Continuous Sirtuin/HDAC (histone deacetylase) activity assay using thioamides as PET (Photoinduced Electron Transfer)–based fluorescence quencher

Zessin, Matthes; Meleshin, Marat; Simic, Zeljko; Kalbas, Diana; Arbach, Miriam; Gebhardt, Philip; Melesina, Jelena; Liebscher, Sandra; Bordusa, Frank; Sippl, Wolfgang; Bařinka, Cyril; Schutkowski, Mike

doi:10.1016/j.bioorg.2021.105425

Cited by 8 publications

(18 citation statements)

References 97 publications

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“…One unit is defined as 1 pmol/min at 37 °C using 100 μM of the FLOUR DE LYS-SIRT1 deacetylase substrate. Sirtuins 2, 3, 5, and 6 were cloned, expressed, and purified as described …”

Section: Methodsmentioning

confidence: 99%

“…Sirtuins 2, 3, 5, and 6 were cloned, expressed, and purified as described. 58 HPLC-Based Deacylation Assay. Reactions were performed in a total volume of 70 μL in HDAC assay buffer containing 50 mM HEPES (pH 7.4), 140 mM NaCl, 10 mM KCl, 1 mM TCEP, and 0.2 mg mL −1 BSA for HDAC1, -2, -3, -4, -5, -6, -7, -8, -9, and -11 or in SIRT assay buffer containing 20 mM Tris-HCl (pH 7.8), 150 mM NaCl, 5 mM MgCl 2 , and 0.2 mg mL −1 BSA for SIRT2, SIRT3, SIRT5, and SIRT6.…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

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Uncovering Robust Delactoylase and Depyruvoylase Activities of HDAC Isoforms

et al. 2022

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Zinc-dependent histone deacetylases (HDACs) and sirtuins (SIRT) represent two different classes of enzymes which are responsible for deacylation of modified lysine side chains. The repertoire of acyl residues on lysine side chains identified in vivo is rapidly growing, and very recently lysine lactoylation was described to be involved in metabolic reprogramming. Additionally, lysine pyruvoylation represents a marker for aging and liver cirrhosis. Here, we report a systematic analysis of acyl-specificity of human zinc-dependent HDAC and sirtuin isoforms. We identified HDAC3 as a robust delactoylase with several-thousand-fold higher activity as compared to SIRT2, which was claimed to be the major in vivo delactoylase. Additionally, we systematically searched for enzymes, capable of removing pyruvoyl residues from lysine side chains. Using model peptides, we uncovered high depyruvoylase activity for HDAC6 and HDAC8. Interestingly, such substrates have extremely low K M values for both HDAC isoforms, pointing to possible in vivo functions.

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“…One unit is defined as 1 pmol/min at 37 °C using 100 μM of the FLOUR DE LYS-SIRT1 deacetylase substrate. Sirtuins 2, 3, 5, and 6 were cloned, expressed, and purified as described …”

Section: Methodsmentioning

confidence: 99%

Section: ■ Experimental Sectionmentioning

confidence: 99%

Uncovering Robust Delactoylase and Depyruvoylase Activities of HDAC Isoforms

et al. 2022

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“…Several enzymatic assays have been developed to monitor the lysine deacylase activity of HDACs/SIRTs as reviewed in [ 2 , 3 ]. Most of these activity assays are discontinuous (HPLC-based or mass spectrometry-based assays), or suffer from complexity due to coupled enzymatic [ 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 ] or chemical reactions [ 12 ] (reviewed in [ 13 , 14 ]).…”

Section: Introductionmentioning

confidence: 99%

Continuous Fluorescent Sirtuin Activity Assay Based on Fatty Acylated Lysines

Zessin

Meleshin

Hilscher

et al. 2023

IJMS

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Lysine deacetylases, like histone deacetylases (HDACs) and sirtuins (SIRTs), are involved in many regulatory processes such as control of metabolic pathways, DNA repair, and stress responses. Besides robust deacetylase activity, sirtuin isoforms SIRT2 and SIRT3 also show demyristoylase activity. Interestingly, most of the inhibitors described so far for SIRT2 are not active if myristoylated substrates are used. Activity assays with myristoylated substrates are either complex because of coupling to enzymatic reactions or time-consuming because of discontinuous assay formats. Here we describe sirtuin substrates enabling direct recording of fluorescence changes in a continuous format. Fluorescence of the fatty acylated substrate is different when compared to the deacylated peptide product. Additionally, the dynamic range of the assay could be improved by the addition of bovine serum albumin, which binds the fatty acylated substrate and quenches its fluorescence. The main advantage of the developed activity assay is the native myristoyl residue at the lysine side chain avoiding artifacts resulting from the modified fatty acyl residues used so far for direct fluorescence-based assays. Due to the extraordinary kinetic constants of the new substrates (KM values in the low nM range, specificity constants between 175,000 and 697,000 M−1s−1) it was possible to reliably determine the IC50 and Ki values for different inhibitors in the presence of only 50 pM of SIRT2 using different microtiter plate formats.

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“…Fmoc-based solid phase peptide chemistry was used for assembly of peptide derivatives. Nosyl-protected lysine building blocks enabled selective on-resin modification of the respective lysine side chain . All compounds have purity >95%, as determined by HPLC at 220 nm and showed the expected molecular mass (Figures S53–S61).…”

Section: Resultsmentioning

confidence: 98%

“…Nosyl-protected lysine building blocks enabled selective on-resin modification of the respective lysine side chain. 63 All compounds have purity >95%, as determined by HPLC at 220 nm and showed the expected molecular mass (Figures S53−S61). All butyrylated derivatives were synthesized as a mixture of stereoisomers regarding the ß-carbon of the acyl residue (Figure 3A).…”

Section: O�cn([h])ccccc(c(�o)n([h])-c(c(�o)n[h])c(c)o[h])n([h])c(�o)c...mentioning

confidence: 98%

Small Changes Make the Difference for SIRT2: Two Different Binding Modes for 3-Arylmercapto-Acylated Lysine Derivatives

et al. 2022

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Sirtuins are protein deacylases regulating metabolism and stress responses and implicated in aging-related diseases. Modulators of the human sirtuins 1–7 are sought as chemical tools and potential therapeutics, for example, for treatment of cancer. We were able to show that 3-aryl-mercapto-succinylated- and 3-benzyl-mercapto-succinylated peptide derivatives yield selective Sirt5 inhibitors with low nM K i values. Here, we synthesized and characterized 3-aryl-mercapto-butyrylated peptide derivatives as effective and selective sirtuin 2 inhibitors with K D values in the low nanomolar range. According to kinetic measurements and microscale thermophoresis/surface plasmon resonance experiments, the respective inhibitors bind with the 3-aryl-mercapto moiety in the selectivity pocket of Sirtuin 2, inducing a rearrangement of the active site. In contrast, 3-aryl-mercapto-nonalyl or palmitoyl derivatives are characterized by a switch in the binding mode blocking both the hydrophobic channel by the fatty acyl chain and the nicotinamide pocket by the 3-aryl-mercapto moiety.

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Continuous Sirtuin/HDAC (histone deacetylase) activity assay using thioamides as PET (Photoinduced Electron Transfer)–based fluorescence quencher

Cited by 8 publications

References 97 publications

Uncovering Robust Delactoylase and Depyruvoylase Activities of HDAC Isoforms

Uncovering Robust Delactoylase and Depyruvoylase Activities of HDAC Isoforms

Continuous Fluorescent Sirtuin Activity Assay Based on Fatty Acylated Lysines

Small Changes Make the Difference for SIRT2: Two Different Binding Modes for 3-Arylmercapto-Acylated Lysine Derivatives

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