Proteomic Analysis of m.8296A>G Variation in the Mitochondrial tRNA Lys Gene

Abstract

Variation in the mitochondrial tRNA ^Lys gene at position 8296 was previously found to be associated with maternally inherited diabetes mellitus and deafness, hypertrophic cardiomyopathy, myoclonic epilepsy with ragged-red fibers and mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes. The pathogenicity of the m.8296A>G variation is unclear. In this study, we aimed to analyze the mitochondrial proteome in a patient with m.8296A>G variation to elucidate the effects of this mutation at the protein level. Whole-exome sequencing and mitochondrial genome analysis were performed in a patient with sensorineural hearing impairment, cognitive impairment, leukodystrophy, migraine-like headaches, and gastrointestinal dysmotility. Mitochondrial genome analysis identified a homoplasmic m.8296A>G variation in the mitochondrial tRNA ^Lys gene in the proband and unaffected mother. Global mitochondrial proteome analysis was carried out in the muscle mitochondria of the index patient and a control subject. Comparative muscle mitochondrial proteome analysis revealed a total of 13 nuclear-encoded mitochondrial proteins differently expressed with respect to the control. Ten of the 13 proteins were downregulated. Most of the proteins were involved in ATP synthesis and Krebs cycle and have strong interactions with each other. We considered the m.8296A>G variation to be pathogenic with variable penetrance for our patient's phenotype, and this variation led to different expressions of nuclear-encoded proteins involved in energy metabolism.

Keywords: A8296G mutation; Leukodystrophy; Mitochondrial proteome; Muscle mitochondria; Sensorineural hearing impairment.

Fig. 1

Cranial MRI of the patient. a T2-weighted axial image. b T2-weighted sagittal image. c Fluid-attenuated inversion recovery (FLAIR) sequence. d T1-weighted image with gadolinium injection. There are symmetrical lesions (hyperintense in the T2-weighted and FLAIR sequence and hypointense in the T1-weighted image, without contrast enhancement) of the white matter with parieto-occipital predominance (arrows). Cerebellar white matter is spared.

Fig. 2

Levels of oxidative phosphorylation (OXPHOS) complexes. Fibroblast mitochondria were prepared from the control (C) and the patient (P). Mitochondria underwent Western blotting and were probed with an mAb against OXPHOS complexes I, II, III, IV, and V, which are components of the inner membrane, and Hsp60 and β-actin (as controls for equal loading). a Representative Western blot analysis. The levels of complex I (NDUFB8), complex II (SDHB), and complex IV (MTCO1) were reduced in the patient compared with the control (100%). b Relative amounts of the respiratory complexes in the mitochondrial fractions of the samples (the y axis shows the % of band intensities). The patient had 45, 46, and 39% less respiratory complexes I, II, and III, respectively. The results are normalized to control mitochondria.

Fig. 3

Comparative proteome analysis of the patient and the control. Muscle biopsy samples were subjected to mitochondrial protein isolation and loaded onto IPG strips at pH 3 to pH 10 for the first dimension and 12% SDS-PAGE gels for the separation of the second dimension and stained with colloidal Coomassie blue for 24 h after fixation for 24 h. The gel is representative for the selected protein spots and 13 were identified. The gel image was created using PDQuest Advance software to determine the number of spots that differentiated every member. The numbers are the numbers of the identified protein assigned to each spot by the researchers. These spots were cut from the gels and were subjected to MALDI-TOF/TOF analysis. Protein identification was performed by peptide mass fingerprinting by MASCOT.

Fig. 4

Classification of the proteins that were identified by MALDI-TOF/TOF analysis. The pie charts present the distribution of the 13 proteins identified based on their molecular function, biological processes, cellular component, protein class, and pathway. Assignments were made based on information from PANTHER analysis (http://www.pantherdb.org/) as well as NCBI (http://www.ncbi.nlm.nih.gov/pubmed) and Swiss-Prot/TrEMBL annotations (http://www.expasy.org/).

Fig. 5

String interaction analysis (http://string-db.org/; version 9.1) of the proteins identified in the MALDI-TOF/TOF analysis [Szklarczyk et al., 2019].