The small ubiquitin related modifier SUMO is a posttranslational modifier that functions in a wide range of cellular processes like intracellular transport, cell cycle regulation, DNA repair and regulation of transcription. SUMO is an 11 kDa protein and is ligated to its target proteins using a mechanism involving three regulated enzymatic steps that are catalyzed by an E1, E2 and an E3 enzyme. Since many crucial biological processes are regulated by SUMO modification, it is evident that the SUMO pathway can play a role in disease as well. In fact, a role for SUMO modification has been implicated in cancer, neurodegenerative diseases and viral infection. The work in this thesis provides insight into the molecular mechanism of sumoylation by analysis of SUMO modification using a combination of biochemistry and structural biology. The E2 enzyme is a central player in the sumoylation cascade since it accepts SUMO from the E1 enzyme and transfers it to the target. Surprisingly, we found that the SUMO protein itself can regulate the function of the E2 enzyme, Ubc9, in two different ways. The noncovalent interaction of SUMO with Ubc9 is important for the formation of SUMO chains possibly through a mechanism that is also used in K63-linked ubiquitin chain formation. The covalent complex of SUMO and Ubc9 is structurally and functionally different and is involved in target discrimination, some targets are enhanced in SUMO modification while others are inhibited in modification. Generally, an E3 enzyme is required for enhancement of sumoylation, one of these SUMO E3 enzymes is the nucleoporin RanBP2. We have studied the catalytic mechanism of RanPB2, and shown that it accelerates SUMO transfer by an allosteric mechanism. Finally we have studied the consequences of sumoylation of a newly identified SUMO target, the ubiquitin conjugating enzyme E2-25K. Here sumoylation inhibits the basic enzymatic activity of the E2. Even though the ubiquitin E2 enzyme E2-25K is similar to the SUMO E2 Ubc9, the effect of their sumoylation is completely different. In this thesis we describe various aspects of both regulation by sumoylation as well as the regulation of the process of sumoylation itself. We found how SUMO can regulate the process of sumoylation and determined the function of sumoylation of two distinct targets.

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NWO-MW (Nederlandse organisatie voor wetenschappelijk onderzoek - medische wetenschappen), NWO-Pionier, CBG (Centre for Biomedical Genetics), Rubicon Network of excellence (EU).
T.K. Sixma (Titia)
Erasmus University Rotterdam
Erasmus MC: University Medical Center Rotterdam