1. CBS-INSERM 2013 Montpellier, France Sophie Giguère Class of 2015

2. The Project… The COP9 signalosome (CSN) complex is a well-conserved eukaryotic protein complex that acts as a regulator in numerous metabolic processes, including gene expression, cell proliferation, cell cycle, and DNA repair CSN plays an important role in the regulation of the cullin- RING ligase (CRL) family of ubiquitin E3 ligases involved in the ubitiquitin-proteasome pathway: It functions as an isopeptidase, cleaving the post-translational Nedd8 modification from the cullin of the CRL ubiquitin ligase (a process known as deneddylation), deactivating the CRL complex

3. Within the COP9 signalosome, the MPN-containing CSN5 subunit is responsible for the catalytic metalloisopeptidase activity However, CSN5 has no isopeptidase activity when isolated from the COP9 holocomplex Prior to my arrival, the laboratory recently found a number of CSN5 variants, notably CSN5 R106T, that do in fact have catalytic isopeptidase activity when isolated from the CSN holocomplex In addition, the laboratory recently discovered that the COP9 signalosome – in addition to its known isopeptidase activity – also has peptidase activity, and, in vitro, is capable of processing the precursor of Nedd8, proNedd8, into its mature conjugable form

4. The laboratory is also investigating the effect of CSN6, another subunit of the CSN complex, and one whose purpose is unknown, on the enzymatic activity of CSN5 The laboratory is also investigating the effect of CSN6, another subunit of the CSN complex, and one whose purpose is unknown, on the enzymatic activity of CSN5 It has no enzymatic ability, and was deemed unnecessary in the minimal deneddylase core of the CSN complex. However, it has been found that both CSN5 and CSN6 are necessary for development and viability, and that CSN6 may play a role in maintaining the stability and structural integrity of the complex

5. Characterization of the CSN5-CSN6 Interface with NMR Spectroscopy

7. My work… I helped a PhD student in the lab, Melissa Birol, investigate the novel pro-Nedd8 processing activity of CSN5 and investigate the effect of CSN6 on the enzymatic efficiency of CSN5 by… Comparing in vitro proNedd8 processing assays of CSN5 + CSN6, CSN5 R106T + CSN6, CSN5 and CSN5 R106T Generating CSN6 variants with polymorphisms found within the CSN6-CSN5 interface or within the CSN6- Nedd8 interface

8. What we found… CSN5 R106T, in a complex with CSN6, processes half of proNedd8 in 45 minutes, while CSN5 in a complex with CSN6 requires 90 minutes

9. Determination of the effect of CSN6- (1) on the enzymatic activity of CSN5 We studied the effect of four CSN6 mutants that we had generated (CSN6 H14A, CSN6 N55K, CSN6 E58A, and CSN6 V85E) on the formation, stability, and activity of the CSN5-CSN6 subcomplex using… In vitro time course proNedd8 processing assays Chemical crosslinking assays, pull-down assays, and analytical gel filtration assays

10. We found… CSN6 H14A and CSN6 V85E severely reduce CSN5 peptidase activity; CSN6 N55K reduces CSN5 peptidase activity somewhat, and CSN6 V85E has seemingly no effect

11. Problems we had… The -80ºC freezer broke down the weekend before I arrived, and the lab lost much of its protein stocks, so we had to spend weeks replenishing them The -80ºC freezer broke down the weekend before I arrived, and the lab lost much of its protein stocks, so we had to spend weeks replenishing them We had a lot of difficulty purifying nondegraded forms of CSN6 and CSN5 R106T We had a lot of difficulty purifying nondegraded forms of CSN6 and CSN5 R106T We had to repeat the proNedd8 processing assays multiple times before we were obtained the accurate pipetting and nondegraded proteins we needed for accurate data We had to repeat the proNedd8 processing assays multiple times before we were obtained the accurate pipetting and nondegraded proteins we needed for accurate data

12. What I Learned… Lab techniques Lab techniques Cell culture Cell culture Transformation Transformation Gel filtration chromatography protein purification Gel filtration chromatography protein purification Chemical crosslinking Chemical crosslinking Analytical gel filtration Analytical gel filtration Circular dichroism Circular dichroism Protein crystallization for X-Ray Crystallography Protein crystallization for X-Ray Crystallography That even the best designed science experiments may have to be done over and over again! That even the best designed science experiments may have to be done over and over again!

13. My Travels! My internship in Montpellier was wonderful, not only for the research that I carried out, but also for the trips I was able to make… My internship in Montpellier was wonderful, not only for the research that I carried out, but also for the trips I was able to make…

14. Cities I visited… Paris Paris Carcassonne Carcassonne Marseille Marseille Barcelona Barcelona Avignon Avignon Nice Nice Arles Arles

16. In Conclusion I would like to thank I would like to thank Dr. Aude Echalier-Glazer, for accepting me into her lab and giving me a wonderful project to work on Dr. Aude Echalier-Glazer, for accepting me into her lab and giving me a wonderful project to work on Melissa Birol, for taking me on as an assistant, teaching me numerous lab techniques, and putting up with all of my mistakes Melissa Birol, for taking me on as an assistant, teaching me numerous lab techniques, and putting up with all of my mistakes IIP, for giving me this great opportunity IIP, for giving me this great opportunity