Proteomics approaches to investigate the role of lysine acetylation in type 2 diabetes mellitus and in breast cancer

Protein post-translational modifications (PTMs) play a key role in the field of functional proteomic because they regulate activity, localization, and interaction with other cellular molecules, such as proteins, nucleic acids, lipids and cofactors. One of these modifications is named lysine acetylation and, although has been known from 50 years, it is still not completely understood, given its low stoichiometry and technical challenges in identifying modified sites. This PTM involves the transfer of an acetyl group from the donor acetyl-CoA to a protein and it is capable of affecting protein functions, regulating enzymatic activity and protein subcellular localization. The cornerstone of this thesis is the investigation of post-translational lysine acetylation, in proteins involved in lipotoxicity in type 2 diabetes mellitus and in proteins involved in fibroblast activation in the context of breast cancer. The main research project involves the investigation of acetylation in pancreatic β cells in the context of type 2 diabetes mellitus (T2DM) and experiments have been carried out in the Department of Pharmacy of the University of Pisa, under the guide of Prof. Lucacchini. A second project, carried out at the Beatson Institute for Cancer Research in Glasgow under the supervision of Dr Zanivan, examines acetylation in normal fibroblasts and cancer-associated fibroblasts in the context of breast cancer. Type 2 diabetes mellitus is a complex metabolic disorder, characterized above all by insulin resistance in muscle, liver and adipose tissue. One typical predisposition factor is obesity and our aim in this project is to investigate the effect of a diet rich in saturated fatty acids on pancreatic islets in type 2 diabetes. Palmitate is the most common saturated free fatty acid in human plasma and has been used for in vitro and in vivo studies on lipotoxicity. We used palmitate to study the mechanisms of lipotoxicity in rat β cell lines first and in human pancreatic islets then. Palmitate has been found to cause impairment of insulin secretion through mitochondrial alterations. However, the relationship between type 2 diabetes and mitochondrial dysfunctions is still under investigation and requires further studies. On this basis, by two dimensional gel electrophoresis, western blot and liquid chromatography-mass spectrometry we evaluated the effects of palmitate on mitochondrial proteins from INS-1E cells and on proteins from human pancreatic islets (HPI) after 24h palmitate exposure. The second project aimed to set up a quantitative proteomic approach to study lysine acetylation in normal (NFs) and cancer associated fibroblasts (CAFs), derived from human mammary tissues. Extensive comparative proteomic studies on human mammary fibroblasts, carried out in the Proteomic group of Dr Zanivan, revealed that the levels of some metabolic proteins and enzymes are altered in CAFs upon activation. In particular, it has been found that such proteins are involved in the generation of acetyl-CoA, resulting in higher acetyl-CoA levels in CAFs than in normal fibroblasts. Acetyl-CoA is the building block for protein acetylation reactions as well as for fatty acid and cholesterol synthesis. To investigate whether acetyl-CoA can be used to acetylate proteins and in this way regulating cellular functions in a differential manner between NFs and CAFs, we approached to study the acetylome of these cells using a bottom-up proteomics approach. Stable isotope labelling by amino acids in cell culture (SILAC) has been used to accurately quantify the acetylome of NFs and CAFs and an antibody-based strategy allowed the enrichment of acetylated peptides. Findings linking lysine acetylation to fibroblast activation, and acquisition of a proinvasive phenotype, could allow us to better understand mechanisms underlying this activation and discover novel therapeutic targets in cancer.