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Extension of the generic amyloid hypothesis to nonproteinaceous metabolite assemblies.

Shaham-Niv S, Adler-Abramovich L, Schnaider L, Gazit E - Sci Adv (2015)

Bottom Line: Although the formation of these supramolecular entities has previously been associated with proteins and peptides, it was later demonstrated that even phenylalanine, a single amino acid, can form fibrils that have amyloid-like biophysical, biochemical, and cytotoxic properties.Moreover, the generation of antibodies against these assemblies in phenylketonuria patients and the correlating mice model suggested a pathological role for the assemblies.The formation of amyloid-like assemblies by metabolites implies a general phenomenon of amyloid formation, not limited to proteins and peptides, and offers a new paradigm for metabolic diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.

ABSTRACT
The accumulation of amyloid fibrils is the hallmark of several major human diseases. Although the formation of these supramolecular entities has previously been associated with proteins and peptides, it was later demonstrated that even phenylalanine, a single amino acid, can form fibrils that have amyloid-like biophysical, biochemical, and cytotoxic properties. Moreover, the generation of antibodies against these assemblies in phenylketonuria patients and the correlating mice model suggested a pathological role for the assemblies. We determine that several other metabolites that accumulate in metabolic disorders form ordered amyloid-like ultrastructures, which induce apoptotic cell death, as observed for amyloid structures. The formation of amyloid-like assemblies by metabolites implies a general phenomenon of amyloid formation, not limited to proteins and peptides, and offers a new paradigm for metabolic diseases.

No MeSH data available.


Related in: MedlinePlus

Cytotoxicity of the metabolite assemblies as determined by XTT assay.Metabolites were dissolved at 90°C in cell medium followed by gradual cooling of the solution. The control (zero concentration of metabolites) reflects medium with no metabolites, which was treated in the same manner. Treated SH-SY5Y cells were incubated with medium containing metabolites for 6 hours, following the addition of the XTT reagent. After 2.5 hours of incubation, absorbance was determined at 450 nm. The results represent three biological repeats; error bars represent 95% confidence interval (P < 0.01). (A) Adenine. (B) Orotic acid. (C) Uracil. (D) Tyrosine. (E) Phenylalanine. (F) Alanine.
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Figure 3: Cytotoxicity of the metabolite assemblies as determined by XTT assay.Metabolites were dissolved at 90°C in cell medium followed by gradual cooling of the solution. The control (zero concentration of metabolites) reflects medium with no metabolites, which was treated in the same manner. Treated SH-SY5Y cells were incubated with medium containing metabolites for 6 hours, following the addition of the XTT reagent. After 2.5 hours of incubation, absorbance was determined at 450 nm. The results represent three biological repeats; error bars represent 95% confidence interval (P < 0.01). (A) Adenine. (B) Orotic acid. (C) Uracil. (D) Tyrosine. (E) Phenylalanine. (F) Alanine.

Mentions: The metabolite assemblies displayed a dose-dependent cytotoxic effect on the cells, as indicated by the 2,3-bis-(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-carboxanilide (XTT) cell viability assay (Fig. 3, A to E). Metabolites in medium alone served as a control, showing that the change in the absorbance was due to the change in cell viability and was not affected by the structures formed by the metabolites. For this reason, cystine assemblies could not be examined using this assay, because they absorb at the same wavelength as the XTT reagent, thus affecting the absorbance even without the presence of any cells. Additionally, we were interested in determining the concentrations that resulted in ~50% reduction in cell viability, and thus, a uracil concentration of 10 mg/ml was examined as well, because lower concentrations did not trigger such an effect. The highest concentration of adenine had the most cytotoxic effect out of all tested metabolites, inducing a decrease in cell viability to about 33% (Fig. 3A). The highest concentration of the other investigated metabolites resulted in a decrease in cell viability to about 50% (Fig. 3, B to E). To examine whether the observed toxic effect was not an outcome of osmotic stress or any other colligative properties, but indeed due to the metabolite structures, the alanine amino acid was used as a negative control. Alanine was not reported to accumulate in any metabolic disorder, and when analyzed under the same set of concentrations and conditions, no structure formation was observed. Moreover, alanine did not demonstrate any toxic effect even in the highest concentration of 10 mg/ml (Fig. 3F), supporting the notion that the toxic effect was due to the formation of supramolecular metabolite structures.


Extension of the generic amyloid hypothesis to nonproteinaceous metabolite assemblies.

Shaham-Niv S, Adler-Abramovich L, Schnaider L, Gazit E - Sci Adv (2015)

Cytotoxicity of the metabolite assemblies as determined by XTT assay.Metabolites were dissolved at 90°C in cell medium followed by gradual cooling of the solution. The control (zero concentration of metabolites) reflects medium with no metabolites, which was treated in the same manner. Treated SH-SY5Y cells were incubated with medium containing metabolites for 6 hours, following the addition of the XTT reagent. After 2.5 hours of incubation, absorbance was determined at 450 nm. The results represent three biological repeats; error bars represent 95% confidence interval (P < 0.01). (A) Adenine. (B) Orotic acid. (C) Uracil. (D) Tyrosine. (E) Phenylalanine. (F) Alanine.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4643800&req=5

Figure 3: Cytotoxicity of the metabolite assemblies as determined by XTT assay.Metabolites were dissolved at 90°C in cell medium followed by gradual cooling of the solution. The control (zero concentration of metabolites) reflects medium with no metabolites, which was treated in the same manner. Treated SH-SY5Y cells were incubated with medium containing metabolites for 6 hours, following the addition of the XTT reagent. After 2.5 hours of incubation, absorbance was determined at 450 nm. The results represent three biological repeats; error bars represent 95% confidence interval (P < 0.01). (A) Adenine. (B) Orotic acid. (C) Uracil. (D) Tyrosine. (E) Phenylalanine. (F) Alanine.
Mentions: The metabolite assemblies displayed a dose-dependent cytotoxic effect on the cells, as indicated by the 2,3-bis-(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-carboxanilide (XTT) cell viability assay (Fig. 3, A to E). Metabolites in medium alone served as a control, showing that the change in the absorbance was due to the change in cell viability and was not affected by the structures formed by the metabolites. For this reason, cystine assemblies could not be examined using this assay, because they absorb at the same wavelength as the XTT reagent, thus affecting the absorbance even without the presence of any cells. Additionally, we were interested in determining the concentrations that resulted in ~50% reduction in cell viability, and thus, a uracil concentration of 10 mg/ml was examined as well, because lower concentrations did not trigger such an effect. The highest concentration of adenine had the most cytotoxic effect out of all tested metabolites, inducing a decrease in cell viability to about 33% (Fig. 3A). The highest concentration of the other investigated metabolites resulted in a decrease in cell viability to about 50% (Fig. 3, B to E). To examine whether the observed toxic effect was not an outcome of osmotic stress or any other colligative properties, but indeed due to the metabolite structures, the alanine amino acid was used as a negative control. Alanine was not reported to accumulate in any metabolic disorder, and when analyzed under the same set of concentrations and conditions, no structure formation was observed. Moreover, alanine did not demonstrate any toxic effect even in the highest concentration of 10 mg/ml (Fig. 3F), supporting the notion that the toxic effect was due to the formation of supramolecular metabolite structures.

Bottom Line: Although the formation of these supramolecular entities has previously been associated with proteins and peptides, it was later demonstrated that even phenylalanine, a single amino acid, can form fibrils that have amyloid-like biophysical, biochemical, and cytotoxic properties.Moreover, the generation of antibodies against these assemblies in phenylketonuria patients and the correlating mice model suggested a pathological role for the assemblies.The formation of amyloid-like assemblies by metabolites implies a general phenomenon of amyloid formation, not limited to proteins and peptides, and offers a new paradigm for metabolic diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.

ABSTRACT
The accumulation of amyloid fibrils is the hallmark of several major human diseases. Although the formation of these supramolecular entities has previously been associated with proteins and peptides, it was later demonstrated that even phenylalanine, a single amino acid, can form fibrils that have amyloid-like biophysical, biochemical, and cytotoxic properties. Moreover, the generation of antibodies against these assemblies in phenylketonuria patients and the correlating mice model suggested a pathological role for the assemblies. We determine that several other metabolites that accumulate in metabolic disorders form ordered amyloid-like ultrastructures, which induce apoptotic cell death, as observed for amyloid structures. The formation of amyloid-like assemblies by metabolites implies a general phenomenon of amyloid formation, not limited to proteins and peptides, and offers a new paradigm for metabolic diseases.

No MeSH data available.


Related in: MedlinePlus