Biomedical subjects

Paola Ghisellini

Publications and source records attributed to Paola Ghisellini.

7 recordsLinked to original sources

PubMed2005

Site-directed mutations (Asp405Ile and Glu124Ile) in cytochrome P450scc: effect on adrenodoxin binding.

Cytochrome P450scc, mitochondrial adrenodoxin (Adx), and adrenodoxin reductase (AdR) are an essential components in a steroid hydroxylation system. In particular, mytochondrial cytochrome P450scc enzyme catalyses the first step in steroid hormones biosynthesis, represented by the conversion of cholesterol to pregnenolone. In order to study the effect of single mutations on the Adx binding a model of bovine cytochrome P450scc, previously optimized by molecular modeling, was utilized. It was hypothesized by molecular docking that two residues (Asp405 and Glu124) are involved in Adx binding. By site-directed mutagenesis, two mutants of cytochrome P450scc (Asp405Ile and Glu124Ile) expressed in Escherichia coli, were realized by replacing with isoleucines. The site-directed mutations effect on Adx binding was evaluated by differential spectral titration. The apparent dissociation constant values for Asp405Ile and Glu124Ile cytochrome P450scc show that the mutated residues seem to be at the interaction domain with Adx or at least close to it, as predicted by molecular modeling study. Finally, the engineered enzymes were characterized by biochemical and biophysical techniques such as circular dichroism (CD), UV/Vis spectroscopy, and electrochemical analysis.

Adrenodoxin↗

PubMed2004

Recombinant cytochrome p450 immobilization for biosensor applications.

As a result of the very attractive pleiotropic properties of the heme-enzymes, three P450 cytochrome isoforms (P4501A2, P4502B4, P450SCC) have been utilized to identify a general optimal procedure to biodevice assembly for sensing a wide range of organic substances. The Langmuir-Blodgett films appears to yield the best stable working conditions as shown by UV-vis spectrophotometry, nanogravimetry, circular dichroism, and electrochemical characterization, to identify the ordered nanostructures of P450 cytochromes optimal for clozapine, styrene, and cholesterol sensing. Only in the presence of low purity grade protein, as in the case of P4501A2, a gel-matrix was needed to warrant the optimal clozapine sensing. By the combination of proper immobilization, transducer and nanostructured mutants of high-grade stable and selective P450-based sensors appear capable to detect the interaction with a wide range of organic substrates such as fatty acids, drugs, and toxic compounds.

Biosensing Techniques↗

PubMed2004

P450scc mutant nanostructuring for optimal assembly.

Molecular modeling and protein engineering were synergically employed to improve the fabrication of cytochrome P450scc mutant nanostructures for biodevice assembly. The optimization of protein three-dimensional structure by molecular modeling was performed using two models: in vacuum and simulating the presence of a polar solvent. Calculations were performed on a model to predict a P450scc mutant which could improve the process of molecules' immobilization onto solid supports. Engineerized cytochrome P450scc thin films were prepared and characterized by various biophysical techniques such as pi-A isotherms, surface potential measurements, Brewster angle microscopy, UV-vis spectroscopy, circular dichroism, nanogravimetry, and electrochemical analysis. This paper takes into consideration biomolecules modified by protein engineering that represent a new and powerful approach for obtaining synthetic simpler artificial structures with new or improved properties (i.e., specificity, stability, sensitivity, etc.) useful for biosensors development.

Amino Acid Sequence↗

PubMed2002

Unique activation mechanism of protein kinase CK2. The N-terminal segment is essential for constitutive activity of the catalytic subunit but not of the holoenzyme.

CK2 is an essential, ubiquitous, and highly pleiotropic protein kinase whose catalytic subunits (alpha and alpha') and holoenzyme (composed by two catalytic and two regulatory beta-subunits) are both constitutively active, a property that is suspected to contribute to its pathogenic potential. Extensive interactions between the N-terminal segment and the activation loop are suspected to underlie the high constitutive activity of the isolated catalytic subunit. Here we show that a number of point mutations (Tyr(26) --> Phe, Glu(180) --> Ala, Tyr(182) --> Phe) and deletions (Delta 2-6, Delta 2-12, Delta 2-18, Delta 2-24, Delta 2-30) expected to affect these interactions are more or less detrimental to catalytic activity of the alpha-subunit of human CK2, the deleted mutants Delta 2-24 and Delta 2-30 being nearly inactive under normal assay conditions. Kinetic analyses showed that impaired catalytic activity of mutants Delta 2-12, Delta 2-18, Delta 2-24, and Y182F is mainly accounted for by dramatic increases in the K(m) values for ATP, whereas a drop in K(cat) with K(m) values almost unchanged was found with mutants Y26F and E180A. Holoenzyme reconstitution restored the activity of mutants Delta 2-12, Delta 2-18, Y26F, E180A, and Y182F to wild type level and also conferred catalytic activity to the intrinsically inactive mutants, Delta 2-24 and Delta 2-30. These data demonstrate that specific interactions between the N-terminal segment and the activation loop are essential to provide a fully active conformation to the catalytic subunits of CK2; they also show that these interactions become dispensable upon formation of the holoenzyme, whose constitutive activity is conferred by the beta-subunit through a different mechanism.

Adenosine Triphosphate↗

PubMed2002

Expression, purification, and structural characterization of human histone H4.

Recombinant human histone H4 (hH4) was produced in milligrams quantities in Escherichia coli, without altering the codons of the original cDNA sequence. The hH4 cDNA was subcloned into the pQE30 expression vector, in frame with a sequence encoding an N-terminal stretch of six histidine residues. Purification to electrophoretic homogeneity was obtained by nickel-chelating chromatography, followed by gel filtration. The final yield of the entire expression and purification process was about 1 mg of pure histone H4 per liter of bacterial culture. SDS-PAGE analysis showed for the recombinant H4 a molecular weight corresponding to the expected one (12,535 Da). Circular dichroism spectroscopy was used to estimate the secondary structural composition of recombinant histone, when it is isolated from the physiological core particle. It was observed that under these conditions histone H4 exhibits an altered secondary conformation. In order to induce the recombinant histone to assume a conformation more similar to the one measured when it is organized inside the nucleosome, we resuspended it in buffers at increasing ionic strengths and in the presence of different concentrations of trifluoroethanol. We tried also to mimic the physiological situation of histone H4 by adding an equimolar amount of a commercial DNA to the protein solution. Finally, an estimation of protein thermal stability was evaluated by spectropolarimetry.

Circular Dichroism↗

PubMed2002

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.

Biocompatible Materials↗

PubMedDate not supplied

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.

Animals↗