. Although the UBA-containing DCNL1 and DCNL2 are likely functional homologues of yeast Dcn1, DCNL3 also interacts with human Culs and is able to complement the neddylation defect of yeast dcn1 Δ cells. DCNL3 down-regulation by RNAi decreases Cul neddylation, and overexpression of a Cul3 mutant deficient in DCNL3 binding interferes with Cul3 function in vivo. Interestingly, DCNL3 accumulates at the plasma membrane through a conserved, lipid-modified motif at the N terminus. Membrane-bound DCNL3 is able to recruit Cul3 to membranes and is functionally important for Cul3 neddylation in vivo. We conclude that DCNL proteins function as nonredundant Cul Nedd8-E3 ligases. Moreover, the diversification of the N termini in mammalian Dcn1 homologues may contribute to substrate specificity by regulating their subcellular localization.

Penicillin-binding protein 5 (PBP5), a product of the Escherichia coli gene dacA, possesses some β-lactamase activity. On binding to penicillin or related antibiotics via an ester bond, it deacylates and destroys them functionally by opening the β-lactam ring. This process takes several minutes. We exploited this process and showed that a fragment of PBP5 can be used as a reversible and monomeric affinity tag. At ambient temperature (e.g., 22°C), a PBP5 fragment binds rapidly and specifically to ampicillin Sepharose. Release can be facilitated either by eluting with 10mM ampicillin or in a ligand-free manner by incubation in the cold (1–10°C) in the presence of 5% glycerol. The “Dac-tag”, named with reference to the gene dacA, allows the isolation of remarkably pure fusion protein from a wide variety of expression systems, including (in particular) eukaryotic expression systems.