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
