“EpigeNADics” – Understanding the role of the cofactor NAD in epigenetic alterations
Cancer cells employ a variety of metabolic adaptations to maintain their growth and proliferation. One of those adaptations is the increased production of the key cellular cofactor Nicotinamide adenine dinucleotide (NAD). NAD is a highly versatile biomolecule and participates in many energy- and redox-related reactions. It also serves as substrate for several enzyme families such as sirtuins (SIRTs), NAD-dependent histone deacetylases, and is thus involved in epigenetic and transcriptional control mechanisms. Histone acetylation at specific lysine residues influences chromatin structure and gene expression patterns. Currently, seven mammalian SIRT family members have been identified with distinct functions regarding genome stability and tumorigenesis. To further explore the specific changes in SIRT activity in response to altered NAD levels, we set out to develop a mass-spectrometry (MS)-based method for the global detection of NAD-dependent acetylation sites and epigenetic marks, which we coined “EpigeNADics”. Together with the Protein Core Facility (PCF) we therefore established a tandem mass tag (TMT) approach for the robust detection of the NAD-dependent “Acetyl-ome”. We established the workflow using glioblastoma (GB) cells, cultured in absence and presence of an NAD precursor. We optimized total protein extraction and compared two different protocols for cell lysis. We furthermore tested three acetylation-specific antibodies from different manufactures and optimized the input amounts for affinity purification of labeled peptides.
Overall, our EpigeNADics approach yielded more than 8000 acetylated peptides and we are currently validating several identified acetylation sites with specific antibodies via immunoblotting. Our data suggest that specific histone lysine residues are significantly deacetylated in response to NAD precursor starvation, indicating an altered epigenetic profile upon nutrient stress. In-depth investigation of these alterations may potentially help to open up novel avenues for pharmacological intervention of NAD-related (signaling) pathways, in particular for the activity of NAD-dependent SIRTs that are commonly deregulated in cancer.