scholarly journals The Fluorescent Dye 1,6-Diphenyl-1,3,5-Hexatriene Binds to Amyloid Fibrils Formed by Human Amylin and Provides a New Probe of Amylin Amyloid Kinetics

2021 ◽  
Author(s):  
Ming-Hao Li ◽  
Lakshan Manathunga ◽  
Erwin London ◽  
Daniel Raleigh
Keyword(s):  
Time Course ◽  
Amyloid Fibrils ◽  
Fold Increase ◽  
Fluorescent Dye ◽  
Thioflavin T ◽  
Lag Phase ◽  
Amyloid Formation ◽  
Islet Amyloid ◽  
Human Amylin ◽  
Mutant Forms

The fluorescent dye 1,6-diphenyl-1,3,5-hexatriene (DPH) is widely used as a probe of membrane order. We show that DPH also interacts with amyloid fibrils formed by human amylin (also known as islet amyloid polypeptide) in solution and this results in a 100-fold increase in DPH fluorescence for a sample of microM human amylin and 0.25 microM DPH. No increase in DPH fluorescence is observed with the non-amyloidogenic rat amylin or with freshly dissolved, non-fibrillar human amylin. The time course of amyloid formation by amylin was followed by monitoring the fluorescence of added DPH as a function of time and was similar to that monitored by the standard fluorescent probe thioflavin-T. The inclusion of DPH in the buffer did not perturb the time course of amyloid formation under the conditions examined and the time course was independent of the range of DPH concentrations tested (0.25 to 5 microM). Maximum final fluorescence intensity is observed at substoichiometric ratios of DPH to amylin. No significant increase in fluorescence was observed during the lag phase of amyloid formation, and the implications for the structure of amylin pre-fibril oligomers are discussed. Human amylin contains three aromatic residues. A triple aromatic to leucine mutant forms amyloid and DPH binds to the resulting fibrils, indicating that interactions with aromatic side chains are not required for DPH amylin amyloid interactions. DPH may be especially useful for studies on mutant amylins and other polypeptides in which changes in charged residues might complicate interpretation of thioflavin-T fluorescence.

scholarly journals Mechanistic Contributions of Biological Cofactors in Islet Amyloid Polypeptide Amyloidogenesis

2015 ◽  
Vol 2015 ◽  
pp. 1-13 ◽  
Author(s):  
Phuong Trang Nguyen ◽  
Nagore Andraka ◽  
Carole Anne De Carufel ◽  
Steve Bourgault
Keyword(s):  
Type Ii Diabetes ◽  
Amyloid Fibrils ◽  
Driving Forces ◽  
Islet Amyloid Polypeptide ◽  
Type Ii Diabetes Mellitus ◽  
Major Protein ◽  
Local Concentration ◽  
Amyloid Formation ◽  
Type Ii ◽  
Islet Amyloid

Type II diabetes mellitus is associated with the deposition of fibrillar aggregates in pancreatic islets. The major protein component of islet amyloids is the glucomodulatory hormone islet amyloid polypeptide (IAPP). Islet amyloid fibrils are virtually always associated with several biomolecules, including apolipoprotein E, metals, glycosaminoglycans, and various lipids. IAPP amyloidogenesis has been originally perceived as a self-assembly homogeneous process in which the inherent aggregation propensity of the peptide and its local concentration constitute the major driving forces to fibrillization. However, over the last two decades, numerous studies have shown a prominent role of amyloid cofactors in IAPP fibrillogenesis associated with the etiology of type II diabetes. It is increasingly evident that the biochemical microenvironment in which IAPP amyloid formation occurs and the interactions of the polypeptide with various biomolecules not only modulate the rate and extent of aggregation, but could also remodel the amyloidogenesis process as well as the structure, toxicity, and stability of the resulting fibrils.

Extended life span is associated with insulin resistance in a transgenic mouse model of insulinoma secreting human islet amyloid polypeptide

2004 ◽  
Vol 286 (3) ◽  
pp. E418-E424 ◽  
Author(s):  
Sofianos Andrikopoulos ◽  
Rebecca L. Hull ◽  
C. Bruce Verchere ◽  
Feng Wang ◽  
Shani M. Wilbur ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Life Span ◽  
Amyloid Fibrils ◽  
Islet Amyloid Polypeptide ◽  
Transgenic Animals ◽  
Human Islet ◽  
Amyloid Formation ◽  
Islet Amyloid ◽  
Glucose Levels ◽  
Human Islet Amyloid Polypeptide

Pancreatic amyloid is found in patients with insulinomas and type 2 diabetes. To study mechanisms of islet amyloidogenesis, we produced transgenic mice expressing the unique component of human islet amyloid, human islet amyloid polypeptide (hIAPP). These mice develop islet amyloid after 12 mo of high-fat feeding. To determine whether we could accelerate the rate of islet amyloid formation, we crossbred our hIAPP transgenic animals with RIP-Tag mice that develop islet tumors and die at 12 wk of age from hypoglycemia. At 12 wk of age, this new line of hIAPP×RIP-Tag mice was heavier (29.7 ± 1.0 vs. 25.0 ± 1.3 g, P < 0.05) and had increased plasma glucose levels (4.6 ± 0.4 vs. 2.9 ± 0.6 mmol/l, P < 0.05) compared with littermate RIP-Tag mice. However, the hIAPP×RIP-Tag mice did not display islet amyloid or amyloid fibrils despite high circulating hIAPP levels (24.6 ± 7.0 pmol/l). Interestingly, hIAPP×RIP-Tag mice had a longer life span than RIP-Tag mice (121 ± 8 vs. 102 ± 5 days, P < 0.05). This increase in life span in hIAPP×RIP-Tag was positively correlated with body weight ( r = 0.48, P < 0.05) and was associated with decreased insulin sensitivity compared with RIP-Tag mice. hIAPP×RIP-Tag mice did not develop amyloid during their 4-mo life span, suggesting that increased hIAPP secretion is insufficient for islet amyloid formation within such a short time. However, hIAPP×RIP-Tag mice did have an increase in life span that was associated with insulin resistance, suggesting that hIAPP has extrapancreatic effects, possibly on peripheral glucose metabolism.

scholarly journals Suppression by polycyclic compounds of the conversion of human amylin into insoluble amyloid

2003 ◽  
Vol 374 (3) ◽  
pp. 779-784 ◽  
Author(s):  
Jacqueline F. AITKEN ◽  
Kerry M. LOOMES ◽  
Barbara KONARKOWSKA ◽  
Garth J. S. COOPER
Keyword(s):  
Acridine Orange ◽  
Congo Red ◽  
Type Ii Diabetes Mellitus ◽  
Significant Inhibition ◽  
Molar Ratio ◽  
Amyloid Formation ◽  
Islet Amyloid ◽  
Polycyclic Compounds ◽  
Molar Excess ◽  
Human Amylin

There is a significant correlation between the occurrence of pancreatic islet amyloid and β-cell failure in advanced type II diabetes mellitus. Islet amyloid is composed primarily of the fibrillar form of the pancreatic hormone, amylin. Using thioflavin-T fluorescence binding and radioprecipitation assays, we investigated whether or not a series of small tricyclic compounds, tetracycline or Congo Red could interfere with the conversion of synthetic human amylin into its insoluble amyloid form. Of the compounds investigated, incubation of human amylin with a 20-fold molar excess of either Congo Red or Acridine Orange resulted in significant inhibition in the rate of amyloid formation. With Congo Red, maximal inhibition effectively occurred at a 1:1 molar ratio or greater over human amylin, whereas inhibition by Acridine Orange was dose-dependent. A 20-fold molar excess of the compound tetracycline also decreased insoluble amyloid content after extended incubation periods of approx. 20 h. Amyloid fibril morphology in the presence of tetracycline, as measured by transmission electron microscopy, was characterized by short fragmented fibrils compared with the longer and denser appearance of fibrils formed by amylin alone. These findings show that polycyclic compounds can suppress the formation of amyloid by human amylin, providing support for an alternative approach to peptide-based strategies by which islet amyloid formation could be modulated.

scholarly journals Effect of Polyamines on Shoot Multiplication and Furanocoumarin Production in Ruta graveolens Cultures

2012 ◽  
Vol 7 (7) ◽  
pp. 1934578X1200700 ◽  
Author(s):  
Renuka Diwan ◽  
Nutan Malpathak
Keyword(s):  
Time Course ◽  
Shoot Multiplication ◽  
Multiple Shoots ◽  
Fold Increase ◽  
Lag Phase ◽  
Multiplication Rate ◽  
Shoot Cultures ◽  
Ruta Graveolens ◽  
Promotive Effect ◽  
Exogenous Addition

The influence of the polyamines putrescine (Put), spermine (Spr) and spermidine (Spd) on growth and furanocoumarin production was investigated by exogenous addition, at different concentrations, to shoot cultures of Ruta graveolens at different phases of growth. Preliminary studies indicated that addition of Put (20 μM) and Spr (80 μM) had a promotive effect on shoot multiplication rate and number of multiple shoots formed. Spd was toxic, even at lower concentrations. The growth-phase of the culture at the time of exogenous addition of polyamines was found to be an important factor. Put was most effective when added at the lag phase, while Spr was most effective when added in the log phase. Time course studies of growth and furanocoumarin content were carried out for each polyamine and phase of addition. It was seen that maximum production of furanocoumarins (256.8 mg/10 g DW) occurred in the second week when Put was added in the lag phase and 260.5 mg/10 g DW in the fourth week when Spr was added in the log phase. Put addition resulted in a 3.10 fold increase in psoralen, 6.12 in xanthotoxin and 1.46 fold in bergapten production. Spr addition resulted in a 1.31 fold increase in psoralen, 4.11 fold in xanthotoxin and 1.49 fold in bergapten production. Results indicate that alteration of growth and furanocoumarin production kinetics is a combined outcome of choice of polyamine and the phase of culture at the time of exogenous addition. Polyamine addition enabled significant enhancement in production of pharmaceutically important bergapten and xanthotoxin in shoot cultures of Ruta graveolens, which could be explored for commercial production.

scholarly journals Structural Features of Amyloid Fibrils Formed from the Full-Length and Truncated Forms of Beta-2-Microglobulin Probed by Fluorescent Dye Thioflavin T

2018 ◽  
Vol 19 (9) ◽  
pp. 2762 ◽  
Author(s):  
Anna Sulatskaya ◽  
Natalia Rodina ◽  
Dmitry Polyakov ◽  
Maksim Sulatsky ◽  
Tatyana Artamonova ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amyloid Fibrils ◽  
Biological Fluids ◽  
Fluorescent Dye ◽  
Full Length ◽  
Thioflavin T ◽  
Protein Amino Acid ◽  
Terminal Amino ◽  
Beta 2 ◽  
Beta 2 Microglobulin

The persistence of high concentrations of beta-2-microglobulin (β2M) in the blood of patients with acute renal failure leads to the development of the dialysis-related amyloidosis. This disease manifests in the deposition of amyloid fibrils formed from the various forms of β2M in the tissues and biological fluids of patients. In this paper, the amyloid fibrils formed from the full-length β2M (β2m) and its variants that lack the 6 and 10 N-terminal amino acids of the protein polypeptide chain (ΔN6β2m and ΔN10β2m, respectively) were probed by using the fluorescent dye thioflavin T (ThT). For this aim, the tested solutions were prepared via the equilibrium microdialysis approach. Spectroscopic analysis of the obtained samples allowed us to detect one binding mode (type) of ThT interaction with all the studied variants of β2M amyloid fibrils with affinity ~104 M−1. This interaction can be explained by the dye molecules incorporation into the grooves that were formed by the amino acids side chains of amyloid protofibrils along the long axis of the fibrils. The decrease in the affinity and stoichiometry of the dye interaction with β2M fibrils, as well as in the fluorescence quantum yield and lifetime of the bound dye upon the shortening of the protein amino acid sequence were shown. The observed differences in the ThT-β2M fibrils binding parameters and characteristics of the bound dye allowed to prove not only the difference of the ΔN10β2m fibrils from other β2M fibrils (that can be detected visually, for example, by transmission electron microscopy (TEM), but also the differences between β2m and ΔN6β2m fibrils (that can not be unequivocally confirmed by other approaches). These results prove an essential role of N-terminal amino acids of the protein in the formation of the β2M amyloid fibrils. Information about amyloidogenic protein sequences can be claimed in the development of ways to inhibit β2M fibrillogenesis for the treatment of dialysis-related amyloidosis.

scholarly journals Hexafluoroisopropanol Induces Amyloid Fibrils of Islet Amyloid Polypeptide by Enhancing Both Hydrophobic and Electrostatic Interactions

2011 ◽  
Vol 286 (27) ◽  
pp. 23959-23966 ◽  
Author(s):  
Kotaro Yanagi ◽  
Mizue Ashizaki ◽  
Hisashi Yagi ◽  
Kazumasa Sakurai ◽  
Young-Ho Lee ◽  
...  
Keyword(s):  
Electrostatic Interactions ◽  
Amyloid Fibrils ◽  
Hydrophobic Interactions ◽  
Islet Amyloid Polypeptide ◽  
Helical Structure ◽  
Thioflavin T ◽  
General View ◽  
Islet Amyloid ◽  
Neutral Ph ◽  
Low Concentrations

Although amyloid fibrils deposit with various proteins, the comprehensive mechanism by which they form remains unclear. We studied the formation of fibrils of human islet amyloid polypeptide associated with type II diabetes in the presence of various concentrations of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) under acidic and neutral pH conditions using CD, amyloid-specific thioflavin T fluorescence, fluorescence imaging with thioflavin T, and atomic force microscopy. At low pH, the formation of fibrils was promoted by HFIP with an optimum at 5% (v/v). At neutral pH in the absence of HFIP, significant amounts of amorphous aggregates formed in addition to the fibrils. The addition of HFIP suppressed the formation of amorphous aggregates, leading to a predominance of fibrils with an optimum effect at 25% (v/v). Under both conditions, higher concentrations of HFIP dissolved the fibrils and stabilized the α-helical structure. The results indicate that fibrils and amorphous aggregates are different types of precipitates formed by exclusion from water-HFIP mixtures. The exclusion occurs through the combined effects of hydrophobic interactions and electrostatic interactions, both of which are strengthened by low concentrations of HFIP, and a subtle balance between the two types of interactions determines whether the fibrils or amorphous aggregates dominate. We suggest a general view of how the structure of precipitates varies dramatically from single crystals to amyloid fibrils and amorphous aggregates.

scholarly journals Amyloidogenicity and cytotoxicity of des-Lys-1 human amylin provides insight into amylin self-assembly and highlights the difficulties of defining amyloidogenicity

2019 ◽  
Vol 32 (2) ◽  
pp. 87-93 ◽  
Author(s):  
Kyung-Hoon Lee ◽  
Alexander Zhyvoloup ◽  
Daniel Raleigh
Keyword(s):  
Self Assembly ◽  
Amyloid Formation ◽  
Wild Type ◽  
Islet Amyloid ◽  
High Charge Density ◽  
Human Amylin ◽  
Phosphate Buffered Saline

Abstract The polypeptide amylin is responsible for islet amyloid in type 2 diabetes, a process which contributes to β-cell death in the disease. The role of the N-terminal region of amylin in amyloid formation is relatively unexplored, although removal of the disulfide bridged loop between Cys-2 and Cys-7 accelerates amyloid formation. We examine the des Lys-1 variant of human amylin (h-amylin), a variant which is likely produced in vivo. Lys-1 is a region of high charge density in the h-amylin amyloid fiber. The des Lys-1 polypeptide forms amyloid on the same time scale as wild-type amylin in phosphate buffered saline, but does so more rapidly in Tris. The des Lys-1 variant is somewhat less toxic to cultured INS cells than wild type. The implications for the in vitro mechanism of amyloid formation and for comparative analysis of amyloidogenicity are discussed.

scholarly journals The β-cell assassin: IAPP cytotoxicity

2017 ◽  
Vol 59 (3) ◽  
pp. R121-R140 ◽  
Author(s):  
Daniel Raleigh ◽  
Xiaoxue Zhang ◽  
Benoît Hastoy ◽  
Anne Clark
Keyword(s):  
Amyloid Fibrils ◽  
Molecular Conformation ◽  
Cell Protein ◽  
Amyloid Formation ◽  
Β Cell ◽  
Islet Amyloid ◽  
The Unfolded Protein Response ◽  
Β Sheet

Islet amyloid polypeptide (IAPP) forms cytotoxic oligomers and amyloid fibrils in islets in type 2 diabetes (T2DM). The causal factors for amyloid formation are largely unknown. Mechanisms of molecular folding and assembly of human IAPP (hIAPP) into β-sheets, oligomers and fibrils have been assessed by detailed biophysical studies of hIAPP and non-fibrillogenic, rodent IAPP (rIAPP); cytotoxicity is associated with the early phases (oligomers/multimers) of fibrillogenesis. Interaction with synthetic membranes promotes β-sheet assembly possibly via a transient α-helical molecular conformation. Cellular hIAPP cytotoxicity can be activated from intracellular or extracellular sites. In transgenic rodents overexpressing hIAPP, intracellular pro-apoptotic signals can be generated at different points in β-cell protein synthesis. Increased cellular trafficking of proIAPP, failure of the unfolded protein response (UPR) or excess trafficking of misfolded peptide via the degradation pathways can induce apoptosis; these data indicate that defects in intracellular handling of hIAPP can induce cytotoxicity. However, there is no evidence for IAPP overexpression in T2DM. Extracellular amyloidosis is directly related to the degree of β-cell apoptosis in islets in T2DM. IAPP fragments, fibrils and multimers interact with membranes causing disruption in vivo and in vitro. These findings support a role for extracellular IAPP in β-sheet conformation in cytotoxicity. Inhibitors of fibrillogenesis are useful tools to determine the aberrant mechanisms that result in hIAPP molecular refolding and islet amyloidosis. However, currently, their role as therapeutic agents remains uncertain.

scholarly journals Mechanisms of Transthyretin Inhibition of IAPP AmyloidFormation

Biomolecules ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 411
Author(s):  
Sanduni Wasana Jayaweera ◽  
Solmaz Surano ◽  
Nina Pettersson ◽  
Elvira Oskarsson ◽  
Lovisa Lettius ◽  
...  
Keyword(s):  
Type Ii Diabetes ◽  
Type Ii Diabetes Mellitus ◽  
Low Ph ◽  
Lag Phase ◽  
Amyloid Formation ◽  
Human Pancreas ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Islet Amyloid ◽  
Potential Therapeutic Target

Amyloid-formation by the islet amyloid polypeptide (IAPP), produced by the β-cells in the human pancreas, has been associated with the development of type II diabetes mellitus (T2DM). The human plasma-protein transthyretin (TTR), a well-known amyloid-inhibiting protein, is interestingly also expressed within the IAPP producing β-cells. In the present study, we have characterized the ability of TTR to interfere with IAPP amyloid-formation, both in terms of its intrinsic stability as well as with regard to the effect of TTR-stabilizing drugs. The results show that TTR can prolong the lag-phase as well as impair elongation in the course of IAPP-amyloid formation. We also show that the interfering ability correlates inversely with the thermodynamic stability of TTR, while no such correlation was observed as a function of kinetic stability. Furthermore, we demonstrate that the ability of TTR to interfere is maintained also at the low pH environment within the IAPP-containing granules of the pancreatic β-cells. However, at both neutral and low pH, the addition of TTR-stabilizing drugs partly impaired its efficacy. Taken together, these results expose mechanisms of TTR-mediated inhibition of IAPP amyloid-formation and highlights a potential therapeutic target to prevent the onset of T2DM.

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