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. 2019 Dec 3;7(1):197.
doi: 10.1186/s40478-019-0853-9.

A metastable subproteome underlies inclusion formation in muscle proteinopathies

Prajwal Ciryam  1 , Matthew Antalek  2 , Fernando Cid  3 , Gian Gaetano Tartaglia  3 , Christopher M Dobson  4 , Anne-Katrin Guettsches  5 , Britta Eggers  6 , Matthias Vorgerd  5 , Katrin Marcus  6 , Rudolf A Kley  7 , Richard I Morimoto  2 , Michele Vendruscolo  4 , Conrad C Weihl  8
Affiliations

A metastable subproteome underlies inclusion formation in muscle proteinopathies

Prajwal Ciryam et al. Acta Neuropathol Commun. .

Abstract

Protein aggregation is a pathological feature of neurodegenerative disorders. We previously demonstrated that protein inclusions in the brain are composed of supersaturated proteins, which are abundant and aggregation-prone, and form a metastable subproteome. It is not yet clear, however, whether this phenomenon is also associated with non-neuronal protein conformational disorders. To respond to this question, we analyzed proteomic datasets from biopsies of patients with genetic and acquired protein aggregate myopathy (PAM) by quantifying the changes in composition, concentration and aggregation propensity of proteins in the fibers containing inclusions and those surrounding them. We found that a metastable subproteome is present in skeletal muscle from healthy patients. The expression of this subproteome escalate as proteomic samples are taken more proximal to the pathologic inclusion, eventually exceeding its solubility limits and aggregating. While most supersaturated proteins decrease or maintain steady abundance across healthy fibers and inclusion-containing fibers, proteins within the metastable subproteome rise in abundance, suggesting that they escape regulation. Taken together, our results show in the context of a human conformational disorder that the supersaturation of a metastable subproteome underlies widespread aggregation and correlates with the histopathological state of the tissue.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
A protein aggregates when its concentration exceeds its solublity, thus becoming supersaturated. Supersaturated proteins tend to be abundantly expressed despite having a relatively low solubility. Disruptions in the protein homostasis system favor protein aggregate formation whereas an enhancement in protein homeostasis favors protein solubility (dashed lines). The translational repression of abundant aggregation-prone proteins favors a non-aggregated proteomic state. Protein inclusions in diseased tissue are heterogenous and composed of multiple proteins that have exceeded their solubility threshold
Fig. 2
Fig. 2
Proteins in rimmed vacuoles from protein aggregation myopathies are supersaturated. Representative images of: (a) healthy control myofibers (HC), control unaffected myofibers in diseased samples (DC), surrounding tissues of affected fibers (AF), and rimmed vacuoles (RV) from human subjects with inclusion body myositis, and (c) DC and AF samples from human subjects with myotilin mutations. Outlines represent areas for LMD. In (c), prior to LMD, muscle was immunostained with an antibody directed to myotilin (green) to identify aggregate containing fibers (AF). b, d, e Comparison of the unfolded supersaturation scores (σu) of the proteome (Prt) (N = 15,954) and (b) proteins enriched in RVs (RV) (N = 50), amyloid plaques (Plq) (N = 26), neurofibrillary tangles (NFT) (N = 76), proteins found in TDP-43 inclusions (TDP) (N = 32); (d) proteins enriched in affected fibers from any of three protein aggregation myopathies (hPAM) (N = 50); and (e) proteins enriched in affected fibers from individual protein aggregation myopathies involving desmin (Desm) (N = 6), filamin (Fil) (N = 16), and myotilin (Myot) (N = 46) mutations. f, g Comparison of the folded supersaturation scores (σf) for the proteome (Prt) (N = 1605) and (f) the proteins enriched in affected fibers from any of three protein aggregation myopathies (hPAM) (N = 46) and (g) the proteins enriched in affected fibers from individual protein aggregation myopathies involving desmin (Desm) (N = 5), filamin (Fil) (N = 15), and myotilin (Myot) (N = 43) mutations. The fold change (Δ) represents the fold difference in the median σu or σf scores between each inclusion type and the proteome. The median σu or σf supersaturation score for the proteome is normalized to 0. Boxes range from the 25th percentile to 75th percentile, while whiskers extend to maximum and minimum data points up to 1.5x interquartile range above and below the limits of the boxes. Remaining outliers are plotted as open circles. Statistical significance determined by one-tailed Wilcoxon/Mann-Whitney test with Holm-Bonferroni correction. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
Fig. 3
Fig. 3
Protein supersaturation in hereditary protein aggregate myopathies. Comparison of folded supersaturation scores (σf) between the proteome and proteins enriched in aggregate-containing myofibers (AF) for: (a, b, c) desminopathy (DC: Prt N = 611, Agg N = 6; AF: Prt N = 1387, Agg N = 6), (d, e, f) filaminopathy (DC: Prt N = 333, Agg N = 16; AF: Prt N = 507, Agg N = 16), and (g, h, i) myotillinopathy (DC: Prt N = 680, Agg N = 46; AF: Prt N = 742, Agg N = 46). Box plots and statistical tests as in Fig. 1. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
Fig. 4
Fig. 4
Escalating supersaturation in inclusion body myositis. Comparison of folded supersaturation scores (σf) for the proteome (Prt) and proteins enriched in rimmed vacuoles (RV) relative to diseased control myofibers. a Healthy control myofiber (HC) (Prt N = 1605, RV N = 47), b control myofibers unaffected in diseased samples (DC) (Prt N = 1988, RV N = 52), c aggregate-containing affected myofibers (AF) (Prt N = 2396, RV N = 52), and (d) rimmed vacuoles (RV) (Prt N = 2104, RV N = 52). e Comparison of the fold difference in median σf between RV and Prt. Box plots and statistical tests as in Fig. 1. *p < 0.05, ***p < 0.001, ****p < 0.0001
Fig. 5
Fig. 5
Protein supersaturation is associated with downregulation. In this analysis, only proteins that are detected in HC, DC, AF, and RV, and for which there are defined σf scores in HC are included. a Folded supersaturation scores (σf) for the proteome (Prt) (N = 830) and proteins downregulated from HC to AF (N = 50). Box plots and statistical tests as in Fig. 1. b Percentage of proteins downregulated in the proteome (Prt) (48/830), proteins enriched in rimmed vacuoles (RV) (0/47), and top 5% most supersaturated proteins (based on HC context) (Top σf) (7/41). Significance determined by the Fisher Exact test, with Holm-Bonferroni correction. c Protein abundances in HC, DC, AF, and RV are plotted for the 47 proteins enriched in RVs included in the subset analyzed in this figure. Desmin is highlighted in yellow, the only RV-enriched protein whose abundance is increased between HC and AF. d Protein abundances in HC, DC, AF, and RV are plotted for the 47 proteins with the highest supersaturation scores (top 5%). Desmin again is highlighted in yellow, the only highly supersaturated protein with rising abundance. Those proteins that are significantly downregulated between HC and AF are highlighted in blue. In (c) and (d), the background black line and grey bar represent median and 25th–75th percentile range for the 830 proteins in the proteome in this subset. *p < 0.05, ****p < 0.0001
Fig. 6
Fig. 6
Escalating supersaturation of an aggregation-prone subproteome puts affected fibers at risk of inclusion formation in inclusion body myositis. 1) Supersaturation of the aggregate proteome increases to the point of aggregate formation at muscle inclusion bodies (gray regions). 2) The most highly supersaturated proteins decrease in abundance upon approaching the inclusion body. In contrast, the abundance of the aggregate/RV enriched supersaturated proteome increases and escapes downregulation
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