Počet záznamů: 1
Continuous Fluorescent Sirtuin Activity Assay Based on Fatty Acylated Lysines
- 1.
SYSNO ASEP 0571923 Druh ASEP J - Článek v odborném periodiku Zařazení RIV J - Článek v odborném periodiku Poddruh J Článek ve WOS Název Continuous Fluorescent Sirtuin Activity Assay Based on Fatty Acylated Lysines Tvůrce(i) Zessin, M. (DE)
Meleshin, M. (DE)
Hilscher, S. (DE)
Schiene-Fischer, C. (DE)
Bařinka, Cyril (BTO-N) RID, ORCID
Jung, M. (DE)
Schutkowski, M. (DE)Celkový počet autorů 7 Číslo článku 7416 Zdroj.dok. International Journal of Molecular Sciences. - : MDPI
Roč. 24, č. 8 (2023)Poč.str. 19 s. Jazyk dok. eng - angličtina Země vyd. CH - Švýcarsko Klíč. slova histone deacetylases ; sirtuins ; fluorescence quenching ; sirtuin inhibitors Vědní obor RIV EB - Genetika a molekulární biologie Obor OECD Biochemistry and molecular biology CEP GA21-31806S GA ČR - Grantová agentura ČR Způsob publikování Open access Institucionální podpora BTO-N - RVO:86652036 UT WOS 000977440400001 EID SCOPUS 85157998321 DOI 10.3390/ijms24087416 Anotace 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-M values in the low nM range, specificity constants between 175,000 and 697,000 M(-1)s(-1)) it was possible to reliably determine the IC50 and K-i values for different inhibitors in the presence of only 50 pM of SIRT2 using different microtiter plate formats. Pracoviště Biotechnologický ústav Kontakt Monika Kopřivová, Monika.Koprivova@ibt.cas.cz, Tel.: 325 873 700 Rok sběru 2024 Elektronická adresa https://www.mdpi.com/1422-0067/24/8/7416
Počet záznamů: 1