| Summary: | This thesis is focused on using SAMDI-MS to study the how the lysine deacetylases (KDACs) are regulated under different cellular context, at molecular level via changes in cofactor concentrations, in cells during differentiation and in animal tissue.
First part describes the differential regulation of SIRTs 1-3 through NADH inhibition. The SIRT1-3 deacetylases play a central role in metabolic regulation by having activities that depend on the cell redox state as defined by NAD+/NADH. However, biochemical studies have not established the basis for this sensitivity or the extent to which the various SIRT isotypes respond differently to levels of the two molecules. In this part, we show that SIRT1, but not SIRT2/3, is inhibited by NADH and suggests that differential sensitivity of the SIRTs to NADH is important in metabolic regulation.
Second part describes profiling deacetylase activities in cell lysates with peptide arrays and SAMDI Mass spectrometry. The combination of peptide arrays and SAMDI-MS provides a label-free approach for identifying patterns of enzyme activities in cell lysates. The approach is demonstrated by profiling KDAC activities in cell lysates of the CHRF megakaryocytic (Mk) cell line. Class-specific deacetylase inhibitors were used to show that terminal Mk differentiation of CHRF cells is marked by a pronounced decrease in sirtuin activity and by little change in activity of KDACs 1-11. This work establishes a platform that can be used to identify changes in global activity profiles of cell lysates for a wide variety of enzymatic activities.
The third part describes the use of SAMDI-MS assay towards studying KDAC activity in animal tissue. This part includes two examples of how the SAMDI-MS assay can be applied to report endogenous activity of SIRTs in animal tissue samples under different cellular context. The first example shows the clock-driven NAD+ oscillations can regulate SIRT3 activity in mice liver extracts. The second example shows the SIRT deacetylase activity is decreased in the skin of diabetic mice.
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