Cell-signaling processes mediated by ubiquitinylation, the post-translational covalent conjugation of ubiquitin molecules, are of prime importance for cellular activity and particularly for protein turnover. Ubiquitin-ligase enzymes, E3s, are responsible for the last step of the ubiquitinylation reaction. The E3 cullin-RING ubiquitin ligases (CRLs) represent the main ubiquitin ligase family. Among several factors that regulate CRL activity, cullin neddylation/deneddylation cycles are central (1).
The COP9 signalosome (CSN), a large multiprotein complex that resembles the 19S lid of the 26S proteasome, plays a central role in the regulation of the E3-cullin RING ubiquitin ligases (CRLs). Due to the fact that a large number of proteins are ubiquitinylated by CRLs, the COP9 signalosome (CSN) is implicated in the control of a significant proportion of the proteome, including pro-oncogenes (for example Myc), tumor suppressors (for example p53) and other important cellular protagonists. Different biological and biochemical functions of the CSN complex have been studied over the years, but by far the most studied is its role as a CRL deneddylase. The catalytic activity of the CSN complex, carried by subunit 5 (CSN5/Jab1), resides in the deneddylation of the CRLs, that is the hydrolysis of the cullin-Nedd8 isopeptide bond. Structurally, the CSN is an eight-subunit complex of about 320 kDa (six PCI (proteasome COP9 eIF3)-based subunits and two Mpr1-Pad1-N-terminal [MPN]-containing subunits). Subunit 5 (CSN5), one of the MPN-containing subunits, carries a zinc-dependent isopeptidase catalytic centre that contains a JAMM (Jab1/MPN/Mov34) motif (also known as MPN+ motif; (2)). Recent detailed studies suggested that the organization of the CSN complex resembles that of the 26S proteasome lid (3), with the deubiquitinase enzyme Rpn11 being the equivalent of the deneddylating subunit CSN5 (2, 4).
The CSN, implicated in various cellular functions, ranging from cell cycles, to circadian rhythm, to immunity, is a very well conserved multi-protein complex in eukaryotes, from plants to mammalian cells. Its importance in cellular functions has been highlighted by genetic studies (5). The physiology of the CSN in normal cells has been well researched, and many studies have found a strong link between the CSN and cancers (6). Intriguingly, the CSN cancer implication is attributable to mainly CSN5, which is located on human chromosome 8q—itself often amplified in cancers.
Smaller forms of the holo-CSN complex, with variable compositions, have been found in vivo (7-11). Although important in cell cycle progression, these sub-CSN complexes have not yet been fully functionally characterized (12). It is interesting that, as alluded to for Rpn11 in the context of the proteasome lid (4), CSN5 is found in two forms, a holo-CSN-associated form that is catalytically active and a holo-CSN-independent state void of isopeptidase activity (2, 3). The modularity and topology of the CSN complex have been explored in vitro by non-denaturing mass spectrometry (MS), which revealed that CSN5 is a peripheral subunit that can homo-dimerize outside of the CSN complex and interacts mostly with the other MPN-containing subunit, CSN6, in the context of the CSN complex (3). The potential interactions of CSN5 with other CSN subunits, namely CSN1, CSN2, CSN4 and CSN7, have been highlighted in earlier reports (1, 8, 13, 14).
Whereas CSN-dependent CSN5 displays isopeptidase activity, it is intrinsically inactive in other physiologically relevant forms. To elucidate the molecular regulation of CSN5 activity, the inventors structurally and functionally characterized it in its CSN-independent form by X-ray crystallography, molecular dynamics (MD) simulations, and in vitro studies. Furthermore, the invention provides a preliminary glimpse into the rational screening of small molecules, antibodies, peptides, pseudopeptide, and polypeptides inhibitors of CSN5 isopeptidase activity.