Sirtuins play a critical role in post-translational modification of proteins by catalyzing the NAD+-dependent deacetylation of ε-N-acetyl lysine residues. Compared to mammals, much less is known about plant sirtuins. Only recently, a sirtuin-mediated fine-tuning of mitochondrial energy metabolism has been suggested in Arabidopsis. Nevertheless, to date, a direct assessment of sirtuin activity has never been reported in plant cell extracts and/or subcellular organelles. Here, a HTRF®-based assay was properly adapted and applied for the first time to measure sirtuin activity in highly purified mitochondria from durum wheat (DWM). A NAD+-dependent deacetylase activity equal to 268±10 mU∙mg-1 of protein was measured, resulting i) linearly dependent on DWM protein, ii) abolished by boiling DWM and iii) completely inhibited by nicotinamide, a specific sirtuin inhibitor. Moreover, DWM-sirtuin activity was not significantly affected by resveratrol and quercetin, both reported as activators of the well-studied human sirtuin 1 isoform. Overall, results obtained in this study demonstrate that the adapted HTRF® assay may represent a useful tool to study native plant sirtuin activity.

Direct measurement of native plant sirtuin activity using a Homogeneous Time-Resolved Fluorescence (HTRF®)-based assay: first application to durum wheat mitochondria

M. Soccio;
2018

Abstract

Sirtuins play a critical role in post-translational modification of proteins by catalyzing the NAD+-dependent deacetylation of ε-N-acetyl lysine residues. Compared to mammals, much less is known about plant sirtuins. Only recently, a sirtuin-mediated fine-tuning of mitochondrial energy metabolism has been suggested in Arabidopsis. Nevertheless, to date, a direct assessment of sirtuin activity has never been reported in plant cell extracts and/or subcellular organelles. Here, a HTRF®-based assay was properly adapted and applied for the first time to measure sirtuin activity in highly purified mitochondria from durum wheat (DWM). A NAD+-dependent deacetylase activity equal to 268±10 mU∙mg-1 of protein was measured, resulting i) linearly dependent on DWM protein, ii) abolished by boiling DWM and iii) completely inhibited by nicotinamide, a specific sirtuin inhibitor. Moreover, DWM-sirtuin activity was not significantly affected by resveratrol and quercetin, both reported as activators of the well-studied human sirtuin 1 isoform. Overall, results obtained in this study demonstrate that the adapted HTRF® assay may represent a useful tool to study native plant sirtuin activity.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11369/372528
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