The evolutionary rewiring of ubiquitination targets has reprogrammed the regulation of carbon assimilation in the pathogenic yeast Candida albicans

Date of Acceptance: 13/11/2012 This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Correction for Sandai et al., The Evolutionary Rewiring of Ubiquitination Targets Has Reprogrammed the Regulation of Carbon Assimilation in the Pathogenic Yeast Candida albicans published 20-01-2015 DOI: 10.1128/mBio.02489-14Peer reviewedPublisher PD

Syntaphilin Ubiquitination Regulates Mitochondrial Dynamics and Tumor Cell Movements.

Syntaphilin (SNPH) inhibits the movement of mitochondria in tumor cells, preventing their accumulation at the cortical cytoskeleton and limiting the bioenergetics of cell motility and invasion. Although this may suppress metastasis, the regulation of the SNPH pathway is not well understood. Using a global proteomics screen, we show that SNPH associates with multiple regulators of ubiquitin-dependent responses and is ubiquitinated by the E3 ligase CHIP (or STUB1) on Lys111 and Lys153 in the microtubule-binding domain. SNPH ubiquitination did not result in protein degradation, but instead anchored SNPH on tubulin to inhibit mitochondrial motility and cycles of organelle fusion and fission, that is dynamics. Expression of ubiquitination-defective SNPH mutant Lys111!Arg or Lys153!Arg increased the speed and distance traveled by mitochondria, repositioned mitochondria to the cortical cytoskeleton, and supported heightened tumor chemotaxis, invasion, and metastasis in vivo. Interference with SNPH ubiquitination activated mitochondrial dynamics, resulting in increased recruitment of the fission regulator dynamin-related protein-1 (Drp1) to mitochondria and Drp1-dependent tumor cell motility. These data uncover nondegradative ubiquitination of SNPH as a key regulator of mitochondrial trafficking and tumor cell motility and invasion. In this way, SNPH may function as a unique, ubiquitination-regulated suppressor of metastasis

Hepatic cytochromes P450: structural degrons and barcodes, posttranslational modifications and cellular adapters in the ERAD-endgame.

The endoplasmic reticulum (ER)-anchored hepatic cytochromes P450 (P450s) are enzymes that metabolize endo- and xenobiotics i.e. drugs, carcinogens, toxins, natural and chemical products. These agents modulate liver P450 content through increased synthesis or reduction via inactivation and/or proteolytic degradation, resulting in clinically significant drug-drug interactions. P450 proteolytic degradation occurs via ER-associated degradation (ERAD) involving either of two distinct routes: Ubiquitin (Ub)-dependent 26S proteasomal degradation (ERAD/UPD) or autophagic lysosomal degradation (ERAD/ALD). CYP3A4, the major human liver/intestinal P450, and the fast-turnover CYP2E1 species are degraded via ERAD/UPD entailing multisite protein phosphorylation and subsequent ubiquitination by gp78 and CHIP E3 Ub-ligases. We are gaining insight into the nature of the structural determinants involved in CYP3A4 and CYP2E1 molecular recognition in ERAD/UPD [i.e. K48-linked polyUb chains and linear and/or "conformational" phosphodegrons consisting either of consecutive sequences on surface loops and/or disordered regions, or structurally-assembled surface clusters of negatively charged acidic (Asp/Glu) and phosphorylated (Ser/Thr) residues, within or vicinal to which, Lys-residues are targeted for ubiquitination]. Structural inspection of select human liver P450s reveals that such linear or conformational phosphodegrons may indeed be a common P450-ERAD/UPD feature. By contrast, although many P450s such as the slow-turnover CYP2E1 species and rat liver CYP2B1 and CYP2C11 are degraded via ERAD/ALD, little is known about the mechanism of their ALD-targeting. On the basis of our current knowledge of ALD-substrate targeting, we propose a tripartite conjunction of K63-linked Ub-chains, P450 structural "LIR" motifs and selective cellular "cargo receptors" as plausible P450-ALD determinants

Selective targeting of activating and inhibitory Smads by distinct WWP2 ubiquitin ligase isoforms differentially modulates TGFβ signalling and EMT

Ubiquitin-dependent mechanisms have emerged as essential regulatory elements controlling cellular levels of Smads and TGFß-dependent biological outputs such as epithelial–mesenchymal transition (EMT). In this study, we identify a HECT E3 ubiquitin ligase known as WWP2 (Full-length WWP2-FL), together with two WWP2 isoforms (N-terminal, WWP2-N; C-terminal WWP2-C), as novel Smad-binding partners. We show that WWP2-FL interacts exclusively with Smad2, Smad3 and Smad7 in the TGFß pathway. Interestingly, the WWP2-N isoform interacts with Smad2 and Smad3, whereas WWP2-C interacts only with Smad7. In addition, WWP2-FL and WWP2-C have a preference for Smad7 based on protein turnover and ubiquitination studies. Unexpectedly, we also find that WWP2-N, which lacks the HECT ubiquitin ligase domain, can also interact with WWP2-FL in a TGFß-regulated manner and activate endogenous WWP2 ubiquitin ligase activity causing degradation of unstimulated Smad2 and Smad3. Consistent with our protein interaction data, overexpression and knockdown approaches reveal that WWP2 isoforms differentially modulate TGFß-dependent transcription and EMT. Finally, we show that selective disruption of WWP2 interactions with inhibitory Smad7 can stabilise Smad7 protein levels and prevent TGFß-induced EMT. Collectively, our data suggest that WWP2-N can stimulate WWP2-FL leading to increased activity against unstimulated Smad2 and Smad3, and that Smad7 is a preferred substrate for WWP2-FL and WWP2-C following prolonged TGFß stimulation. Significantly, this is the first report of an interdependent biological role for distinct HECT E3 ubiquitin ligase isoforms, and highlights an entirely novel regulatory paradigm that selectively limits the level of inhibitory and activating Smads

BRCA1 is a histone-H2A-specific ubiquitin ligase

The RING domain proteins BRCA1 and BARD1 comprise a heterodimeric ubiquitin (E3) ligase that is required for the accumulation of ubiquitin conjugates at sites of DNA damage and for silencing at DNA satellite repeat regions. Despite its links to chromatin, the substrate and underlying function of the BRCA1/BARD1 ubiquitin ligase remain unclear. Here, we show that BRCA1/BARD1 specifically ubiquitylates histone H2A in its C-terminal tail on lysines 127 and 129 in vitro and in vivo. The specificity for K127-129 is acquired only when H2A is within a nucleosomal context. Moreover, site-specific targeting of the BRCA1/BARD1 RING domains to chromatin is sufficient for H2Aub foci formation in vivo. Our data establish BRCA1/BARD1 as a histone-H2A-specific E3 ligase, helping to explain its localization and activities on chromatin in cells

Sequential primed kinases create a damage-responsive phosphodegron on Eco1.

Sister-chromatid cohesion is established during S phase when Eco1 acetylates cohesin. In budding yeast, Eco1 activity falls after S phase due to Cdk1-dependent phosphorylation, which triggers ubiquitination by SCF(Cdc4). We show here that Eco1 degradation requires the sequential actions of Cdk1 and two additional kinases, Cdc7-Dbf4 and the GSK-3 homolog Mck1. These kinases recognize motifs primed by previous phosphorylation, resulting in an ordered sequence of three phosphorylation events on Eco1. Only the latter two phosphorylation sites are spaced correctly to bind Cdc4, resulting in strict discrimination between phosphates added by Cdk1 and by Cdc7. Inhibition of Cdc7 by the DNA damage response prevents Eco1 destruction, allowing establishment of cohesion after S phase. This elaborate regulatory system, involving three independent kinases and stringent substrate selection by a ubiquitin ligase, enables robust control of cohesion establishment during normal growth and after stress

Structural insight into SUMO chain recognition and manipulation by the ubiquitin ligase RNF4

The small ubiquitin-like modifier (SUMO) can form polymeric chains that are important signals in cellular processes such as meiosis, genome maintenance and stress response. The SUMO-targeted ubiquitin ligase RNF4 engages with SUMO chains on linked substrates and catalyses their ubiquitination, which targets substrates for proteasomal degradation. Here we use a segmental labelling approach combined with solution nuclear magnetic resonance (NMR) spectroscopy and biochemical characterization to reveal how RNF4 manipulates the conformation of the SUMO chain, thereby facilitating optimal delivery of the distal SUMO domain for ubiquitin transfer

Ubiquitination site preferences in anaphase promoting complex/cyclosome (APC/C) substrates.

Ordered progression of mitosis requires precise control in abundance of mitotic regulators. The anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase plays a key role by directing ubiquitin-mediated destruction of targets in a temporally and spatially defined manner. Specificity in APC/C targeting is conferred through recognition of substrate D-box and KEN degrons, while the specificity of ubiquitination sites, as another possible regulated dimension, has not yet been explored. Here, we present the first analysis of ubiquitination sites in the APC/C substrate ubiquitome. We show that KEN is a preferred ubiquitin acceptor in APC/C substrates and that acceptor sites are enriched in predicted disordered regions and flanked by serine residues. Our experimental data confirm a role for the KEN lysine as an ubiquitin acceptor contributing to substrate destruction during mitotic progression. Using Aurora A and Nek2 kinases as examples, we show that phosphorylation on the flanking serine residue could directly regulate ubiquitination and subsequent degradation of substrates. We propose a novel layer of regulation in substrate ubiquitination, via phosphorylation adjacent to the KEN motif, in APC/C-mediated targeting