Upregulation of cytochrome c oxidase subunit 6b1 (Cox6b1) and formation of mitochondrial supercomplexes: implication of Cox6b1 in the effect of calorie restriction

Abstract

Calorie restriction (CR), a non-genetic intervention that promotes longevity in animals, may exert anti-aging effects by modulating mitochondrial function. Based on our prior mitochondrial proteome analysis, we focused on the potential roles of cytochrome c oxidase (Cox or Complex IV) subunit 6b1 on formation of mitochondrial supercomplexes comprised of Complex I, III, and IV. Blue native polyacrylamide gel electrophoresis followed by immunoblotting showed that the amount of Cox6b1 and the proportion of high molecular weight supercomplexes (SCs) comprised of Complexes I, III, and IV were increased in the liver of mice subjected to 30 % CR, compared with the liver of mice fed ad libitum. In in vitro experiments, in Cox6b1-overexpressing NIH3T3 (Cox6b1-3T3) cells, Cox6b1 was increased in the SC, III2IV1, and III2IV2 complexes and Cox was concomitantly recruited abundantly into the SC, compared with control (Con)-3T3 cells. The proportions of III2IV1, and III2IV2, relative to IV monomer were also increased in Cox6b1-3T3 cells. Cox6b1-3T3 cells showed increased oxygen consumption rates, Cox activity, and intracellular ATP concentrations, indicating enhanced mitochondrial respiration, compared with Con-3T3 cells. Despite the increased basal level of mitochondrial reactive oxygen species (ROS), cell viability after inducing oxidative stress was greater in Cox6b1-3T3 cells than in Con-3T3 cells, probably because of prompt activation of protective mechanisms, such as nuclear translocation of nuclear factor E2-related factor-2. These in vivo and in vitro studies show that Cox6b1 is involved in regulation of mitochondrial function by promoting the formation of SC, suggesting that Cox6b1 contributes to the anti-aging effects of CR.

Fig. 1

Effect of CR on the expression of Cox6b1 mRNA and mitochondrial proteins. a Cox6b1 mRNA expression in the liver of 12-month-old mice fed AL or 30 % CR was determined by qPCR. The mRNA expression of β-actin was used as an internal control. The results are presented as means ± SEM (n = 6, *p < 0.05). b Immunoblotting analysis of mitochondrial subunits using the indicated antibodies. c Densitometry of the immunoblots. The complex II subunit (30 kDa), SDHB, was immunodetected as a loading control. The results are presented as means ± SEM (n = 3, *p < 0.05)

Fig. 2

CR promotes the assembly of mitochondrial supercomplexes. a Blue native polyacrylamide gel electrophoresis (BN-PAGE) of liver tissue samples from 12-month-old mice fed AL or 30 % CR. Coomassie blue staining of 1D BN-PAGE gel (left panel) and subsequent immunoblotting with anti-Cox6b1 antibody (right panel). SDS-PAGE and immunoblotting with anti-VDAC/Porin antibody was also performed to confirm equivalent protein loading of mitochondria lysates (lower panel). b Densitometric analysis of immunoblots. The results are presented as mean ± SD (n = 6, **p < 0.01, ***p < 0.001, vs. AL group). c 2D BN/SDS-PAGE of AL and CR liver samples. Immunoblotting was performed using the anti-ATP5A antibody for Complex V, the anti-Core2 antibody for Complex III, the anti-SDHB antibody for Complex II, and the anti-Cox1 and the anti-Cox6b1 antibodies for Complex IV. The position SC-a corresponds to the mitochondrial supercomplexes containing I/III₂/IV_n (n = 1, 2, 3, or 4); the position SC-b corresponds to I/III₂; The position SC-c corresponds to Complex V dimers. The positions of Complex V monomer (CV), Complex III monomer (CIII), Complex III dimers + Complex IV monomers or dimers (III₂IV_n, n = 1 or 2), Complex IV monomer or dimers (CIV or IV₂), and Complex II monomers (CII) are also indicated. CV and III₂IV_n spots are overlapping. The values represent the proportion of signal intensity in each spot in a row detected by a specific antibody

Fig. 3

Establishment of Cox6b1-3T3 cells. a Quantitative PCR analysis of the mRNA expression levels of Cox subunits, normalized for β-actin mRNA, in control NIH-3T3 cells (Con-3T3) and Cox6b1-overexpressing NIH-3T3 (Cox6b1-3T3) cells. The results are presented as mean ± SEM (n = 6, *p < 0.05 vs. Con-3T3). b Immunoblotting for Cox1, Cox4, and Cox6b1 in the cytosolic and mitochondrial fractions of Con-3T3 and Cox6b1-3T3 cells. β-actin and GAPDH were used as cytosolic markers and VDAC/Porin was used as a mitochondrial marker. c Relative expression of Cox6b1 as determined by densitometry of the immunoblots. Results are presented as mean ± SEM (n = 4, ***p < 0.001 vs. Con-3T3). The values were normalized to those of VDAC/Porin. d Localization of Cox6b1 and MitoTracker Red signals in Cox6b1-3T3 cells. Confocal microscopic images of Cox6b1-3T3 cells stained with MitoTracker Red (mitochondria) and TO-PRO-3 (nuclei). Microscopic images of MitoTracker Red signals, Cox6b1 cells, and the merged signals (TO-PRO-3 and MitoTracker Red) are shown in the left, middle, and right panels, respectively

Fig. 4

Effects of Cox6b1 overexpression on the recruitment of Cox6b1 to mitochondrial supercomplexes. BN-PAGE and immunoblotting of mitochondrial fractions from Con-3T3 and Cox6b1-3T3 cells. a 1D gels were stained with Coomassie R-250 (left panel) or immunoblotted with the anti-Cox6b1 antibody. SDS-PAGE and immunoblotting with the anti-VDAC/Porin antibody was also performed to confirm equivalent protein loading of mitochondria lysates (lower panel). Gels were transferred for the high molecular weight supercomplexes (right panel). b Densitometric analysis of immunoblots. The results are presented as mean ± SD (n = 4, *p < 0.05, **p < 0.01, vs. Con-3T3). c 2D SDS-PAGE of mitochondria fractions from Con-3T3 and Cox6b1-3T3 cells. Immunoblotting was performed with anti-ATP5A antibody for Complex V, anti-Core2 antibody for Complex III, anti-SDHB antibody for Complex II, and anti-Cox1 and anti-Cox4 antibodies for Complex IV. The positions corresponding to the mitochondrial supercomplexes are as follows: SC-a, Complex V trimer or tetramer (Vn, n = 3 or 4); SC-b, I/III₂/IV_n (n = 1, 2, 3, or 4); SC-c, Complex I + Complex III (I/III₂); SC-d, Complex V dimer (V₂). The positions of Complex V monomers (CV), Complex III dimers + Complex IV monomers or dimers (III₂IV_n, n = 1 or 2), predicted Complex IV dimers (IV₂ ^*), Complex IV monomers (CIV), and Complex II monomers (CII) are shown. The values represent the proportion of signal intensity in each spot in a row detected by a specific antibody

Fig. 5

Effect of Cox6b1 overexpression on mitochondrial function. a Oxygen consumption rate (OCR) was measured in Con-3T3 and Cox6b1-3T3 cells under basal conditions and after exposure to the indicated compounds using a XFe96 Extracellular Flux Analyzer. b Basal respiration (Basal), coupled respiration (Coupled), spare respiration capacity (SRC), and uncoupled respiration (Uncoupled) were calculated from the original XFe96 Extracellular Flux Analyzer data shown in Fig. 5a. The results are presented as mean ± SD (n = 5; *p < 0.05, **p < 0.01, ***p < 0.001 vs. Con-3T3). c Cytochrome c oxidase activity in isolated mitochondria from Con-3T3 and Cox6b1-3T3 cells. The results are presented as mean ± SEM (n = 6; **p < 0.01). Citrate synthase activity was used as an internal control. d Relative ATP concentrations were measured using a luminescence assay and were normalized for the protein concentration in each sample. Results are presented as mean ± SEM (n = 6; **p < 0.01). f Relative cellular viability following exposure to hydrogen peroxide (H₂O₂). Cell viability was measured using a WST-1 assay. Cells were exposed to 500 or 700 μM H₂O₂ for 24 h. The results are presented as mean ± SEM (n = 12; ***p < 0.001)

Fig. 6

The effect of Cox6b1 knockdown (KD) on mitochondrial function. a OCR was measured in control siRNA (consiRNA) and Cox6b1 KD cells under basal conditions and after exposure to the indicated compounds using a XFe96 Extracellular Flux Analyzer. b Basal respiration (Basal), coupled respiration (Coupled), spare respiration capacity (SRC), and uncoupled respiration (Uncoupled) were calculated from the original XFe96 Extracellular Flux Analyzer data in Fig. 6a. The results are presented as mean ± SD (n = 5; *p < 0.05, ***p < 0.001 vs. consiRNA). c Cytochrome c oxidase activity measured in isolated mitochondria from consiRNA and Cox6b1 KD cells. Results are presented as mean ± SEM, n = 6 (*p < 0.05; citrate synthase activity (CS) was used as internal control). d Relative ATP production was detected using a luminescence assay. Results are presented as mean ± SEM, n = 6 (*p < 0.05; each sample was normalized by protein concentration). e The relative cell viability in response to hydrogen peroxide was measured by WST-1 assay. The cells were exposed to different concentrations of H₂O₂ (500, 700 μM) for 24 h. Data represent mean ± SEM (n = 12, ***p < 0.001)

Fig. 7

Cox6b1 overexpression induces Nrf2 nuclear translocation and upregulates antioxidant enzymes. a Nuclear and cytosolic levels of Nrf2 in Con-3T3 and Cox6b1-3T3 cells. Cells were exposed to 25 μM of tBHQ for 60 or 120 min, and nuclear extracts were used for immunoblotting analysis. Lamin B1 and β-actin were used as internal controls for nuclear and cytosolic proteins, respectively. Fold changes of nuclear Nrf2 and cytosolic Nrf2, relative to those of Con-3T3 cells at 0 min, are indicated. b Quantitative PCR analysis of the mRNA expression levels of Nrf2-target genes, those are associated with oxidative stress responses (NQO1, HO-1, γGCS, GST-α1, SOD2) in control NIH-3T3 cells (Con-3T3) and Cox6b1-overexpressing NIH-3T3 cells (Cox6b1-3T3) in response to vehicle (DMSO only) or 25 μM of tBHQ for 6 h. β-actin was used as a control housekeeping gene. The results are presented as mean ± SEM (n = 4, *p < 0.05, **p < 0.01, ***p < 0.001). c Nrf2-dependent antioxidant enzyme expression is increased in Cox6b1-3T3 cells. Con-3T3 and Cox6b1-3T3 were incubated with 25 tBHQ for 18 h, and the total cell lysates were subjected to immunoblotting for NQO1 and HO-1. β-actin was used as an internal control. Fold changes of NQO-1 and HO-1, relative to those of Con-3T3 at 0 μM, are indicated