Paola Ghisellini

Toward the rational design of protein kinase casein kinase-2 inhibitors.

Casein kinase-2 (CK2) probably is the most pleiotropic member of the protein kinase family, with more than 200 substrates known to date. Unlike the great majority of protein kinases, which are tightly regulated enzymes, CK2 is endowed with high constitutive activity, a feature that is suspected to underlie its oncogenic potential and possible implication in viral infections. This makes CK2 an attractive target for anti-neoplastic and antiviral drugs. Here, we present an overview of our present knowledge about CK2 inhibitors, with special reference to the information drawn from two recently solved crystal structures of CK2alpha in complex with emodin and with 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), this latter being the most specific CK2 inhibitor known to date. A comparison with a series of anthraquinone and xanthenone derivatives highlights the crucial relevance of the hydroxyl group at position 3 for inhibition by emodin, and discloses the possibility of increasing the inhibitory potency by placing an electron withdrawing group at position 5. We also present mutational data corroborating the relevance of two hydrophobic residues unique to CK2, Val66 and Ile174, for the interactions with emodin and TBB, but not with the flavonoid inhibitors quercetin and fisetin. In particular, the CK2alpha mutant V66A displays 27- and 11-fold higher IC(50) values with emodin and TBB, respectively, as compared with the wild-type, while the IC(50) value with quercetin is unchanged. The data presented pave the road toward the rational design of more potent and selective inhibitors of CK2 and the generation of CK2 mutants refractory to inhibition, useful to probe the implication of CK2 in specific cellular functions.

Biochemical evidence that the N-terminal segments of the α subunit and the β 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 α subunit has been validated by generating two mutants (Y26F and Δ2–6) 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 β subunit, in conjunction with the observation that synthetic peptides reproducing the N‐terminal segment (1–30) and the activation loop (175–201) of CK2α counteract the functional effects of the C‐terminal domain of the β subunit, is consistent with a mechanism of activation of CK2 where the N‐terminal domain of α and the C‐terminal domain of β play interchangeable roles.

Electrochemical study of the interaction between cytochrome P450sccK201E and cholesterol

Cytochrome P450sccK201E, mutated form of cytochrome P450scc native recombinant (P450sccNR), was employed to study the enzyme-substrate interaction. The detection of the cholesterol was performed by electrochemical method using cyclic voltammetry (CV) and chronoamperometry measurements. The biochemical analysis was realized to observe the electrochemical responses of the engineerized enzyme to three different forms of cholesterol: free, low-density lipoprotein (LDL) and high-density lipoproteins (HDL). Compared to cytochrome P450sccNR, the cytochrome P450sccK201E displays a different behavior in the interaction with the substrate detection. The results show that the engineerized enzyme can be utilized for the cholesterol detection in biosensor field.

Pollutant sensing layer based on cytochrome P450

Abstract In this work, cytochrome P4502B4 fusion protein was utilized to realize sensing layers for detection of styrene in atmosphere. Glutathione S-transferase (GST) fusion protein was utilized to realize thin films by Langmuir–Blodgett (LB) horizontal transfer technique. The interaction between the styrene and the sensing layer was monitored by spectrophotometric and by gravimetric measurements. A shift of the Soret peak of cytochrome P450 in the presence of the substrate was found when absorbance measurements were performed. Index of spin state equilibrium variation of P4502B4 was calculated in order to verify the interaction between styrene and cytochrome. A saturation trend in mass density was found when quartzes nanobalance was utilized.

Photoreversibility and photostability in films of octopus rhodopsin isolated from octopus photoreceptor membranes

In this work, a new biomaterial resulting from the isolation of octopus rhodopsin (OR) starting from octopus photoreceptor membranes is presented. Mass spectroscopic characterization was employed in order to verify the presence of rhodopsin in the extract. Photoreversibility and photochromic properties were investigated using spectrophotometric measurements and pulsed light. Thin films of OR were realized using the gel-matrix entrapment method in polyvinyl alcohol solution. The results indicate that the photoreversibility and the photostability of the OR in gel-matrices are maintained. Several measurements were performed to test the stability of the resulting biomaterial in time and at room temperature. Preliminary tests demonstrate that the photoreversibility and the photostability are still found after few days from the biomaterial preparation and after the exposure for several hours at room temperature.

Generation of mutants of CK2α which are dependent on the β-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 CK2α 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 Δ2-12 displays under standard conditions an almost complete loss of catalytic activity accounted for by a dramatic rise in its Km for ATP (from 10 to 206 μM) 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 CK2α were restored upon association with the regulatory β-subunit, suggesting that constitutive activity is conferred to CK2α 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 CK2α alone vs. a CK2α–β peptide complex) may result in the ability of the activation loop to adopt its proper conformation independently of interactions with the N-terminal segment.

Development of immobilization techniques of cytochrome P450-GST fusion protein

Abstract Glutathione S-transferase (GST) fusion cytochrome P4502B4 enzyme obtained by genetic engineering was used in order to optimize the immobilization of the proteins on solid supports. Langmuir–Schaefer, ‘layer-by-layer (LbL)’ and self-assembling techniques were used to form thin films on solid surfaces. In particular, it was studied the possibility to realize alternated structures stabilized by binding affinity between GST-fusion protein and glutathione (GSH) using ‘LbL’ techniques. The characterization of the films was performed by means of π-A isotherms, Brewster angle microscopy and spectrophotometry. Preliminary analysis of the P4502B4 films functionality was realized monitoring the spin-state of the cytochrome P450 by spectrophotometric measurements.

DNASER. II. Novel surface patterning for biomolecular microarray

A new matrix-integral part of the new DNA microarray instrumentation DNA analyzer (DNASER) is introduced based on a novel DNA patterning on the solid support surface. Such patterning found the way to modify a glass surface for a precise positioning of small droplets of aqueous DNA solutions, without special robots (arrayers), within the boundaries of the modified regions. The physically heterogeneous surface consists of highly hydrophilic spots surrounded by a highly hydrophobic area leading to the surface patterning needed for a DNA microarray: a matrix of hydrophilic spots properly activated for immobilization of oligonucleotides has been fabricated on absolutely passive hydrophobic surface. The optimal efficiency of the above functionalitation technology of a glass-substrate in obtaining DNA microarray was confirmed by the Cy3-dCTP-labeled DNA sample, as shown by charge-coupled device images of the DNASER previously described.

Spin state transitions in Langmuir-Blodgett films of recombinant cytochrome P450scc and adrenodoxin

Abstract Langmuir–Blodgett (LB) films of recombinant cytochrome P450scc, of P450scc–adrenodoxin (Adx) complex and alternated layers of Adx and P450scc have been obtained. Spectral properties of these proteins in thin films were investigated by UV–Vis absorption spectroscopy. It has been found that cytochrome P450scc exists in LB films only in low-spin state while before the deposition it was in high-spin state. The data suggest that transferring the hemoprotein or its complex with redox partner results in the modification of the spin state by a conformational transition. In order to investigate further the P450scc and Adx interaction, the mass density of the films formed from these molecules has been studied by nanogravimetric measurements. Comparative study between nanogravimetric and spectral characterisation was performed. The results indicate that the protein–protein interaction is disrupted, when the complex is organised in thin film.

Synchrotron study of heat induced order in protein Langmuir–Blodgett films

Abstract Langmuir–Blodgett films of different proteins, such as immunoglobuline, thioredoxin, cytochrome P450 and bacteriorhodopsin were deposited. The structure of the films was studied by synchrotron X-ray scattering. It was found that thermal treatment of the films, performed after depositing each successive monolayer, results in a pronounced increase of the film order. Bragg reflections were registered for films thus prepared for all the proteins investigated.