Abstract
The COP9 (Constitutive photomorphogenesis 9) signalosome (CSN) is an eight-subunit-containing multiprotein complex (320 kDa) implicated in diverse cellular processes including cell cycle progression, gene expression and DNA repair via its function in the ubiquitin-proteasome pathway. It is a highly evolutionary conserved protein complex in higher eukaryotes for which its activity is essential. Over years of biochemical and biological studies to elucidate the role of the CSN, its most studied and best understood function is linked to the control of protein ubiquitylation (post-translational modification corresponding to the covalent conjugation of an ubiquitin molecule) by a class of E3 ubiquitin ligases. The CSN exhibits catalytic activity to regulate E3-cullin RING ubiquitin ligases (CRLs) by the removal of an ubiquitin-like protein, Nedd8 (cullin-neural precursor cell expressed developmentally downregulated gene 8), from CRLs. Cycles of neddylation/deneddylation are essential for CRL function and the CSN is central in this process through its activity as a CRL deneddylase.Structural and functional similarities link the CSN, the 19S lid of the 26S proteasome and the eukaryotic initiation factor-3 (elF3). These multi-subunit assemblies comprise six PCI (proteasome COP9 eIF3) domain subunits and two MPN (Mpr1–Pad1–N-terminal) domain-containing subunits. The catalytic activity of the CSN is centred on its subunit 5 (CSN5/Jab1), which hydrolyses the Nedd8-CRL isopeptide bond. CSN5 contains a zinc-dependent isopeptidase catalytic centre constituted of a JAMM (Jab1/MPN/Mov34) motif. CSN5 incorporation within the CSN complex unleashes its isopeptidase activity, whereas it remains inactive in isolation. The work presented in this manuscript led to five main findings. (i) Having elucidated the crystal structure of CSN5 catalytic domain, biochemical and in silico investigations that furthered the understanding of CSN5 molecular regulation, led to the identification of a potential molecular trigger enabling CSN5 to be active and the design of a constitutively active CSN5 variant form. (ii) The ability of CSN5 to homodimerise was investigated in solution, in silico and in cellular extracts and brought information that could be important for its function. (iii) Further to that work, to address CSN5 activity within the CSN, the contribution of another CSN subunit, mainly CSN6, shown to interact directly with CSN5 was evaluated and this led to the identification of CSN6 as the CSN5 activating subunit. (iv) The biochemical and biophysical characterisation of the CSN5-CSN6 complex was exploited to explore at the molecular level this complex in the context of its binding to Nedd8 and of its integration within the holo-CSN assembly through an integrated approach that includes biochemical, structural, biophysical and computational techniques. (v) Finally the CSN5-CSN6 complex was shown to be able in vitro to pursue peptidase activity on the Nedd8 precursor protein, pro-Nedd8 by cleaving its C-terminal extension (-G75G76GGLRQ) and preliminary results relating to the exploration of this new activity are presented.Overall this work allowed to gain an in-depth understanding of the activity determinants and of the regulatory mechanisms that the CSN catalytic subunit CSN5 is subjected to.