Irreversible Oxidative Modification of Cysteines Resulting from a Prolonged High Fat Diet Model — ASN Events
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  3. Irreversible Oxidative Modification of Cysteines Resulting from a Prolonged High Fat Diet Model

Irreversible Oxidative Modification of Cysteines Resulting from a Prolonged High Fat Diet Model (#226)

Lauren E Smith 1 , Kasper Engholm-Keller 2 , Jana Paulech 3 , Stuart J Cordwell 1 3 , Melanie Y White 1 3
  1. Discipline of Pathology, University of Sydney, Sydney, NSW, Australia
  2. Children's Medical Research Institute, Westmead, NSW
  3. School of Molecular Bioscience, University of Sydney, Sydney, NSW

The extent of oxidative stress on the myocardial molecular environment due to obesity is yet to be fully understood. The cellular response to increased oxidative stress is regulated in part, by highly reactive thiol groups on cysteine residues undergoing various oxidative modifications that regulate protein function. Pathologies such as obesity lead to the formation of aberrant oxidative modifications on proteins, such as the largely irreversible sulfinic and sulfonic acids. This study aims to utilise a novel enrichment strategy to identify proteins that undergo these oxidative modifications as a result of obesity.
Rats were fed a CHOW (12% fat) or high fat (HF) (42% fat) diet for 6 months. To quantify alterations in protein abundance, samples were isobarically tagged prior to mass spectrometry (MS) analysis. In parallel, irreversibly oxidised peptides were negatively selected utilising low pH loading conditions for strong cation exchange (SCX), whereby the negative charge imparted by the over-oxidation of Cys limits interaction with the chromatography. The resulting SCX fractions were further partitioned by hydrophilic interaction chromatography prior to MS analysis.
Irreversibly oxidised Cys peptides were increased 2.5 fold in the HF diet, with 10 unique irreversibly oxidised Cys-containing peptides observed in the CHOW diet (10 unique proteins) compared with 22 observed in the HF diet (arising from 20 unique proteins). 55 discrete proteins were observed to be significantly changing in abundance as a result of the HF diet. These proteins were largely found to be associated with fatty acid metabolism, degradation and oxidation. The most prevalent of these proteins identified as oxidised due to the HF diet was β-actin. This irreversible oxidation may cause stiffening of the cytoskeleton, impacting the arrangement and functionality of the z-disk, as shown in hypertrophic models. Given that hypertrophy is a comorbidity associated with obesity, we suggest that this alteration to beta-actin has functional implications in this model.
This study is the first application of this novel method of enrichment for irreversibly oxidised Cys proteins to obesity, identifying key proteins associated with important functional groups. Understanding these modifications and their impact on myocardial function may provide novel mechanisms of treatment to improve recovery from AMI in the high-risk obese population.