Biomedical subjects

P Amodeo

Publications and source records attributed to P Amodeo.

At least 19 recordsLinked to original sources

PubMed2001

Thiol/disulfide interconversion in bovine lens aldose reductase induced by intermediates of glutathione turnover.

The effectiveness of cysteine and cysteinylglycine to act as protein thiolating agents was investigated using bovine lens aldose reductase (ALR2) as the protein target. Disulfides of both thiol compounds appear to be very effective as ALR2 thiolating agents. Cysteine- and CysGly-modified ALR2 forms (Cys-ALR2 and CysGly-ALR2, respectively) are characterized by the presence of a mixed disulfide bond involving Cys298, as demonstrated by a combined electrospray mass spectrometry and Edman degradation approach. Both Cys-ALR2 and CysGly-ALR2 essentially retain the ability to reduce glyceraldehyde but lose the susceptibility to inhibition by Sorbinil and other ALR2 inhibitors. Cys-ALR2 and CysGly-ALR2 are easily reduced back to the native enzyme form by dithiothreitol and GSH treatment; on the contrary, Cys and 2-mercaptoethanol appear to act as protein trans-thiolating agents, rather than reducing agents. The treatment at 37 degrees C of both Cys-ALR2 and CysGly-ALR2, unlikely what observed for glutathionyl-modified ALR2 (GS-ALR2), promotes the generation of an intramolecular disulfide bond between Cys298 and Cys303 residues. A rationale for the special susceptibility of Cys-ALR2 and CysGly-ALR2, as compared to GS-ALR2, to the thermally induced intramolecular rearrangement is given on the basis of a molecular dynamic and energy minimization approach. A pathway of thiol/disulfide interconversion for bovine lens ALR2 induced, in oxidative conditions, by physiological thiol compounds is proposed.

Aldehyde Reductase↗

PubMed2001

Modularity and homology: modelling of the titin type I modules and their interfaces.

Titin is a giant muscle protein with a highly modular architecture consisting of multiple repeats of two sequence motifs, named type I and type II. Type I motifs are homologous to members of the fibronectin type 3 (Fn3) superfamily, one of the motifs most widespread in modular proteins. Fn3 domains are thought to mediate protein-protein interactions and to act as spacers. In titin, Fn3 modules are present in two different super-repeated patterns, likely to be involved in sarcomere assembly through interactions with A-band proteins. Here, we discuss results from homology modelling the whole family of Fn3 domains in titin. Homology modelling is a powerful tool that will play an increasingly important role in the post-genomic era. It is particularly useful for extending experimental structure determinations of parts of multidomain proteins that contain multiple copies of the same motif. The 3D structures of a representative titin type I domain and of other extracellular Fn3 modules were used as a template to model the structures of the 132 copies in titin. The resulting models suggest residues that contribute to the fold stability and allow us to distinguish these from residues likely to have functional importance. In particular, analysis of the models and mapping of the consensus sequence onto the 3D structure suggest putative surfaces of interaction with other proteins. From the structures of isolated modules and the pattern of conservation in the multiple alignment of the whole titin Ig and Fn3 families, it is possible to address the question of how tandem modules are assembled. Our predictions can be validated experimentally.

Amino Acid Motifs↗

PubMed2001

Kinase recognition by calmodulin: modeling the interaction with the autoinhibitory region of human cardiac titin kinase.

Calmodulin (CaM)-protein interactions are usually described by studying complexes between synthetic targets of ca 25 amino acids and CaM. To understand the relevance of contacts outside the protein-binding region, we investigated the complex between recombinant human CaM (hCaM) and P7, a 38-residue peptide corresponding to the autoinhibitory domain of human cardiac titin kinase (hTK). To expedite the structure determination of hCaM-P7 we relied upon the high degree of similarity with other CaM-kinase peptide complexes. By using a combined homonuclear NMR spectroscopy and molecular modeling approach, we verified for the bound hCaM similar trends in chemical shifts as well as conservation of NOE patterns, which taken together imply the conservation of CaM secondary structure. P7 was anchored to the protein with 52 experimental intermolecular contacts. The hCaM-P7 structure is very similar to known CaM complexes, but the presence of NOE contacts outside the binding cavity appears to be novel. Comparison with the hTK crystal structure indicates that the P7 charged residues all correspond to accessible side-chains, while the putative anchoring hydrophobic side-chains are partially buried. To test this finding, we also modeled the early steps of the complex formation between Ca(2+)-loaded hCaM and hTK. The calculated trajectories strongly suggest the existence of an "electrostatic funnel", driving the long-range recognition of the two proteins. On the other hand, on a nanosecond time scale, no intermolecular interaction is formed as the P7 hydrophobic residues remain buried inside hTK. These results suggest that charged residues in hTK might be the anchoring points of Ca(2+)/hCaM, favoring the intrasteric regulation of the kinase. Furthermore, our structure, the first of CaM bound to a peptide derived from a kinase whose three-dimensional structure is known, suggests that special care is needed in the choice of template peptides to model protein-protein interactions.

Amino Acid Sequence↗

PubMed2001

Modulation of aldose reductase activity through S-thiolation by physiological thiols.

The glutathionyl-modified aldose reductase (GS-ALR2) is unique, among different S-thiolated enzyme forms, in that it displays a lower specific activity than the native enzyme (ALR2). Specific interactions of the bound glutathionyl moiety (GS) with the ALR2 active site, were predicted by a low perturbative molecular modelling approach. The outcoming GS allocation, involving interactions with residues relevant for catalysis and substrate allocation, explains the rationale behind the observed differences in the activity between GS-ALR2 and other thiol-modified enzyme forms. The reversible S-glutathionylation of ALR2 observed in cultured intact bovine lens undergoing an oxidative/non oxidative treatment cycle is discussed in terms of the potential of ALR2/GS-ALR2 inter-conversion as a response to oxidative stress conditions.

Aldehyde Reductase↗

PubMed2000

Pain peptides. Solution structure of orphanin FQ2.

Orphanin FQ2 (OFQ2) is a novel heptadecapeptide generated from prepronociceptin (PPNOC), the same precursor of nociceptin/orphanin FQ and nocistatin. OFQ2 is a potent analgesic when administered both supraspinally and spinally. In order to clarify the structural relationship with all peptides generated from PPNOC, we have undertaken the conformational study of OFQ2 in water and in structure-promoting solvent media. Nuclear magnetic resonance data and theoretical calculations are consistent with a well defined helical structure from Met(5) to Ser(16). The uniform distribution of hydrophobic residues along the helix suggests that OFQ2 may interact with the transmembrane helices of a receptor akin to those of nociceptin and opioids.

Amino Acid Sequence↗

PubMed1999

Exploring protein interiors: the role of a buried histidine in the KH module fold.

The K-homology (KH) module is a novel RNA-binding motif. The structures of a representative KH motif from vigilin (vig-KH6) and of the first KH domain of fmr1 have been recently solved by nuclear magnetic resonance (NMR) and automated assignment-refinement techniques (ARIA). While a hydrophobic residue is found at position 21 in most of the KH modules, a buried His is conserved in all the 15 KH repeats of vigilin. This position must therefore have a key structural role in stabilizing the hydrophobic core. In the present work, we have addressed the following questions in order to obtain a detailed description of the role of His 21: i) what is the exact role of the histidine in the hydrophobic core of vig-KH6? ii) can we define the interactions that allow a conserved buried position to be occupied by a histidine both in vig-KH6 and in the whole vigilin KH sub-family? iii) how is the structure and stability of vig-KH6 influenced by the state of protonation of this histidine? To answer these questions, we have carried out an extensive refinement of the vig-KH6 structure using both an improved ARIA protocol starting from different initial structures and successively running restrained and unrestrained trajectories in water. An analysis of the stability of secondary structural elements, solvent accessibility, and hydrogen bonding patterns allows hypothesis on the structural role of residue His 21 and on the interactions that this residue forms with the environment. The importance of the protonation state of His 21 on the stability of the KH fold was addressed and validated by experimental results.

Carrier Proteins↗

PubMed1999

A new cacospongionolide derivative from the sponge Fasciospongia cavernosa.

Cacospongionolide F (4a), a new bioactive cacospongionolide-related sesterterpene, has been isolated from the Northern Adriatic sponge Fasciospongia cavernosa. The structure was proposed on the basis of spectroscopic data and chemical transformations. The absolute configuration was established using the modified Mosher's method. A molecular mechanics study of the dehydrodecalin ring explained the observed differences in dynamic behavior between cacospongionolide F and mamanuthaquinone, a related compound. Antimicrobial activity, brine shrimp and fish lethalities of this new compound are reported.

4-Butyrolactone↗

PubMed1999

Conformational flexibility in calcitonin: the dynamic properties of human and salmon calcitonin in solution.

We have studied the dynamic properties of human (h) and salmon (s) calcitonin (CT) in solution. For both hormones, distance geometry in torsion-angle space has been used to generate three-dimensional structures consistent with NMR data obtained in sodium dodecyl sulfate micelles. For sCT and hCT we used, respectively, 356 and 275 interproton distances together with hydrogen-bonds as restraints. To better characterize their flexibility and dynamic properties two fully unrestrained 1100-ps molecular dynamics (MD) simulations in methanol were performed on the lowest-energy structures of both hormones. Statistical analyses of average geometric parameters and of their fluctuations performed in the last 1000 ps of the MD run show typical helical values for residues 9-19 of sCT during the whole trajectory. For hCT a shorter helix was observed involving residues 13-21, with a constant helical region in the range 13-19. Angular order parameters S(phi) and S(psi) indicate that hCT exhibits a higher flexibility, distributed along the whole chain, including the helix, while the only flexible amino acid residues in sCT connect three well-defined domains. Finally, our study shows that simulated annealing in torsion-angle space can efficiently be extended to NMR-based three-dimensional structure calculations of helical polypeptides. Furthermore, provided that a sufficient number of NMR restraints describes the system, the method allows the detection of equilibria in solution. This identification occurs through the generation of 'spurious' high-energy structures, which, for right-handed alpha-helices, are likely to be represented by left-handed alpha-helices.

Amino Acid Sequence↗

PubMed1998

Solution structure of human calcitonin in membrane-mimetic environment: the role of the amphipathic helix.

The 32 amino acid hormone human calcitonin was studied at pH 3.7 and 7.4 by multidimensional NMR spectroscopy in sodium dodecyl sulfate micelles at 310K. The secondary structure was obtained from nuclear Overhauser enhancement spectroscopy (NOESY), 3JHNalpha coupling constants, and slowly exchanging amide data. Three-dimensional structures consistent with NMR data were generated by using distance geometry calculations. A set of 265 interproton distances derived from NOESY experiments, hydrogen-bond constraints obtained from amide exchange, and coupling constants were used. From the initial random conformations, 30 distance geometry structures with minimal violations were selected for further refinement with restrained energy minimization. In micelles, at both pHs, the hormone assumes an amphipathic alpha-helix from Leu9 to Phel6, followed by a type-I beta-turn between residues Phel6 and Phel9. From His20 onward the molecule is extended and no interaction with the helix was observed. The relevance of the amphipathic helix for the structure-activity relationship, the possible mechanisms of interaction with the receptor, as well as the formation of fibrillar aggregates, is discussed.

Amino Acid Sequence↗

PubMed1998

Topology of the calmodulin-melittin complex.

The topology of the Ca2+-calmodulin-melittin ternary complex has been investigated by a combined strategy which integrates limited proteolysis and cross-linking experiments with mass spectrometric methodologies. The rationale behind the methods is that the interface regions of two interacting proteins are accessible to the solvent in the isolated molecules, whereas they become protected following the formation of the complex. Therefore, when limited proteolysis experiments are carried out on both the isolated proteins and the complex, differential peptide maps are obtained from which the interface regions can be inferred. Alternatively, cross-linking reactions performed under strictly controlled conditions lead to the identification of spatially closed amino acid residues in the complex. Mass spectrometry can be employed in both procedures for the definition of the cleavage sites and to identify covalently linked residues. Our results show that melittin interacts with calmodulin by adopting a parallel orientation, i.e. the N and C-terminal halves of the peptide are anchored to the amino and carboxy-terminal domains of the protein, respectively. This orientation is inverted with respect to all the peptide substrates examined so far. A model of the complex was designed and refined on the basis of the experimental results, supporting the above conclusions. This finding reveals a further dimension to the already remarkable capability of calmodulin in binding different protein substrates, providing this protein with the capability of regulating an even larger number of enzymes.

Amino Acid Sequence↗

PubMed1998

Conformational sampling of bioactive conformers: a low-temperature NMR study of 15N-Leu-enkephalin.

Conformational studies of enkephalins are hampered by their high flexibility which leads to mixtures of quasi-isoenergetic conformers in solution and makes NOEs very difficult to detect in NMR spectra. In order to improve the quality of the NMR data, Leu-enkephalin was synthesized with 15N-labelled uniformly on all amide nitrogens and examined in a viscous solvent medium at low temperature. HMQC NOESY spectra of the labelled Leu-enkephalin in a DMSOd6/H2O) mixture at 275 K do show numerous NOEs, but these are not consistent with a single conformer and are only sufficient to describe the conformational state as a mixture of several conformers. Here a different approach to the structure-activity relationships of enkephalins is presented: it is possible to analyse the NMR data in terms of limiting canonical structures (i.e. beta- and gamma-turns) and finally to select only those consistent with the requirements of delta selective agonists and antagonists. This strategy results in the prediction of a family of conformers that may be useful in the design of new delta selective opioid peptides.

Cold Temperature↗

PubMed1998

Solution conformational analysis of sodium complexed [Gly6]- and [Gly9]-antamanide analogs.

To investigate the conformational flexibility of metal-complexed cyclodecapeptides, we synthesized and studied two antamanide analogs, in which the phenylalanine residue in position 6 or 9 of the sequence was substituted by Gly. Previous conformational studies on antamanide suggested that these backbone regions are affected by conformational variation. The NMR conformational study showed a high degree of flexibility for the two analogs. With sodium ions, on the other hand, [Gly9]-antamanide was able to form a fairly stable equimolar complex, whereas [Gly6]-antamanide showed a conformational heterogeneity, with one prevailing conformer. For the [Gly9]-antamanide analog, the whole NMR data, combined with extensive theoretical calculations, were consistent with the presence of 1) two beta-turns of type I, centered on Gly9-Phe10 and Ala4-Phe5, respectively; 2) a central cavity with a six-carbonyl oxygen cage, optimal for a Na+ hexacoordination; 3) strongly H-bonded amide protons for residues 1 and 6, both involved in the formation of the two type I beta-turns, which, however, exhibited some fluctuations during the molecular dynamics simulations. For the [Gly6]-antamanide-Na+ complex the prevailing conformer was consistent with a more open structure, with the partial solvent exposure of all the amide protons; that is, the Gly residue in position 6 increases the flexibility of this critical site more than does the Gly in position 9. These data in some way parallel the results of the cytotoxicity tests on B16-F10 transformed cells for the two analogs: [Gly9]-antamanide is cytotoxic after 48 h exposure, whereas [Gly6]-antamanide is almost inactive. On the contrary, both analogs are practically inactive in vivo against phalloidin.

Amino Acid Substitution↗

PubMed1997

A new triple-stranded alpha-helical bundle in solution: the assembling of the cytosolic tail of MHC-associated invariant chain.

BACKGROUND: The invariant chain (li) is a transmembrane protein that associates with the major histocompatibility complex class II (MHC II) molecules in the endoplasmic reticulum. The cytosolic tail of li contains two leucine-based sorting motifs and is involved in sorting the MHC II molecules to the endosomal pathway where the peptide antigen is bound. This region of li also contributes to phenotypical changes in cells, such as the formation of large endocytic structures. RESULTS: We report here the three-dimensional structure of a 27 amino acid peptide corresponding to the cytosolic tail of li. The structure was determined by nuclear magnetic resonance (NMR) spectroscopy using a computational strategy. At high concentration, this structure reveals a new triple-stranded alpha-helical bundle in which the helices, two parallel and one antiparallel, are almost coplanar. Trimerization is mediated by electrostatic interactions intercalated by three hydrophobic layers. CONCLUSIONS: The new trimer fold, the first to be identified by NMR data alone, can be used to improve understanding of protein-protein interactions and to model multiple-helical transmembrane proteins and receptors. We suggest that interactions of the li cytosolic tails may form part of a mechanism that could cause the endosomal retention and enlarged endosomes induced by li.

Amino Acid Sequence↗

PubMed1997

Design of mu selective opioid dipeptide antagonists.

We have recently designed potent delta selective opioid antagonist dipeptides on the basis of a simple conformational analysis. Following a similar procedure we found a mu selective dipeptide antagonist, 2,6-dimethyl-Tyr-D-Phe-NH2. Although its selectivity is not as high as those of the quoted delta selective dipeptides it has good in vitro activity and looks very promising for further development since the 2,6-dimethyl-Tyr-D-Phe message, like the delta selective 2,6-dimethyl-Tyr-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid counterpart, seems able to impart antagonism to longer peptides.

Animals↗

PubMed1995

Conformational analysis of potent and very selective delta opioid dipeptide antagonists.

The delta selectivity and antagonism of peptides containing L-tetrahydro-3-isoquinoline carboxylic acid (Tic) in second position can be attributed mainly to the Tyr-Tic unit. These properties can be further enhanced by substituting Tyr1 with 2,6-dimethyl-L-tyrosyl (Dmt). Dmt-Tic-NH2, Dmt-Tic-OH, Dmt-Tic-Ala-NH2 and Dmt-Tic-Ala-OH are all more active and/or selective than the corresponding [Tyr1]-parent peptides. In fact the selectivities of Dmt-Tic-OH and Dmt-Tic-Ala-OH are the highest ever recorded for opioid molecules. 1H NMR spectra in a DMSO/water mixture at 278 K reveal the presence of two similar conformers, characterised by a cis or trans Dmt-Tic bond, in all four peptides. A detailed conformational analysis in solution of Dmt-Tic-NH2 shows that these conformers have a shape very similar to that of the bioactive conformation of Tyr-Tic-NH2 and to that of naltrindole.

Dipeptides↗

PubMed1995

Assignment and secondary-structure determination of monomeric bovine seminal ribonuclease employing computer-assisted evaluation of homonuclear three-dimensional 1H-NMR spectra.

Monomeric bovine seminal ribonuclease (mBS-RNase), the subunit of dimeric bovine seminal ribonuclease (BS-RNase), is an unusual monomer: for its structural stability, its catalytic activity, which is even higher than that of the parent dimeric enzyme, and for its role as an intermediate in the refolding of dimeric BS-RNase. Here we present the proton NMR assignment and secondary-structure determination of mBS-RNase, with a comparison of its structure to the structure of its parent protein, and to the structure of RNase A, a homologue with more than 80% identity in amino acid sequence. Proton NMR assignment was performed using a computer-assisted procedure, through a partially automated analysis of homonuclear three-dimensional spectra [Oschkinat, H., Holak, T. A. & Cieslar, C. (1991) Biopolymers 31, 699-712]. The secondary structures of mBS-RNase, of the A chain of dimeric BS-RNase, and of RNase A, are found to be similar. Significant differences are found instead, between mBS-RNase and RNase A in the more flexible stretches of the molecule, where a higher number of substitutions is present. Furthermore, a preliminary tertiary-structure model is reported, showing that the overall folding of mBS-RNase is closer to that of RNase A rather than that of (dimeric) BS-RNase.

Amino Acid Sequence↗

PubMed1994

Multiple conformations and proline cis-trans isomerization in salmon calcitonin: a combined nuclear magnetic resonance, distance geometry, and molecular mechanics study.

The relationship between multiple conformations and proline cis-trans isomerization in salmon calcitonin (sCT) has been investigated by 1H-NMR, distance geometry, and molecular mechanics. In different solvents, the isomerization of Pro23 induces resonance heterogeneity for amino acids adjacent to it. NOESY experiments have related such heterogeneity to two different isoforms of the hormone, which interconvert into each other in polar solvents as well as in SDS micelles. The cis-trans ratio was found to depend upon the structure of the hormone. In water, the random-coil sCT showed a 35% cis population, while 16% cis was observed in the folded SDS-bound hormone. Such a decrease contributes, per se, a Gibbs free energy stabilization of 3.10 kJ mol-1. Except for the 21-25 region, a common NOE pathway was found for both isomers. This is in agreement with the common secondary structure for both isoforms: a central helix and an extended C-terminal segment interacting with it. Calculations indicated that while the trans isomer is helical in the Thr6-Tyr22 region, a shorter helix (Thr6-Lys18) is present in the cis isoform. It is concluded that isomerization of Pro23 does not alter the three-dimensional structure of the hormone, although trans structures show a lower average NOE root mean square deviation and a higher relative stability. Both cis and trans structures satisfactorily reproduce the experimental data, although they do not fulfill the whole set of NOE restraints, pointing out the danger involved in structure calculation whenever the cis-trans equilibrium does not affect the global fold.

Amino Acid Sequence↗

PubMed1994

Conversion of enkephalin and dermorphin into delta-selective opioid antagonists by single-residue substitution.

The properties of di- and tri-peptides containing 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) in second position suggest that the message domain of opioid peptides can be composed of only two residues [Temussi, P. A., Salvadori, S., Amodeo, P., Guerrini, R., Tomatis, R., Lazarus, L. H., Picone, D. & Tancredi, T. (1994) Biochem. Biophys. Res. Commun. 198, 933-939]. As a crucial test of the possibility that the Tyr-Tic segment be a message domain in longer peptide sequences, we have inserted it in the sequences of two typical opioid peptides: [Leu]enkephalin, a non-selective agonist, and dermorphin, a selective mu agonist. Here we report the synthesis and biological activity of [L-Tic2]enkephalin, [L-Tic2]dermorphin, [L-Tic2]dermorphin carboxylic acid and [D-Tic2]dermorphin: all [L-Tic2]peptides were converted from agonists to delta-selective antagonists. The NMR conformational study in a dimethylsulfoxide/water cryoprotective mixture at low temperature shows diagnostic side-chain--side-chain NOEs in the spectra of all [L-Tic2]peptides and hints that the 90 degrees arrangement of the the two aromatic rings found in the cis-Tyr-L-Tic moiety, typical of N-methyl naltrindole and other delta-selective opiate antagonists, is responsible for the antagonist activity of all these peptides.

Amino Acid Sequence↗