Summary of key activities carried out by the Programme

The group is working on the following projects:

CCT

The eukaryotic cytosolic chaperonin CCT (chaperonin containing TCP-1) is the most complex of all chaperonins, an oligomeric structure built from two identical rings, each composed of single copies of eight different 60kDa subunits called α, β, γ, ζ, ε, δ, θ and η. This macromolecular complex of 1MDa has crucial relevance in several essential biological processes and is emerging as a key molecule during mitosis due to its role in the folding of many important molecules residing at the centrosome and involved in cell division (Plk1, p27, Cdc20, PP2a, actin, α, and β- tubulins). In tight collaboration with Dr. Valpuesta we solved the crystal structure of this protein machine in complex with tubulin (see figure below), providing information about the molecular mechanism by which this macromolecular complex aids the tubulin folding process.

Par Complex

In collaboration with Dr. Carazo we co-expressed and purified the three subunits of the Drosophila Par complex. Initial biochemical assays confirm the presence of the three subunits in the complex. Preliminary EM work (negative stain) is promising (see Carazo´s section).

AuroraA-XMAP215-TACC3

The AuroraA-TACC-XMAP215 complex tightly regulates centrosome maturation and microtubule growth, essential mechanisms for cell proliferation. Furthermore the interactions of these three components have been implicated in breast and ovarian cancers. Here at the CNIO, we were able to over-express and purify all three recombinant proteins (from Xenopus). In vitro pull downs and immunoprecipitation assays show the minimum TACC domain required for interactions with XMAP215 and AuroraA, together with its localisation to the spindle poles and aster formations (in collaboration with Dr. Vernos). Crystallization trials of this new domain were set up. Electron microcrographs of the negative stained XMAP215 protein reveal interesting particles: extended filaments and circularise rings often filled with endogenous tubulin (Dr. Valpuesta). Protein-protein interaction studies of XMAP215 in complex with various TACC domains and a putative triple complex (Aurora A- TACC-XMAP215) are currently in progress.

Plk1-Bora-AuroraA

Polo-like kinase 1 (Plk1) and Aurora A kinases are key regulators of the cell cycle. Recent studies shed light into the mechanism by which Plk1 is activated, showing that Aurora A is the trigger kinase supported by the regulatory protein Bora. We identified a minimal domain of Bora that interacts with Aurora A. In particular, due to the flexible nature of Bora, the Bora-Aurora A complex was analyzed by NMR spectroscopy and other biophysical techniques such as Isothermal Titration Calorimetry and Static Light Scattering. The affinity of the interaction is in the micromolar range and we are currently exploring how phosphorylation and Plk1 binding affects this complex.

Plk4

Plk4 is a centriolar protein and plenty of evidence point at it being in charge of controlling the duplication of centrioles once per cell cycle. The full length PLK4 is a multidomain protein which we produced from eukaryotic cells. Experiments are ongoing to obtain pure samples suitable for structural analysis. For the same purpose the single domains were cloned for expression E. coli, although we encountered some difficulties in obtaining soluble products. In the case of the kinase domain, we finally were able to obtain it as a soluble recombinant protein as a fusion with a SUMO-tag. Being PLK4 over-expression or dysregulation causative of aneuploidy and tumorigenesis, this enzyme seems a promising drug target. We are currently trying to crystallize PLK4k.d. alone and bound to generic kinase inhibitors.

Web design: Alfonso Núñez Salgado