A human tRNA synthetase is a potent PARP1-activating effector target for resveratrol

Abstract

Resveratrol is reported to extend lifespan and provide cardio-neuro-protective, anti-diabetic, and anti-cancer effects by initiating a stress response that induces survival genes. Because human tyrosyl transfer-RNA (tRNA) synthetase (TyrRS) translocates to the nucleus under stress conditions, we considered the possibility that the tyrosine-like phenolic ring of resveratrol might fit into the active site pocket to effect a nuclear role. Here we present a 2.1 Å co-crystal structure of resveratrol bound to the active site of TyrRS. Resveratrol nullifies the catalytic activity and redirects TyrRS to a nuclear function, stimulating NAD(+)-dependent auto-poly-ADP-ribosylation of poly(ADP-ribose) polymerase 1 (PARP1). Downstream activation of key stress signalling pathways are causally connected to TyrRS-PARP1-NAD(+) collaboration. This collaboration is also demonstrated in the mouse, and is specifically blocked in vivo by a resveratrol-displacing tyrosyl adenylate analogue. In contrast to functionally diverse tRNA synthetase catalytic nulls created by alternative splicing events that ablate active sites, here a non-spliced TyrRS catalytic null reveals a new PARP1- and NAD(+)-dependent dimension to the physiological mechanism of resveratrol.

Extended Data Figure 1. Resveratrol inhibits TyrRS activation

a, The ATP-PPi exchange assay as described in methods demonstrated the inhibitory effect of resveratrol on TyrRS and (b), resveratrol shifts the Km for tyrosine. c, Resveratrol binds TyrRS better than tyrosine. The apparent Ki for resveratrol was deduced by varying the concentration of RSV and plotting the slope of (1/v vs 1/[Tyr]) versus [RSV] as indicated.

Extended Data Figure 2. Resveratrol induces a distinct conformational change upon binding to active site of TyrRS

a, Comparison of the overall conformational change induced by resveratrol at the active site of TyrRS by structure based superposition (yellow-tyrosine-bound structure and magenta-resveratrol-bound structure). Note the conformational change near the helix region (P331-P342) that connects the linker region with the C-domain. b, Illustration of the extensive interactions of resveratrol with the active site. c, trans-resveratrol (dark blue) docks (manual) into TyrRS active site without significant structural disturbances. d, Generation of a new pocket through a RSV-induced conformational change in TyrRS accommodates the dihydroxy phenolic ring of RSV (otherwise exposed to the destabilizing aqueous environment in the trans form) and hence facilitates the trans (dark blue) to cis (light blue) conversion of RSV.

Extended Data Figure 3. Resveratrol facilitates the TyrRS/PARP-1 interaction in an active-site-dependent manner

a, Both heat shock (42°C for 30 min) and tunicamycin-treatment (10 μg/ml, ER stress) facilitated the nuclear translocation of TyrRS and activation of PARP-1. b, Resveratrol or serum starvation facilitate TyrRS interaction with PARP-1 and Tyr-SA prevents this interaction. ZZ-PARP-1 was immunoprecipiated with IgG from HeLa cells treated with RSV or serum starvation alone or in combination with Tyr-SA. c, Resveratrol or serum starvation mediated PARP-1 activation is blocked only by Tyr-SA and not by Gly-SA. d, TyrRS interacts directly with PARP-1. HeLa cell lysate after RSV treatment (5 μM, 30 min) was divided into three parts and treated with PARG and catalytically inactive PARG-MT. PARP-1 was immunoprecipitated and analyzed for TyrRS interaction. e. Model illustrating the mechanism of RSV mediated TyrRS interaction with PARP-1 and subsequent release after auto-PARylation. f. Ni-NTA pull-down of N- and C-terminal fragments of PARP-1 overexpressed in E. coli demonstrated that TyrRS interacts with the C-terminal region of PARP-1. g, Only the full-length TyrRS (1-528) and none of the various fragments of TyrRS (mini-TyrRS (1-364), ΔN-TyrRS (228-528) or the C-domain (328-528)) interacts with PARP-1. h, Coomassie blue staining of a gel showing the total protein input in for the experiment of Extended Data Figure 3g.

Extended Data Figure 4. Tyrosyl-AMP analogue (Tyr-SA) does not affect DNA-dependent auto-PARylation of PARP-1

a, Silver stained SDS-PAGE gel showing the purity and input of PARP-1 and TyrRS in the in vitro PARylation study of figure 2. b, Quantitation (Image J software) of the band intensity of PARylated PARP-1 in figure 2a top. c, Tyrosyl-AMP analogue (Tyr-SA) does not affect DNA-dependent auto-PARylation of PARP-1. d, Overexpression of nuclear translocation-weakened mutant of TyrRS is less effective in activating PARP-1. e. Y314A-TyrRS is more sensitive to RSV than is TyrRS in facilitating PARP-1 activation.

Extended Data Figure 5. Resveratrol enhances the acetylation of Tip60 and modulates [NAD⁺] in a dose and time dependent manner

a, Treatment of HeLa cells (1 h) with increasing concentration of resveratrol enhances the acetylation level of Tip60. Activation of Tip60 was monitored by histone acetylation status. b, Total NAD⁺ content of serum starved cells or RSV treated samples were compared with untreated samples at 15 min using a commercially available BioVision NAD⁺/NADH quantitation colorimetric kit. c, Total nicotinamide or ADP-ribose produced was deduced from the difference in the amount of NAD⁺ in each sample with respect to the untreated sample (consumption of one mole of NAD⁺ would give rise one mole of nicotinamide and one mole of ADP-ribose). d, Total NAD⁺ content of the serum starved cells or RSV treated samples were compared with untreated samples at 1 hour. (Although the experiments were done in biological triplicates (all samples showing similar results), the error bars in the figure represent the deviations from the mean of the technical triplicates from one representative biological sample.) e,f Time course study of poly-ADP-ribosylation status and associated signaling events after (e) serum starvation (extended time course data of the same image shown in figure 3c) and (f) treatment with 5 μM RSV. Using the respective antibodies, Activation of p53 was monitored by the induction of p21 and SIRT6. Activation of NRF2 was monitored by HO-1 induction.

Extended Data Figure 6. siRNA ( siRNA^TyrRS or siRNA^PARP-1), with and without low RSV (5 μM), does not affect cell viability

HeLa cells (1× 10⁶) were reverse-transfected with siRNA targeted to TyrRS or PARP-1. An siRNA^Con (a scrambled sequence of siRNA^PARP-1) was used as a control. Viability was monitored using the RTCA iCELLigence System (ACEA Biosciences). Samples were treated with RSV (5μM) at 60h and monitoring was continued for another 2h for siRNA^TyrRS (total of 62h of monitoring) and for another 16h for siRNA^Con and siRNA^PARP-1 (total 76h monitoring).

Extended Data Figure 7. siRNA^SIRT1 did not affect downstream signaling events at low RSV (5 μM)

HeLa cells were treated with siRNA^SIRT1 for 60 h to knockdown SIRT1. HeLa cells were treated with RSV (5 μM) for another 4 h and samples were collected intervals as indicated. Samples were analyzed for down stream signaling markers using appropriate antibodies.

Extended Data 8. | siRNA- (siRNA^TyrRS or siRNA^PARP-1) treated cells did not upregulate the levels of NAD⁺ in response to RSV (5 μM) after 1h

HeLa cells (1× 10⁶) were reverse-transfected, separately, with siRNA targeted to PARP-1 or TyrRS. A scrambled sequence of target siRNA was used as a control. Total NAD⁺ content of RSV (5 μM)-treated samples were compared with untreated samples at 1h, using a commercially available BioVision NAD⁺/NADH quantitation colorimetric kit. (Although the experiments were done in biological triplicates (all samples showing similar results), the error bars in the figure represent the deviations from the mean of the technical triplicates from one reprentative biological sample.) The comparator (shown as a dashed bar) is taken from Extended Data Fig. 5d.

Extended Data Figure 9. Resveratrol treatment activates PARP-1 and associated signaling events in the mouse tissues

a, Activation of PARP-1 in mouse muscle tissue treated with resveratrol monitored by increased PARylation and (b) by increased acetylation status. Activation of Tip60 and AMPK were monitored by using α-AcK16-H4 and α-pSer36-H2B, respectively. c, Activation of PARP-1 in mouse heart tissue treated with resveratrol monitored by increased PARylation and (d) by increased acetylation status. e, Resveratrol treatment cause only a transient activation on PARP-1. Immunoblotting of mouse muscle tissue samples after 24 hr of RSV treatment showed no significant difference in the level of PARP-1^PAR with respect control. f, RSV treatment enhances TyrRS interaction with and activation of PARP-1 in the muscle tissue. g and h, Resveratrol-mediated activation of PARP-1 (g, monitored by PARylation status and h, monitored by acetylation status) is blocked by Tyr-SA in the mouse heart tissues.

Figure 1. Resveratrol binds at the active site of TyrRS

a, Cartoon illustration of the domain organization of Hs TyrRS. Both domains are connected by a linker of ~20 amino acids. b, Left, Electron density of co-crystal x-ray structures (2.1 A°) of TyrRS bound to cis-resveratrol and to L-tyrosine. Right, Resveratrol induced a local conformational change relative to bound tyrosine at the active site. c, Both serum starvation and resveratrol treatment (5 μM) facilitated the nuclear translocation of TyrRS with a concomitant increase in the PARylation of PARP-1.

Figure 2. TyrRS facilitates the activation of PARP-1 in an active-site-dependent manner

a, top, TyrRS activates PARP-1 in an in vitro assay. a, middle. Resveratrol potentiates TyrRS mediated activation of PARP-1. a, bottom. Tyr-SA blocks the resveratrol-mediated activation of PARP-1. b, top. TyrRS-V5 overexpression activates PARP-1 in HeLa cells in a concentration-dependent manner. b, middle. Resveratrol treatment activates PARP-1 in HeLa cells and enhances TyrRS interaction with PARP-1. b, bottom. Tyr-SA blocks the resveratrol-mediated interaction of TyrRS and activation of PARP-1. c, Cartoon illustration of the C-domain disposition in TyrRS (left) and Y341ATyrRS (right). d. Y341ATyrRS enhances its interaction and activates PARP-1 compared to WT.

Figure 3. Resveratrol and serum starvation mimic similar downstream signaling events mediated by PARP-1 activation

a, Cartoon illustrating the molecular basis and the integration of different signaling pathways to mediate a TyrRS/PARP-1 activated stress response evoked by either resveratrol or different stress conditions. b, PARP-1 activating conditions enhance Tip60-mediated activation of ATM. c, Time course of poly-ADP-ribosylation status and associated signaling events as depicted in figure 3a after serum starvation and (d) resveratrol (1 μM) treatment.

Figure 4. Resveratrol treatment activates TyrRS-PARP-1 driven signaling events in mouse tissues

a, siRNA of PARP-1 or (b) siRNA of TyrRS abrogates resveratrol-mediated downstream signaling events. HeLa cells were treated with siRNA^PARP-1 or siRNA^TyrRS for 60 h to knockdown PARP-1 or TyrRS expression levels by ~70–80%. Knockdown efficiency was monitored by with α-PARP-1 or α-TyrRS. For, siRNA^TyrRS HeLa cells were treated with either serum starvation or resveratrol (5, 10 25 and 50 μM) for 45 minutes. The dashed line in figure 4a represents the demarcation between siRNA^Con and siRNA^PARP-1; all the samples were run on the same gel. c, NAMPT inhibition abrogates resveratrol-mediated downstream signaling events. HeLa cells were pre-treated with STF-118804 (100 nM), a NAMPT inhibitor, for 16 h and then treated with resveratrol (5 μM), with samples collected at intervals as indicated. Various signaling events, as depicted, were monitored using appropriate antibodies. Acetylation status was monitored using α-acetyl-lysine. The dashed line separates results without and with the inhibitor. d, Resveratrol mediated activation of PARP-1 is blocked by Tyr-SA. Activation of Tip60 and AMPK were monitored by using α-AcK16-H4 and α-pSer36-H2B, respectively. e, Resveratrol-mediated interaction of TyrRS with PARP-1 and acetylation of p53 are blocked by Tyr-SA. Immunoprecipitation of PARP-1 and p53 from muscle tissue demonstrated RSV-mediated TyrRS-PARP-1 interaction and p53 acetylation.