Caffeine Targets SIRT3 to Enhance SOD2 Activity in Mitochondria

Abstract

Caffeine is chemically stable and not readily oxidized under normal physiological conditions but also has antioxidant effects, although the underlying molecular mechanism is not well understood. Superoxide dismutase (SOD) 2 is a manganese-containing enzyme located in mitochondria that protects cells against oxidative stress by scavenging reactive oxygen species (ROS). SOD2 activity is inhibited through acetylation under conditions of stress such as exposure to ultraviolet (UV) radiation. Sirtuin 3 (SIRT3) is the major mitochondrial nicotinamide adenine dinucleotide (NAD⁺)-dependent deacetylase, which deacetylates two critical lysine residues (lysine 68 and lysine 122) on SOD2 and promotes its antioxidative activity. In this study, we investigated whether the antioxidant effect of caffeine involves modulation of SOD2 by SIRT3 using in vitro and in vivo models. The results show that caffeine interacts with SIRT3 and promotes direct binding of SIRT3 with its substrate, thereby enhancing its enzymatic activity. Mechanistically, caffeine bound to SIRT3 with high affinity (K _D = 6.858 × 10^-7 M); the binding affinity between SIRT3 and its substrate acetylated p53 was also 9.03 (without NAD⁺) or 6.87 (with NAD⁺) times higher in the presence of caffeine. Caffeine effectively protected skin cells from UV irradiation-induced oxidative stress. More importantly, caffeine enhanced SIRT3 activity and reduced SOD2 acetylation, thereby leading to increased SOD2 activity, which could be reversed by treatment with the SIRT3 inhibitor 3-(1H-1,2,3-triazol-4-yl) pyridine (3-TYP) in vitro and in vivo. Taken together, our results show that caffeine targets SIRT3 to enhance SOD2 activity and protect skin cells from UV irradiation-induced oxidative stress. Thus, caffeine, as a small-molecule SIRT3 activator, could be a potential agent to protect human skin against UV radiation.

Keywords: SIRT3; SOD2; UV radiation; antioxidant effects; caffeine; skin photoprotection.

FIGURE 1

Caffeine has no antioxidant activity in vitro but enhances SIRT3 activity. (A) The structure of caffeine. (B) Caffeine showed no DPPH (free radical) scavenging activity, as evidenced by the absence of any change in absorbance. (C) Caffeine does not directly enhance SOD2 activity in vitro. (D) Caffeine directly enhances SIRT3 activity in an in vitro system. (E) Caffeine promotes SIRT3-mediated deacetylation of SOD2 in vitro. (F) Caffeine directly binds to SIRT3 with high affinity (K_D = 6.562 × 10^{– 7} M). BLI sensorgrams indicating the interactions between gradient concentrations of caffeine and SIRT3 were measured on an Octet Red 96 system, with association and dissociation for 300 s each. Data are expressed as the mean ± SEM of three independent experiments. **P < 0.01, ***P < 0.001 vs. the control.

FIGURE 2

Caffeine directly binds to SIRT3 to enhance its substrate binding affinity. (A) Enzymatic reaction involving SIRT3. (B) Caffeine binds to SIRT3, with K_D = 6.858 × 10^{– 7} M. (C) NAD⁺ does not directly interact with SIRT3. (D) The acetylated substrate p53-Ac binds to SIRT3, with K_D = 2.434 × 10^{– 5} M. (E) NAD⁺ is the coenzyme for the SIRT3 deacetylation reaction, and its presence enhances the binding affinity of p53-Ac to SIRT3, with K_D = 2.800 × 10^{– 6} M. (F) Addition of caffeine enhances the binding affinity between SIRT3 and its substrate, with K_D = 2.696 × 10^{– 6} M. (G) The binding affinity between SIRT3 and its substrate is increased in the presence of caffeine and NAD⁺, with K_D = 4.076 × 10^{– 7} M. All SPR experiments were carried out using a Biacore S200 instrument (Biacore, GE Healthcare). The data represented here represent one of three independent experiments with similar results.

FIGURE 3

Caffeine pretreatment of HaCaT cells before UV exposure enhances the removal of UV-induced ROS and inhibits apoptosis. (A) Caffeine did not affect the viability of HaCaT cells, as determined with the MTT assay. (B) Caffeine attenuated the growth inhibitory effect of UV irradiation in HaCaT cells. (C) UV-induced intracellular and mitochondrial ROS accumulation in HaCaT cells determined using CM-H₂DCFDA and MitoSOX, respectively. Images were captured at 200× magnification. (D) Western blotting analysis of the HaCaT whole-cell lysates with the indicated antibodies. SIRT3 (E) and SOD2 (F) activities in the HaCaT whole-cell lysates. Data are expressed as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the control; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. UV irradiation only.

FIGURE 4

SIRT3 inhibitor 3-TYP abrogates the protective effect of caffeine in UV-irradiated HaCaT cells. Detection of SIRT3 (A) and SOD2 (B) activities. (C) Western blotting analysis of the HaCaT whole-cell lysates with the indicated antibodies. (D) Detection of mitochondrial ROS. Images were captured at 200× magnification. Data are expressed as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. UV group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. “Caffeine (1.29 mM) + UV” group or “Caffeine (5.15 mM) + UV” group.

FIGURE 5

Caffeine protects mouse skin from UV damage by acting on SIRT3. (A) Appearance of the dorsal skin from mice. (B,C) Histological analysis of skin sections by H&E and Masson’s trichrome staining. Images were captured at 100× magnification. (D) Body weight of mice. SIRT3 (E) and SOD2 (F) activities in mouse skin tissues. (G) Western blotting analysis of the mouse skin tissues with the indicated antibodies. Data are expressed as the mean ± SEM of six mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 vs. UV group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. “Caffeine (1.29 mM) + UV” group or “Caffeine (5.15 mM) + UV” group.