Preliminary analysis of the diagnostic and prognostic value of Heat Shock Proteins and their lysine methylation status in the progression of Type 2 Diabetes Mellitus

Abstract

Type 2 Diabetes Mellitus (T2DM) is a chronic metabolic disorder characterised by hyperglycaemia (high blood sugar), insulin resistance, and inadequate insulin production. T2DM is of significant concern in Europe, having an affected adult population of approximately 3%, and even more so in Malta with a staggering 10%. The significantly high prevalence of T2DM translates into elevated morbidity, mainly through cardiovascular complications, reaching nearly 25% of death under the age of 65. Despite several studies having been devoted to investigating the genetics of T2DM, very little advancement has been achieved in our understanding of the molecular mechanisms behind this disorder which can be applied to diagnostics. Likewise, at the protein level, very little is available especially regarding the role played by post-translational modification (PTMs) in disease progression as a result of aging or other enviromental factors. The aim of the project spanning 3 phases over 5 years is to determine if certain HSPs and their methylation status can be used to detect stress in the body as a result of the progression of T2DM ahead of its current clinical detection through plasma glucose testing, while the body is still below the homeostatic threshold and prior to any clinical presentation. In the current study a custom designed sandwich ELISA for lysine methylation was used to detec the presence this PTM on slected Heat Shock Proteins (HSPs) in the serum of diabetics or healthy controls and relative quantification was used in combiation with demographic stratification to try and identify key areas of focus for further testing. In the pilot phase of the project only male subjects aged 18-85 were recruited and the HSPs selcted for analysis in terms of total protein and lysine methylation status were HSP27 and HSP70. The results of the pilot phase show a significant change in total HSP when all subjects are group together by condition, while the methylation status shows a trend tending towards significance. When stratification by age using brackets of 10 years is applied the significance increases but the stastical power is reduced due to an overall reduction in participant numbers which thus increases variability and error. In the second phase of the project, subjects of both sexes are currently being recruited and the HSPs selected for detection and quantification of both total protein and methlated lysine status include HSP27, HSP60, HSP70, GRP78 and HSP90. It is envisioned that with more internal normalisation being possible over the selected 5 proteins, the overall trends can be strengthened, increasing relability and reducing false correlations due to co-morbidities.