Investigating the Toxicity and Mechanisms of Non-Protein Amino Acids

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Non-protein amino acids (NPAAs) are amino acids not normally used in protein synthesis. However, a small subset of this type of amino acid, known as amino acid analogues, can be mistakenly utilised in protein synthesis due to their structural similarity to a canonical protein amino acid. This process has been implicated in the development of neurodegenerative disease. This research set out to investigate the toxicity and mechanisms of two lesser studied NPAAs which enter the human food chain and have the potential to contribute to the development of disease. These were L-norvaline (Nva), an analogue of the branched chain amino acids that is usually found in dietary supplements consumed by bodybuilders, and L-azetidine-2-carboxylic acid (Aze), an analogue of L-proline that is produced by sugar beets and enters the human food as a dietary supplement and as fodder for livestock. Initial experiments sought to identify whether either Nva or Aze induced cell death using metabolic and imaging assays. Both NPAAs were cytotoxic to human neuroblastoma cells, confirming the rationale behind the present investigation and providing the impetus for further research. Cell death pathways were also investigated, and death was determined to be necrotic following Nva exposure, and both necrotic and apoptotic following treatment with Aze. The toxicity of these NPAAs to mitochondria was then characterised using immunofluorescence microscopy and bioenergetic flux assays. These revealed, for the first time, that Nva and Aze cause mitochondrial dysfunction. Distinct morphological changes and bioenergetic failure were common to both conditions, however, for Nva bioenergetic failure was only observed in the presence of a nitric oxide synthase inhibitor due to Nva’s secondary action as an arginase inhibitor. Finally, a proteomic characterisation of cells exposed to both NPAAs was performed to further elucidate the molecular mechanisms involved in their toxicity. This was the first study to perform this analysis in human cells treated with either NPAA and revealed disruptions to processes that precede protein translation. Changes to proteins involved in the processing of DNA and RNA during cell cycle progression indicate, for the first time, that NPAA exposure exerts toxic effects upstream of protein translation. These results identify Nva and Aze as NPAAs with significant potential for toxicity that should, therefore, be consumed with caution. Furthermore, these results add to the existing knowledge of the mechanisms of both these individual NPAAs, and amino acid analogues in general.
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