Bioactivation of pro‐proteins by limited proteolysis is a general mechanism in the biosynthesis of hormones, receptors and viral protein precursors. This proceeds by cleavage of peptide bonds at the level of single or pairs of basic residues in the proforms. Examination of a number of cleavage loci in various precursors failed to reveal any consensus primary sequence around the dibasic cleavage sites. Thus it has been proposed, on the basis of secondary structure predictions [Rholam, M., Nicolas, P. and Cohen, P. (1986) FEBS Lett., 207, 1–6], that those basic residues which operate as signal loci for the proteolytic enzyme machinery are situated in, or next to, privileged precursor regions most often constituted by flexible and exposed motifs, e.g. beta‐turns and/or loops. Peptides reproducing the N‐terminal processing domain of the hormone precursor, pro‐ocytocin‐neurophysin, were examined by a combination of spectroscopical techniques including circular dichroism, infrared Fourier transform and one‐ and two‐dimensional proton NMR. The results indicate that: (i) the region situated on the N terminus of the Lys‐Arg doublet is organized as a beta‐turn in solution; (ii) the sequential organization of the residues participating in the beta‐turn determines the privileged relative orientation of the basic amino acid side chains and the subtype of turn; (iii) the peptide segment situated on the C‐terminal side of the dibasic, corresponding to the N‐terminal octapeptide of neurophysin, is organized as an alpha‐helix.(ABSTRACT TRUNCATED AT 250 WORDS)
Proline residues located near the processing sites of human prosomatostatin were previously shown to be important for cleavage of the precursor into somatostatin 28 and somatostatin 14[Gomez, S., Boileau, G., Zollinger, L., Nault, C., Rholam, M. & Cohen, P. (1989) EMBU J. 8, 2911 -29161. In this study, site-directed and regional mutagenesis of the human prosomatostatin cDNA coupled with analysis by circular-dichroism and Fourier-transform-infrared spectroscopies of the native and mutated peptide sequences were used to elucidate the role of proline in proteolytic processing. Glycine was substituted for proline a position -5 and the P-turn-promoting sequence Pro-Arg-Glu-Arg, located near the somatostatin-1 4 cleavage site and predicted to form a p-turn structure, was replaced by Ser-Ser-Asn-Arg or Tyr-Lys-Gly-Arg, which have been shown by X-ray diffraction to form p turns in other proteins. Analysis of the prosomatostatin-derived peptides produced by expression of the mutated cDNA species in Neuro2A cells indicated that while Pro-5 + Ala abolished cleavage at the dibasic site, the formation of mutants [Gly-'] prosomatostatin, [Ser-', S e r 4 , Arg-3] prosomatostatin and [ T y P , L y s~~, G~Y -~] prosomatostatin did not affect cleavage at the dibasic site but produced modifications in both the relative proportions of the generated hormones and in precursor processing efficiency. Moreover, spectroscopical analysis showed that whereas these substitutions did not modify the presence of a p turn structure in the corresponding peptide sequences, replacement of Pro-5 + Ala resulted in a dramatic increase in a-helix accompanied by the significant decrease of other structures including fi turn.
Previous studies using selectively modified pro-ocytocin/neurophysin substrate analogues and the purified metalloprotease, pro-ocytocin/neurophysin convertase (magnolysin; EC 3.4 24.62), have shown that dibasic cleavage site processing is associated with a prohormone sequence organized in a b-turn structure. We have used various peptide analogues of the pro-ocytocin-neurophysin processing domain, and recombinant prohormone convertase 1/3, to test the validity of this property towards this member of the family of prohormone convertases (PCs). The enzymatic cleavage analysis and kinetics showed that: (a) with methyl amide (N-Met) modification, a secondary structure b-turn breaker, the enzyme substrate interaction was abolished; (b) cleavage was favoured when the dibasic substrate side-chains were oriented in opposite directions; (c) the amino acid present at the P H 1 position is important in the enzyme±substrate interaction; (d) the flexibility of the peptide substrate is necessary for the interaction; (e) Addition of dimethylsulfoxide to the cleavage assay favoured the cleavage of the pro-ocytocin/neurophysin large substrate over that of the smaller one pGlu-Arg-Thr-Lys-Arg-methyl coumarin amide. These data allowed us to conclude that proteolytic processing of pro-ocytocin-related peptide substrates by PC1/3 as well as by the metalloenzyme, magnolysin, involves selective recognition of precise cleavage site local secondary structure by the processing enzyme. It is hypothesized that this may represent a general property of peptide precursor proteolytic processing systems.
Current treatments for autoimmune disorders rely on non-specific immunomodulatory and global immunosuppressive drugs, which show a variable degree of efficiency and are often accompanied by side effects. In contrast, strategies aiming at inducing antigen-specific tolerance promise an exclusive specificity of the immunomodulation. However, although successful in experimental models, peptide-based tolerogenic “inverse” vaccines have largely failed to show efficacy in clinical trials. Recent studies showed that repetitive T cell epitopes, coupling of peptides to autologous cells, or peptides coupled to nanoparticles can improve the tolerogenic efficacy of peptides, suggesting that size and biophysical properties of antigen constructs affect the induction of tolerance. As these materials bear hurdles with respect to preparation or regulatory aspects, we wondered whether conjugation of peptides to the well-established and clinically proven synthetic material polyethylene glycol (PEG) might also work. We here coupled the T cell epitope OVA 323–339 to polyethylene glycols of different size and structure and tested the impact of these nano-sized constructs on regulatory (Treg) and effector T cells in the DO11.10 adoptive transfer mouse model. Systemic vaccination with PEGylated peptides resulted in highly increased frequencies of Foxp3 + Tregs and reduced frequencies of antigen-specific T cells producing pro-inflammatory TNF compared to vaccination with the native peptide. PEGylation was found to extend the bioavailability of the model peptide. Both tolerogenicity and bioavailability were dependent on PEG size and structure. In conclusion, PEGylation of antigenic peptides is an effective and feasible strategy to improve Treg-inducing, peptide-based vaccines with potential use for the treatment of autoimmune diseases, allergies, and transplant rejection.
Synthetic peptides reproducing the proteolytic processing site of pro-ocytocin were studied by different spectroscopic techniques, including circular dichroism, Fourier transform infrared absorption, and mono and bidimensional nuclear magnetic resonance, in order to ascertain the possible role of three-dimensional structure in the recognition process by maturation enzymes. Experimental results were compared with energy minimization calculations and suggest that: (i) the region situated on the N-terminus of the Lys-Arg doublet may form a beta-turn; (ii) the sequential organization of the residues participating in the beta-turn determines the privileged relative orientation of the basic amino acid sidechains and the subtype of turn; and (iii) the peptide segment situated on the C-terminal side of the dibasic doublet may assume a helix arrangement. These findings, in spite of the limitations connected to the flexibility of linear peptides, seem to substantiate the hypothesis that structural motifs around the cleavage site could be important for recognition and processing. however, a straightforward correlation between details of the secondary structure and the in vitro reactivity toward a putative convertase is not yet possible.
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