Ubiquitin-proteasome system and cell cycle control (Olivier Coux)

  • Research
  • Research group
  • Publications
  • More...

Objectives

Our team analyses the mechanisms of action of the Ubiquitin-Proteasome system (UPS), and its functional interactions with the cellular machinery controlling cell proliferation. Our research axes aim at investigating two main issues: (i) which roles plays UPS in the regulation of cell cycle and of its multiple checkpoints ? (ii) conversely, how the proteasome, which is at the heart of the UPS, is affected during cell cycle progression and in stress conditions, particularly upon genotoxic stress ?

 

UPS and cell cycle control

Regarding the role of UPS in cell cycle regulation, we analyzed over the past years the mechanisms of degradation and regulation of two important cell cycle regulators, CDC25B and Cyclin E.

CDC25B is a key phosphatase playing a critical role in the control of MultipolarSpindles 400mitosis entry. We demonstrated that human CDC25B is rapidly degraded during the metaphase/anaphase transition, in a mechanism involving the protein βTrCP, the substrate-recruiting component of the E3 Ub-ligase SCFβTrCP. Interestingly, the CDC25B-DDA mutant of CDC25B, which cannot anymore interact with βTrCP, is partially stabilized during mitosis, and its ectopic expression in human cells entail mitotic exit abnormalities and formation of multipolar mitotic spindles (Fig.1). This phenotype suggests that CDC25B has during mitosis at least another substrate than the CDKs (Cyclin-dependent kinases), the only known CDC25 substrates. Our current work aims at identifying this substrate.


BrdU engl 400Cyclin E is expressed during the G1/S transition of the cell cycle. In association with the Cdk2 kinase, it is a key actor of the control of replication initiation. We found that addition of a dominant-negative form  of the SUMOylation enzyme Ubc9 (Ubc9dn) before induction of replication in Xenopus egg extracts resulted into an increase of the number of fired replication origins. We then demonstrated that the major SUMOylated protein on chromatin at the beginning of S phase was cyclin E. Altogether, our results show that cyclin E SUMOylation negatively controls the firing of replication origins. Our present work on Cyclin E aims at a better understanding of the regulation of its degradation by the ubiquitin-ligase SCFFbw7.

 

Proteasome and PA28γ

The term proteasome corresponds in fact to a family of complexes (more details here) that share a common proteolytic core (the 20S proteasome or CP) but that differ by the regulatory complexes bound to the ends of the CP.  Our team studied the past years the 26S proteasome, which is the proteasome form responsible for the degradation of ubiquitylated proteins. However, our recent work led us to become more and more interested by the complexes formed by the association of the 20S proteasome with its nuclear regulator PA28γ.

PA28γ is a complex described since many  years but its precise functions are still ill-understood. PA28CxesEngIt is able to bind to the 20S proteasome, either into "20S proteasome/PA28γ" complexes or into complexes called "hybrid proteasomes" in which the 20S proteasome is associated to PA28γ at one end and to the 19S complex at the other end. During the past years, it has become clear that PA28γ plays a critical role in the control of intranuclear dynamics. For example, we demonstrated that it is directly involved in nuclear speckles organization (nuclear speckles are nuclear bodies where splicing factors are accumulated and modified. Other groups have shown that PA28γ is involved in the regulation of other types of nuclear bodies such as Cajal or PML bodies, and that it is important for the degradation of several regulatory proteins controlling cell proliferation.

Our current work aims at a better understanding of the mechanisms of action and of the roles in nuclear dynamics of PA28γ, particularly during the cellular response to DNA damages such as those responsible for cancer development.

 

Equipe 2     

Research group

Veronique BALDIN - Staff Scientist - CNRS
Catherine BONNE ANDREA - Staff Scientist - CNRS
Severine BOULON - Staff Scientist - UM2
Olivier COUX - Staff Scientist - CNRS
Beata JONIK-NOWAK - PhD Student - UM2
Francisca MECHALI - Research Assistant - CNRS
  • A. Loukil, M. Zonca, C. Rebouissou, V. Baldin, O. Coux, M. Biard-Piechaczyk, J.M. Blanchard and M. Peter (2014). High resolution live cell imaging reveals novel cyclin A2 degradation foci involving autophagy J. Cell Sci., in press. pubmed

  • D.E. Mbang-Benet, Y. Sterkers, C. Morelle, N.M. Kebe, L. Crobu, P. Portalès, O. Coux, J.F. Hernandez, S. Meghamla, M. Pagès and P. Bastien (2014). The bacterial-like HslVU protease complex subunits are involved in the control of different cell cycle events in trypanosomatids. Acta Trop. 131:22-31. pubmed

  • C. Bonne-Andrea, M. Kahli, F. Mechali, J.M. Lemaitre, G. Bossis and O. Coux (2013). SUMO2/3 modification of cyclin E contributes to the control of replication origin firing. Nat Commun 4:1850 pubmed

  • M. Biard-Piechaczyk, S. Borel, L. Espert, G. de Bettignies and O. Coux (2012). HIV-1, ubiquitin and ubiquitin-like proteins: the dialectic interactions of a virus with a sophisticated network of post-translational modifications. Biol. Cell 104:165-87. pubmed

  • L. Henry, L. Le Gallic, G. Garcin, O. Coux, N. Jumez, P. Roger, T. Lavabre-Bertrand, J. Martinez, L. Meunier and P.E. Stoebner (2011). Proteolytic activity and expression of the 20S proteasome are increased in psoriasis lesional skin. Br. J. Dermatol. 165:311-20. pubmed

  • O. Coux (2011). « Protéasome, ubiquitine et protéines apparentées à l'ubiquitine (Proteasome, ubiquitin and ubiquitin-like proteins) ». Editions TEC & DOC – Lavoisier, 482 pages (French, 23 chapters).

  • V. Baldin (2011). Localisation intracellulaire des protéasomes. In « Protéasome, ubiquitine et protéines apparentées à l'ubiquitine (Proteasome, ubiquitin and ubiquitin-like proteins) », TEC & DOC - Lavoisier, ed. (Paris, France), pp. 65-81.

  • C. Bonne-Andrea (2011). UPS et réplication. In « Protéasome, ubiquitine et protéines apparentées à l'ubiquitine (Proteasome, ubiquitin and ubiquitin-like proteins) », TEC & DOC - Lavoisier, ed. (Paris, France), pp. 335-369.

  • M. Benkirane, C. Sardet and O. Coux (2010). Lessons from interconnected ubiquitylation and acetylation of p53: think metastable networks. Biochem. Soc. Trans. 38:98-103. pubmed

  • Y. Thomas, O. Coux and V. Baldin (2010). βTrCP-dependent degradation of CDC25B phosphatase at the metaphase-anaphase transition is a pre-requisite for correct mitotic exit. Cell Cycle 9:4338-50. pubmed

  • G. de Bettignies and O. Coux (2010). Proteasome inhibitors: Dozens of molecules and still counting. Biochimie 92:1530-45. pubmed

  • H. Wodrich, D. Henaff, B. Jammart, C. Segura-Morales, S. Seelmeir, O. Coux, Z. Ruzsics, C.M. Wiethoff and E.J. Kremer (2010). A capsid-encoded PPxY-motif facilitates adenovirus entry. PLoS Pathog. 6:e1000808 pubmed

  • A. Y. Le Feuvre, and, and Coux O. (2010). La voie de dégradation ubiquitine dépendante (The ubiquitin-dependant degradation pathway). Online Review (French) for a web site dedicated to school teachers : Expert site "Vie (Life)" of the French Ministry of National Education :. http://www.snv.jussieu.fr/vie/dossiers/ubiquitine/ub0.html

  • A.Y. Le Feuvre, C. Dantas-Barbosa, V. Baldin and O. Coux (2009). High yield bacterial expression and purification of active recombinant PA28alphabeta complex. Protein Expr. Purif. 64:219-24. pubmed

  • R. Farràs, V. Baldin, S. Gallach, C. Acquaviva, G. Bossis, I. Jariel-Encontre and M. Piechaczyk (2008). JunB breakdown in mid-/late G2 is required for down-regulation of cyclin A2 levels and proper mitosis. Mol. Cell. Biol. 28:4173-87. pubmed

  • V. Baldin, M. Militello, Y. Thomas, C. Doucet, W. Fic, S. Boireau, I. Jariel-Encontre, M. Piechaczyk, E. Bertrand, J. Tazi and O. Coux (2008). A novel role for PA28gamma-proteasome in nuclear speckle organization and SR protein trafficking. Mol. Biol. Cell 19:1706-16. pubmed

  • L.K. Linares, R. Kiernan, R. Triboulet, C. Chable-Bessia, D. Latreille, O. Cuvier, M. Lacroix, L. Le Cam, O. Coux and M. Benkirane (2007). Intrinsic ubiquitination activity of PCAF controls the stability of the oncoprotein Hdm2. Nat. Cell Biol. 9:331-8. (Linares, Kiernan : equal contribution; Coux, Benkirane : co-senior authors) pubmed

  • C.Y. Hsu, F. Mechali and C. Bonne-Andrea (2007). Nucleocytoplasmic shuttling of bovine papillomavirus E1 helicase downregulates viral DNA replication in S phase. J. Virol. 81:384-94. pubmed

  • I. Lassot, D. Latreille, E. Rousset, M. Sourisseau, L.K. Linares, C. Chable-Bessia, O. Coux, M. Benkirane and R.E. Kiernan (2007). The proteasome regulates HIV-1 transcription by both proteolytic and nonproteolytic mechanisms. Mol. Cell 25:369-83. (Lassot, Latreille : equal contribution) pubmed

  • D. Nury, C. Doucet and O. Coux (2007). Roles and potential therapeutic targets of the ubiquitin proteasome system in muscle wasting. BMC Biochem. 8 Suppl 1:S7. pubmed

  • L. Le Cam, L.K. Linares, C. Paul, E. Julien, M. Lacroix, E. Hatchi, R. Triboulet, G. Bossis, A. Shmueli, M.S. Rodriguez, O. Coux and C. Sardet (2006). E4F1 is an atypical ubiquitin ligase that modulates p53 effector functions independently of degradation. Cell 127:775-88. (Le Cam, Linares : equal contribution) pubmed

  • C. Doucet, G.J. Gutierrez, C. Lindon, T. Lorca, G. Lledo, C. Pinset and O. Coux (2005). Multiple phosphorylation events control mitotic degradation of the muscle transcription factor Myf5. BMC Biochem. 6:27 pubmed

  • S. Bellanger, S. Blachon, F. Mechali, C. Bonne-Andrea, and F. Thierry (2005). High-risk but not low-risk HPV E2 proteins bind to the APC activators Cdh1 and Cdc20 and cause genomic instability. Cell Cycle. 4:1608-15.

The Ubiquitin-Proteasome System

UbPr System Eng 400

The ubiquitin-proteasome system (UPS) is an extremely complex multienzymatic system, which schematically functions in two distinct steps in the regulated degradation of intracellular proteins.

In a first step, the protein substrate is tagged by covalent addition of a chain formed by the successive conjugation of one or several ubiquitin (Ub) molecule(s), thanks to an enzymatic cascade involving three types of factors named E1 (Ub-activating enzyme), E2 (Ub-conjugating enzyme) and E3 (Ub-ligase). The specificity of the reaction (called ubiquitylation reaction) is mainly due to the E3s, which are responsible for substrate recruitment. Accordingly, numerous (several hundreds)  E3-ligases exist in cells. Many de-ubiquitylation enzymes (about 100 in humans) are able to remove the Ub-chain from the substrates, and thus participate to the fine tuning of the ubiquitylation reaction.

In a second step, the poly-Ub chain is recognized by a giant protease called the 26S proteasome, which degrades the ubiquitylated protein into inactive peptides.

 


 

The proteasome

The 20S proteasome is a hollow cylinder-shaped protease, 20S 400 Engextremely conserved during evolution. It is formed by 28 subunits assembled in four rings of seven subunits each. The two outer rings are formed by α subunits, and the two inner rings by β subunits.

The catalytic sites are enclosed in an internal cavity defined by the β rings. Three types of peptidase activities are described: one chymotrypsin-like activity cleaving after hydrophobic residues, a trypsin-like acivity cleaving after basic residues and a caspase-like activity cleaving after acidic residues.

An important feature of the 20S proteasome is that it is weakly active by itself. Indeed, the catalytic sites are buried into the internal chamber formed by the β rings, and are thus only accessible through axial pores defined by the α subunits and closed by the N-terminal ends of these subunits. Consequently, substrate entry into the catalytic chamber requires the binding to the 20S proteasome of regulatory complexes that open the pores upon binding. A video illustrating this opening process (called 20S proteasome gate opening) upon the binding of a regulator (here PA28αβ, see below) has been realized by Geoffroy de Bettignies, a former member of our team (see the video here).

ProteasomeFamily 400Thanks to the existence of different 20S proteasome regulators, the term 'proteasome' usually used in the scientific literature corresponds in reality to a family of protease complexes, which share a common proteolytic core (the 20S proteasome) but differ by the regulators bound to it. Among these regulators, the best known is the 19S complex or RP (for regulatory complex), which forms with the 20S proteasome the 26S proteasome responsible for the degradation of ubiquitylated proteins. Other regulators with ill-understood functions and mechanisms of action have been described: two nuclear complexes, PA28γ and PA200, and a cytoplasmic complex related to PA28γ and called PA28αβ.