Spatial conformation and topography of the tyrosine aromatic ring in substrate recognition by protein tyrosine kinases.

The side chain orientation of the tyrosine residue included in a peptide, which is an excellent substrate of Syk tyrosine kinase, was fixed in different conformations by either incorporating the tyrosine in cyclic structures (6-OH-Tic, 5-OH-Aic, and Hat derivatives) or adding a sterically bulky substituent in the tyrosine side chain moiety (beta-MeTyr). Synthetic peptides containing tyrosine analogues displaying different side chain orientations were analyzed by NMR techniques and tested as potential substrates of the nonreceptor tyrosine kinases Syk, Csk, Lyn, and Fyn. The "rotamer scan" of the phosphorylatable residue generated optimal substrates in terms of both phosphorylation efficiency and selectivity for Syk tyrosine kinase, while the peptidomimetics were not recognized by the other tyrosine kinases. In particular, l-beta-MeTyr and d-Hat containing peptides resulted to be both suitable substrates for the specific monitoring of Syk and consensus sequence scaffolds for the design of potential inhibitors highly selective for this tyrosine kinase.

Synthesis and evaluation of neuroprotective alpha,beta-unsaturated aldehyde scavenger histidyl-containing analogues of carnosine.

The synthesis, scavenging activity, and cytoprotective profiles of histidyl-containing carnosine analogues bearing hydrazide or 1,2-diol moieties is reported. Some compounds have demonstrated higher aldehyde-sequestering efficiency than carnosine and were also efficient in protecting SH-SY5Y neuroblastoma cells and rat hippocampal neurons from 4-hydroxy-trans-2,3-nonenal (HNE)-mediated death. The cytoprotective efficacy of these compounds suggests their potential use as therapeutic agents for disorders that involve excessive membrane lipids peroxidation and HNE-mediated neuronal toxicity.

Mechanistic studies of amide bond scission during acidolytic deprotection of Pip containing peptide.

Unusual TFA catalyzed cleavage reaction is reported for peptide containing pipecolic acid residues. Although the use of TFA under standard cleavage conditions is sufficiently mild to prevent degradation of the desired products, the amide bond between consecutive pipecolic acid residues is unexpectedly hydrolyzed by standard TFA treatment. The hydrolysis is proposed to proceed via an oxazolinium ion intermediate. This mechanism is supported by H/D exchange as observed by ESI-MS and NMR experiments.

Fluorescent, internally quenched, peptides for exploring the pH-dependent substrate specificity of cathepsin B.

Cathepsin B is a cysteine protease that in tumor tissues is localized in both acidic lysosomes and extracellular spaces. It can catalyze the cleavage of peptide bonds by two mechanisms: endoproteolytic attack with a pH optimum around 7.4, and attack from the C-terminus with a pH optimum at 4.5-5.5. In this work, seven fluorescent, internally quenched, decapeptides have been synthesized using the prototypical cathepsin B selective substrate Z-Phe-Arg-AMC as a lead, and used to identify the structural factors determining the susceptibility of peptides to hydrolysis at acidic and neutral pH values. Each peptide differs from the others in one amino acid (residue 6) and contains a highly fluorescent Nma group linked to the alpha-amino function of the N-terminal Orn residue and a Dnp group linked to the side chain of the Lys(8) residue acting as a quencher. Proteolytic cleavage was monitored by measuring the increase of fluorescence at 440 nm upon excitation at 340 nm, and the cleavage sites were determined by HPLC followed by ESI-MS analysis. Peptides containing Ala or Phe at position 6 are good substrates for the enzyme at both pH 5.0 and 7.4. By contrast, those containing Glu, Asp, Lys or Val are not cleaved at all by cathepsin B at pH 7.4, and are poorly hydrolyzed at pH 5.0. These findings provide new information for the rational design of cathepsin B-activated peptide-containing anticancer drugs.

4-Fluoroproline derivative peptides: effect on PPII conformation and SH3 affinity.

Eukaryotic signal transduction involves the assembly of transient protein-protein complexes mediated by modular interaction domains. Specific Pro-rich sequences with the consensus core motif PxxP adopt the PPII helix conformation upon binding to SH3 domains. For short Pro-rich peptides, little or no ordered secondary structure is usually observed before binding interactions. The association of a Pro-rich peptide with the SH3 domain involves unfavorable binding entropy due to the loss of rotational freedom on forming the PPII helix. With the aim of stabilizing the PPII helix conformation in the Pro-rich HPK1 decapeptide PPPLPPKPKF (P2), a series of P2 analogues was prepared, in which specific Pro positions were alternatively occupied by 4(S)- or 4(R)-4-fluoro-L-proline. The interactions of these peptides with the SH3 domain of the HPK1-binding partner HS1 were quantitatively analyzed by the NILIA-CD approach. A CD thermal analysis of the P2 analogues was performed to assess their propensity to adopt the PPII helix conformation. Contrary to our expectations, the K(d) values of the analogues were lower than that of the parent peptide P2. These results clearly show that the induction of a stable PPII helix conformation in short Pro-rich peptides is not sufficient to increase their affinity toward the SH3 domain and that the effect of 4-fluoroproline strongly depends on the position of this residue in the sequence and the chirality of the substituent in the pyrrolidine ring.

Tat cell-penetrating peptide has the characteristics of a poly(proline) II helix in aqueous solution and in SDS micelles.

Tat cell-penetrating peptide (GRKKRRQRRRPPQG) is able to translocate and carry molecules across cell membranes. Using CD spectroscopy the conformation of this synthetic peptide was studied in aqueous and membrane-mimicking, micellar SDS solutions at different temperatures. The CD spectrum of the Tat cell-penetrating peptide in SDS micellar solution was virtually unchanged from that in aqueous solution, and at low temperature it was close to that of a poly(proline) II helix.

Conformational constraints of tyrosine in protein tyrosine kinase substrates: Information about preferred bioactive side-chain orientation.

The side-chain orientation of a tyrosine residue located in a peptide, which is an excellent substrate of Syk tyrosine kinase (A. M. Brunati, A. Donella-Deana, M. Ruzzene, O. Marin, L. A. Pinna, FEBS Letters, 1995, Vol. 367, pp. 149-152), was fixed in the gauche (+) or gauche (-) conformation by using the 7-hydroxy-1,2,3,4-tetrahydro isoquinoline-3-carboxylic (Htc) structure. The tyrosine trans conformation was blocked by using an aminobenzazepine-type (Hba) structure. The proposed side-chain orientations were confirmed by the analysis of the (1)H-NMR parameters: chemical shifts, coupling constants, and nuclear Overhauser effects to the tyrosine constraints in the different analogs. This "rotamer scan" of the phosphorylatable residue allowed us to generate optimal substrates in terms of both phosphorylation efficiency and selectivity for Syk tyrosine kinase. In contrast, these conformationally restricted tyrosine analogs were not tolerated by the Src-related tyrosine kinases Lyn and c-Fgr.