Poster Presentation 24th Annual Lorne Proteomics Symposium 2019

Analysis of cysteine redox post-translational modifications in a rat model of type II diabetes mellitus   (#126)

Shivanjali J Lingam 1 , Desmond K Li 1 , Alexander W Rookyard 2 , Stuart J Cordwell 3 , Melanie Y White 3
  1. Discipline of Pathology, School of Medical Sciences, Charles Perkins Centre, University of Sydney, Sydney, New South Wales, Australia
  2. School of Life and Environmental Sciences, Charles Perkins Centre, University of Sydney, Sydney, NSW, Australia
  3. Discipline of Pathology, School of Medical Sciences, School of Life and Environmental Sciences, Charles Perkins Centre, University of Sydney, Sydney, New South Wales, Australia

Type 2 diabetes mellitus (T2DM) and associated complications remains a major challenge world-wide. Several animal/human and tissue/cell-based studies have found reactive oxygen and nitrogen species (ROS/RNS) as key mediators of signal transduction during T2DM and T2DM-mediated diseases. It has been suggested that cellular antioxidant defenses are incapable of compensating for T2DM-induced ROS/RNS production. The associated oxidative stress modulates protein function by accelerating redox protein post-translational modifications (PTM) on cysteine residues (e.g. S-glutathionylation, S-nitrosylation and S-sulfenylation). Our group has defined changes in several protein PTMs in T2DM and T2DM-mediated diseases and aim to understand the interplay between these various PTMs. Here, we examined the redox-modified renal proteome in T2DM. Rats were fed a standard CHOW (C) (12% fat) or high fat (HF) (42% fat) diet for 8 weeks, with T2DM induced in 50% of the animals after 4 weeks utilising a low dose of streptozotocin (STZ; 35mg/kg); a pancreatic β-cell toxin. The remaining 50% were injected with a buffer vehicle (Cit). At the cessation of the feeding protocol, 9 animals per group (C Cit; C STZ; HF Cit; HF STZ) were euthanised and kidneys excised. Tissue lysates were subjected to western blotting using anti-glutathione, anti-sulfenic acid, and anti-S-nitrosylation antibodies. Western blots revealed gross changes in glutathionylation, sulfenylation and nitrosylation PTMs associated with diet and pathology. To identify discrete sites targeted by these PTMs we performed isobaric tagging (TMT) and enriched for modified peptides using thiol-affinity chromatography, with subsequent analysis by LC-MS/MS on a Thermo Q-Exactive HF-X. These analyses further the scope of PTMs associated with DM and may further understanding of molecular mechanisms underlying metabolic disturbances of this disease.