Structural and Functional Characterization of the SUMO Proteases SENP6 and SENP7 Kamela Olivya Alegre Universitat Autonoma de Barcelona 1 “There are two ways you can live your life. One is as though nothing is a miracle. The other is as though everything is a miracle.” Albert Einstein 2 3 Structural and Fumctional Analysis of the SUMO Proteases SENP6 and SENP7 Doctoral thesis presented by Kamela Olivya Alegre for the degree of PhD in Biochemistry from the University Autonomous of Barcelona Institute of Biotecnology and Biomedicine, Protein Structure laboratory. Thesis supervised by Dr. David Reverter. 4 Kamela Alegre David Reverter 5 INDEX Abbreviations ............................................................................................................................................ 1-2 General Introduction .............................................................................................................................. 3-44 The Ubiquitin System: Ubiquitin and Ubiquitin-like Proteins ...............................................................5 Introduction to ubiquitin ................................................................................................................5 Mechanism and enzyme cascade ....................................................................................7 Ubiquitin conjugation machinery ...................................................................................9 Introduction to SUMO ..................................................................................................................13 SUMO conjugation machinery ......................................................................................16 SUMO isoforms ...............................................................................................................19 Cellular functions ............................................................................................................19 Lysine-linked chains .......................................................................................................23 Introduction to Ubiquitin and SUMO Isopeptidases ...............................................................27 The SENPs – SENtrin-specific family of Proteases ..................................................................................... 34 SENP1 and SENP2 .........................................................................................................................34 SENP3 and SENP5 .........................................................................................................................40 SENP6 and SENP7 .........................................................................................................................42 Objectives .....................................................................................................................................................47 Experimental Procedures ...........................................................................................................................51 Protein production and purification .........................................................................................................51 Protein constructs ..........................................................................................................................52 SENP7 .............................................................................................................................. 52 SENP2 .............................................................................................................................. 52 SENP6 ...............................................................................................................................53 6 SUMO .............................................................................................................................. 55 RanGAP1 ..........................................................................................................................56 SUMOylation reaction ..................................................................................................................57 RanGAP1-SUMO1 and RanGAP1-SUMO2 ................................................................ 57 diSUMO2 and diSUMO2/1 ...........................................................................................57 Biochemical assays .......................................................................................................................................58 C-terminal hydrolase activity ......................................................................................................58 Deconjugation activity ..................................................................................................................59 Initial rate velocities ......................................................................................................................60 Michaelis-Menten steady-state kinetics .....................................................................................60 Loop3 characterization ................................................................................................................................61 NMR ................................................................................................................................................61 Circular dichroism .........................................................................................................................61 Infrared spectral analysis .............................................................................................................61 Limited proteolysis .......................................................................................................................62 N-terminal sequencing .................................................................................................................62 Mass spectromer analysis .............................................................................................................62 Complex preparation ..................................................................................................................................62 Complex formation/purification via gel filtration .................................................................................63 Results ...........................................................................................................................................................67 Swapping the SUMO isofrom specificity of SENP6/7 ............................................................67 SENP6 is SUMO2/3-specific and inefficient at SUMO processing .........................67 Model for SENP6/7 interaction with SUMO2/3 and identification of interface residues ..............................................................................................69 Swapping SUMO1 and SUMO2 residues in processing reaction ..............71 Swapping SUMO1 and SUMO2 residues in RanGAP1-SUMO in deconjuation reaction .......................................................................................72 7 Swapping SUMO1 and SUMO2 residues in diSUMO2 in deconjugation reaction ...............................................................................................................74 SENP6 loop deletion mutants .........................................................................75 Steady-state kinetic analysis of SUMO1 mutants ........................................79 Loop1 SENP6/7 is responsible for SUMO interface specificity .............................................82 Structure of Loop1 ............................................................................................82 Mutagenic analysis of SENP7 Loop1 .............................................................83 SENP7 Loop1 interaction with SUMO2 ........................................................86 SENP2 Loop1 insertion is highly reactive against diSUMO2 ....................88 Complexes with substrates ..........................................................................................................90 Processing complexes ......................................................................................90 Deconjugation complexes ................................................................................91 SENP63CS forms a stable complex with diSUMO2 ..................................91 SENP623CS forms a stable complex with diSUMO2 ..............................93 SENP2CS forms a stable complex with diSUMO2 ......................................95 Characterization of SENP6 Loop3 ..................................................................97 DISCUSSION ............................................................................................................................................107 BIBLIOGRAPHY ......................................................................................................................................125 CONCLUSIONS .......................................................................................................................................141 APPENDIX .................................................................................................................................................143 8 Alegre, K. O., Reverter, D. (2011). “Swapping the Small Ubiquitin-like Modifier (SUMO) Isoform Specificity of SUMO Proteases SENP6 and SENP7” J Biol Chem 41, 36142-51. ....145 Summary SENP Activities .........................................................................................................157