The ubiquitin-proteasome system and cancer.

The ubiquitin proteasome system (UPS) has emerged from obscurity to be seen as a major player in all regulatory processes in the cell. The concentrations of key proteins in diverse regulatory pathways are controlled by post-translational ubiquitination and degradation by the 26 S proteasome. These regulatory cascades include growth-factor-controlled signal-transduction pathways and multiple points in the cell cycle. The cell cycle is orchestrated by a combination of cyclin-dependent kinases, kinase inhibitors and protein phosphorylation, together with the timely and specific degradation of cyclins and kinase inhibitors at critical points in the cell cycle by the UPS. These processes provide the irreversibility needed for movement of the cycle through gap 1 (G1), DNA synthesis (S), gap 2 (G2) and mitosis (M). The molecular events include cell-size control, DNA replication, DNA repair, chromosomal rearrangements and cell division. It is doubtful whether these events could be achieved without the temporally and spatially regulated combination of protein phosphorylation and ubiquitin-dependent degradation of key cell-cycle regulatory proteins. The oncogenic transformation of cells is a multistep process that can be triggered by mutation of genes for proteins involved in regulatory processes from the cell surface to the nucleus. Since the UPS has critical functions at all these levels of control, it is to be expected that UPS activities will be central to cell transformation and cancer progression.

Advances and Progress in Drug Design - SMi's ninth annual meeting.

SMi's Advances and Progress in Drug Design, held in London, included topics covering new developments in the field of drug design. This conference report highlights selected presentations on targeting GPCRs and kinases, virtual screening for hit and lead identification, and the ChEMBL database for drug discovery. One investigational drug, losmapimod (GlaxoSmithKline plc), was discussed.

The MIT domain of UBPY constitutes a CHMP binding and endosomal localization signal required for efficient epidermal growth factor receptor degradation.

We have identified and characterized a Microtubule Interacting and Transport (MIT) domain at the N terminus of the deubiquitinating enzyme UBPY/USP8. In common with other MIT-containing proteins such as AMSH and VPS4, UBPY can interact with CHMP proteins, which are known to regulate endosomal sorting of ubiquitinated receptors. Comparison of binding preferences for the 11 members of the human CHMP family between the UBPY MIT domain and another ubiquitin isopeptidase, AMSH, reveals common interactions with CHMP1A and CHMP1B but a distinct selectivity of AMSH for CHMP3/VPS24, a core subunit of the ESCRT-III complex, and UBPY for CHMP7. We also show that in common with AMSH, UBPY deubiquitinating enzyme activity can be stimulated by STAM but is unresponsive to its cognate CHMPs. The UBPY MIT domain is dispensable for its catalytic activity but is essential for its localization to endosomes. This is functionally significant as an MIT-deleted UBPY mutant is unable to rescue its binding partner STAM from proteasomal degradation or reverse a block to epidermal growth factor receptor degradation imposed by small interfering RNA-mediated depletion of UBPY.

Ubiquitin and ubiquitin-like proteins as multifunctional signals.

Protein ubiquitylation is a recognized signal for protein degradation. However, it is increasingly realized that ubiquitin conjugation to proteins can be used for many other purposes. Furthermore, there are many ubiquitin-like proteins that control the activities of proteins. The central structural element of these post-translational modifications is the ubiquitin superfold. A common ancestor based on this superfold has evolved to give various proteins that are involved in diverse activities in the cell.

Crystallization of gankyrin, an oncoprotein that interacts with CDK4 and the S6b (rpt3) ATPase of the 19S regulator of the 26S proteasome.

Gankyrin is an oncoprotein overexpressed in hepatocarcinoma cells that binds to the cell-cycle regulator CDK4 and the S6b ATPase subunit of the regulatory component of the proteasome. It belongs to the family of ankyrin-repeat proteins that appear to mediate protein-protein interactions in diverse biochemical processes. Gankyrin has been crystallized from polyethylene glycol solutions and diffraction data have been obtained from these crystals that extend to 2.1 A spacing.

The crystal structure of gankyrin, an oncoprotein found in complexes with cyclin-dependent kinase 4, a 19 S proteasomal ATPase regulator, and the tumor suppressors Rb and p53.

Gankyrin is a 25-kDa hepatocellular carcinoma-associated protein that mediates protein-protein interactions in cell cycle control and protein degradation. It has been reported to form complexes with cyclin-dependent kinase 4, retinoblastoma protein, the S6b ATPase subunit of the 19 S regulator of the 26 S proteasome, and Mdm2, an E3 ubiquitin ligase involved in p53 degradation. It is the first protein described to bind both to the 26 S proteasome and to proteins in other complexes containing cyclin-dependent kinase(s) and p53 ubiquitylating activities, thus providing a mechanism for delivering cell cycle regulating machinery and ubiquitylated substrates to the proteasome for degradation. Gankyrin contains a 33-residue motif known as the ankyrin repeat that occurs five and a half to six times in the sequence. As a step toward understanding gankyrin interactions with its protein partners we have determined its three-dimensional crystal structure to 2.0-A resolution. It reveals that the entire 226-residue gankyrin polypeptide folds into seven ankyrin repeat elements. The ankyrin repeats, consisting of an antiparallel beta-hairpin followed by a perpendicularly oriented helix-loop-helix, pack side-by-side, creating an extended curved structure with a groove running across the long concave surface. Comparison with the structures of other ankyrin repeat proteins suggests that interactions with partner proteins are mediated by residues situated on this concave surface.