Biomedical subjects

A Pastore

Publications and source records attributed to A Pastore.

At least 19 recordsLinked to original sources

PubMed1992

p53 over-expression is an early event in the development of human squamous-cell carcinoma of the larynx: genetic and prognostic implications.

The tumor-suppressor protein p53 is over-expressed in a large fraction of squamous-cell carcinomas of the larynx (LSCCs). p53 overexpression is dependent upon the synthesis of mutated versions of the protein and has been associated with the malignant progression of certain tumor types. In order to examine the prognostic value of p53 immunodetection in LSCCs, we performed a retrospective analysis on a selected series of tumors, using the PAb 1801 and CM1 antibodies. No significant difference in the frequency of p53 over-expression was observed between tumors from patients with early relapse (67%) and those who had been disease-free for more than 5 years (84%). The lack of correlation of p53 immunoreactivity with clinical stage and differentiation grade of LSCCs, together with the coordinated expression of p53 in primary tumors and the corresponding lymph-node metastases, indicate that p53 over-expression is probably unrelated to the biological aggressiveness of these tumors. In addition, the detection of p53 immunostaining in pre-invasive areas as well as in preneoplastic lesions suggests that p53 abnormalities probably constitute a very early event in LSCC development.

Carcinoma in Situ↗

PubMed1992

Probing protein structure by solvent perturbation of nuclear magnetic resonance spectra. Nuclear magnetic resonance spectral editing and topological mapping in proteins by paramagnetic relaxation filtering.

Soluble spin labels, which "bleach" the surface proton resonances of a protein to n.m.r. measurements, can provide useful information about protein conformation and dynamics. The use of the soluble nitroxide, TEMPOL, has been explored to show the correlation of the paramagnetic perturbations of protein two-dimensional n.m.r. data with proton exposure to the free radical in hen egg-white lysozyme. The results demonstrate that the nitroxide approaches the protein randomly, and that the extent of the observed paramagnetic effects reflects the native folding pattern of the protein. A correlation of spectral simplification with the known tertiary structure establishes the feasibility of new strategies for topological mapping of surface and buried protons of the protein. Application to the elucidation of protein structure and to the study of dynamical processes is discussed.

Amino Acid Sequence↗

PubMed1992

Three-dimensional structure of acylphosphatase. Refinement and structure analysis.

We report here the complete determination of the solution structure of acylphosphatase, a small enzyme that catalyses the hydrolysis of organic acylphosphates, as determined by distance geometry methods based on nuclear magnetic resonance information. A non-standard strategy for the distance geometry calculations was used and is described here some detail. The five best structures were then refined by restrained energy minimization and molecular dynamics in order to explore the conformational space consistent with the experimental data. We address the question of whether the solution structure of acylphosphatase follows the general principles of protein structure, i.e. those learned from analysing crystal structures. Static and dynamic features are discussed in detail. An uncommon beta-alpha-beta motif, so far found only in procarboxypeptidase B and in an RNA-binding protein, is present in acylphosphatase.

Acid Anhydride Hydrolases↗

PubMed1992

A method for multiple superposition of structures.

The study of families of protein structures is important in analysing the results of NMR structure determinations and in investigating mechanisms of molecular evolution at the level of conformation. A method is discussed for finding the transformations that mutually superpose an arbitrary number of structures in the least-squares sense given specified atom-to-atom correspondence.

Least-Squares Analysis↗

PubMed1992

Dynamic properties of salmon calcitonin bound to sodium dodecyl sulfate micelles: a restrained molecular dynamics study from NMR data.

We have investigated the conformational behaviour of salmon calcitonin bound to sodium dodecyl sulfate micelles by means of restrained molecular dynamics simulations with both 'static' and time-averaged NMR distance restraints. A more realistic picture of the inherent flexibility of the hormone is obtained when using time averaging. With this approach, long-range NOEs are interpreted better by considering a dynamical exchange among different conformations.

Amino Acid Sequence↗

PubMed1992

The dimerization domain of LFB1/HNF1 related transcription factors: a hidden four helix bundle?

LFB1/HNF1 alpha and LFB3/HNF1 beta bind DNA as dimers and form heterodimers together in vivo and in vitro. The dimerization domain has been located in both proteins in the 32 N-terminal residues. In previous papers we have described the conformational stability as determined by CD and the secondary structure by NMR studies of a peptide with the amino acid sequence of the dimerization domain of LFB1/HNF1 alpha. This study presents a more complete characterization of similar synthetic peptides spanning the LFB3/HNF1 beta dimerization domain and the alpha/beta heterodimer. The HNF1 peptides represent an example of structures which cannot be determined by NOE data alone because they are not sufficient to define one unique solution. An approach is presented which combines NMR data, the protein structure database and structural analyses according to known principles of protein structure. On this basis we are able to determine possible solutions and to identify a four helix bundle as the structure most consistent with the experimental evidence.

DNA-Binding Proteins↗

PubMed1991

Solution conformation of salmon calcitonin in sodium dodecyl sulfate micelles as determined by two-dimensional NMR and distance geometry calculations.

The 32 amino acid hormone salmon calcitonin was studied at pH 3.7 and 7.4 by two-dimensional NMR in sodium dodecyl sulfate (SDS) micelles at 310 K. The spectrum was fully assigned, and the secondary structure was obtained from nuclear Overhauser enhancement spectroscopy (NOESY), 3JHN alpha coupling constants, and slowly exchanging amide data. Three-dimensional structures consistent with NMR data were generated by using distance geometry calculations. A set of 260 interproton distances, derived from NOESY, and hydrogen-bond constraints, obtained from analysis of the amide exchange, were used. From the initial random conformations, 13 distance geometry structures with minimal violations were selected for further refinement with restrained energy minimization. In SDS, at both pHs, the main conformational feature of the hormone is an alpha-helix from Thr6 through Tyr22, thus including the amphipathic 8-22 segment and two residues of the Cys1-Cys7 N-terminal loop. The C-terminal decapeptide forms a loop folded back toward the helix. The biological significance of this conformation is discussed.

Amino Acid Sequence↗

PubMed1991

Solution structure of the basic region from the transcriptional activator GCN4.

The structure of the basic region (i.e., the region responsible for sequence-specific binding to DNA) of the transcriptional activator GCN4 was studied. Two peptide fragments containing either the basic region alone (residues 240-280) or the basic and the dimerization leucine zipper domains (220-280) were synthesized and investigated by nuclear magnetic resonance and circular dichroic spectroscopy. The basic region in the absence of DNA appears as a mobile flexible segment folded into a loose helix. The helical stability increases upon addition of trifluoroethanol and/or lowering of the temperature. Dimerization via the leucine zipper does not affect the three-dimensional structure of the basic region. Possible consequences for the binding to DNA are discussed.

Amino Acid Sequence↗

PubMed1991

Circular dichroism study on the conformational stability of the dimerization domain of transcription factor LFB1.

LFB1, a dimeric DNA binding protein, is a major determinant of hepatocyte-specific transcription. The thermal and chemical equilibrium unfolding of a 32-residue alpha-helical peptide comprising its dimerization domain (B1-Dim) was monitored by circular dichroism spectroscopy. The conformational stability of this peptide is shown to be concentration dependent, and the unfolding reaction is described as a two-state transition between folded dimers and unfolded monomers. The thermodynamic parameters associated with the unfolding reaction were determined under the two-state assumption by the van't Hoff procedure. The enthalpy of unfolding increases linearly with temperature, and the corresponding value of delta Cp, the difference in heat capacity between the unfolded and the folded forms of the peptide, is estimated to be ca. 0.7 kcal mol-1 K-1. The dimeric folded structure of the peptide is stabilized, at 25 degrees C, by a delta G of about 11.5 kcal mol-1, which is equivalent to a dimerization constant greater than 10(8) mol-1. These results indicate that the dimerization domain of LFB1 can fold and dimerize independently of the rest of the protein, with a thermodynamic stability comparable to that of a small globular protein.

Amino Acid Sequence↗

PubMed1991

1H resonance assignment and secondary structure determination of the dimerization domain of transcription factor LFB1.

We have started the structure determination of the dimerization domain of LFB1 in solution by nuclear magnetic resonance in order to elucidate the way that the LFB1 protein dimerizes and then interacts with DNA. A 32 amino acid peptide was synthesized, and full assignment of the NMR resonances in acidic solution was achieved. The secondary structure determination is presented here. Three structurally distinct regions can be distinguished. The N-terminal region from residues 1 to 6 is extended. Two helical regions span from residues 7 to 18 and from 23 to 32. The absence of dipolar effects involving residues more than four positions apart in the sequence excludes the possibilities both of a four-helix bundle formed by two hairpins and of an antiparallel dimer; the domain must therefore be arranged as a parallel dimer formed by kinked monomers. This structural solution presents important differences from the leucine zipper-type structure observed in other transcriptional activators. Although further studies are still necessary to determine the 3D structure of the peptide, we can exclude the possibility of a coiled-coil structure.

Amino Acid Sequence↗

PubMed1991

Topological mirror images in protein structure computation: an underestimated problem.

When calculating three-dimensional structures from NMR data, alternative solutions with very large RMS deviation can be obtained. Sometimes local or global inversions of the protein folding can be observed. We call these different solutions topological mirror images, as they keep the correct amino acid chirality. They are observed when the number of restraints is insufficient and represent different solutions from the same scalar information. Therefore they are common in small peptides where the NMR data are often limited and the secondary structure is not very well defined. They can also be observed in large molecules in regions of higher flexibility. In our experience the observation of topological mirror images is independent of the efficiency of sampling of the algorithm used. We present four examples of proteins with different size and folding. We also discuss ways to distinguish among the different solutions.

Acid Anhydride Hydrolases↗

PubMed1991

Proto-oncogene allelic variations in human squamous cell carcinomas of the larynx.

Proto-oncogene restriction fragment length polymorphisms (RFLPs) were investigated in a group of 23 patients with squamous cell carcinomas of the larynx. The frequency of the rare 5 kb c-mos allele was significantly higher than that observed in control groups of patients with colorectal neoplasms or lymphoproliferative disorders. In addition, the 2 patients heterozygous at the c-mos locus (TC-8 and TC-10) were the only 2 of our series of develop multiple malignancies. Also, the 10 kb L-myc allele was remarkably more represented in patients with laryngeal carcinoma when compared to controls. These findings suggest that c-mos and L-myc RFLPs might be helpful in identifying those individuals who are at a higher risk of developing laryngeal carcinomas. Single allele amplification of L-myc, c-myb and c-mos proto-oncogenes, with no concomitant mRNA hyperexpression, were observed in 3 cases. The results obtained seem to rule out a direct pathogenetic role of these proto-oncogenes and suggest that the amplification of other closely linked genes, located on chromosomes 1, 6 and 8, respectively, may be causally associated with the development of these tumors. No allelic deletions at the c-myb locus were observed, whereas a loss of a c-Ha-ras-1 allele was demonstrated in one of the 11 heterozygous patients. Thus, the analysis of polymorphic proto-oncogenes in laryngeal carcinomas allowed us to identify a group of genetic abnormalities (chromosomes 1, 6 and 8 gene amplifications and c-Ha-ras-1 deletions) which may be involved in the development or progression of these tumors.

Aged↗

PubMed1991

The structure of ColE1 rop in solution.

The structure of the ColE1 repressor of primer (rop) protein in solution was determined from the proton nuclear magnetic resonance data by a combined use of distance geometry and restrained molecular dynamics calculations. A set of structures was determined with low internal energy and virtually no violations of the experimental distance restraints. Rop forms homodimers: Two helical hairpins are arranged as an antiparallel four helix bundle with a left-handed rope-like twist of the helix axes and with left-handed bundle topology. The very compact packing of the side chains in the helix interfaces of the rop coiled-coil structure may well account for its high stability. Overall, the solution structure is highly similar to the recently determined X-ray structure (Banner, D.W., Kokkinidis, M. and Tsernoglou, D. (1987) J. Mol. Biol., 196, 657-675), although there are minor differences in regions where packing forces appear to influence the crystal structure.

Bacterial Proteins↗

PubMed1991

Brave new proteins: what evolution reveals about protein structure.

Synthetic proteins provide important information about the principles of protein structure. They illuminate the processes of natural protein evaluation, but are not limited by these processes. Here, we review studies of several mutant proteins and discuss the general principles that can be derived from them.

Animals↗

PubMed1991

The solution structure of a leucine-zipper motif peptide.

We report the complete structure determination of a 34 residue synthetic peptide with the amino acid sequence of the dimerization domain (leucine zipper) of GCN4. A high resolution structure in solution was obtained by 1H-NMR studies and distance geometry calculations followed by restrained energy minimization. A set of 20 final structures was obtained with an average root mean square deviation of 1.3 A for the backbone atoms (excluding the first and the last two residues). The structure contains an uninterrupted helix. A comparison with a structure previously determined for a larger peptide containing both the DNA-binding region (basic region) and the leucine-zipper motif shows the structural independence of the leucine-zipper domain from the contiguous DNA binding region.

Amino Acid Sequence↗

PubMed1991

Conformational study of cyclolinopeptide A. A distance geometry and molecular dynamics approach.

The conformation of cyclolinopeptide A, c(Pro-Pro-Phe-Phe-Leu-Ile-Ile-Leu-Val), a naturally occurring peptide with remarkable cytoprotective activity, has been investigated by means of distance geometry calculations and molecular dynamics simulations. The starting points for all the calculations were an X-ray structure and other structures obtained from distance geometry calculations based on NMR data. Restrained and unrestrained molecular dynamics simulations are reported in vacuo and in CCl4. Structural and dynamic properties are investigated and compared with those experimentally determined. The conformation obtained from the MD simulations which best reproduces the NMR parameters is at the same time one of the most stable ones and is also fairly similar to the crystal structure. An explanation for the occurrence of multiple conformations in solution at room temperature is given.

Amino Acid Sequence↗

PubMed1990

NMR and molecular dynamics studies of the mKr2 'zinc finger'.

We determined the structure of a 30-amino-acid 'zinc finger' motif from the mouse Kruppel-like gene mKr2 in solution in the absence of metal ions by two-dimensional NMR, distance geometry and restrained molecular dynamics methods. The most prominent secondary structural feature of the peptide is a helix extending from Ser14 to Ile20. The zinc-containing structure of the peptide was simulated by molecular dynamics calculations with restraints derived from the known geometry of the zinc ion and the ligating amino acid residues Cys6, Cys9, His22, His26. The latter structure does not deviate markedly from the set of structures determined for the zinc-free peptide, i.e. this peptide can accommodate a Zn2+ ion without major structural rearrangements. Thus, we propose that the metal ion stabilizes the existing three-dimensional peptide structural motif rather than introducing novel structural features.

Amino Acids↗

PubMed1990

Comparison of the structures of globins and phycocyanins: evidence for evolutionary relationship.

Globins and phycocyanins are two classes of proteins with different function, different ligands, and no substantial sequence similarity, yet the conformations of their polypeptide chains show very similar folding patterns. Does this arise from a genuine, albeit very distant, evolutionary relationship, or does it represent a common solution of a structural problem? We address this question by a very detailed comparison of the structures of the two protein families. An analysis of the helices and their interactions shows many features common to globins and phycocyanins, including some exceptional features of the globins such as a 3-10 C helix and the unusual "crossed-ridge" packing pattern at the B/E helix interfaces. We conclude that the evidence supports the hypothesis of distant evolutionary relationship between globins and phycocyanins.

Amino Acid Sequence↗