Tier 4 — mechanistic

Structural basis for the activation and inhibition of Sirtuin 6 by quercetin and its derivatives

Weijie You, Wei Zheng, Sandra Weiss, Katrin F. Chua, Clemens Steegborn
Scientific Reports 2019 Volume 9, article 19176

Bibliography

PubMed
PMID 31844103
PubMed Central
PMC6914789
DOI
10.1038/s41598-019-55654-1
Funding
Deutsche Forschungsgemeinschaft grant STE1701/15 (to C.S.). NIH/NIA grant R01AG050997 and Department of Veterans Affairs Merit Award (to K.F.C.). Publication funded by the German Research Foundation (DFG) and the University of Bayreuth through the funding program Open Access Publishing.
Competing interests
The authors declare no competing interests.

Study snapshot

DesignIn vitro biochemistry across five Sirtuin isoforms (SIRT1, SIRT2, SIRT3, SIRT5, SIRT6) plus X-ray crystallography of five compound complexes; complementary U2OS cell histone acetylation assays.
ModelRecombinant human Sirtuin proteins for activity and crystallography; U2OS cells (WT and SIRT6-KO) for cellular histone acetylation.
Samplen=3 replicates per activity assay condition; five crystal structures solved (SIRT6/quercetin, SIRT6/isoquercetin, SIRT6/catechin gallate, SIRT6/cyanidin, SIRT2/quercetin).
InterventionQuercetin and derivatives (luteolin, cyanidin, isoquercetin, catechin gallate) at 0–10 mM in MS-based deacetylation assays with H3K9Ac peptide substrate; 25–100 μM quercetin in cellular H3 acetylation assays.
DurationEnzyme assays: 5 min to 2 h depending on assay; cellular assays not applicable to duration framing.
EndpointsSIRT6 deacetylation of H3K9Ac peptide; SIRT6 activity on purified nucleosomes and free histones (H3K18ac, H3K9ac); SIRT1/2/3/5 deacetylation activity; SIRT6-dependent demyristoylation; cellular H3 acetylation in U2OS WT vs SIRT6-KO.

What the study showed, in plain terms

Quercetin is a plant flavonoid found in onions, apples, and berries that has been widely promoted as an anti-ageing supplement. Earlier reports had produced conflicting results about whether quercetin activates or inhibits SIRT6, a longevity-related enzyme, partly because standard laboratory assays gave misleading readings when quercetin was present. This paper resolved the contradiction by testing quercetin and its close relatives with a more robust mass spectrometry assay.

The authors show that quercetin is a low-potency activator of SIRT6, roughly doubling its ability to remove acetyl groups from histone H3. Related molecules behaved differently: cyanidin (found in black rice and blackcurrants) activated more strongly, while catechin gallate (from tea) inhibited SIRT6. Crystal structures showed all three bind to the same pocket on SIRT6 but in slightly different orientations, and the presence or absence of a specific double bond in the molecule appears to decide whether the compound activates or inhibits the enzyme.

Quercetin also inhibited the other Sirtuin isoforms tested (SIRT1, SIRT2, SIRT3, SIRT5) by binding to a completely different site at the active-site entrance. Isoquercetin, which carries a bulky sugar group, could not fit into this second site and therefore activated SIRT6 selectively without inhibiting the other isoforms — a useful starting point for designing more specific SIRT6 activators.

Key findings

  • Quercetin is a low-potency SIRT6 activator with more than 2-fold maximum stimulation and an EC50 of approximately 1.2 mM in mass spectrometry assays with H3K9Ac peptide substrate.
  • Quercetin activated SIRT6 on purified HeLa nucleosomes (deacetylation of H3K18ac) and on free full-length histones (H3K18ac and H3K9ac), which are otherwise poor SIRT6 substrates.
  • Cyanidin activated SIRT6 with slightly greater potency than quercetin, showing significant activation at 20–80 μM.
  • Catechin gallate inhibited SIRT6 deacetylation with an IC50 of approximately 80 μM in the MS assay, contrary to the activation pattern of related flavonoids.
  • Crystal structures placed the catechol moiety of all quercetin derivatives at the distal end of the SIRT6 acyl channel, anchored by hydrogen bonds to Pro62; the chromen-4-one system orientation differed between activators and inhibitors, correlating with C-ring saturation.
  • Quercetin inhibited SIRT1, SIRT2, SIRT3, and SIRT5 by binding at an alternative site at the active site entrance, as shown by a crystal structure of the SIRT2/quercetin complex; peptide substrate competition confirmed this site as competitive with substrate binding.
  • Isoquercetin selectively activated SIRT6 without affecting SIRT1, SIRT2, SIRT3, or SIRT5, because its bulky sugar group sterically clashes with Tyr-114 in the alternative Sirtuin binding site.
  • Treatment of U2OS cells with 25–100 μM quercetin caused a dose-dependent decrease in H3 acetylation that was abolished in SIRT6-deficient cells, demonstrating a direct SIRT6-dependent cellular effect.

What this study can and cannot tell us

All activity data are in vitro or in a single cancer cell line (U2OS); there is no in vivo evidence that dietary quercetin activates SIRT6 in intact tissues at physiologically achievable concentrations.

The EC50 for quercetin activation is approximately 1.2 mM — orders of magnitude above the low-micromolar plasma concentrations achieved from typical dietary intake or standard supplementation. The cellular experiments used 25–100 μM, still well above realistic dietary exposure. The direct SIRT6 activation shown here is therefore unlikely to explain most observed health effects of oral quercetin.

Quercetin affects many other cellular targets, including protein kinases and CD38. Any downstream benefit attributed to SIRT6 activation must be separated from these off-target effects, which the paper acknowledges but does not resolve.

Common Sirtuin activity assays (Fluor-de-Lys and the coupled enzymatic assay) produced misleading results with quercetin due to fluorescence quenching and downstream enzyme interference. Earlier literature claiming quercetin inhibition of SIRT6 at low concentrations is likely artefactual.

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Dr. Khaled Mahmoud, MBBChCritical Care Medicine, Longevity Science & Editorial Direction Dr. Abdulla Mostafa, PhDCellular Senescence, Neurological, Mitochondrial & Brain-Ageing Biology Dr. Amr Yasser, PhDCardiometabolic, Musculoskeletal, Gut & Bioavailability