Proteomic shifts in embryonic stem cells with gene dose modifications suggest the presence of balancer proteins in protein regulatory networks

Abstract

Large numbers of protein expression changes are usually observed in mouse models for neurodegenerative diseases, even when only a single gene was mutated in each case. To study the effect of gene dose alterations on the cellular proteome, we carried out a proteomic investigation on murine embryonic stem cells that either overexpressed individual genes or displayed aneuploidy over a genomic region encompassing 14 genes. The number of variant proteins detected per cell line ranged between 70 and 110, and did not correlate with the number of modified genes. In cell lines with single gene mutations, up and down-regulated proteins were always in balance in comparison to parental cell lines regarding number as well as concentration of differentially expressed proteins. In contrast, dose alteration of 14 genes resulted in an unequal number of up and down-regulated proteins, though the balance was kept at the level of protein concentration. We propose that the observed protein changes might partially be explained by a proteomic network response. Hence, we hypothesize the existence of a class of "balancer" proteins within the proteomic network, defined as proteins that buffer or cushion a system, and thus oppose multiple system disturbances. Through database queries and resilience analysis of the protein interaction network, we found that potential balancer proteins are of high cellular abundance, possess a low number of direct interaction partners, and show great allelic variation. Moreover, balancer proteins contribute more heavily to the network entropy, and thus are of high importance in terms of system resilience. We propose that the "elasticity" of the proteomic regulatory network mediated by balancer proteins may compensate for changes that occur under diseased conditions.

Figure 1. Representative protein expression pattern of mouse embryonic cell lines as revealed by large-gel 2D-electrophoresis.

Over 5500 proteins (including protein isoforms) were resolved on a single gel. Highlighted spots correspond to spot ID of candidate balancer proteins detailed in Table 2.

Figure 2. Proteins that showed altered expression in transgenic ES cell lines.

(A) Number of altered proteins in each transgenic cell line, expressed as percentile of total number of altered proteins. (B) Amount of proteins that underwent altered expression in each cell line, represented as percent of total spot volume that was up or down-regulated in transgenic cell lines. Dose alteration of 14 genes could no longer be balanced by an equivalent number of variant proteins. However, a balance remained at the level of protein concentration.

Figure 3. Comparison of observed number of co-changed proteins against a theoretical calculation of co-changed proteins across six different transgenic cell lines.

It was assumed that a total of 800 protein spots were investigated, among which 10% of the proteins change in their expression profile. This comparison shows that the occurrence of the same protein alteration in more than three cell lines is unlikely to be coincidental.

Figure 4. Cumulative fraction plots of “balancer”-“cell line-specific protein” comparison.

(A) Entropic contribution.; (B) Number of direct protein interaction partners. Compared to cell line-specific proteins, balancers possess significantly higher values of entropic contribution and a low number of direct interaction partners.

Figure 5. A protein-protein interaction subgraph showing the proteasome subunits, where nodes denote proteins and the edges describe protein-protein interaction.

Two local hub proteins of this subgraph (Psma2 and Psma3) belong to cell line-specific proteins, while a candidate balancer protein (Psmb6) represents a connection between these two modules (see discussion for details). This supports our assumption that balancer proteins could be connective hubs between different modules. Protein marked in green: Psma2; yellow: Psmb6; magenta: Psma3.