Abstract
The amino acid tyrosine forms cytotoxic amyloid-like fibrils by molecular self-assembly. However, the production of antibodies towards tyrosine assemblies, reflecting their presentation to the immune system, was not demonstrated yet. Here, we describe the production of antibodies that specifically recognize tyrosine in its fibrillated form. The antibodies were demonstrated to specifically bind self-assembled tyrosine, in contrast to its non-aggregated form or disintegrated fibrils. The antibodies could be used for immunostaining of tyrosine fibrils in cultured cells. Furthermore, confocal microscopy allowed a demonstration of the intracellular presence of the metabolite amyloids in a neuroblastoma cell model. Finally, pre-incubation of tyrosine fibrils with the antibodies resulted in significant reduction in their cytotoxicity. Taken together, we provide an experimental proof for the immunogenicity of tyrosine amyloid fibrillary assemblies. These specific antibodies against tyrosine structures could be further used as a research tool to study the dynamics, toxicity and cellular localization of the assemblies.
Highlights
The self-assembly of proteins and polypeptides into amyloid fibrils is a major hallmark of various degenerative diseases, including Alzheimer’s disease, Parkinson’s disease and Type 2 diabetes [1,2,3]
To generate Tyr amyloid-like fibrils, Tyr (2 mg/mL) was first dissolved in PBS, and the solution was heated to 90 ◦ C to ascertain the monomeric state gradually cooled to allow the formation of amyloid-like assemblies, as previously described [13]
The production of anti-Tyr antibodies raised against Tyr amyloid-like assemblies is presented
Summary
The self-assembly of proteins and polypeptides into amyloid fibrils is a major hallmark of various degenerative diseases, including Alzheimer’s disease, Parkinson’s disease and Type 2 diabetes [1,2,3]. formed by a structurally diverse group of proteins, all amyloid fibrils share similar biophysical and structural properties [4,5,6]. Over the last two decades, a reductionist approach using increasingly shorter amyloid peptide fragments, including minimal dipeptides, has demonstrated the formation of typical amyloid fibrils These fibrils shows the same biophysical and structural features characteristic of the assemblies formed by full length proteins and polypeptides [5,7,8,9]. It has been recently demonstrated that several metabolites, including both single amino acids and nucleic bases, can form well-ordered amyloid-like fibrillar assemblies These fibrils were shown to bind the amyloid-specific, dyes Thioflavin T (ThT) and Congo red, and to trigger cytotoxicity by inducing apoptotic cell death as observed for protein and polypeptide amyloid structures [10,11,12,13,14]. These discoveries extended the metabolite amyloids hypothesis, suggesting that small, monomeric metabolites can self-assemble to form amyloid-like fibrils showing similar properties to proteinaceous amyloids
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