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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

Apoptotic activity of the assemblies studied by annexin V and PI assay.Metabolites were dissolved at 90°C in cell medium followed by gradual cooling of the solution. The control reflects medium with no metabolites, which was treated in the same manner. Treated SH-SY5Y cells were incubated with medium containing metabolites in the stated concentrations for 24 hours. Control cells were incubated with medium without any addition of metabolites. After incubation, annexin V–FITC (fluorescein isothiocyanate) and PI reagents were added to the cell cultures following the measurement of the cell samples by flow cytometry using single laser emitting excitation light at 488 nm. Early apoptosis is represented in blue, late apoptosis in red, and necrosis in green. (A) Schematic depiction of metabolites that self-assemble into amyloid-like structures, which triggers apoptotic effect. (B) Adenine. (C) Cystine. (D)Tyrosine. (E) Orotic acid. (F) Phenylalanine. (G) Uracil. (H) Alanine.
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Figure 4: Apoptotic activity of the assemblies studied by annexin V and PI assay.Metabolites were dissolved at 90°C in cell medium followed by gradual cooling of the solution. The control reflects medium with no metabolites, which was treated in the same manner. Treated SH-SY5Y cells were incubated with medium containing metabolites in the stated concentrations for 24 hours. Control cells were incubated with medium without any addition of metabolites. After incubation, annexin V–FITC (fluorescein isothiocyanate) and PI reagents were added to the cell cultures following the measurement of the cell samples by flow cytometry using single laser emitting excitation light at 488 nm. Early apoptosis is represented in blue, late apoptosis in red, and necrosis in green. (A) Schematic depiction of metabolites that self-assemble into amyloid-like structures, which triggers apoptotic effect. (B) Adenine. (C) Cystine. (D)Tyrosine. (E) Orotic acid. (F) Phenylalanine. (G) Uracil. (H) Alanine.

Mentions: To explore whether the metabolite assemblies trigger the process of apoptotic cell death, as was reported for amyloid fibrils (22–28), we used the annexin V and propidium iodide (PI) apoptosis assay. The effect of the metabolites was studied using the same SH-SY5Y cell model, at a concentration that was found to result in about 50% decrease in cell viability by the XTT cell viability assay. Cystine was not soluble in a concentration higher than 2 mg/ml and thus was examined at this concentration. The tested metabolite assemblies induced a clear apoptotic effect on cultured cells, as observed in all analyses as the major route for cell toxicity (Fig. 4A). Cystine had the strongest apoptotic effect resulting in 62% cell apoptosis (Fig. 4C), phenylalanine addition resulted in 55% cell apoptosis (Fig. 4F), whereas adenine tyrosine and uracil triggered 40 to 50% cell apoptosis (Fig. 4, B, D, and G). Orotic acid demonstrated a slightly lower effect causing about 30% cell apoptosis (Fig. 4E). The negative control, using alanine as before, did not present any apoptotic response on examined cells (Fig. 4H).


Extension of the generic amyloid hypothesis to nonproteinaceous metabolite assemblies.

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

Apoptotic activity of the assemblies studied by annexin V and PI assay.Metabolites were dissolved at 90°C in cell medium followed by gradual cooling of the solution. The control reflects medium with no metabolites, which was treated in the same manner. Treated SH-SY5Y cells were incubated with medium containing metabolites in the stated concentrations for 24 hours. Control cells were incubated with medium without any addition of metabolites. After incubation, annexin V–FITC (fluorescein isothiocyanate) and PI reagents were added to the cell cultures following the measurement of the cell samples by flow cytometry using single laser emitting excitation light at 488 nm. Early apoptosis is represented in blue, late apoptosis in red, and necrosis in green. (A) Schematic depiction of metabolites that self-assemble into amyloid-like structures, which triggers apoptotic effect. (B) Adenine. (C) Cystine. (D)Tyrosine. (E) Orotic acid. (F) Phenylalanine. (G) Uracil. (H) Alanine.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Apoptotic activity of the assemblies studied by annexin V and PI assay.Metabolites were dissolved at 90°C in cell medium followed by gradual cooling of the solution. The control reflects medium with no metabolites, which was treated in the same manner. Treated SH-SY5Y cells were incubated with medium containing metabolites in the stated concentrations for 24 hours. Control cells were incubated with medium without any addition of metabolites. After incubation, annexin V–FITC (fluorescein isothiocyanate) and PI reagents were added to the cell cultures following the measurement of the cell samples by flow cytometry using single laser emitting excitation light at 488 nm. Early apoptosis is represented in blue, late apoptosis in red, and necrosis in green. (A) Schematic depiction of metabolites that self-assemble into amyloid-like structures, which triggers apoptotic effect. (B) Adenine. (C) Cystine. (D)Tyrosine. (E) Orotic acid. (F) Phenylalanine. (G) Uracil. (H) Alanine.
Mentions: To explore whether the metabolite assemblies trigger the process of apoptotic cell death, as was reported for amyloid fibrils (22–28), we used the annexin V and propidium iodide (PI) apoptosis assay. The effect of the metabolites was studied using the same SH-SY5Y cell model, at a concentration that was found to result in about 50% decrease in cell viability by the XTT cell viability assay. Cystine was not soluble in a concentration higher than 2 mg/ml and thus was examined at this concentration. The tested metabolite assemblies induced a clear apoptotic effect on cultured cells, as observed in all analyses as the major route for cell toxicity (Fig. 4A). Cystine had the strongest apoptotic effect resulting in 62% cell apoptosis (Fig. 4C), phenylalanine addition resulted in 55% cell apoptosis (Fig. 4F), whereas adenine tyrosine and uracil triggered 40 to 50% cell apoptosis (Fig. 4, B, D, and G). Orotic acid demonstrated a slightly lower effect causing about 30% cell apoptosis (Fig. 4E). The negative control, using alanine as before, did not present any apoptotic response on examined cells (Fig. 4H).

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