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Published on: April 11, 2014
Structural characterization of a L-dehydroascorbic acid-L-homocysteine thiolactone reaction product: Intracellular
Ghizlane Loubane1, Gabriel Robert2, Syed Benazir Firdaus1
1Département de Pharmacologie et Physiologie Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, 3001 12 Ave N, Sherbrooke, Quebec J1H5N4, Canada.
Insights
Homocysteine thiolactone (HTL) can cause protein aggregation. Dehydroascorbate (DHA), derived from ascorbate, reacts with L-HTL to form a product that prevents protein N-homocysteinylation, potentially protecting against neurodegenerative diseases.
Area of Science:
- Biochemistry
- Neuroscience
- Oxidative Stress Research
Background:
- Homocysteine thiolactone (HTL) is linked to cardiovascular and neurological diseases due to its ability to bind protein lysine residues, potentially causing aggregation.
- Ascorbate (Vitamin C) combats oxidative stress and can be oxidized to dehydroascorbate (DHA), which can be reduced by thiols.
- Understanding HTL's interactions and potential protective mechanisms is crucial for disease prevention.
Purpose of the Study:
- To investigate the reaction products of L-HTL with aldehydes and DHA.
- To elucidate the structural characteristics of these reaction products using advanced analytical techniques.
- To assess the protective potential of the DHA-L-HTL reaction product against protein N-homocysteinylation and aggregation.
Main Methods:
- Purification and structural determination of reaction products using 1D and 2D-nuclear magnetic resonance (NMR).
- High-resolution accurate electrospray ionization tandem mass spectrometry (ESIMS/MS) for structural confirmation.
- In vitro assays to assess the inhibition of N-homocysteinylation of cytochrome c.
- Cell-based studies using human neuroblastoma cells (SH-SY5Y).
Main Results:
- Novel reaction products were identified from L-HTL with formaldehyde, propionaldehyde, and DHA.
- The DHA-L-HTL adduct features a six-member thiazinane ring with a carboxylic acid group.
- Formation of the DHA-L-HTL reaction product inhibited N-homocysteinylation of cytochrome c by L-HTL.
- The DHA-L-HTL adduct was observed in SH-SY5Y cells exposed to L-HTL and DHA/ascorbate, suggesting in vivo formation.
Conclusions:
- The reaction between DHA and L-HTL yields a stable adduct that prevents protein N-homocysteinylation.
- DHA, through its reaction with L-HTL, shows potential as a protective agent against HTL-induced protein aggregation and associated pathologies.
- These findings highlight a novel protective role for ascorbate/DHA in mitigating the detrimental effects of homocysteine thiolactone.
Abstract:
Homocysteine thiolactone (HTL) has been implicated in cardiovascular and neurological pathologies. While homocysteine can S-homocysteinylate thiol groups in proteins, the chemical properties of HTL facilitates covalent binding to protein ε-amino groups on lysine residues, which can initiate protein aggregation. Ascorbate plays an important role in the prevention of oxidative stress. Ascorbate is easily oxidized by the loss of two electrons to dehydroascorbate (DHA), which can be reduced back to ascorbate by thiol-containing smaller and larger molecules. In the present study, reaction products between two aldehydes, formaldehyde and propionaldehyde as well as DHA with the physiological relevant isomer of homocysteine thiolactone i.e. L-HTL were purified, and their structure was determined by 1D and 2D-nuclear magnetic resonance. In all three cases the reaction products are likely formed by initial imine condensation, subsequent formation of a hemiaminal product followed by HTL ring opening and intramolecular nucleophilic attack of the resulting thiol anion to form a six-member thiazinane ring with a carboxylic acid group. The structure of the DHA, L-HTL reaction product was confirmed by high resolution accurate ESIMS/MS in negative mode. Formation of the reaction product between DHA and HTL prevented N-homocysteinylation of cytochrome c by HTL, confirming earlier observations. The reaction product is formed in human neuroblastoma cells (SH-SY5Y) when exposed to HTL and DHA or ascorbate, potentially preventing protein aggregation. The consequences associated with formation of a reaction product between DHA and HTL suggest that DHA could protect against protein N-homocysteinylation.
