Proteome-wide observation of the phenomenon of life on the edge of solubility

Abstract

To function effectively proteins must avoid aberrant aggregation, and hence they are expected to be expressed at concentrations safely below their solubility limits. By analyzing proteome-wide mass spectrometry data of Caenorhabditis elegans, however, we show that the levels of about three-quarters of the nearly 4,000 proteins analyzed in adult animals are close to their intrinsic solubility limits, indeed exceeding them by about 10% on average. We next asked how aging and functional self-assembly influence these solubility limits. We found that despite the fact that the total quantity of proteins within the cellular environment remains approximately constant during aging, protein aggregation sharply increases between days 6 and 12 of adulthood, after the worms have reproduced, as individual proteins lose their stoichiometric balances and the cellular machinery that maintains solubility undergoes functional decline. These findings reveal that these proteins are highly prone to undergoing concentration-dependent phase separation, which on aging is rationalized in a decrease of their effective solubilities, in particular for proteins associated with translation, growth, reproduction, and the chaperone system.

Keywords: protein aggregation; protein homeostasis; protein misfolding diseases.

Fig. 1.

Comparison between the cellular concentrations and the critical concentrations of proteins in adult C. elegans. Density plot of the total abundance (T) and soluble abundance (S), in logarithmic scale, for the 1,163 proteins quantified as at least at their solubility limits (Materials and Methods). Each point is a protein colored from a heat map scale (black to yellow) according to the density of neighboring points, where black indicates an isolated protein, corresponding to a density value close to 0, while yellow indicates a proteins that is surrounded by many others in that area, corresponding to a density value close to 1. (Inset) The density values are obtained with a standard Gaussian kernel density estimator and are reported in 3D. The gray bisector line in the scatterplot corresponds to the solubility limit. Protein IDs are indicated for the proteins found to be further from the solubility limit. These proteins are intermediate filaments proteins, collagen, and 2 uncharacterized proteins (Q9NES7, O02141).

Fig. 2.

Variations in total and aggregated cellular proteome mass on aging in C. elegans. The sums of the relative abundances of cellular proteins were determined at various points with respect to day 1 of adulthood. (A) Cellular total load variation on aging; 3,078 proteins were detected and quantified in the total fraction at all of the points shown. (B) Cellular aggregate load variation on aging; 965 proteins were detected and quantified in the insoluble fraction at all points measured. (C) Corresponding cellular total load variation on aging calculated only for the subset of proteins detectable in pellets fraction (965 proteins). Errors were calculated with a bootstrap method. Bar plots are colored according to the proteins involved in the calculation: black for the 3,078 proteins and red for the 965 proteins.

Fig. 3.

Proteins aggregating in aging worms are prone to undergo liquid–liquid phase separation. (A) Proteins with low complexity regions in age-dependent aggregates. A total of 73% of the proteins found forming aggregates from days 1 to 12 (Fig. 2B) have at least one low complexity region compared with 67% in the total aging proteome (Fig. 2A), resulting in a relative increase of aggregated compared with the rest of the proteome of 14.2%, with a P value < 10⁻⁸ (Fisher exact test). (B) Boxplot of the distribution of propensities of granule formation for the set of proteins forming aggregates from days 1 to 12 (dark red) compared with the total intracellular aging proteome (dark gray). P value calculated with median test, ****P < 10⁻⁴.

Fig. 4.

Functional analysis of proteins that aggregates in aging worms. Functional annotation of the proteins forming aggregates shown in Fig. 2B. Bar plot of functional terms of gene ontology biological processes (GO BP), cellular components (GO CC), molecular functions (GO MF), of protein families and domains (INTERPRO) and of KEGG pathways, with number of protein members, fold enrichment, and significant Bonferroni-corrected P values (stars) for the 965 cellular proteins forming aggregates from days 1 to 12 of adulthood. Functional terms have been colored according to 5 major groups: translation-related terms (green), reproduction and embryo development (pink), cell-cycle (orange), metabolism (blue), and remaining terms (gray). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 10⁻⁴. The number of proteins belonging to the given term is shown on the right of the bar plot. The 3,078 cellular proteins (Fig. 2A) quantified from days 1 to 12 were used as the background for enrichment calculations.