Search for New Aggregable Fragments of Human Insulin

Swiontek, Monika; Frączyk, Justyna; Waśko, Joanna; Chaberska, Agata; Pietrzak, Łukasz; Kamiński, Zbigniew J.; Szymański, Łukasz; Wiak, Sławomir; Kolesińska, Beata

doi:10.3390/molecules24081600

Cited by 11 publications

(8 citation statements)

References 37 publications

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“…On the CD spectra of the HSA-insulin complex ( Figure 9 ), a regular increase was observed in signal intensity at 194 nm and a stronger minimum at 210 and 224 nm. This means that, in the presence, of the native form of insulin does not tend to form amyloid fibrils, for which the β-sheet structure is the main growth factor [ 60 , 67 ]. We can suppose that possible interactions between HSA and the native hormone inhibit undesirable insulin aggregation and increase the content of the α-helix structure in the sample.…”

Section: Resultsmentioning

confidence: 99%

“…In the sample containing only HSA ( Figure 11 a), we recorded a crystal structure characteristic for proteins. Hot spot fragments 5 and 6 after incubation at 37 °C formed typical amyloid fibers ( Figure 11 b,d), which were very clearly visible after staining with CR [ 60 ]. In the presence of HSA, we observed significantly less content of fibers characteristic for amyloid deposits ( Figure 11 c,e).…”

Section: Resultsmentioning

confidence: 99%

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Human Serum Albumin Binds Native Insulin and Aggregable Insulin Fragments and Inhibits Their Aggregation

et al. 2020

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The purpose of this study was to investigate whether Human Serum Albumin (HSA) can bind native human insulin and its A13–A19 and B12–B17 fragments, which are responsible for the aggregation of the whole hormone. To label the hormone and both hot spots, so that their binding positions within the HSA could be identified, 4-(1-pyrenyl)butyric acid was used as a fluorophore. Triazine coupling reagent was used to attach the 4-(1-pyrenyl)butyric acid to the N-terminus of the peptides. When attached to the peptides, the fluorophore showed extended fluorescence lifetimes in the excited state in the presence of HSA, compared to the samples in buffer solution. We also analyzed the interactions of unlabeled native insulin and its hot spots with HSA, using circular dichroism (CD), the microscale thermophoresis technique (MST), and three independent methods recommended for aggregating peptides. The CD spectra indicated increased amounts of the α-helical secondary structure in all analyzed samples after incubation. Moreover, for each of the two unlabeled hot spots, it was possible to determine the dissociation constant in the presence of HSA, as 14.4 µM (A13–A19) and 246 nM (B12–B17). Congo Red, Thioflavin T, and microscopy assays revealed significant differences between typical amyloids formed by the native hormone or its hot-spots and the secondary structures formed by the complexes of HSA with insulin and A13–A19 and B12–B17 fragments. All results show that the tested peptide-probe conjugates and their unlabeled analogues interact with HSA, which inhibits their aggregation.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Human Serum Albumin Binds Native Insulin and Aggregable Insulin Fragments and Inhibits Their Aggregation

et al. 2020

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“…After 7 days of incubation, a solution of CR in phosphate buffer and/or Thioflavin T was added to the samples. Standard procedures were followed [38–40] …”

Section: Resultsmentioning

confidence: 99%

“…Standard procedures were followed. [38][39][40] Five of the tested peptides were found to have an inhibitory effect on the aggregating (18)(19)(20)(21)(22) HÀ HSSNNÀ OH fragment ( Figure 5). No inhibitory effect was noted for HÀ F(NÀ Me)GA(NÀ Me)ILÀ OH (6) in a complex with (18-22) HÀ HSSNNÀ OH.…”

Section: Resultsmentioning

confidence: 99%

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

Rożniakowski

Gałęcki

Wietrzyk

et al. 2020

Chemistry & Biodiversity

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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 HÀ F(NÀ Me)GA(NÀ Me) ILÀ 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) HÀ HSSNNÀ OH fragment and by the HÀ F(N-Me)GA(NÀ Me)ILÀ OH (6) fragment derived from the 23-27 amylin hot spot. Fragment (33-37) HÀ GSNTYÀ NH 2 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 HÀ F(NÀ Me)GA(NÀ Me)ILÀ 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.

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“…The first aim of the study was investigation on predisposition of peptides 1 – 7 to aggregate. Native fragments of insulin (hot-spots A and B) were used as an internal control [23]. To mimic natural conditions inside the human body, the aggregation of peptides was carried out at pH 7.2 phosphate buffer in 37 °C.…”

Section: Resultsmentioning

confidence: 99%

Insulin Hot-Spot Analogs Formed with N-Methylated Amino Acid Residues Inhibit Aggregation of Native Hormone

et al. 2019

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In this study, N-methylated analogs of hot-spots of insulin were designed and synthesized, in the expectation that they would inhibit the aggregation of both insulin hot-spots and the entire hormone. Synthesis of insulin “amyloidogenic” analogs containing N-methylated amino acid residues was performed by microwave-assisted solid phase according to the Fmoc/tert-Bu strategy. As a coupling reagent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium toluene-4-sulfonate (DMT/NMM/TosO-) was used. Three independent methods were applied in aggregation studies of the complexes of insulin with its N-methylated peptides. Additionally, circular dichroism (CD) measurements were used to confirm that aggregation processes did not occur in the presence of the N-methylated analogs of hot-spot insulin fragments, and that insulin retains its native conformation. Of the seven N-methylated analogs of the A- and B-chain hot-spots of insulin, six inhibited insulin aggregation (peptides 1 and 3–7). All tested peptides were found to have a lower ability to inhibit the aggregation of insulin hot-spots compared to the capability to inhibit native hormone aggregation.

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Search for New Aggregable Fragments of Human Insulin

Cited by 11 publications

References 37 publications

Human Serum Albumin Binds Native Insulin and Aggregable Insulin Fragments and Inhibits Their Aggregation

Human Serum Albumin Binds Native Insulin and Aggregable Insulin Fragments and Inhibits Their Aggregation

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

Insulin Hot-Spot Analogs Formed with N-Methylated Amino Acid Residues Inhibit Aggregation of Native Hormone

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