Investigating the Role of Sirtuin-3 in Nicotinamide N-Methyltransferase-Mediated Increase of Complex I Activity

Saturday, February 16, 2013
Auditorium/Exhibit Hall C (Hynes Convention Center)
Nicole L. Mendoza , Department of Biomedical Engineering, University of California, Irvine, Irvine, CA
Richard B. Parsons , Institute of Pharmaceutical Sciences, King's College, London, London, United Kingdom
Significantly increased levels of Nicotinamide N-methyltransferase (NNMT) have been found in a number of cancers and in the brains of patients who have died of Parkinson’s disease. NNMT is thought to increase Complex I activity, as supported by previous research of our laboratory. Both ATP concentration and Complex I activity were reported to be increased in SH-SY5Y cells transfected with NNMT. This study aimed to elucidate the mechanism by which elevated NNMT expression results in increased Complex I activity. Sirtuin-3 (SirT3) is a NAD dependent deacetylase found in the mitochondria. SirT3 deacetylates Complex I proteins and thus activates Complex I activity. We focused our attention to SirT3 mediation by NNMT. SH-SY5Y cells, along with those cells expressing NNMT, were cultured in vitro, and their mitochondria were extracted using a mitochondria isolation kit. Western blots were used to validate the mitochondrial isolation procedure and slight changes were applied to the protocol to increase yield of mitochondrial protein content. After the procedure was optimized and mitochondrial purity confirmed, Western blots were used to compare the acetylation status between the SH-SY5Y cells and the S.NNMT.LB cells. We observed a difference in acetylation status, indicating that acetylation by SirT3 activity may depend on NNMT expression. These findings may provide an explanation for the elevated energy production in cells that overexpress NNMT. In summary, the expression of NNMT in SH-SY5Y cells appears to alter acetylation status, which may be attributed to NNMT-mediated Sirt3 activity.