Characterisation of the Cullin-3 mutation that causes a severe form of familial hypertension and hyperkalaemia

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

A mechanism for the suppression of homologous recombination in G1 cells

DNA repair by homologous recombination (HR)(1) is highly suppressed in G1 cells(2,3) to ensure that mitotic recombination occurs solely between sister chromatids(4). Although many HR factors are cell cycle-regulated, the identity of the events that are both necessary and sufficient to suppress recombination in G1 cells is unknown. Here we report that the cell cycle controls the interaction of BRCA1 with PALB2-BRCA2 in order to constrain BRCA2 function to the S/G2 phases. We found that the BRCA1-interaction site on PALB2 is targeted by an E3 ubiquitin ligase composed of KEAP1, a PALB2-interacting protein(5), in complex with CUL3-RBX1(6). PALB2 ubiquitylation suppresses its interaction with BRCA1 and is counteracted by the deubiquitylase USP11, which is itself under cell cycle control. Restoration of the BRCA1-PALB2 interaction combined with the activation of DNA end resection is sufficient to induce HR in G1, as measured by RAD51 recruitment, unscheduled DNA synthesis and a CRISPR/Cas9-based gene targeting assay. We conclude that the mechanism prohibiting HR in G1 minimally consists of the suppression of DNA end resection coupled to a multi-step block to BRCA2 recruitment to DNA damage sites that involves the inhibition of BRCA1-PALB2-BRCA2 complex assembly. We speculate that the ability to induce HR in G1 cells with defined factors could spur the development of gene targeting applications in non-dividing cells

Opposing effects of cancer-type-specific SPOP mutants on BET protein degradation and sensitivity to BET inhibitors.

It is generally assumed that recurrent mutations within a given cancer driver gene elicit similar drug responses. Cancer genome studies have identified recurrent but divergent missense mutations affecting the substrate-recognition domain of the ubiquitin ligase adaptor SPOP in endometrial and prostate cancers. The therapeutic implications of these mutations remain incompletely understood. Here we analyzed changes in the ubiquitin landscape induced by endometrial cancer-associated SPOP mutations and identified BRD2, BRD3 and BRD4 proteins (BETs) as SPOP-CUL3 substrates that are preferentially degraded by endometrial cancer-associated SPOP mutants. The resulting reduction of BET protein levels sensitized cancer cells to BET inhibitors. Conversely, prostate cancer-specific SPOP mutations resulted in impaired degradation of BETs, promoting their resistance to pharmacologic inhibition. These results uncover an oncogenomics paradox, whereby mutations mapping to the same domain evoke opposing drug susceptibilities. Specifically, we provide a molecular rationale for the use of BET inhibitors to treat patients with endometrial but not prostate cancer who harbor SPOP mutations

Structural Studies of the C0P9 Signalosome

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

INFLUENCE OF WHITE MATTER LESION FILLING ON VOLUMETRIC ASSESSMENT IN MULTIPLE SCLEROSIS

Background: The continuous progress of information technology has made possible the creation of tools for post processing of magnetic resonance and other imaging modalities, including software programmes aimed at volumetric studies of the brain. They have the potential to enrich visual data with precise numeric values but have to be used with caution because of their possible susceptibility to errors if scans with specific pathology are fed in. Objective: The purpose of the present study is to assess whether filling white matter lesions on magnetic resonance scans of multiple sclerosis patients would influence volumetric values. Methods: MS lesions were filled on T1 3D images of 49 patients by the lesion-filling algorithm of FSL, using previously created lesion masks. Volumes of brain grey and white matter, peripheral grey matter and ventricle CSF were calculated using SIENAX for the filled and non-filled series, which were then compared. Results: There were statistically significant differences for white matter volume before and after lesion filling (p<0.05). No other volumes were significantly different. Conclusion: Filling of white matter lesions may be time-consuming, but can improve the accuracy of SIENAX by reducing bias due to misidentification of tissue intensity. Sometimes though, improvement of specific values may not reach statistical significance

Cortical dynamics during cell motility are regulated by CRL3 KLHL21 E3 ubiquitin ligase

Directed cell movement involves spatial and temporal regulation of the cortical microtubule (Mt) and actin networks to allow focal adhesions (FAs) to assemble at the cell front and disassemble at the rear. Mts are known to associate with FAs, but the mechanisms coordinating their dynamic interactions remain unknown. Here we show that the CRL3KLHL21 E3 ubiquitin ligase promotes cell migration by controlling Mt and FA dynamics at the cell cortex. Indeed, KLHL21 localizes to FA structures preferentially at the leading edge, and in complex with Cul3, ubiquitylates EB1 within its microtubule-interacting CH-domain. Cells lacking CRL3KLHL21 activity or expressing a non-ubiquitylatable EB1 mutant protein are unable to migrate and exhibit strong defects in FA dynamics, lamellipodia formation and cortical plasticity. Our study thus reveals an important mechanism to regulate cortical dynamics during cell migration that involves ubiquitylation of EB1 at focal adhesions.ISSN:2041-172

Multilayered regulation of autophagy by the Atg1 kinase orchestrates spatial and temporal control of autophagosome formation

Autophagy is a conserved intracellular degradation pathway exerting various cytoprotective and homeostatic functions by using de novo double-membrane vesicle (autophagosome) formation to target a wide range of cytoplasmic material for vacuolar/lysosomal degradation. The Atg1 kinase is one of its key regulators, coordinating a complex signaling program to orchestrate autophagosome formation. Combining in vitro reconstitution and cell-based approaches, we demonstrate that Atg1 is activated by lipidated Atg8 (Atg8-PE), stimulating substrate phosphorylation along the growing autophagosomal membrane. Atg1-dependent phosphorylation of Atg13 triggers Atg1 complex dissociation, enabling rapid turnover of Atg1 complex subunits at the pre-autophagosomal structure (PAS). Moreover, Atg1 recruitment by Atg8-PE self-regulates Atg8-PE levels in the growing autophagosomal membrane by phosphorylating and thus inhibiting the Atg8-specific E2 and E3. Our work uncovers the molecular basis for positive and negative feedback imposed by Atg1 and how opposing phosphorylation and dephosphorylation events underlie the spatiotemporal regulation of autophagy

Structural Basis for a Reciprocal Regulation between SCF and CSN

Skp1-Cul1-Fbox (SCF) E3 ligases are activated by ligation to the ubiquitin-like protein Nedd8, which is reversed by the deneddylating Cop9 signalosome (CSN). However, CSN also promotes SCF substrate turnover through unknown mechanisms. Through biochemical and electron microscopy analyses, we determined molecular models of CSN complexes with SCFSkp2/Cks1 and SCFFbw7 and found that CSN occludes both SCF functional sites—the catalytic Rbx1-Cul1 C-terminal domain and the substrate receptor. Indeed, CSN binding prevents SCF interactions with E2 enzymes and a ubiquitination substrate, and it inhibits SCF-catalyzed ubiquitin chain formation independent of deneddylation. Importantly, CSN prevents neddylation of the bound cullin, unless binding of a ubiquitination substrate triggers SCF dissociation and neddylation. Taken together, the results provide a model for how reciprocal regulation sensitizes CSN to the SCF assembly state and inhibits a catalytically competent SCF until a ubiquitination substrate drives its own degradation by displacing CSN, thereby promoting cullin neddylation and substrate ubiquitination

Structural and Biochemical Characterization of the Cop9 Signalosome CSN5/CSN6 Heterodimer

The Cop9 signalosome complex (CSN) regulates the functional cycle of the major E3 ubiquitin ligase family, the cullin RING E3 ubiquitin ligases (CRLs). Activated CRLs are covalently modified by the ubiquitin-like protein Nedd8 (neural precursor cell expressed developmentally down-regulated protein 8). CSN serves an essential role in myriad cellular processes by reversing this modification through the isopeptidase activity of its CSN5 subunit. CSN5 alone is inactive due to an auto-inhibited conformation of its catalytic domain. Here we report the molecular basis of CSN5 catalytic domain activation and unravel a molecular hierarchy in CSN deneddylation activity. The association of CSN5 and CSN6 MPN (for Mpr1/Pad1 N-terminal) domains activates its isopeptidase activity. The CSN5/CSN6 module, however, is inefficient in CRL deneddylation, indicating a requirement of further elements in this reaction such as other CSN subunits. A hybrid molecular model of CSN5/CSN6 provides a structural framework to explain these functional observations. Docking this model into a published CSN electron density map and using distance constraints obtained from cross-linking coupled to mass-spectrometry, we find that the C-termini of the CSN subunits could form a helical bundle in the centre of the structure. They likely play a key scaffolding role in the spatial organization of CSN and precise positioning of the dimeric MPN catalytic core.ISSN:1932-620