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Biomedical subjects

Ernest Giralt

Publications and source records attributed to Ernest Giralt.

At least 55 records · Page 3Linked to original sources
PubMed2004

Ionic self-complementarity induces amyloid-like fibril formation in an isolated domain of a plant copper metallochaperone protein.

BACKGROUND: Arabidopsis thaliana copper metallochaperone CCH is a functional homologue of yeast antioxidant ATX1, involved in cytosolic copper transport. In higher plants, CCH has to be transported to specialised cells through plasmodesmata, being the only metallochaperone reported to date that leaves the cell where it is synthesised. CCH has two different domains, the N-terminal domain conserved among other copper-metallochaperones and a C-terminal domain absent in all the identified non-plant metallochaperones. The aim of the present study was the biochemical and biophysical characterisation of the C-terminal domain of the copper metallochaperone CCH. RESULTS: The conformational behaviour of the isolated C-domain in solution is complex and implies the adoption of mixed conformations in different environments. The ionic self-complementary peptide KTEAETKTEAKVDAKADVE, derived from the C-domain of CCH, adopts and extended conformation in solution with a high content in beta-sheet structure that induces a pH-dependent fibril formation. Freeze drying electron microscopy studies revealed the existence of well ordered amyloid-like fibrils in preparations from both the C-domain and its derivative peptide. CONCLUSION: A number of proteins related with copper homeostasis have a high tendency to form fibrils. The determinants for fibril formation, as well as the possible physiological role are not fully understood. Here we show that the plant exclusive C-domain of the copper metallochaperone CCH has conformational plasticity and forms fibrils at defined experimental conditions. The putative influence of these properties with plant copper delivery will be addressed in the future.

Amino Acid Sequence↗
PubMed2004

A new class of foldamers based on cis-gamma-amino-L-proline.

A synthetic method for the preparation of conformationally constrained gamma-peptides derived from gamma-amino-L-proline is described. The methodology allows the independent buildup of the peptide backbone and the introduction of sequential variations by reactions with the alpha-amino group of gamma-aminoproline. Both alkyl- and acyl-substituted gamma-peptides have been prepared and studied by CD and NMR. Conformational restrictions due to the cyclic structure of the monomer give rise to long-range interactions that are indicative of secondary structures even in aqueous solution. Interresidue NOEs suggest a concatenation of turns that, in a permissive solvent, could give rise to an isolated hydrogen bond ribbon, flanked and protected by proline rings.

Aminobutyrates↗
PubMed2004

Exploring the interaction of the surfactant N-terminal domain of gamma-Zein with soybean phosphatidylcholine liposomes.

Zeins are maize storage proteins that accumulate inside large vesicles called protein bodies. gamma-Zein lines the inner surface of the protein body membrane, and its N-terminal, proline-rich, repetitive domain with the sequence (VHLPPP)(8) appears to be necessary for the accumulation of the protein within the organelle. Synthetic (VHLPPP)(8) adopts an amphipathic polyproline II conformation and forms cylindrical micelles in aqueous solution. Here we explore the interaction of (VHLPPP)(8) with soybean phosphatidylcholine unilamellar lipid vesicles and examine its effect on the stability and permeability of the liposome membrane. The amphipathic N-terminal domain of gamma-zein interacts with the membrane and assembles to form extended domains over the phospholipid membrane. The interaction between the peptide and the membrane increases the stability and permeability of the liposome membrane. The spontaneous amphipathic aggregation of (VHLPPP)(8) on the membrane suggests a mechanism of gamma-zein deposition inside maize protein bodies.

Endoplasmic Reticulum↗
PubMed2004

Amphipathic peptides and drug delivery.

The discovery of cell-penetrating peptides as gene delivery systems and the interest in the mechanism by which these vectors cross the cell membrane have generated a large number of studies. Among the parameters involved in the translocation process, controversy has arisen about the role of the amphipathicity of the carriers in the interaction and reorganization of the cell membrane. In this review we have summarized the vectors with primary or secondary amphipathicity related to secondary structure. Some of the insights into the relationship between the aggregation state of the peptide at the concentrations used for internalization studies and its interaction with the cell membrane result from our contribution to the field with a new family of amphipathic proline-rich peptides.

Amino Acid Sequence↗
PubMed2004

Solid-phase synthesis of second-generation polyproline dendrimers.

Second-generation dendrimers have been prepared on solid phase by successive additions of branched polyproline building blocks starting from two different branching units anchored to the solid support. The preparation of Pro-rich building blocks was carried out by stepwise solid-phase synthesis and their iterative addition was performed by a convergent approach, also using solid-phase synthesis. cis-4-Amino-L-proline and imidazolidine-2-carboxylic acid were used as branching units due to their structural resemblance to proline. The optimized strategy allowed the target compounds to be obtained with high purities without the need for purification steps.

Chromatography, High Pressure Liquid↗
PubMed2004

Computational study of the conformational preferences of the (R)-8-amino-pentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecane-8-carboxylic acid monopeptide.

alpha-Amino acids are important building blocks for the synthesis of a large number of bioactive compounds and pharmaceutical drugs. However, a literature survey revealed that no theoretical conformational study of alpha-amino acids with cage carbon frameworks has been performed to date. This paper reports the results of a conformational study on the (R)-8-amino-pentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecane-8-carboxylic acid monopeptide (cage monopeptide), using molecular mechanics and ab initio methods. The in vacuo Ramachandran maps computed using the different parameterizations of the AMBER force field show the C7eq structure as the most favourable conformation, in contrast to the C7ax structure, that is the lowest energy conformation at the ab initio level. Analysis of these maps reveals the helical preference for the monopeptide and provides the potential for the cage residue to be incorporated into constrained peptide analogues.

Adamantane↗
PubMed2003

Constrained derivatives of stylostatin 1. 1. Synthesis and biological evaluation as potential anticancer agents.

Hydroxyaminolactams have been used as constrained surrogates of the Ser-Leu dipeptide in the synthesis of analogues of the cycloheptapeptide stylostatin 1 (2). The rate of cyclization through formation of the Ile-Pro amide bond allowed us to prove that the valerolactams used induced a turn in the linear precursor. Ring closure at the Pro-Phe amide bond was much quicker and provided access to larger amounts of the target structures, with high purity. The conformation of psi-stylostatin 4 was compared to that of native stylostatin 1 using NMR analysis. The ability of three psi-stylostatins and the native stylostatin 1 to inhibit growth of cancer cell lines was tested. None of the compounds showed activity below 1 microM. A possible relationship between the decrease in activity and the presence of the piperidone Ser-Leu surrogate is considered.

Animals↗
PubMed2003

Analysis of conformational equilibria in aplidine using selective excitation 2D NMR spectroscopy and molecular mechanics/dynamics calculations.

Aplidine (dehydrodidemnin B), a natural product with potent antitumor activity currently in multicenter phase II clinical trials, exists in DMSO as a mixture of four slowly interconverting conformations in a ratio of 47:33:13:7. NMR spectroscopy shows that these arise as a consequence of cis/trans isomerization about the NMe-Leu(7)-Pro(8) and Pro(8)-Pyr amide bonds of the molecule's side chain. Two major conformations account for 47% and 33% of the total population, a ratio of 60:40 between the two. They correspond to the cis- and trans-isomers, respectively, about the Pro(8)-Pyr amide bond. Two minor conformers arise as a consequence of similar isomerism about the Pro(8)-Pyr amide bond, but in structures in which the NMe-Leu(7)-Pro(8) amide bond is cis rather than trans. These account for approximately 13% and 7% of the total population, corresponding to a ratio of 65:35 cis/trans, respectively. Molecular dynamics simulations show that the three-dimensional structures of all four conformational isomers are similar in the macrocycle and that all are essentially unchanged with respect to the macrocycle of didemnin B. Significant differences in the conformation of the molecule's side chain are, however, observed between major and minor pairs. Analysis of hydrogen-bonding patterns shows that each major conformer exhibits a beta-turn like structure and is stabilized by hydrogen bonding between a different carbonyl group of the pyruvyl unit of the molecule's side chain and the NH of the Thr(6) residue. The minor isomers have a cis-amide bond between the NMe-Leu(7) and Pro(8) residues that obliges the side chain to adopt an extended disposition where hydrogen bonding to the macrocycle is absent. These results suggest that the ability of the molecule's side chain to adopt a beta-turn-like conformation may not be a prerequisite for biological activity in the didemnins and that conformations having an extended side-chain may play a role in the biological activity of aplidine.

Amides↗
PubMed2003

Synthesis of 3-aminolactams as X-Gly constrained pseudodipeptides and conformational study of a Trp-Gly surrogate.

3-Amino-delta-valerolactams trans-11a-c were synthesized through conjugate addition and Curtius rearrangement and converted into Fmoc-[Trp-Gly], Fmoc-[Ile-Gly], and Fmoc-[Phe-Gly] pseudodipeptides. Conformational analyses of tripeptide analogues Ac-[Trp-Gly]-Leu-NH(2) 17a and 17b by NMR experiments and molecular modeling calculations showed that diastereomer 17a adopted a gamma-turn/distorted type II beta-turn structure, whereas diastereomer 17b adopted mainly a gamma-turn structure.

Amines↗
PubMed2003

Tentoxin as a scaffold for drug discovery. Total solid-phase synthesis of tentoxin and a library of analogues.

[reaction: see text] A solid-phase method for the synthesis of tentoxin has been developed. Two key steps-dehydration and N-alkylation-are carried out while the peptide is anchored to the resin. The method, which has been validated by the preparation of a library of tentoxin analogues, should be applicable to the generation of further libraries that have the tentoxin scaffold structure, as well as other structures containing N-alkylated didehydroamino acids.

Amino Acid Sequence↗
PubMed2003

Synthesis and NMR structure of p41icf, a potent inhibitor of human cathepsin L.

The total synthesis and structural characterization of the MHCII-associated p41 invariant chain fragment (P41icf) is described. P41icf plays a crucial role in the maturation of MHC class II molecules and antigen processing, acting as a highly selective cathepsin L inhibitor. P41icf synthesis was achieved using a combined solid-phase/solution approach. The entire molecule (65 residues, 7246 Da unprotected) was assembled in solution from fully protected peptides in the size range of 10 residues. After deprotection, oxidative folding in carefully adjusted experimental conditions led to the completely folded and functional P41icf with a disulfide pairing identical to that of native P41icf. CD, NMR, and surface plasmon resonance (SPR) were used for the structural and functional characterization of synthetic P41icf. CD thermal denaturation showed clear cooperative behavior. Tight cathepsin L binding was demonstrated by SPR. (1)H NMR spectroscopy at 800 MHz of unlabeled P41icf was used to solve the three-dimensional structure of the molecule. P41icf behaves as a well-folded protein domain with a topology very close to the crystallographic cathepsin L-bound form.

Amino Acid Sequence↗
PubMed2003

Solution structure of the antitumor candidate trunkamide A by 2D NMR and restrained simulated annealing methods.

Trunkamide A (1) is a cyclic heptapeptide extracted from the ascidian Lissoclinum sp. and has shown very promising cytotoxic activity. This compound incorporates several of the motifs commonly observed in the Patellin family, including dimethylallyl (Dma) Thr and Ser side chains and a thiazoline heterocycle. Given that little is known about the structures adopted by the cyclopeptides of the Patellin family, and with the aim of establishing structure-activity relationships, we have carried out the conformational analysis of trunkamide A by a combination of 2D NMR experiments and simulated annealing calculations. Our results show that the conformation of 1 is very rigid and is dominated by the volume of the dimethylallyl side chains and two trans-annular hydrogen bonds. We have also studied the conformation of 2, the l-Phe diastereoisomer of 1, the analysis of which provides a possible rationale for its epimerization to 1, a process that is observed in solution. Finally, we show how a thorough NMR characterization can be used, in combination with simulated annealing methods, to confirm the configuration of a stereogenic center in the backbone of a rigid cyclic peptide such as trunkamide A (1).

Amino Acid Sequence↗
PubMed2003

The fuc1 gene product (20 kDa FUC1) of Pisum sativum has no alpha-L-fucosidase activity.

An alpha-L-fucosidase purified from pea (Pisum sativum L. cv Alaska) epicotyl was previously described as a cell wall enzyme of 20 kDa that hydrolyses terminal alpha-L-fucosidic linkages from oligosaccharide fragments of xyloglucan. cDNA and genomic copies were further isolated and sequenced. The predicted product of the cDNA and the genomic clone (fuc1), was a 20 kDa protein containing a signal peptide and five cysteines. This was the first alpha-L-fucosidase gene to be cloned in plants but its fucosidase activity has not been demonstrated. Here, our biochemical and immuno analyses suggest that fuc1 does not encode an alpha-L-fucosidase. Pea fuc1 expressed in Escherichia coli, insect cells and Arabidopsis thaliana produced recombinant proteins without alpha-L-fucosidase activity. Pea plants had endogenous alpha-L-fucosidase activity, but the enzyme was not recognised by an antibody produced against recombinant FUC1 protein expressed in E. coli. In contrast, the antibody immunoprecipitated a 20 kDa protein which was inactive. By chromatographic analysis of pea protein extracts, we separated alpha-L-fucosidase-active fractions from the 20 kDa protein fractions. We conclude that the alpha-L-fucosidase activity is not attributable to the 20 kDa FUC1 protein. A new function for fuc1 gene product, now named PIP20 (for protease inhibitor from pea) is proposed.

Animals↗
PubMed2003

NMR-based methods and strategies for drug discovery.

Nuclear Magnetic Resonance (NMR) spectroscopy has long been a favourite tool of chemists interested in host-guest systems because it permits access to a wealth of information about the molecular recognition reaction. NMR has evolved dramatically in the last 15 years and, in parallel with the development of NMR methods for the determination of protein structure, a variety of tools aimed at detecting protein ligand interactions have been proposed and are being now used both in industrial and academic laboratories as valuable tools for structure-based drug discovery. Very recent developments have considerably increased the fraction of therapeutic targets that can be tackled by NMR and significantly reduced the amount of sample required for analysis; in this tutorial review we outline the essential NMR-based techniques and describe some examples of their implementation as part of drug discovery programmes.

Animals↗
PubMed2002

Spirolactams as conformationally restricted pseudopeptides: synthesis and conformational analysis.

The synthesis of 1-(tert-butoxycarbonyl)-7-[1-(tert-butoxycarbonyl)-3-methylbutyl]-6-oxo-1,7-diazaspiro[4.5]decanes (S,S)-1a and (S,R)-1b is described. Derivatives 17a,b and 19a are prepared for use in peptide synthesis as constrained surrogates of the Pro-Leu and Gly-Leu dipeptides. The Ac-[Gly-Leu]-Met-NH(2) derivatives (S,S,S)-2a and (S,R,S)-2b, with the tripeptidic C-terminal region present in tachykinins, are also synthesized. Conformational analyses of these tripetide analogues by NMR experiments and molecular modeling calculations show that both (S,S,S)-2a and (S,R,S)-2b epimers are gamma-turn/distorted type II beta-turn mimetics.

Hydrogen Bonding↗
PubMed2002

A new method for the preparation of unprotected peptides on biocompatible resins with application in combinatorial chemistry.

[formula: see text] A synthetic strategy for the preparation of side chain free peptides on biocompatible solid supports is described. Final peptide detachment is afforded in mild basic conditions with no presence of scavengers or other additives, thus allowing single peptide-resin beads to be cleaved in mass spectrometry sample plates for direct sequencing using MALDI-TOF post-source decay. This methodology offers clear advantages for the development of one-bead--one-compound combinatorial libraries in addition to parallel and regular synthesis of peptides.

Amino Acid Sequence↗