Interactions among mitochondrial proteins altered in glioblastoma

Abstract

Mitochondrial dysfunction is putatively central to glioblastoma (GBM) pathophysiology but there has been no systematic analysis in GBM of the proteins which are integral to mitochondrial function. Alterations in proteins in mitochondrial enriched fractions from patients with GBM were defined with label-free liquid chromatography mass spectrometry. 256 mitochondrially-associated proteins were identified in mitochondrial enriched fractions and 117 of these mitochondrial proteins were markedly (fold-change ≥ 2) and significantly altered in GBM (p ≤ 0.05). Proteins associated with oxidative damage (including catalase, superoxide dismutase 2, peroxiredoxin 1 and peroxiredoxin 4) were increased in GBM. Protein-protein interaction analysis highlighted a reduction in multiple proteins coupled to energy metabolism (in particular respiratory chain proteins, including 23 complex-I proteins). Qualitative ultrastructural analysis in GBM with electron microscopy showed a notably higher prevalence of mitochondria with cristolysis in GBM. This study highlights the complex mitochondrial proteomic adjustments which occur in GBM pathophysiology.

Fig. 1

Overview of proteomic data from mitochondrial fractions. Hierarchical clustering of the 902 proteins (normalised protein intensities) detected by LC–MS in mitochondrial fractions of GBM (T) and peritumoural control brain (C). Each column (in greyscale) represents the proteomic profile (intensities of the 902 proteins) in a single sample. Protein intensities were extracted from Progenesis software. The dendrogram (x-axis) provides a visual representation of sample–sample correlations, with correlated samples grouped in branches. Note there are two main branches to this dendrogram which precisely correspond to the two experimental groupings GBM and peritumoural control. The data highlight that there are global differences in the mitochondrial enriched proteome in GBM compared to peritumoural control. Illustrative changes in 3 proteins associated with oxidative damage (catalase, peroxiredoxin 1 and glutathione peroxidase 4) and 3 proteins associated with the Electron Transport Chain (NDUFA4, NDUFB10 and NDUFV3) in GBM relative to peritumoural controls. Each point represents an individual patient

Fig. 2

Interactome analysis of mitochondrial proteins altered in GBM. Putative localization of many of the proteins altered in GBM within the mitochondria (based upon the mitochondrial canonical pathway in Ingenuity Pathway Analysis (IPA); www.ingenuity.com). Note particularly the large number of proteins that are significantly less abundant in green (p < 0.05 and a ratio GBM/control <0.5) in GBM which are localised to the Electron Transport Chain Complex 1 (23 proteins). Proteins highlighted in red are significantly more abundant in GBM. Protein–protein interactions between mitochondrial proteins altered in GBM. Each node (shape) represents a protein and its association with other proteins is represented by a line. Nodes have different shapes that represent different molecule types, for example transcription factors, enzymes, kinases and phosphatases (refer to Ingenuity Systems Software for detailed node information). A high scoring interactome generated by IPA with the nuclear transcription factor HNF4A as an inserted hub protein (no colour). HNF4A linking 10 of the proteins increased (red) in GBM and 5 of the proteins decreased (green) in GBM. For details of all of the protein–protein interactomes generated by IPA from the 117 mitochondrial proteins altered, see supplementary information S6 (protein lists) and S7 (diagrammatic representations of networks)

Fig. 3

Morphology of mitochondria in GBM. The morphology of ~150 mitochondria was assessed in each of 6 GBM and 7 peritumoural control samples using Electron Microscopy (EM). a Percentage of normal mitochondria (i.e. where cristae are visible throughout the mitochondria, or in at least 50 % of the mitochondrial interior area) in peritumoural control and GBM samples (each bar represents one sample; *** p-value = 0.0001); b Percentage of abnormal mitochondria (i.e. with very few cristae, interior matrix condensed and dark or round swollen with interior missing) in peritumoural control and GBM samples (please see supplementary information S3 for more details; *** p-value = 0.0001). c, d Representative EM images of normal and abnormal mitochondria respectively. The scale bars represent 0.5 μm