Continuous Fluorescent Sirtuin Activity Assay Based on Fatty Acylated Lysines
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. Int...
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| Format: | Article |
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MDPI AG
2023-04-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/24/8/7416 |
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| author | Matthes Zessin Marat Meleshin Sebastian Hilscher Cordelia Schiene-Fischer Cyril Barinka Manfred Jung Mike Schutkowski |
| author_facet | Matthes Zessin Marat Meleshin Sebastian Hilscher Cordelia Schiene-Fischer Cyril Barinka Manfred Jung Mike Schutkowski |
| author_sort | Matthes Zessin |
| collection | DOAJ |
| description | 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 (K<sub>M</sub> values in the low nM range, specificity constants between 175,000 and 697,000 M<sup>−1</sup>s<sup>−1</sup>) it was possible to reliably determine the IC<sub>50</sub> and K<sub>i</sub> values for different inhibitors in the presence of only 50 pM of SIRT2 using different microtiter plate formats. |
| first_indexed | 2024-03-11T04:55:48Z |
| format | Article |
| id | doaj.art-198a48185cf74616a0dc7b18a61f561d |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-11T04:55:48Z |
| publishDate | 2023-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-198a48185cf74616a0dc7b18a61f561d2023-11-17T19:40:06ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-04-01248741610.3390/ijms24087416Continuous Fluorescent Sirtuin Activity Assay Based on Fatty Acylated LysinesMatthes Zessin0Marat Meleshin1Sebastian Hilscher2Cordelia Schiene-Fischer3Cyril Barinka4Manfred Jung5Mike Schutkowski6Department of Medicinal Chemistry, Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, 06120 Halle, GermanyDepartment of Enzymology, Charles Tanford Protein Center, Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, 06120 Halle, GermanyDepartment of Medicinal Chemistry, Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, 06120 Halle, GermanyDepartment of Enzymology, Charles Tanford Protein Center, Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, 06120 Halle, GermanyInstitute of Biotechnology, Czech Academy of Sciences, BIOCEV, Prumyslova 595, 25250 Vestec, Czech RepublicInstitute of Pharmaceutical Sciences, University of Freiburg, Albertstraße 25, 79104 Freiburg, GermanyDepartment of Enzymology, Charles Tanford Protein Center, Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, 06120 Halle, GermanyLysine 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 (K<sub>M</sub> values in the low nM range, specificity constants between 175,000 and 697,000 M<sup>−1</sup>s<sup>−1</sup>) it was possible to reliably determine the IC<sub>50</sub> and K<sub>i</sub> values for different inhibitors in the presence of only 50 pM of SIRT2 using different microtiter plate formats.https://www.mdpi.com/1422-0067/24/8/7416histone deacetylasessirtuinsfluorescence quenchingsirtuin inhibitorsmyristoylated substratescontinuous activity assay |
| spellingShingle | Matthes Zessin Marat Meleshin Sebastian Hilscher Cordelia Schiene-Fischer Cyril Barinka Manfred Jung Mike Schutkowski Continuous Fluorescent Sirtuin Activity Assay Based on Fatty Acylated Lysines International Journal of Molecular Sciences histone deacetylases sirtuins fluorescence quenching sirtuin inhibitors myristoylated substrates continuous activity assay |
| title | Continuous Fluorescent Sirtuin Activity Assay Based on Fatty Acylated Lysines |
| title_full | Continuous Fluorescent Sirtuin Activity Assay Based on Fatty Acylated Lysines |
| title_fullStr | Continuous Fluorescent Sirtuin Activity Assay Based on Fatty Acylated Lysines |
| title_full_unstemmed | Continuous Fluorescent Sirtuin Activity Assay Based on Fatty Acylated Lysines |
| title_short | Continuous Fluorescent Sirtuin Activity Assay Based on Fatty Acylated Lysines |
| title_sort | continuous fluorescent sirtuin activity assay based on fatty acylated lysines |
| topic | histone deacetylases sirtuins fluorescence quenching sirtuin inhibitors myristoylated substrates continuous activity assay |
| url | https://www.mdpi.com/1422-0067/24/8/7416 |
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