The replication of viruses depends on the cell cycle status of the infected cells. Viruses have evolved functions that alleviate restrictions imposed on their replication by the host. Vpr, an accessory factor of primate lentiviruses, arrests cells at the DNA damage checkpoint in G 2 phase of the cell cycle, but the mechanism underlying this effect has remained elusive. Here we report that Vpr proteins of both the human (HIV-1) and the distantly related simian (

Isopeptidases are essential regulators of protein ubiquitination and sumoylation. However, only two families of SUMO isopeptidases are at present known. Here, we report an activity-based search with the suicide inhibitor haemagglutinin (HA)-SUMOvinylmethylester that led to the identification of a surprising new SUMO protease, ubiquitin-specific protease-like 1 (USPL1). Indeed, USPL1 neither binds nor cleaves ubiquitin, but is a potent SUMO isopeptidase both in vitro and in cells. C13orf22l-an essential but distant zebrafish homologue of USPL1-also acts on SUMO, indicating functional conservation. We have identified invariant USPL1 residues required for SUMO binding and cleavage. USPL1 is a low-abundance protein that colocalizes with coilin in Cajal bodies. Its depletion does not affect global sumoylation, but causes striking coilin mislocalization and impairs cell proliferation, functions that are not dependent on USPL1 catalytic activity. Thus, USPL1 represents a third type of SUMO protease, with essential functions in Cajal body biology.Keywords: SUMO protease; ubiquitin-specific protease family; USPL1; zebrafish C13orf22l; Cajal body EMBO reports (2012) 13, 930-938.

To measure rates of sumoylation and isopeptidase cleavage in vitro, we developed an enzyme assay that is based on fluorescence resonance energy transfer (FRET). FRET is a process by which the excited state energy of a fluorescent donor molecule is transferred to an acceptor molecule. Efficient energy transfer requires very close proximity, and can therefore be used as a read-out for covalent and non-covalent protein interactions. The assay described here uses bacterially expressed and purified YFP-SUMO-1 and CFP-RanGAP1 as model substrates that are covalently coupled in the presence of recombinant SUMO E1 and E2 enzymes and ATP. Reactions of 25 microl volume, set up in 384-wells plates, give sufficient signal for analysis. Consequently, this assay requires very low amounts of recombinant proteins and allows measurement of time courses in high-throughput format.

The first person that I would like to acknowledge is my advisor Prof. Frauke Melchior. Without her, this entire project would have never happened. She invited me to her laboratory, inspired and guided me along the way. She was a demanding, patient and helpful mentor. She has been optimistic and encouraging, not avoiding criticism. I am truly grateful for her help and support.I would like to express my gratitude to the members of my doctoral committee Prof. Gerhard Braus and Prof. Detlef Doenecke for the inspiring discussions and support.I am truly grateful to Dr. Steffen Burkhardt for his help and involvement. This thesis is a result of collaboration and help of many people. I'd like to thank them all for this. Prof. Erich Wanker for providing the library, Dr. Reinert Hitt for performing the screen, Dr. Huib Ovaa for the vinylmethylester, Dr. Henning Urlaub for the MS anlysis and Dr. Kai Hoffman the for bioinformatics analysis I am grateful to Dr. Ralp Kehlenbach for discussions and reagents.A big part of this work is based on the labeling technology that Dr. Erik Meulmeester introduced me to. I would like to thank him for that as well as for fruitful discussion and his help during this project.Dr. Ruth Geiss-Friedlander has been very encouraging throughout this project, but not avoiding criticism. This allowed me seeing things from a different angle, which was very important. She has also been great friend. I am very grateful to her.The time at the laboratory would have not been the same without my labmates: