Multidimensional Proteomics Identifies Declines in Protein Homeostasis and Mitochondria as Early Signals for Normal Aging and Age-associated Disease in Drosophila

Abstract

Aging is characterized by a gradual deterioration in proteome. However, how protein dynamics that changes with normal aging and in disease is less well understood. Here, we profiled the snapshots of aging proteome in Drosophila, from head and muscle tissues of post-mitotic somatic cells, and the testis of mitotically-active cells. Our data demonstrated that dysregulation of proteome homeostasis, or proteostasis, might be a common feature associated with age. We further used pulsed metabolic stable isotope labeling analysis to characterize protein synthesis. Interestingly, this study determined an age-modulated decline in protein synthesis with age, particularly in the pathways related to mitochondria, neurotransmission, and proteostasis. Importantly, this decline became dramatically accelerated in Pink1 mutants, a Drosophila model of human age-related Parkinson's disease. Taken together, our multidimensional proteomic study revealed tissue-specific protein dynamics with age, highlighting mitochondrial and proteostasis-related proteins. We suggest that declines in proteostasis and mitochondria early in life are critical signals prior to the onset of aging and aging-associated diseases.

Keywords: Drosophila melanogaster; aging; mitochondria function or biology; protein synthesis; proteome homeostasis; quantification.

Graphical abstract

Fig. 1.

Tissue-specific quantitative proteomic analysis of aging in WT Drosophila. A, Experimental scheme of quantitative aging proteome analysis. Three tissues including the post-mitotic head and muscle tissues, and the mitotic testis, were harvested at three different age time points. A tandem mass tag (TMT)-based quantitative method was employed. For each analysis, nine samples derived from three aging time points with three biological replicates for each tissue were labeled with different TMT reagents, mixed, and analyzed by the shotgun method. B, Volcano plots of tissue-specific quantitative aging proteomes between 5d and 60d. The numbers of significantly regulated proteins were indicated on the plots. C, Venn plot of proteins with significantly changed abundance (p value < 0.05) of three tissues. D, Pathway analysis for proteins with significantly changed abundance. The enriched pathways were shown for either tissue-specific proteins or proteins present in more than one tissues.

Fig. 2.

Tissue-specific functional analysis of aging in WT Drosophila. A, Clustering of proteins using the fuzzy c-means method according to their abundance changes during the aging process. The proteins localized in the middle cluster were generally stable. In contrast, the proteins in cluster 1 and cluster 5 changed dramatically with age, whereas proteins in cluster 2 and cluster 4 changed mildly. B, Pathway analysis for proteins in different clusters. Many tissue-specific and common pathways were identified for proteins in different clusters.

Fig. 3.

Protein synthesis decreases during aging in Drosophila. A, Experimental scheme of quantitative proteomic synthesis. Wild-type, Pink1^B9 mutant or Pcl^c421/+;Su(z)12^c253 double mutant flies at different age time points were fed heavy stable isotope labeled lysine food for a fixed 5 d period. The newly synthesized proteins during this labeling period incorporated the heavy isotope lysine, thus, the percentage of each protein containing heavy lysine reflected its synthesis rate. B, Protein synthesis decreases during aging in Drosophila. The proportions of heavy isotope labeled proteins reflected protein synthesis rates at a given aging time point. A median turnover rate decreased from ∼30%/5 d at day 5 to ∼16%/5 d at day 60. (The median values were represented at the center) C, Clustering of proteins with different synthesis change trends. Only a small fraction of proteins in cluster 1 exhibited slightly increased synthesis with age. D, Pathway analysis for proteins in different clusters.

Fig. 4.

Factors that may impact the protein synthesis rate. A, Pearson correlation between protein abundance changes and protein synthesis changes. The respective ratios between day 5 and day 60 were compared. B, Protein synthesis affects protein abundance. Proteins in clusters 1 and 2 of Fig. 2A showed decreased abundance, and proteins belonging to clusters 4 and 5 in Fig. 2A showed increased abundance. Proteins in cluster 3 in Fig. 2A had stable abundance. C, Protein complexes stabilize protein synthesis. The proteins in cluster 2 exhibited the most stable synthesis during aging. A total of 37% of proteins in the cluster 2 were associated with a protein complex. D, Protein coexpression stabilizes protein synthesis. The proteins in cluster 2 were more likely to be coexpressed with other proteins. The median values were marked at the center.

Fig. 5.

Early-onset collapse of protein synthesis in Pink1 mutant. A, PCA analysis of proteomic synthesis data of both WT and Pink1^B9 mutant flies. B, Comparison of protein synthesis of WT and Pink1^B9 mutant flies of different ages. The most dramatic difference was observed on day 5. C, Proteostasis and mitochondrial function are affected in the Pink1^B9 mutant. The EIF2 pathway was a representative pathway in protein synthesis. The most significant difference was observed on day 5. The median values were marked at the center. Two-side student t-tests were used to compare WT and Pink1^B9 mutant (B, C).

Fig. 6.

Protein translation shows different changes with shrinkage or extension of the lifespan. A, Volcano plot and functional analysis of protein synthesis between 5 d WT and Pink1^B9 mutant flies. Many proteins in the Pink1^B9 mutant had decreased protein synthesis compared with the WT levels, and these proteins functioned in proteostasis and mitochondria (Fisher's exact test). B, Protein synthesis is deceased in both aging and Pink1^B9 mutant flies for some specific pathways. Proteins in clusters 2, 3 and 4 of Fig. 3C showed decreased protein synthesis with aging. C, Volcano plot of protein synthesis between 3 d WT and Pcl^c421/+;Su(z)12^c253 mutant flies.