Proteomic analysis of the ubiquitin landscape in the type II diabetic rat model

Lingam S1,2, Smith L1,2, Cordwell S1,2,3 and White M1,2,3

  1. Charles Perkins Centre, University of Sydney, Australia.
  2. Discipline of Pathology, School of Medical Sciences, University of Sydney, Australia.
  3. School of Life and Environmental Sciences, University of Sydney, Australia.

Type II diabetes mellitus (DM) remains one of the major causes of morbidity and mortality in the industrialized world. Several animal and human studies have identified increased generation of reactive oxygen species (ROS), a consequence of metabolic abnormalities in DM, as one of the key mediators of signal transduction during DM and DM-mediated diseases. In addition to redox-specific post-translational modifications (PTM), accumulating evidence suggests the involvement of ROS in accelerating non-specific ubiquitination PTMs of cardiac proteins as a label for protein degradation. Our group has defined changes in several protein PTMs in DM and begun to understand how these various PTMs cross-talk with each other. Here, we examined the ubiquitin-modified proteome in key metabolic tissues; adipose, brain, heart, liver, pancreas, kidney and skeletal muscle in DM. Rats were fed a standard chow citrate (CC) (12% fat) or high fat (HF) (42% fat) diet for 8 weeks, with DM induced in 50% of the animals after 4 weeks utilising a low dose of streptozotocin (STZ; 55mg/kg); a pancreatic β-cell toxin. At the cessation of the feeding protocol, animals were euthanised and organs were excised. Tissue lysates were subjected to western blotting using anti-ubiquitin antibodies. Western blots revealed gross changes in ubiquitination associated with diet and pathology. To identify sites targeted by these PTMs we next used immunoprecipitation to enrich for modified peptides, which were subsequently analysed by LC-MS/MS. These analyses further the scope of PTMs associated with DM and may further understanding of molecular mechanisms underlying metabolic disturbances of this disease.