Network clustering revealed the systemic alterations of mitochondrial protein expression

Abstract

The mitochondrial protein repertoire varies depending on the cellular state. Protein component modifications caused by mitochondrial DNA (mtDNA) depletion are related to a wide range of human diseases; however, little is known about how nuclear-encoded mitochondrial proteins (mt proteome) changes under such dysfunctional states. In this study, we investigated the systemic alterations of mtDNA-depleted (ρ(0)) mitochondria by using network analysis of gene expression data. By modularizing the quantified proteomics data into protein functional networks, systemic properties of mitochondrial dysfunction were analyzed. We discovered that up-regulated and down-regulated proteins were organized into two predominant subnetworks that exhibited distinct biological processes. The down-regulated network modules are involved in typical mitochondrial functions, while up-regulated proteins are responsible for mtDNA repair and regulation of mt protein expression and transport. Furthermore, comparisons of proteome and transcriptome data revealed that ρ(0) cells attempted to compensate for mtDNA depletion by modulating the coordinated expression/transport of mt proteins. Our results demonstrate that mt protein composition changed to remodel the functional organization of mitochondrial protein networks in response to dysfunctional cellular states. Human mt protein functional networks provide a framework for understanding how cells respond to mitochondrial dysfunctions.

Figure 1. Analysis of the human mt proteomics data.

(A) Reliability evaluation of mt proteomics data and functional module identification. (B) Compositions of the mt proteins from the cICAT proteomics data. (C) Distributions of protein abundance ratios (ρ⁰/ρ⁺) in reliable mt proteins. Down-regulated, not significantly changed, and up-regulated proteins were shown.

Figure 2. Human mt protein functional network.

(A) Global functional network of human mt proteins. Nodes are color coded according to the ρ⁰/ρ⁺ ratio. Green and red nodes represent up-regulated mt proteins and down-regulated mt proteins under the dysfunctional ρ⁰ state, respectively. (B) Number of links according to the link types. An intraregulatory link is a link between proteins with the same regulatory pattern: up- and up-regulated or down- and down-regulated proteins. An interregulatory link is a link between up- and down-regulated proteins. (C) The fraction of link types per single protein. (D) The shortest path length according to the link types.

Figure 3. Thirteen significantly changed functional modules under mtDNA-depleted dysfunctional state.

(A) Five up-regulated functional modules (green boxes) and eight down-regulated functional modules (red boxes) were shown. (B) Distributions of protein abundance ratios in five up-regulated functional modules. (C) Distributions of protein abundance ratios in eight down-regulated functional modules.

Figure 4. Mitochondrial proteome-transcriptome profiles in the dysfunctional ρ⁰ state.

(A) Expression patterns of mt proteins and mRNAs. As abundance ratios of proteins and mRNAs increased, the colors changed to blue (mt protein) and yellow (mRNA). (B) Box-plots of protein and mRNA abundance ratios for the up-regulated functional modules. Abundance ratios of protein and mRNA were colored as blue and light blue, respectively. The error bars indicate the standard deviations of protein and mRNA abundance ratios. The black dots represent the average protein and mRNA abundance ratios. (C) Box-plots of protein and mRNA abundance ratios for the down-regulated functional modules.

Figure 5. Validations of expression changes of mt proteins.

Expression of (A) eight up-regulated proteins and (B) nine down-regulated proteins from the different functional modules were examined. Mitochondrial lysates (10 µg) were resolved using 12% SDS-PAGE and analyzed by western blot. Numbers in parenthesis are the protein abundance ratios. β-actin served as a loading control.

Figure 6. Validating mitochondrial localizations of mt proteins.

SK-Hep1 cells expressing DsRed2-mito were transfected with GFP-hybrid plasmids of ZCD1, GPT2, PYCR2, CTSD, and HSPBP1. The transfected cells were fixed, mounted, and imaged using a confocal microscope. The functional module of each protein is presented on the top. Merged images of EGFP and DsRed signals represent the mt localization of the proteins. Scale bar = 10 µm.