Nanostructuring of heme-proteins for biodevice applications.

Proteins represent versatile building blocks for the realisation of nanostructured materials to be applied in the nanobiotechnological field. The Langmuir-Blodgett (LB) technique was utilised as a means to develop nanobiodevices based on protein molecules. Namely, engineered Cytochrome P450 thin films were fabricated and characterised. The possibility to employ LB-based protein structures to use in biosensors has been exploited. The characterisation process was performed in order to verify the best working parameters. As a first step' the protein films were studied at the air-water interface and then transferred into a solid support for further characterisation. The films were characterised by different techniques such as: UV-vis spectroscopy, nanogravimetry, atomic force microscopy and biochemical assays. The results showed that it was possible to form active cytochrome P450s nanostructures by the LB technique.

Preliminary electrochemical characterisation of cytochrome P4501A2-clozapine interaction.

Cytochromes P450 are a large superfamily of heme-thiolate enzymes involved in the metabolism of many different organic substrates such as drugs, fatty acids and toxic compounds. The aim of this work is to analyse the binding between the cytochrome P4501A2, in solution and in gel-matrix, and its substrate (clozapine), utilising voltammetric tests. The interaction measurements were carried out using two different screen printed electrodes (rhodium-graphite and graphite-riboflavin), and the results were compared. It was demonstrated that it is possible to realise a biosensor prototype to detect the presence of clozapine indirectly by chronoamperometry.

Generation of mutants of CK2alpha which are dependent on the beta-subunit for catalytic activity.

To shed light on the structural features underlying high constitutive activity of protein kinase CK2 a number of mutants of the human CK2alpha-subunit altered in the interactions between the N-terminal segment and the activation loop have been generated and shown to be defective in catalytic activity. In particular the truncated mutant delta2-12 displays under standard conditions an almost complete loss of catalytic activity accounted for by a dramatic rise in its Km forATP (from 10 to 206 microM) and a reduced Kcat. Such a drop in efficiency is paralleled by conformational disorganization, as judged from Superdex 75 gel filtration profile. Both catalytic properties and gel filtration behaviour similar to those of wild type CK2alpha were restored upon association with the regulatory beta-subunit, suggesting that constitutive activity is conferred to CK2alpha and to CK2 holoenzyme through different molecular mechanisms. In the holoenzyme an assumable release of tension at the backbone of Ala-193 (as seems to be indicated by a comparison of the crystal structures of maize CK2alpha alone vs. a CK2alpha-beta peptide complex) may result in the ability of the activation loop to adopt its proper conformation independently of interactions with the N-terminal segment.

Interstitial and large chromosome 1p deletion occurs in localized and disseminated neuroblastomas and predicts an unfavourable outcome.

We studied chromosome 1p loss of heterozygosity (1p-LOH) in 53 neuroblastomas (NBs) using 15 (CA)n repeat loci, which covered a region of 90 cM. We also assessed chromosome 1p36 deletion by fluorescence in situ hybridization (FISH) on interphase nuclei. 1p-LOH was found in 19 (36%, 95% confidence interval (CI) 23-50%) NBs. We detected interstitial and large deletion in both localized and disseminated tumours and in one tumour of a patient at stage 4S. Allelic loss was frequently observed in 1p36 and 1p32 regions. In patients older than 1 year of age (53 versus 13%, P < 0.002) we detected significant chromosome 1p deletion and it was associated with MYCN amplification (P = 0.001). Overall survival (OS) analysis showed that 1p-LOH is predictive of a poor outcome (odds ratio 16.5, 95% CI 5.4-50.9%); therefore, 1p-LOH should be regarded as an additional tumour progression marker in neuroblastoma.

Biochemical evidence that the N-terminal segments of the alpha subunit and the beta subunit play interchangeable roles in the activation of protein kinase CK2.

The concept that the amino-terminal segment plays a role in conferring high basal activity to protein kinase CK2 alpha subunit has been validated by generating two mutants (Y26F and delta2-6) which are defective both in catalytic activity and in thermal stability. The additional finding that the activity of the two mutants is fully restored upon association with the regulatory beta subunit, in conjunction with the observation that synthetic peptides reproducing the N-terminal segment (1-30) and the activation loop (175-201) of CK2alpha counteract the functional effects of the C-terminal domain of the beta subunit, is consistent with a mechanism of activation of CK2 where the N-terminal domain of alpha and the C-terminal domain of beta play interchangeable roles.