Deficient tRNA posttranscription modification dysregulated the mitochondrial quality controls and apoptosis

Abstract

Mitochondria are dynamic organelles in cellular metabolism and physiology. Mitochondrial DNA (mtDNA) mutations are associated with a broad spectrum of clinical abnormalities. However, mechanisms underlying mtDNA mutations regulate intracellular signaling related to the mitochondrial and cellular integrity are less explored. Here, we demonstrated that mt-tRNA^Met 4435A>G mutation-induced nucleotide modification deficiency dysregulated the expression of nuclear genes involved in cytosolic proteins involved in oxidative phosphorylation system (OXPHOS) and impaired the assemble and integrity of OXPHOS complexes. These dysfunctions caused mitochondrial dynamic imbalance, thereby increasing fission and decreasing fusion. Excessive fission impaired the process of autophagy including initiation phase, formation, and maturation of autophagosome. Strikingly, the m.4435A>G mutation upregulated the PARKIN dependent mitophagy pathways but downregulated the ubiquitination-independent mitophagy. These alterations promoted intrinsic apoptotic process for the removal of damaged cells. Our findings provide new insights into mechanism underlying deficient tRNA posttranscription modification regulated intracellular signaling related to the mitochondrial and cellular integrity.

Keywords: Cell biology; Molecular physiology; Properties of biomolecules.

Graphical abstract

Figure 1

Western blot analysis of mitochondrial proteins (A and D) Twenty micrograms of total cellular proteins from various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted, and hybridized with antibodies for 17 subunits (CO3 encoded by mtDNA and 16 encoded by nuclear genes) (A) 4 assembly factors (D) of OXPHOS, and TOM20 as a loading control, respectively. (B and E) Quantification of 17 subunits (B) and 4 assembly factors (E) of OXPHOS. Average relative each polypeptide content per cell was normalized to the average content per cell of TOM20 in each cell line. The values for the latter are expressed as percentages of the average values for the control cell line. (C) Average levels of subunits from each complex of OXPHOS (6 of complexes I, 2 of II, 3 of III, 3 of IV, and 3 of V). The calculations were based on three independent determinations. The error bars indicate two standard error of the mean (SEM) of the means. p indicates the significance, according to the t test, of the differences between mutant and control cell lines. ∗p < 0.05; ∗∗p < 0.001; ∗∗∗p < 0.0001; #, not significant.

Figure 2

Defective assembly and activity of OXPHOS complexes (A) The steady-state levels of five OXPHOS complexes by blue native gel electrophoresis. Twenty micrograms of mitochondrial proteins from various cell lines were electrophoresed through a Blue Native gel, electroblotted, and hybridized with antibodies specific for subunits of five OXPHOS complexes (NDUFS2 for complex I, SDHB for complex II, UQCRC2 for complex III, COX5A for complex IV, and ATP5A a for complex V), and with TOM20 as a loading control. (B) Quantification of levels of complexes I, II, III, IV, and V in mutant and control cell lines. The calculations were based on three independent experiments. (C) In-gel activity of complexes I, II, IV, and V. The activities of OXPHOS complexes from various cell lines after BN-PAGE were measured in the presence of specific substrates [NADH and NTB for complex I, sodium succinate, phenazine methosulfate, and NTB for complex II, DAB and cytochrome c for complex IV, glycine, MgSO4, ATP, and Pb(NO3)2 for complex V]. (D) Quantification of in-gel activities of complexes I, II, IV, and V. The calculations were based on three independent determinations in each cell line. Graph details and symbols are explained in the legend to Figure 1.

Figure 3

Assessment of mitochondrial dynamics (A) Immunofluorescence analysis. The distributions of cytochrome c from mutant II-9.5 and control C59.8 cybrids were visualized by immunofluorescent staining with mitochondrial dye MitoTracker and labeling with DRP1 antibody conjugated to Alex Fluor 488 (green) analyzed by confocal microscopy. Scale bars: 10 μm. (B) Quantification of mitochondrial morphology. Mitochondrial morphology was scored as follows: fragmented, mainly small and round; normal, mixture of round and shorter tubulated; and elongated, long and higher interconnectivity. The percentage of cells with indicated mitochondrial morphologies was determined as a percentage of the total number of cells counted (≥100 cells per experiment). n = 3 independent experiments. (C) Quantification of levels of DRP1 fluorescence intensity. Three independent determinations were done in each cell line. (D) Western blot analysis of mitochondrial fission-associated proteins (DRP1, MFF, and FIS1) and fusion-associated proteins (OPA1, MFN1, and MFN2) in six cell lines with β-actin as a loading control. (E) Quantification of mitochondrial fission-associated proteins (DRP1, MFF, and FIS1) and fusion-associated proteins (OPA1, MFN1, and MFN2). Three independent experiments were made for each cell line. Graph details and symbols are explained in the legend to Figure 1.

Figure 4

Analysis of autophagy (A) Immunofluorescence analysis. The distributions of LAMP1 from cybrids (C59.12 and II.9-4) were visualized by immunofluorescent labeling with LAMP1 antibody conjugated to Alex Fluor 488 (green) and LC3 antibody conjugated to Alex Fluor 594 (red) analyzed by confocal microscopy. DAPI-stained nuclei were shown by the blue fluorescence. Scale bars: 10 μm. (B and D) Western blot analysis of autophagy-associated proteins (B) and autophagosome formation and maturation associated proteins (D) in six cell lines with β-actin as a loading control. (C and E) Quantification of markers of autophagy (C) and autophagosome formation and maturation associated proteins (E). Three independent determinations were done in each cell line. (F) Cells from mutant and control cybrids were examined by transmission electron microscopy of initial autophagic vacuoles (white), degradative autophagic vacuole (black); M: mitochondria; N: nucleus; P: phagophore; scale bar: 500 nm. Ultrathin sections were stained with uranyl acetate and alkaline lead citrate. 50,000× magnifications were used. Graph details and symbols are explained in the legend to Figure 1.

Figure 5

Mitophagy assays (A and B) Immunofluorescence assays using live cell imaging under normal or energy starved conditions in the absence (A) and presence (B) of HBSS. The distributions of LC3 from mutant and control cybrids were visualized by GFP-LC3 (green) and immunofluorescent staining with mitochondrial dye MitoTracker (red) analyzed by confocal microscopy. Scale bars: 10 μm. (C and D) Immunofluorescence analysis. The distributions of PARKIN and BNIP3L from mutant and control cybrids were visualized by immunofluorescent staining with mitochondrial dye MitoTracker (red) and labeling with PARKIN (C), and BNIP3L (D) antibody conjugated to Alex Fluor 488 (green) analyzed by confocal microscopy. (E and F) Western blot analysis of PARKINdependent mitophagy proteins (E) and ubiquitination-independent mitophagy proteins (F) in six cell lines with β-actin as a loading control.

Figure 6

Upregulated intrinsic apoptosis (A) Annexin V/PI apoptosis assay by flow cytometry. Cells were harvested and stained with Annexin V and 1 μL of propidium iodide. The percentage of Annexin V-positive cells were assessed. (B) Relative Annexin V-positive cells from various cell lines. Three independent determinations were done in each cell line. (C) Immunofluorescence analysis. The distributions of cytochrome c from mutant II-9.2 and control C59.8 cybrids were visualized by immunofluorescent staining with mitochondrial dye MitoTracker and labeling with cytochrome c antibody conjugated to Alex Fluor 488 (green) analyzed by confocal microscopy. DAPI stained nuclei were identified by their blue fluorescence. Scale bars: 10 μm. (D) The levels of cytochrome c in cytosol in mutant and control cell lines were measured by fractioning the cells into mitochondrial and cytosolic fractions and western blot analysis using cytochrome c, Tom20 for mitochondrial protein and Vinculin for cytosolic protein. Total, total cell lysate; Cyto, cytosol; Mito, mitochondria. (E) Western blotting analysis of apoptosis-associated proteins. Total cellular proteins (20 μg) from various cell lines were electrophoresed, electroblotted and hybridized with several apoptosis-associated protein antibodies: cytochrome c, Bcl-xL, BAD, BAX, uncleaved caspases 9, caspases 3 and caspases 7, and cleaved caspases 3, 7, and 9, with β-actin as a loading control. (F) Quantification of apoptosis-associated proteins: cytochrome c, Bcl-xL, BAD, BAX, uncleaved caspases 3, 7, and 9, and cleaved caspases 3, 7, and 9. The levels of apoptosis-associated proteins in various cell lines were determined as described elsewhere. Three independent experiments were made for each cell line. Graph details and symbols are explained in the legend to Figure 1.