Continuous Fluorescent Sirtuin Activity Assay Based on Fatty Acylated Lysines

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F86652036%3A_____%2F23%3A00571923" target="_blank" >RIV/86652036:_____/23:00571923 - isvavai.cz</a>

Výsledek na webu

<a href="https://www.mdpi.com/1422-0067/24/8/7416" target="_blank" >https://www.mdpi.com/1422-0067/24/8/7416</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.3390/ijms24087416" target="_blank" >10.3390/ijms24087416</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Continuous Fluorescent Sirtuin Activity Assay Based on Fatty Acylated Lysines

Popis výsledku v původním jazyce

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.

Název v anglickém jazyce

Continuous Fluorescent Sirtuin Activity Assay Based on Fatty Acylated Lysines

Popis výsledku anglicky

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.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

<a href="/cs/project/GA21-31806S" target="_blank" >GA21-31806S: Ovlivnění strukturních a funkčních charakteristik chaperonu HSP90 prostřednictvím reverzibilní acetylace</a><br>

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2023

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

International Journal of Molecular Sciences

ISSN

1422-0067

e-ISSN

1422-0067

Svazek periodika

24

Číslo periodika v rámci svazku

8

Stát vydavatele periodika

CH - Švýcarská konfederace

Počet stran výsledku

19

Strana od-do

7416

Kód UT WoS článku

000977440400001

EID výsledku v databázi Scopus

2-s2.0-85157998321