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A Pastore

Publications and source records attributed to A Pastore.

At least 109 records · Page 6Linked to original sources
PubMed1995

The C-terminal domain of alpha-spectrin is structurally related to calmodulin.

An alignment of amino acid sequences suggests that the spectrin domain, which contains two EF-hand calcium-binding motifs, is structurally related to calmodulin. It is possible to align approximately 160 residues at the C-terminus of alpha-spectrin with the entire calmodulin sequence. We have expressed this domain in Escherichia coli and purified it. Circular dichroic and nuclear magnetic resonance spectroscopy show that the protein is folded and mostly helical. The conformation of the protein, as monitored spectroscopically, is sensitive to calcium at 0.1-1.0 mM. Equilibrium dialysis shows that there are two binding sites within this domain, with affinities in the 0.5 mM range. The domain can be split into N-terminal and C-terminal halves which fold independently. Only the N-terminal subdomain binds calcium. These data suggest that the C-terminus of alpha-spectrin has a domain with a calmodulin fold and two calcium-binding sites. Sequence alignments suggest that the related domains in alpha-actinin, and possibly in dystrophin, may share the same calmodulin-like structure. However, only non-muscle alpha-actinins appear to have one or two EF-hand(s) with the calcium-binding consensus sequence, and a strict consensus is not found in the muscle alpha-actinins or dystrophins.

Amino Acid Sequence↗
PubMed1995

Probing protein structure by solvent perturbation of NMR spectra. A comparison with photochemically induced dynamic nuclear polarization techniques applied to native alpha-lactalbumin.

We have suggested elsewhere the use of surface mapping by spin label probes (Esposito et al., 1992). According to this approach, soluble nitroxides are added to a protein solution. Resonances of protons that are accessible to the nitroxide are broadened and bleached out of the spectrum, while resonances in the protein interior remain unaffected. This approach is, in principle, complementary to another technique, photochemically induced dynamic nuclear polarization, which maps the position of aromatic protons on the protein surface. A detailed comparison between the two techniques is necessary for a confident use of the more recent suggested nitroxide perturbation approach. In the present study, we show that the results obtained by the two techniques for the native state of bovine alpha-lactalbumin are fully consistent and may therefore be combined for the study of protein surfaces.

Animals↗
PubMed1995

Probing protein structure by solvent perturbation of NMR spectra. Photochemically induced dynamic nuclear polarization and paramagnetic perturbation techniques applied to the study of the molten globule state of alpha-lactalbumin.

We have characterized the high-temperature molten globule state of bovine alpha-lactalbumin by a combined use of photochemically induced dynamic nuclear polarization and nitroxide surface perturbation. Both techniques are extremely well suited to follow the progressive increase of exposed surfaces in the native state and the appearance of partially or completely unfolded species. Our results suggest that the molten globule state obtained at high temperature and pH 7, and the state obtained at pH 2 are not only thermodynamically but also structurally very similar.

Animals↗
PubMed1995

Secondary structure determination by NMR spectroscopy of an immunoglobulin-like domain from the giant muscle protein titin.

We present the complete 15N and 1H NMR assignment and the secondary structure of an immunoglobulin-like domain from the giant muscle protein titin. The assignment was obtained using homonuclear and 15N heteronuclear 2D and 3D experiments. The complementarity of 3D TOCSY-NOESY and 3D 15N NOESY-HSQC experiments, using WATERGATE for water suppression, allowed an efficient assignment of otherwise ambiguous cross peaks and was helpful in overcoming poor TOCSY transfer for some amino acids. The secondary structure is derived from specific NOEs between backbone alpha- and amide protons, secondary chemical shifts of alpha-protons and chemical exchange for the backbone amide protons. It consists of eight beta-strands, forming two beta-sheets with four strands each, similar to the classical beta-sandwich of the immunoglobulin superfamily, as previously predicted by sequence analysis. Two of the beta-strands are connected by type II beta-turns; the first beta-strand forms a beta-bulge. The whole topology is very similar to the only intracellular immunoglobulin-like domain for which a structure has been determined so far, i.e., telokin.

Amino Acid Sequence↗
PubMed1995

The folding and stability of titin immunoglobulin-like modules, with implications for the mechanism of elasticity.

Titin (first known as connectin) is a vast modular protein found in vertebrate striated muscle. It is thought to assist myofibrillogenesis and to provide a passive elastic restoring force that helps to keep the thick filaments properly centered in the sarcomere. We show that representative titin modules do indeed fold independently, and report their stabilities (i.e., delta G of unfolding and melting temperature) as measured by circular dichroism, fluorescence, and nuclear magnetic resonance spectroscopies. We find that there is a region-dependent variation in stability, although we find no evidence to support a proposed elastic mechanism based on a molten-globular-like equilibrium folding intermediate, nor do our calculations support any mechanism based on the configurational entropy of the molecule itself; instead we suggest a model based on hydrophobic hinge regions that would not be strongly dependent on the precise folding pattern of the chain.

Animals↗
PubMed1995

[The problem of lymph nodes in malignant epithelial tumors of the parotid gland].

Cervical lymph-node treatment in parotid gland epithelial malignancies is still debated. According to Literature, three different strategies (surgery, radiotherapy, "wait and see") have all been proposed theoretically, particularly when dealing with N0 cases. The present study was designed to evaluate the results of different lymphonode treatment strategies in 57 parotid gland carcinomas followed at the ENT Clinic of the University of Ferrara. The most frequent hystological patterns appeared to be the adenoidcystic carcinoma (33.3%) and the mucoepidermoid tumor (21.1%). Total parotidectomy was the treatment of choice in all cases. Ipsilateral neck dissection was performed in 14 cases (24.5%), 5 cases being N0. In 27 patients (47.4%) postsurgical radiotherapy was applied: in 7 cases on T and in 20 on both T and N.T recurrences were 7, while those of N and of both T and N were respectively 2 and 2. No occult metastases were found in N0 dissected patients. The results obtained led the Authors to the following conclusions: -neck dissection is fundamental in treatment of clinical adenopathies in any parotid gland malignancy: -postsurgical radiotherapy on the neck is the treatment of choice in all N0 carcinomas except in cases of acinic cell and mucoepidermoid carcinomas, where a "wait and see" policy seems to be more suitable.

Adolescent↗
PubMed1994

The dimerization stability of the HLH-LZ transcription protein family is modulated by the leucine zippers: a CD and NMR study of TFEB and c-Myc.

In the HLH-LZ protein family, the helix-loop-helix DNA-binding dimerization domain is followed in the sequence by a leucine zipper motif. The precise function of this second dimerization domain is still unclear, since the HLH motif of a subset of this family has been shown to be necessary and sufficient for dimerization. However, deletion and mutagenesis studies of the leucine zipper in various HLH-LZ proteins have shown a clear influence of this motif on homo- and heterodimerization. In this paper, we present a structural characterization of synthetic peptides encompassing the leucine zipper sequences of c-Myc and TFEB, using circular dichroism, analytical ultracentrifugation, and nuclear magnetic resonance. We show that the different ability of the synthetic leucine zippers of c-Myc and TFEB to homodimerize at neutral pH reflects the different dimerization properties reported for the entire proteins. The TFEB protein is known to form homodimers. c-Myc, on the other hand, does not homodimerize in vivo, but is mostly found in heterodimeric complexes with Max, another protein of the HLH-LZ family. Accordingly, our results show that the TFEB peptide homodimerizes at neutral pH whereas the Myc peptide dimerizes to a comparable amount only at acidic pH and high ionic strength. Both synthetic peptides are far less stable than leucine zippers of the b-ZIP family. The relative stability of the two leucine zippers and the factors which stabilize the dimer formation are discussed.

Amino Acid Sequence↗
PubMed1994

Immunoglobulin-type domains of titin are stabilized by amino-terminal extension.

We have recently suggested that similarly folded titin modules located at different sarcomeric regions have distinct molecular properties and stability. Could our selection of module boundaries have potentially influenced our conclusions? To address this question we expressed amino-terminally extended versions of the same modules and determined, with the use of CD and Fluorescence techniques, key thermodynamic parameters characterizing their stability. We present here our results which confirm our previous observations and show that, while amino-terminal extension has a profound effect on the stability of individual modules, it does not affect at all their folding pattern or their relative stabilities. Moreover, our data suggest that the selection of module boundaries can be of critical importance for the structural analysis of modular proteins in general, especially when a well-defined intron-exon topography is absent and proteolytic methods are inconclusive.

Amino Acid Sequence↗
PubMed1994

Immunoglobulin-type domains of titin: same fold, different stability?

Titin is a 3-MDa protein thought to form a fibrous intracellular system in vertebrate striated muscle and to play an important role in sarcomere alignment during muscle contraction. It has also been implicated as a "molecular ruler", regulating the assembly and the precise length of the thick filaments [Whiting, A. J., Wardale, J., & Trinick, J. (1989) J. Mol. Biol. 205, 163-169]. Partial sequencing of titin-encoding cDNAs suggests that the protein is organized in a modular fashion, containing two classes of approximately 100-residue repeats [Labeit, S., Barlow, D. P., Gautel, M., Gibson, T., Holt, J., Hsieh, C. L., Francke, U., Leonard, K., Wardale, J., Whiting, A., & Trinick, J. (1990) Nature 345, 273-276]. These motifs, referred to as type I and type II modules, show sequence homology to the fibronectin III and immunoglobulin C2 superfamilies, respectively. Since the type II modules represent the most widely occurring motifs along the titin molecule, we expressed in Escherichia coli three domains of this type spanning different regions of the sarcomere (A-band and M-line) and studied their structure and stability. Using circular dichroism, nuclear magnetic resonance, and fluorescence spectroscopy, we showed that all the fragments examined are independently folded in solution and possess a beta-sheet conformation. Furthermore, employing NMR analysis, we identified an overall folding pattern present in all modules and related to the Ig fold, as previously suggested by theoretical predictions. The stability of the modules over a range of conditions was investigated by measuring key thermodynamic parameters for both thermal and chemical denaturation and by monitoring amide proton exchange as a function of time.(ABSTRACT TRUNCATED AT 250 WORDS)

Amides↗
PubMed1994

Nebulin, a helical actin binding protein.

Nebulin, a giant protein (molecular mass 800 kDa) specific for the skeletal muscle of vertebrates, has been suggested to be involved in the length regulation of the thin filament as a 'molecular ruler'. Despite its size, nebulin appears to be composed mainly of small repeats of approximately 35 amino acids. We have characterized in this study the conformational and functional properties of single repeats. Complete repeats were found to bind to F-actin while a truncated one did not. One repeat is therefore the smallest unit for nebulin--actin interaction. Circular dichroism and nuclear magnetic resonance spectra measured for the peptides in water indicated a transient helical conformation. The folded region is located for them all around the conserved sequence SDxxYK. The helical conformation is strongly stabilized by anionic detergents and trifluoroethanol while uncharged or positively charged detergents have no effect. Since the surface of the actin filament is known to contain clusters of negative charges, anionic detergents may mimic the effect of an actin environment. 3D structures were calculated for three representative peptides in SDS. In vivo, the nebulin helices should form a complex with the actin filament. Based on the assumed importance of charge interactions between nebulin and actin, we propose a model for the structure of the F-actin-nebulin complex in vivo. According to that, two nebulin molecules occupy symmetrical positions along the central cleft of the actin filament bridging the two strands of the actin two-start helix. The consistency of this model with experimental data is discussed.

Actins↗
PubMed1994

Conformation and molecular dynamics calculations on uteroglobin fragment 18-47.

The conformational properties of fragment 18-47 of rabbit uteroglobin in aqueous solution containing SDS micelles were investigated by two-dimensional nmr spectroscopy and molecular dynamics calculations. The fragment comprises helices II and III and the beta-turn connecting the two helices. The nmr results and nmr-restrained molecular dynamics calculations showed that in the isolated fragment the elements of secondary structure present in the intact protein are preserved only in part. Specifically, a well-defined alpha-helix was found in the sequence 33-44, corresponding to helix III of uteroglobin, while the regions of helix II and beta-turn are characterized by high flexibility in the fragment.

Amino Acid Sequence↗
PubMed1994

A novel chromosomal abnormality detected in a case of acute myelomonocytic leukemia.

A not yet recorded translocation, t(3:17)(q21;q23), detected in a case of acute myelomonocytic leukemia, is reported. In spite of an aggressive cytotoxic chemotherapy, the disease showed a rapid fatal course, confirming the dismal prognostic significance of structural, 'primary' chromosomal abnormalities in acute leukemias.

Chromosomes, Human, Pair 17↗
PubMed1993

Conformational states of human placental glutathione transferase as probed by limited proteolysis.

Limited proteolysis experiments have been carried out on human placental glutathione transferase in its different forms. The reduced enzyme, as well as the oxidized form and that inactivated with cystamine were all sensitive to 10% (w/w) trypsin, under nondenaturing conditions. The proteolytic cleavage was accompanied by a concomitant loss of enzymatic activity. On the contrary, the presence of glutathione or glutathione conjugates strongly protected the reduced enzyme against inactivation and from the proteolytic attack. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and peptide sequence analysis showed that only the peptide bond between Lys 44 and Ala 45 was cleaved. Since Lys 44 has been demonstrated to be involved in the glutathione binding, it is suggested that the region surrounding this amino acid residue (alpha B helix) could be more exposed to the solvent, in the absence of glutathione. Crystallographic data also indicated that this region is flexible, supporting the idea that it may be involved in the observed conformational change upon glutathione binding.

Alanine↗
PubMed1993

Probing protein structure by solvent perturbation of NMR spectra. II. Determination of surface and buried residues in homologous proteins.

The experimental assignment of most residues in a protein to the surface or interior is in principle possible without prior solution of a complete three-dimensional structure. The method described is based on nmr measurements that determine the amino acid composition of the surface of a protein [A. Petros, L. Mueller, and K.D. Kopple (1990) Biochemistry, Vol. 29, pp. 10041-10048; G. Esposito, A. M. Lesk, H. Molinari, A. Motta, N. Niccolai, and A. Pastore (1992) Journal of Molecular Biology, Vol. 224, pp. 659-670]. If these measurements are carried out on several homologous proteins of known sequence, it is possible to combine the results to determine, in most cases, which positions in the sequence contain exposed residues.

Amino Acid Sequence↗
PubMed1993

Homology model building of the thyroid transcription factor 1 homeodomain.

A possible structure for the homeodomain of rat thyroid transcription factor 1 is proposed on the basis of the homology with other homeodomains whose structures have been solved by X-ray crystallography. A structure very similar to the reference ones is feasible and may account for the observed DNA-binding specificity. Structural features of the model, which are likely to be shared by other homeodomains, are discussed and may help researchers in the field to analyse their own experimental data.

Amino Acid Sequence↗
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↗