Bioluminescence Assay of Lysine Deacylase Sirtuin Activity

Lysine acylation can direct protein function, localization, and interactions. Sirtuins deacylate lysine towards maintaining cellular homeostasis, and their aberrant expression contributes to the pathogenesis of multiple pathological conditions, including cancer. Measuring sirtuins’ activity is essential to exploring their potential as therapeutic targets, but accurate quantification is challenging. We developed ‘SIRTify’, a high-sensitivity assay for measuring sirtuin activity in vitro and in vivo. SIRTify is based on a split-version of the NanoLuc® luciferase consisting of a truncated, catalytically inactive N-terminal moiety (LgBiT) that complements with a high-affinity C-terminal peptide (p86) to form active luciferase. Acylation of two lysines within p86 disrupts binding to LgBiT and abates luminescence. Deacylation by sirtuins reestablishes p86 and restores binding, generating a luminescence signal proportional to sirtuin activity. Measurements accurately reflect reported sirtuin specificity for lysine acylations and confirm the effects of sirtuin modulators. SIRTify effectively quantifies lysine deacylation dynamics and may be adaptable to monitoring additional post-translational modifications.

[1]  M. Savitski,et al.  Control of protein stability by post-translational modifications , 2023, Nature Communications.

[2]  J. Michaelis,et al.  Succinylation of H3K122 destabilizes nucleosomes and enhances transcription , 2021, EMBO reports.

[3]  Matthew J. Rardin,et al.  SUCLA2 mutations cause global protein succinylation contributing to the pathomechanism of a hereditary mitochondrial disease , 2020, Nature Communications.

[4]  Brian C. Smith,et al.  Development of activity-based probes for the protein deacylase Sirt1. , 2020, Bioorganic chemistry.

[5]  L. K. Nguyen,et al.  Asparagine Hydroxylation is a Reversible Post-translational Modification , 2020, Molecular & Cellular Proteomics.

[6]  P. Cole,et al.  The Chemical Biology of Reversible Lysine Post-translational Modifications. , 2020, Cell chemical biology.

[7]  R. Cohen,et al.  Improved mass spectrometry-based activity assay reveals oxidative and metabolic stress as sirtuin-1 regulators , 2019, Redox biology.

[8]  E. Apartsin,et al.  Topological Aspects of the Design of Nanocarriers for Therapeutic Peptides and Proteins , 2019, Pharmaceutics.

[9]  C. Leonetti,et al.  Pharmacological activation of SIRT6 triggers lethal autophagy in human cancer cells , 2018, Cell Death & Disease.

[10]  O. Muzik,et al.  Molecular Imaging of Sirtuin1 Expression-Activity in Rat Brain Using Positron-Emission Tomography-Magnetic-Resonance Imaging with [18F]-2-Fluorobenzoylaminohexanoicanilide. , 2018, Journal of medicinal chemistry.

[11]  J. Ovádi,et al.  New chemical tools for probing activity and inhibition of the NAD+-dependent lysine deacylase sirtuin 2 , 2018, Philosophical Transactions of the Royal Society B: Biological Sciences.

[12]  D. Sinclair,et al.  Sirtuin activators and inhibitors: Promises, achievements, and challenges. , 2018, Pharmacology & therapeutics.

[13]  P. Barnes,et al.  The dynamic shuttling of SIRT1 between cytoplasm and nuclei in bronchial epithelial cells by single and repeated cigarette smoke exposure , 2018, PloS one.

[14]  Y. Cen,et al.  Development of Activity-Based Chemical Probes for Human Sirtuins. , 2018, ACS chemical biology.

[15]  C. Steegborn,et al.  Mechanism-Based Inhibitors of the Human Sirtuin 5 Deacylase: Structure-Activity Relationship, Biostructural, and Kinetic Insight. , 2017, Angewandte Chemie.

[16]  Stephen P. Jackson,et al.  Deubiquitylating enzymes and drug discovery: emerging opportunities , 2017, Nature Reviews Drug Discovery.

[17]  Peter G. Schultz,et al.  Genetically Encoded Fluorescent Probe for Detecting Sirtuins in Living Cells. , 2017, Journal of the American Chemical Society.

[18]  Laura G. Dubois,et al.  A Class of Reactive Acyl-CoA Species Reveals the Non-enzymatic Origins of Protein Acylation. , 2017, Cell metabolism.

[19]  C. Chen,et al.  Quantitative global proteome and lysine succinylome analyses provide insights into metabolic regulation and lymph node metastasis in gastric cancer , 2017, Scientific Reports.

[20]  X. Niu,et al.  Desuccinylation of pyruvate kinase M2 by SIRT5 contributes to antioxidant response and tumor growth , 2016, Oncotarget.

[21]  H. Nakagawa,et al.  A Fluorescent Probe for Imaging Sirtuin Activity in Living Cells, Based on One‐Step Cleavage of the Dabcyl Quencher , 2016, Chembiochem : a European journal of chemical biology.

[22]  D. Sinclair,et al.  Slowing ageing by design: the rise of NAD+ and sirtuin-activating compounds , 2016, Nature Reviews Molecular Cell Biology.

[23]  Hening Lin,et al.  The Substrate Specificity of Sirtuins. , 2016, Annual review of biochemistry.

[24]  Brock F. Binkowski,et al.  NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells. , 2016, ACS chemical biology.

[25]  Calum A. MacRae,et al.  Zebrafish as tools for drug discovery , 2015, Nature Reviews Drug Discovery.

[26]  C. Steegborn,et al.  A Novel Continuous Assay for the Deacylase Sirtuin 5 and Other Deacetylases. , 2015, Journal of medicinal chemistry.

[27]  Matthew J. Rardin,et al.  SIRT5 Regulates both Cytosolic and Mitochondrial Protein Malonylation with Glycolysis as a Major Target. , 2015, Molecular cell.

[28]  Yiyu Cheng,et al.  Specific Turn-On Fluorescent Probe with Aggregation-Induced Emission Characteristics for SIRT1 Modulator Screening and Living-Cell Imaging. , 2015, Analytical chemistry.

[29]  Yingming Zhao,et al.  Metabolic Regulation by Lysine Malonylation, Succinylation, and Glutarylation* , 2015, Molecular & Cellular Proteomics.

[30]  Wolfgang Sippl,et al.  Selective Sirt2 inhibition by ligand-induced rearrangement of the active site , 2015, Nature Communications.

[31]  J. Denu,et al.  Site-Specific Reactivity of Nonenzymatic Lysine Acetylation , 2015, ACS chemical biology.

[32]  B. O’Rourke,et al.  Metabolism leaves its mark on the powerhouse: recent progress in post-translational modifications of lysine in mitochondria , 2014, Front. Physiol..

[33]  Matthew J. Rardin,et al.  SIRT5 regulates the mitochondrial lysine succinylome and metabolic networks. , 2013, Cell metabolism.

[34]  Frank Fischer,et al.  An acetylome peptide microarray reveals specificities and deacetylation substrates for all human sirtuin isoforms , 2013, Nature Communications.

[35]  G. Donmez,et al.  SIRT1 and SIRT2: emerging targets in neurodegeneration , 2013, EMBO molecular medicine.

[36]  J. Denu,et al.  Sirtuin Catalysis and Regulation* , 2012, The Journal of Biological Chemistry.

[37]  P. Canonico,et al.  Identification of a sirtuin 3 inhibitor that displays selectivity over sirtuin 1 and 2. , 2012, European journal of medicinal chemistry.

[38]  C. Olsen,et al.  Substrates for efficient fluorometric screening employing the NAD-dependent sirtuin 5 lysine deacylase (KDAC) enzyme. , 2012, Journal of medicinal chemistry.

[39]  Johan Auwerx,et al.  Sirtuins as regulators of metabolism and healthspan , 2012, Nature Reviews Molecular Cell Biology.

[40]  A. Vaquero,et al.  The dual role of sirtuins in cancer. , 2011, Genes & cancer.

[41]  U. Haberkorn,et al.  The pharmacokinetics of cell-penetrating peptides. , 2010, Molecular pharmaceutics.

[42]  L. Gentilucci,et al.  Chemical modifications designed to improve peptide stability: incorporation of non-natural amino acids, pseudo-peptide bonds, and cyclization. , 2010, Current pharmaceutical design.

[43]  Shu-Chen Lu,et al.  Resveratrol is Not a Direct Activator of SIRT1 Enzyme Activity , 2009, Chemical biology & drug design.

[44]  Brian C. Smith,et al.  A continuous microplate assay for sirtuins and nicotinamide-producing enzymes. , 2009, Analytical biochemistry.

[45]  Yigong Shi.  Serine/Threonine Phosphatases: Mechanism through Structure , 2009, Cell.

[46]  Takashi Nakagawa,et al.  SIRT5 Deacetylates Carbamoyl Phosphate Synthetase 1 and Regulates the Urea Cycle , 2009, Cell.

[47]  Yang Shi,et al.  Histone lysine demethylases: emerging roles in development, physiology and disease , 2007, Nature Reviews Genetics.

[48]  K. Shimamoto,et al.  Nucleocytoplasmic Shuttling of the NAD+-dependent Histone Deacetylase SIRT1* , 2007, Journal of Biological Chemistry.

[49]  Brian K. Kennedy,et al.  Sirtuins in Aging and Age-Related Disease , 2006, Cell.

[50]  P. Distefano,et al.  Inhibition of SIRT1 Catalytic Activity Increases p53 Acetylation but Does Not Alter Cell Survival following DNA Damage , 2006, Molecular and Cellular Biology.

[51]  P. Distefano,et al.  Microplate filtration assay for nicotinamide release from NAD using a boronic acid resin. , 2005, Methods.

[52]  S. Fields,et al.  Substrate-specific Activation of Sirtuins by Resveratrol* , 2005, Journal of Biological Chemistry.

[53]  K. Glaser,et al.  Fluorescence assay of SIRT protein deacetylases using an acetylated peptide substrate and a secondary trypsin reaction. , 2004, Analytical biochemistry.

[54]  William C Hahn,et al.  Lentivirus-delivered stable gene silencing by RNAi in primary cells. , 2003, RNA.

[55]  E. Verdin,et al.  The human silent information regulator (Sir)2 homologue hSIRT3 is a mitochondrial nicotinamide adenine dinucleotide–dependent deacetylase , 2002, The Journal of cell biology.

[56]  L. Guarente.  Sirtuins, Aging, and Medicine , 2011 .