Development of a mitochondrial sirtuin 4 FRET assay based on its activity for removing 3-hydroxy-3-methylglutaryl (HMG) modification

SIRT4 belongs to one of three mitochondrial sirtuins, which plays important roles in regulating many biological processes and has significant implications for treating several human diseases. However, the development of those small compounds that can modulate SIRT4 activities is limited because there is no efficient SIRT4 assay available for screening its modulators. Thanks to recent discoveries of several enzymatic activities and substrates for SIRT4, we have developed a FRET-based assay suitable for screening SIRT4 modulators based on its activity of removing 3-hydroxy-3-methylglutaryl (HMG) lysine modification, which could be further coupled with a secondary FRET-based assay for other sirtuins to identify SIRT4-specific inhibitors or activators.