Enzolution(R) SIRT3 TR-FRET Assay System

The Enzolution(R) SIRT3 Assay System is intended for use with the Transcreener(R) OAADPr SIRT Assay Kits (Parts #3046 and #3047) to measure the enzymatic activity of SIRT3 (NAD-dependent protein deacetylase sirtuin 3). SIRT3 catalyzes transfer of acetyl groups from acetylated histones to NAD, yielding the unacetylated peptide, O-acetyl-ADP ribose (OAADPr), and nicotinamide as products (Figure 1). The assay relies on a Coupling Enzyme (CE) to convert OAADPr into AMP, which is then detected using a far-red, competitive fluorescence polarization (FP) or time-resolved Försterresonance-energy-transfer (TR-FRET) assay. The assay has been optimized and extensively validated for high throughput screening (HTS) and inhibitor dose response measurements using most multimode plate readers. The Enzolution SIRT3 Assay System provides all reagents required to screen and profile SIRT3 inhibitors when used with the Transcreener OAADPr SIRT Assay Kits, including purified, human SIRT3 (aa 101399) and acetylated peptide, H3K9Ac (1-21). The protocol is configured for 384-well plates, use of different multi-well plate formats will require adjustment of reagent concentrations utilized in the assay. Key Applications:  Screening for SIRT3 inhibitors (or activators)  Generating dose response curves Protein Function: NAD-dependent protein deacetylase (PubMed:12186850, PubMed:12374852, PubMed:16788062, PubMed:18680753, PubMed:18794531, PubMed:19535340, PubMed:23283301, PubMed:24121500, PubMed:24252090). Activates or deactivates mitochondrial target proteins by deacetylating key lysine residues (PubMed:12186850, PubMed:12374852, PubMed:16788062, PubMed:18680753, PubMed:18794531, PubMed:23283301, PubMed:24121500, PubMed:24252090, PubMed:38146092). Known targets include ACSS1, IDH, GDH, SOD2, PDHA1, LCAD, SDHA, MRPL12 and the ATP synthase subunit ATP5PO (PubMed:16788062, PubMed:18680753, PubMed:19535340, PubMed:24121500, PubMed:24252090, PubMed:38146092). Contributes to the regulation of the cellular energy metabolism (PubMed:24252090). Important for regulating tissue-specific ATP levels (PubMed:18794531). In response to metabolic stress, deacetylates transcription factor FOXO3 and recruits FOXO3 and mitochondrial RNA polymerase POLRMT to mtDNA to promote mtDNA transcription (PubMed:23283301). Acts as a regulator of ceramide metabolism by mediating deacetylation of ceramide synthases CERS1, CERS2 and CERS6, thereby increasing their activity and promoting mitochondrial ceramide accumulation (By similarity). Regulates hepatic lipogenesis (By similarity). Uses NAD(+) substrate imported by SLC25A47, triggering downstream activation of PRKAA1/AMPK-alpha signaling cascade that ultimately downregulates sterol regulatory element-binding protein (SREBP) transcriptional activities and ATP-consuming lipogenesis to restore cellular energy balance (By similarity). In addition to protein deacetylase activity, also acts as a protein-lysine deacylase by mediating delactylation of proteins, such as CCNE2 and 'Lys-16' of histone H4 (H4K16la) (PubMed:36896611, PubMed:37720100) [The Uniprot Consortium]