The regulation of global deSUMOylation by human SUMO- specific proteases

SUMOylation - the covalent tagging of a target protein with a SUMO (small ubiquitin-related modifier) molecule - has recently emerged as an important post-translational modification in regulating a wide array of biological events. SUMOylation is a dynamics process, where SUMO- specific proteases (SENPs) process proSUMO molecules and also deconjugate them from substrates. My dissertation focuses on the deconjugating activity of SENPs and addresses the role they play in regulating the global SUMOylation "status quo" of eukaryotic cells. SENPs are among the most specific proteases and only cleave SUMO molecules, yet they exhibit substantial substrate specificity among SUMO paralogs. To address the deconjugating specificities of human SENPs, I developed two different proteomics strategies to profile these proteases. One method has the potential to identify lysines of SUMOylation, besides the SUMOylation substrate itself, while the other can differentiate SUMOylated substrates from SUMO-interactors. To this end, I demonstrated the proof-of-principle of both methods and have identified polySUMOylated substrates. Such polySUMOylation (ie: modification of proteins by SUMO chains) has been an elusive phenomenon to study. In fact, so far, mainly mass spectrometry has shed light on the nature of SUMO chains through identifying SUMO-SUMO isopeptides. Thus, in the bulk of my dissertation, using a conjugatable, but not deconjugatable SUMO, I demonstrate the abundance of SUMO chains in vivo, make them visually appreciable and uncover the rapid dynamics of SUMOylation through their deconjugation. I propose that the balance of the dynamics is largely due to the action of SENPs in constantly cleaving SUMO from its targets, whether from a target protein or from another SUMO. This way, SENPs guard the SUMOylation status of the proteome, which by definition includes keeping in check modification of substrates by SUMO chains. I also establish the mechanism of SUMO chain deconjugation, which I show to be stochastic. Lastly, I reiterate the importance of deSUMOylation in vivo, by showing that the lack of deSUMOylation is adversary for the growth of Schizosaccharomyces pombe under replicative stress and for human cell

The regulation of global deSUMOylation by human SUMO- specific proteases

SUMOylation - the covalent tagging of a target protein with a SUMO (small ubiquitin-related modifier) molecule - has recently emerged as an important post-translational modification in regulating a wide array of biological events. SUMOylation is a dynamics process, where SUMO- specific proteases (SENPs) process proSUMO molecules and also deconjugate them from substrates. My dissertation focuses on the deconjugating activity of SENPs and addresses the role they play in regulating the global SUMOylation "status quo" of eukaryotic cells. SENPs are among the most specific proteases and only cleave SUMO molecules, yet they exhibit substantial substrate specificity among SUMO paralogs. To address the deconjugating specificities of human SENPs, I developed two different proteomics strategies to profile these proteases. One method has the potential to identify lysines of SUMOylation, besides the SUMOylation substrate itself, while the other can differentiate SUMOylated substrates from SUMO-interactors. To this end, I demonstrated the proof-of-principle of both methods and have identified polySUMOylated substrates. Such polySUMOylation (ie: modification of proteins by SUMO chains) has been an elusive phenomenon to study. In fact, so far, mainly mass spectrometry has shed light on the nature of SUMO chains through identifying SUMO-SUMO isopeptides. Thus, in the bulk of my dissertation, using a conjugatable, but not deconjugatable SUMO, I demonstrate the abundance of SUMO chains in vivo, make them visually appreciable and uncover the rapid dynamics of SUMOylation through their deconjugation. I propose that the balance of the dynamics is largely due to the action of SENPs in constantly cleaving SUMO from its targets, whether from a target protein or from another SUMO. This way, SENPs guard the SUMOylation status of the proteome, which by definition includes keeping in check modification of substrates by SUMO chains. I also establish the mechanism of SUMO chain deconjugation, which I show to be stochastic. Lastly, I reiterate the importance of deSUMOylation in vivo, by showing that the lack of deSUMOylation is adversary for the growth of Schizosaccharomyces pombe under replicative stress and for human cell

Still standing: how European firms weathered the crisis - The third EFIGE policy report. Bruegel Blueprint 15, 22 December 2011

This research output confirms the strength of the approach underpinning the EFIGE project, which is based on the recognition that firms are heterogeneous in the extent and the pattern of their internationalisation, as they are in many other respects. The project provides more, and more precise, evidence of what makes firms successful and therefore also what makes countries successful in the context of globalisation. Internationalisation, however, also makes firms vulnerable to shocks affecting international trade and may transform them into agents of propagation of global downturns. At the time of the Great Recession of 2009, there was intense speculation about the reasons why trade collapsed much more than output. It was sometimes claimed that global supply chains were not only propagators, but also multipliers of international fluctuations. This report by László Halpern and his colleagues makes use of the fact that the EFIGE survey was – by accident – conducted in 2009 and – by design – included questions about the firms’ response to the global crisis. It provides a fascinating account of what happened to them in an especially turbulent environment. The stylised facts presented in this report are important to bear in mind at a time when Europe is heading for another severe downturn

The COP9 signalosome inhibits Cullin-RING E3 ubiquitin ligases independently of its deneddylase activity

The COP9 signalosome inhibits the activity of Cullin-RING E3 ubiquitin ligases by removing Nedd8 modifications from their Cullin subunits. Neddylation renders these complexes catalytically active, but deneddylation is also necessary for them to exchange adaptor subunits and avoid auto-ubiquitination. Although deneddylation is thought to be the primary function of the COP9 signalosome, additional activities have been ascribed to some of its subunits. We recently showed that COP9 subunits protect the transcriptional repressor and tumor suppressor Capicua from two distinct modes of degradation. Deneddylation by the COP9 signalosome inactivates a Cullin 1 complex that ubiquitinates Capicua following its phosphorylation by MAP kinase in response to Epidermal Growth Factor Receptor signaling. The CSN1b subunit also stabilizes unphosphorylated Capicua to control its basal level, independently of the deneddylase function of the complex. Here we further examine the importance of deneddylation for COP9 functions in vivo. We use an uncleavable form of Nedd8 to show that preventing deneddylation does not reproduce the effects of loss of COP9. In contrast, in the presence of COP9, conjugation to uncleavable Nedd8 renders Cullins unable to promote the degradation of their substrates. Our results suggest that irreversible neddylation prolongs COP9 binding to and inhibition of Cullin-based ubiquitin ligases

Deubiquitinases as a Signaling Target of Oxidative Stress

Deubiquitinating enzymes (DUBs) constitute a large family of cysteine proteases that have a broad impact on numerous biological and pathological processes, including the regulation of genomic stability. DUBs are often assembled onto multiprotein complexes to assist in their localization and substrate selection, yet it remains unclear how the enzymatic activity of DUBs is modulated by intracellular signals. Herein, we show that bursts of reactive oxygen species (ROS) reversibly inactivate DUBs through the oxidation of the catalytic cysteine residue. Importantly, USP1, a key regulator of genomic stability, is reversibly inactivated upon oxidative stress. This, in part, explains the rapid nature of PCNA monoubiquitination-dependent DNA damage tolerance in response to oxidative DNA damage in replicating cells. We propose that DUBs of the cysteine protease family act as ROS sensors in human cells and that ROS-mediated DUB inactivation is a critical mechanism for fine-tuning stress-activated signaling pathways

SENP8 limits aberrant neddylation of nedd8 pathway components to promote cullin-RING ubiquitin ligase function

NEDD8 is a ubiquitin-like modifier most well-studied for its role in activating the largest family of ubiquitin E3 ligases, the cullin-RING ligases (CRLs). While many non-cullin neddylation substrates have been proposed over the years, validation of true NEDD8 targets has been challenging, as overexpression of exogenous NEDD8 can trigger NEDD8 conjugation through the ubiquitylation machinery. Here, we developed a deconjugation-resistant form of NEDD8 to stabilize the neddylated form of cullins and other non-cullin substrates. Using this strategy, we identified Ubc12, a NEDD8-specific E2 conjugating enzyme, as a substrate for auto-neddylation. Furthermore, we characterized SENP8/DEN1 as the protease that counteracts Ubc12 autoneddylation, and observed aberrant neddylation of Ubc12 and other NEDD8 conjugation pathway components in SENP8-deficient cells. Importantly, loss of SENP8 function contributes to accumulation of CRL substrates and defective cell cycle progression. Thus, our study highlights the importance of SENP8 in maintaining proper neddylation levels for CRL-dependent proteostasis

DUB-Resistant Ubiquitin to Survey Ubiquitination Switches in Mammalian Cells

The ubiquitin-modification status of proteins in cells is highly dynamic and maintained by specific ligation machineries (E3 ligases) that tag proteins with ubiquitin or by deubiquitinating enzymes (DUBs) that remove the ubiquitin tag. The development of tools that offset this balance is critical in characterizing signaling pathways that utilize such ubiquitination switches. Herein, we generated a DUB-resistant ubiquitin mutant that is recalcitrant to cleavage by various families of DUBs both in vitro and in mammalian cells. As a proof-of-principle experiment, ectopic expression of the uncleavable ubiquitin stabilized monoubiquitinated PCNA in the absence of DNA damage and also revealed a defect in the clearance of the DNA damage response at unprotected telomeres. Importantly, a proteomic survey using the uncleavable ubiquitin identified ubiquitinated substrates, validating the DUB-resistant ubiquitin expression system as a valuable tool for interrogating cell signaling pathways