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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

Formation of amyloid-like structures by metabolite self-assembly.Skeletal formula, TEM micrographs of elongated metabolite fibrils, confocal fluorescence microscopy images of metabolite stained with ThT, and ThT fluorescence emission spectra images. All metabolites were dissolved at 90°C in PBS. Columns from left to right (as indicated above): TEM micrographs. Scale bars, 500 nm. Confocal microscopy images were taken immediately after the addition of the ThT reagent at a 1:1 ratio with the metabolites, with excitation and emission wavelengths of 458 and 485 nm, respectively. Scale bars, 20 μm. ThT emission spectra (excitation at 430 nm) were collected for each of the metabolites. Aged samples of each metabolite were added to 40 μM ThT in PBS to a final concentration of 20 μM ThT. (A) Adenine—TEM (1 mg/ml), ThT microscopy (4 mg/ml) and spectra (2 mg/ml). (B) Orotic acid—TEM (1 mg/ml), ThT microscopy and spectra (2 mg/ml). (C) Cystine—TEM (1 mg/ml), ThT microscopy and spectra (2 mg/ml). (D) Tyrosine—TEM (1 mg/ml), ThT microscopy and spectra (4 mg/ml). (E) Uracil—TEM (5 mg/ml), ThT microscopy and spectra (10 mg/ml). (F) Phenylalanine—TEM (2 mg/ml), ThT microscopy and spectra (4 mg/ml). AU, arbitrary units.
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Figure 2: Formation of amyloid-like structures by metabolite self-assembly.Skeletal formula, TEM micrographs of elongated metabolite fibrils, confocal fluorescence microscopy images of metabolite stained with ThT, and ThT fluorescence emission spectra images. All metabolites were dissolved at 90°C in PBS. Columns from left to right (as indicated above): TEM micrographs. Scale bars, 500 nm. Confocal microscopy images were taken immediately after the addition of the ThT reagent at a 1:1 ratio with the metabolites, with excitation and emission wavelengths of 458 and 485 nm, respectively. Scale bars, 20 μm. ThT emission spectra (excitation at 430 nm) were collected for each of the metabolites. Aged samples of each metabolite were added to 40 μM ThT in PBS to a final concentration of 20 μM ThT. (A) Adenine—TEM (1 mg/ml), ThT microscopy (4 mg/ml) and spectra (2 mg/ml). (B) Orotic acid—TEM (1 mg/ml), ThT microscopy and spectra (2 mg/ml). (C) Cystine—TEM (1 mg/ml), ThT microscopy and spectra (2 mg/ml). (D) Tyrosine—TEM (1 mg/ml), ThT microscopy and spectra (4 mg/ml). (E) Uracil—TEM (5 mg/ml), ThT microscopy and spectra (10 mg/ml). (F) Phenylalanine—TEM (2 mg/ml), ThT microscopy and spectra (4 mg/ml). AU, arbitrary units.

Mentions: We initially composed a comprehensive list of enzymatic products that were found to accumulate in genetic inborn error of metabolism disorders (Fig. 1B) (16). Next, we explored possible conditions under which these soluble metabolites may undergo self-association. While attempting to mimic physiological conditions, we dissolved the metabolites in phosphate-buffered saline (PBS) to reflect physiological pH and ionic strength. We first dissolved the metabolites at 90°C in physiological buffer to obtain a homogenous monomeric solution. This was followed by gradual cooling of the solution, which resulted in the formation of ultrastructures. Each metabolite was assayed at various concentrations to determine the conditions under which it forms ordered supramolecular entities. Several of the studied metabolites were found to form elongated and fibrillar structures with nanoscale order, including adenine, orotic acid, cystine (an amino acid formed by the oxidation of two cysteine molecules that covalently link via a disulfide bond), tyrosine, uracil, and phenylalanine (Fig. 2). Comprehensive analysis of the propensity for amyloid fibril formation, by all coded amino acids, in the context of peptides and proteins, had identified phenylalanine, cysteine, and tyrosine as residues with the highest aggregation potential (17, 18). Therefore, it appears to be that the observed behavior of the various amino acids in the polypeptide chain may also be comparable as free amino acids.


Extension of the generic amyloid hypothesis to nonproteinaceous metabolite assemblies.

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

Formation of amyloid-like structures by metabolite self-assembly.Skeletal formula, TEM micrographs of elongated metabolite fibrils, confocal fluorescence microscopy images of metabolite stained with ThT, and ThT fluorescence emission spectra images. All metabolites were dissolved at 90°C in PBS. Columns from left to right (as indicated above): TEM micrographs. Scale bars, 500 nm. Confocal microscopy images were taken immediately after the addition of the ThT reagent at a 1:1 ratio with the metabolites, with excitation and emission wavelengths of 458 and 485 nm, respectively. Scale bars, 20 μm. ThT emission spectra (excitation at 430 nm) were collected for each of the metabolites. Aged samples of each metabolite were added to 40 μM ThT in PBS to a final concentration of 20 μM ThT. (A) Adenine—TEM (1 mg/ml), ThT microscopy (4 mg/ml) and spectra (2 mg/ml). (B) Orotic acid—TEM (1 mg/ml), ThT microscopy and spectra (2 mg/ml). (C) Cystine—TEM (1 mg/ml), ThT microscopy and spectra (2 mg/ml). (D) Tyrosine—TEM (1 mg/ml), ThT microscopy and spectra (4 mg/ml). (E) Uracil—TEM (5 mg/ml), ThT microscopy and spectra (10 mg/ml). (F) Phenylalanine—TEM (2 mg/ml), ThT microscopy and spectra (4 mg/ml). AU, arbitrary units.
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Related In: Results  -  Collection

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Figure 2: Formation of amyloid-like structures by metabolite self-assembly.Skeletal formula, TEM micrographs of elongated metabolite fibrils, confocal fluorescence microscopy images of metabolite stained with ThT, and ThT fluorescence emission spectra images. All metabolites were dissolved at 90°C in PBS. Columns from left to right (as indicated above): TEM micrographs. Scale bars, 500 nm. Confocal microscopy images were taken immediately after the addition of the ThT reagent at a 1:1 ratio with the metabolites, with excitation and emission wavelengths of 458 and 485 nm, respectively. Scale bars, 20 μm. ThT emission spectra (excitation at 430 nm) were collected for each of the metabolites. Aged samples of each metabolite were added to 40 μM ThT in PBS to a final concentration of 20 μM ThT. (A) Adenine—TEM (1 mg/ml), ThT microscopy (4 mg/ml) and spectra (2 mg/ml). (B) Orotic acid—TEM (1 mg/ml), ThT microscopy and spectra (2 mg/ml). (C) Cystine—TEM (1 mg/ml), ThT microscopy and spectra (2 mg/ml). (D) Tyrosine—TEM (1 mg/ml), ThT microscopy and spectra (4 mg/ml). (E) Uracil—TEM (5 mg/ml), ThT microscopy and spectra (10 mg/ml). (F) Phenylalanine—TEM (2 mg/ml), ThT microscopy and spectra (4 mg/ml). AU, arbitrary units.
Mentions: We initially composed a comprehensive list of enzymatic products that were found to accumulate in genetic inborn error of metabolism disorders (Fig. 1B) (16). Next, we explored possible conditions under which these soluble metabolites may undergo self-association. While attempting to mimic physiological conditions, we dissolved the metabolites in phosphate-buffered saline (PBS) to reflect physiological pH and ionic strength. We first dissolved the metabolites at 90°C in physiological buffer to obtain a homogenous monomeric solution. This was followed by gradual cooling of the solution, which resulted in the formation of ultrastructures. Each metabolite was assayed at various concentrations to determine the conditions under which it forms ordered supramolecular entities. Several of the studied metabolites were found to form elongated and fibrillar structures with nanoscale order, including adenine, orotic acid, cystine (an amino acid formed by the oxidation of two cysteine molecules that covalently link via a disulfide bond), tyrosine, uracil, and phenylalanine (Fig. 2). Comprehensive analysis of the propensity for amyloid fibril formation, by all coded amino acids, in the context of peptides and proteins, had identified phenylalanine, cysteine, and tyrosine as residues with the highest aggregation potential (17, 18). Therefore, it appears to be that the observed behavior of the various amino acids in the polypeptide chain may also be comparable as free amino acids.

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