Continuous Fluorescent Sirtuin Activity Assay Based on Fatty Acylated Lysines
The result's identifiers
Result code in 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>
Result on the web
<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>
Alternative languages
Result language
angličtina
Original language name
Continuous Fluorescent Sirtuin Activity Assay Based on Fatty Acylated Lysines
Original language 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-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.
Czech name
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Czech description
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Classification
Type
J<sub>imp</sub> - Article in a specialist periodical, which is included in the Web of Science database
CEP classification
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OECD FORD branch
10608 - Biochemistry and molecular biology
Result continuities
Project
<a href="/en/project/GA21-31806S" target="_blank" >GA21-31806S: Modulation of structural and functional properties of the HSP90 chaperone machinery by reversible acetylation</a><br>
Continuities
I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace
Others
Publication year
2023
Confidentiality
S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů
Data specific for result type
Name of the periodical
International Journal of Molecular Sciences
ISSN
1422-0067
e-ISSN
1422-0067
Volume of the periodical
24
Issue of the periodical within the volume
8
Country of publishing house
CH - SWITZERLAND
Number of pages
19
Pages from-to
7416
UT code for WoS article
000977440400001
EID of the result in the Scopus database
2-s2.0-85157998321