Non-Aggregating Amylin Fragments as an Inhibitors of the Aggregation Process of Susceptible to Aggregation Fragments 18-22, 23-27, and 33-37 of Hormone.

Amylin aggregation is one of the factors in the development of diabetes mellitus, which is classified as a civilization disease. The aim of this research was to find whether non-aggregating fragments 1-7, 8-12, 13-17 and 28-32 of amylin would inhibit the aggregation of the amyloidogenic cores 18-22, 23-27, 33-37 of hormone. In the study of the inhibitory potential of non-aggregating amylin fragments, a set of independent methods were used to study aggregation properties (spectroscopic and fluorescence studies with the use of indicators, microscopic studies, circular dichroism studies) and the method of prediction of aggregation properties. The performed research allowed to select the cyclic fragment (1-7) H-KCNTATC-OH with disulfide bond as an inhibitor capable of inhibiting the aggregation of all amyloidogenic cores 18-22, 23-27, 33-37 of the hormone. Additionally, it was found that this peptide inhibits insulin hot spot aggregation, which may indicate its universal utility in inhibiting the process of aggregation of hormones regulating carbohydrate metabolism directly related to the development of diabetes. Research on the possibility of the extensive use of the cyclic fragment (1-7) of H-KCNTATC-OH as a peptide inhibitor of the polypeptide/protein aggregation process is ongoing.

N-Methylated Analogs of hIAPP Fragments 18-22, 23-27, 33-37 Inhibit Aggregation of the Amyloidogenic Core of the Hormone.

Amylin (hIAPP) aggregation leads to the formation of insoluble deposits and is one of the factors in the development of type II diabetes. The aim of this research was to find N-methylated analogs of the aggregating amylin fragments 18-22, 23-27, and 33-37, which would not themselves be susceptible to aggregation and would inhibit the aggregation of the amyloidogenic cores of the hormone. None of the analogs of fragment 18-22 containing one or two N-methylated amino acid residues showed any tendency to aggregate. Only the peptide H-F(N-Me)GA(N-Me) IL-OH (6) derived from the 23-27 hIAPP hot spot did not form fibrous structures. All analogs of the 33-37 amylin fragment were characterized by the ability to form aggregates, despite the presence of N-methylated amino acids in their structures. N-Methylated peptides 1-5 demonstrated inhibitory properties against the aggregation of fragment 18-22. Aggregation of the amyloidogenic core of 23-27 was significantly inhibited by N-methylated peptides 1-3 derived from the (18-22) H-HSSNN-OH fragment and by the H-F(N-Me)GA(N-Me)IL-OH (6) fragment derived from the 23-27 amylin hot spot. Fragment (33-37) H-GSNTY-NH2 was found to be inhibited in the presence of N-methylated peptides 1-3 derived from the 18-22 fragment and by the double methylated peptide H-F(N-Me)GA(N-Me)IL-OH (6). Research on the possibility of using N-methylated analogs of amyloidogenic amylin cores as inhibitors of hormone aggregation is ongoing, with a focus on finding the minimum concentration of N-methylated peptides capable of inhibiting the aggregation of hIAPP hot spots.

New Human Islet Amyloid Polypeptide Fragments Susceptible to Aggregation.

Human Islet Amyloid Polypeptide (hIAPP) plays a key role in the pathogenesis of type II diabetes. The aim of this research was to search for new amyloidogenic fragments of hIAPP. An initial attempt to predict the amyloidogenic cores of polypeptides/proteins using five different computer programs did not provide conclusive results. Therefore, we synthesized hIAPP fragments covering the entire hormone. The fragments were assessed for their aggregation ability, using recommended methods to search for the amyloidogenic fragments of the polypeptides/proteins. It was found that fragments (18-22) H-HSSNN-OH and (33-37) H-GSNTY-NH2 aggregate and form stable amyloid-like structures. Both of these fragments have a much higher antiproliferative activity relative to the RIN-5F cell compared to the (23-27) H-FGAIL-OH fragment widely regarded as the amyloidogenic core of amylin. The analog of (33-37) H-GSNTY-NH2 containing a free carboxy group on the C-terminal amino acid (H-GSNTY-OH) does not have amyloidogenic properties and can therefore be considered as a potential inhibitor of amylin aggregation. Research on the use of non-aggregating amylin fragments as potential hormone aggregation inhibitors is ongoing.

Search for Fibrous Aggregates Potentially Useful in Regenerative Medicine Formed under Physiological Conditions by Self-Assembling Short Peptides Containing Two Identical Aromatic Amino Acid Residues.

This study investigates the propensity of short peptides to self-organize and the influence of aggregates on cell cultures. The dipeptides were derived from both enantiomers of identical aromatic amino acids and tripeptides were prepared from two identical aromatic amino acids with one cysteine or methionine residue in the C-terminal, N-terminal, or central position. The formation or absence of fibrous structures under physiological conditions was established using Congo Red and Thioflavine T assays as well as by microscopic examination using normal and polarized light. The in vitro stability of the aggregates in buffered saline solution was assessed over 30 days. Materials with potential for use in regenerative medicine were selected based on the cytotoxicity of the peptides to the endothelial cell line EA.hy 926 and the wettability of the surfaces of the films, as well as using scanning electron microscopy. The criteria were fulfilled by H-dPhedPhe-OH, H-dCysdPhedPhe-OH, H-CysTyrTyr-OH, H-dPhedPhedCys-OH, H-TyrTyrMet-OH, and H-TyrMetTyr-OH. Our preliminary results suggest that the morphology and cell viability of L919 fibroblast cells do not depend on the stereochemistry of the self-organizing peptides.

The quest for the shortest fragments of A (13-19) and B (12-17) responsible for the aggregation of human insulin.

AIM: To identify the shortest components of A13-A19, B12-B17 fragments capable for fibrillation and to validate the dependability of aggregation on the presence of hydroxyl group engaged in the 'tyrosine kissing'. MATERIALS & METHODS: Fragments A13-A19 and B12-B17 of insulin and all shortened analogues were obtained by using DMT/NMM/TosO(-) as a coupling reagent. The aggregation was studied by three independent tests. RESULTS: Studies on the susceptibility to aggregation of truncated analogs of insulin amyloidogenic core show three groups of peptides. CONCLUSION: Truncation of A13-A419 fragment shows that fibrous structures are formed by all peptides bearing (13)H-LeuTyr-OH(14). Propensity to aggregation was found for (16)H-TyrLeu-OH(17) B12-B17 fragment. Tyrosine residue modification by incorporation of tert-butyl group on hydroxyl function gave analogues still predisposed to aggregation.

Heavy atom induced phosphorescence study on the influence of internal structural factors on the photophysics of tryptophan in aqueous solutions.

In this study the effect of alanyl residue insertion into tryptophan and to some extent the effect of peptide bond on the photophysics of tryptophan chromophore has been studied. The photophysical parameters crucial in triplet state decay mechanism of aqueous AW, WA and AWA peptides have been determined applying our previously proposed methodology based on the heavy atom effect and compared with the previously reported values for tryptophan (Kowalska-Baron et al., 2012). The obtained results clearly indicated that the presence of alanyl residue and the peptide bond results in the changes in the fluorescence and phosphorescence decay kinetics of tryptophan. The fluorescence decays of the oligopeptides studied at pH 7 were biexponential. The longer lifetime component of WA arises from anionic form of this dipeptide, while the shorter one may be assigned to the zwitterionic form of WA. The observed invariance of the lifetimes of anionic and zwitterionic forms of WA throughout the pH studied supports the idea that these two components of WA fluorescence decay correspond to nearly independent species, possibly interconverting but at a rate slower than the fluorescence decay rates. Comparing the determined phosphorescence spectra of the oligopeptides studied with that of tryptophan, a slight blue-shift and more evident red-shift was observed in the spectrum of AW and WA, respectively. On the basis of the results of the phosphorescence measurements performed at pH 10, the 170 µs lifetime of WA, observed even at pH 7, may be assigned to the anionic form of the compound. It may be suggested that at pH 7 during the excited triplet state lifetime of WA there is a shift in the equilibrium towards the anionic form of this dipeptide. In the case of AW and AWA at pH 7 the obtained monoexponential decay kinetics, most probably, arise from zwitterionic forms of these peptides. The determined triplet quantum yield of AWA is slightly lower than that of tryptophan, while the quantum yield of AW is twofold lower than that of tryptophan. The highest value of the determined triplet quantum yield of WA confirms the presence of anionic form of this dipeptide at pH 7.