Cyclophilin D over-expression increases mitochondrial complex III activity and accelerates supercomplex formation

Abstract

Cyclophilin D (CyPD), a mitochondrial matrix protein, has been widely studied for its role in mitochondrial-mediated cell death. Unexpectedly, we previously discovered that overexpression of CyPD in a stable cell line, increased mitochondrial membrane potentials and enhanced cell survival under conditions of oxidative stress. Here, we investigated the underlying mechanisms responsible for these findings. Spectrophotometric measurements in isolated mitochondria revealed that overexpression of CyPD in HEK293 cells increased respiratory chain activity, but only for Complex III (CIII). Acute treatment of mitochondria with the immumosupressant cyclosporine A did not affect CIII activity. Expression levels of the CIII subunits cytochrome b and Rieske-FeS were elevated in HEK293 cells overexpressing CyPD. However, CIII activity was still significantly higher compared to control mitochondria, even when normalized by protein expression. Blue native gel electrophoresis and Western blot assays revealed a molecular interaction of CyPD with CIII and increased levels of supercomplexes in mitochondrial protein extracts. Radiolabeled protein synthesis in mitochondria showed that CIII assembly and formation of supercomplexes containing CIII were significantly faster when CyPD was overexpressed. Taken together, these data indicate that CyPD regulates mitochondrial metabolism, and likely cell survival, by promoting more efficient electrons flow through the respiratory chain via increased supercomplex formation.

Keywords: Chaperone; Metabolic regulation; Mitochondrial permeability transition (MPT); Mitochondrial respiratory chain complex; Prolyl isomerase.

Fig. 1. Overexpression of CyPD leads to enhanced cellular respiration and ATP production

Oxygen consumption of HEK293 control cells (transfected with pcDNA3 vector) and stables cell lines overexpressing pcDNA3-CyPD were performed using StratKelvin oxygen consumption electrode. It is shown as a time course (a) and as absolute oxygen rate (b); representative traces of slope plots corresponding to control and oligomycin condition after control/vehicule (Water) and ATP addition (b1); absolute oxygen rate after control/vehicule (Water) and ATP addition (b2). HEK293 control cells and stables cell lines overexpressing CyPD were obtained. The ATPlite^™ Luminescence Assay System was used to determine ATP concentrations (c). Western blot analysis of CyPD levels in HEK293 control cells (transfected with pcDNA3 vector) and overexpressed with pcDNA3-CyPD (CyPD) (d). Densitometry histogram is normalized with actin (e). All experiments were performed in triplicate. Oxygen consumption a representative experiment is shown of three performed separately, and ATP production data represent the mean of three independent experiments and are represented as mean ± SEM.

Fig. 2. Cyclophilin D over-expression increases CIII enzymatic activity

Isolated mitochondria from HEK293 control cells (transfected with pcDNA3 vector) and stable cells lines differentially overexpressing CyPD (CyPD1, CyPD2 and CyPD3) were obtained. The enzymatic activity of individual respiratory complexes (I–IV) was measured by spectrophotometric assays as is indicated in Experimental Procedure (a–d). Data represent the mean either of two independent experiments (CI, CII and CIV enzymatic activities) or three (CIII enzymatic activity) and are represented as mean ± SEM.). Western blot analysis of CyPD levels in HEK293 control cells (transfected with pcDNA3 vector) and overexpressed with pcDNA3-CyPD (CyPD1, CyPD2 and CyPD3) and densitometry histogram normalized with actin (e). Western blot data are the mean of three independent experiment represented as mean ± SEM.

Fig. 3. The effect of Cyclophilin D over-expression on enzymatic CIII activity is peptidyl-prolyl isomerase (PPIase) independent

Isolated mitochondria from HEK293 control cells (transfected with pcDNA3 vector) and stable transfected cells line overexpressing CyPD (CyPD3) were obtained. The enzymatic activity of CIII was measured by spectrophotometric assay in the absence and presence of CsA at 6 μM and 20 μM incubated for 30 min. Data represent the mean of four independent experiments using different sets of isolated mitochondria and are represented as mean ± SEM.

Fig 4. Cyclophilin D over-expression increases protein levels of CIII subunits and increases CIII specific enzymatic activity

Mitochondrial protein extracts were obtained from HEK293 control cells (transfected with pcDNA3 vector) and stable transfected cells line overexpressing CyPD and analyzed by Western blot using antibodies against cytochrome b and the Rieske subunit of CIII (a), then the PVDF membrane was stripped and re-probed with antibodies against Core II and CyP-D (b). The PVDF membrane is shown stained with ponceau S, this was performed inmediately after transfer, before immunostainig (c). Values for CIII enzymatic activity were normalized either by total protein, ponceau S stained (d) or by levels of individual CIII subunits (e–g). Data represent the mean of four independent experiments and are represented as mean ± SEM.

Fig 5. CyPD interacts with individual CIII and supercomplex

Mitochondrial protein were obtained from HEK-293 control cells (transfected with pcDNA3 vector) and stable transfected cell line overexpressing CyPD were loaded onto BNGs; mitochondrial protein extracts from bovine heart were used as control (a). The mitochondrial extracts were loaded onto BNGs (100ug), run, transferred to PVDF membrane and probed using antibodies against Core II (b). The PVDF membrane was stripped and re-probed with antibodies against CyPD (c). Representative experiment is shown of three performed separately. (d) Densitometry histogram of Core II (upper, middle and lower bands) from CyPD transfected cell line are normalized with the corresponding value of control cells. (e) Densitometry histogram of CyPD (upper, middle and lower bands) from CyPD transfected cell line are normalized with the corresponding value of control cells.

Fig. 6. Cyclophilin D over-expression induces changes in supercomplex stoichiometry

Mitochondrial protein extracts from control (a) and CyPD-overexpressing HEK293 cells (b) were loaded onto BNGs. Bands from each cell line were excised and ran in second dimension on SDS-PAGE, then transferred to PVDF membrane and analyzed by Western blot using an antibody cocktail which probes for individual subunits of all five complexes. Complex I (NDUFA9), complex II (subunit 70kDa), complex III (Core II), complex IV (subunit IV), and complex V (subunit alpha). Representative experiment is shown of four performed separately.

Fig. 7. Radiolabeling of mitochondrial proteins reveals complex III is assembled faster in cells overexpressing CyPD

In vivo labeling of mitochondrial translation products was performed. Control HEK293 cells, transfected with pcDNA 3 vector (a) and stable transfected cells line overexpressing CyPD (b) were pre-incubated in the presence of chloramphenicol, for 20 h. Then the cells were subsequently washed and incubated with 0.2 mCi of [³⁵S]methionine-cysteine for 2 h, in the presence of cycloheximide. Cells were then chased with unlabeled medium for 1, 2, 4, 8, 24, 48 and 72 h, in the absence of cycloheximide. Mitochondrial protein extracts were loaded onto BNGs; the gel was fixed, dried, and proteins were visualized by autoradiography. Quantification of bands from radiolabeling reveals relative CIII, CV+III/IV and supercomplexes amounts on multiple time points before and after 35S labeling (b, d). Data represent the mean of three independent experiments and are represented as mean ± SEM.