Post-Translational Deimination of Immunological and Metabolic Protein Markers in Plasma and Extracellular Vesicles of Naked Mole-Rat (Heterocephalus glaber)

Abstract

Naked mole-rats are long-lived animals that show unusual resistance to hypoxia, cancer and ageing. Protein deimination is an irreversible post-translational modification caused by the peptidylarginine deiminase (PAD) family of enzymes, which convert arginine into citrulline in target proteins. Protein deimination can cause structural and functional protein changes, facilitating protein moonlighting, but also leading to neo-epitope generation and effects on gene regulation. Furthermore, PADs have been found to regulate cellular release of extracellular vesicles (EVs), which are lipid-vesicles released from cells as part of cellular communication. EVs carry protein and genetic cargo and are indicative biomarkers that can be isolated from most body fluids. This study was aimed at profiling deiminated proteins in plasma and EVs of naked mole-rat. Key immune and metabolic proteins were identified to be post-translationally deiminated, with 65 proteins specific for plasma, while 42 proteins were identified to be deiminated in EVs only. Using protein-protein interaction network analysis, deiminated plasma proteins were found to belong to KEEG (Kyoto Encyclopedia of Genes and Genomes) pathways of immunity, infection, cholesterol and drug metabolism, while deiminated proteins in EVs were also linked to KEEG pathways of HIF-1 signalling and glycolysis. The mole-rat EV profiles showed a poly-dispersed population of 50-300 nm, similar to observations of human plasma. Furthermore, the EVs were assessed for three key microRNAs involved in cancer, inflammation and hypoxia. The identification of post-translational deimination of critical immunological and metabolic markers contributes to the current understanding of protein moonlighting functions, via post-translational changes, in the longevity and cancer resistance of naked mole-rats.

Keywords: extracellular vesicles (EVs); immunity; metabolism; miR155; miR210); microRNA (miR21; naked mole-rat (Heterocephalus glaber); peptidylarginine deiminases (PADs); protein deimination.

Figure 1

Peptidylarginine deiminases (PADs) and deiminated proteins in naked mole-rat plasma and plasma-extracellular vesicles (EVs). (A) PAD positive bands were identified at the expected size of approximately 70–75 kDa using the human PAD2, PAD3 and PAD4 specific antibodies in naked mole-rat plasma. (B) Total deiminated proteins were identified in naked mole-rat plasma (n = 4) using the F95 pan-deimination specific antibody. (C) Total deiminated proteins were identified in naked mole-rat plasma-EVs using the F95 pan-deimination specific antibody (EV pools from plasma of 4 individuals are shown, respectively). (D) The F95-enriched IP fraction from mole-rat plasma (from a pool of 5 individual mole-rat plasma; F95_IP) is shown. The molecular weight marker is indicated next to each blot.

Figure 2

Deiminated proteins identified in naked mole-rat plasma and plasma-EVs. Species specific hits identified for deiminated proteins in naked mole-rat plasma and EVs showed 112 total proteins identified in plasma and 80 in EVs, respectively. Of these, 48 protein hits were overlapping, while 64 proteins were specific for whole plasma and 32 for plasma-EVs only, respectively.

Figure 3

Protein-protein interaction networks of deiminated proteins identified in naked mole-rat plasma. Reconstruction of protein-protein interactions based on known and predicted interactions using STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) analysis. (A) Coloured nodes represent query proteins and first shell of interactors. (B) KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways relating to the identified proteins and reported in STRING are highlighted as follows: red = complement and coagulation cascade; dark green = Staphylococcus aureus infection; purple = pertussis; yellow = platelet activation; light blue = systemic lupus erythematosus (SLE); orange = prion diseases; dark blue = cholesterol metabolism; light green = vitamin digestion and absorption; dark red = ferroptosis; pink = drug metabolism. Coloured nodes represent query proteins and first shell of interactors; white nodes are second shell of interactors. Coloured lines indicate whether protein interactions are identified via known interactions (curated databases, experimentally determined), predicted interactions (gene neighbourhood, gene fusion, gene co-occurrence) or via text mining, co-expression or protein homology (see the colour key for connective lines included in the figure).

Figure 3

Protein-protein interaction networks of deiminated proteins identified in naked mole-rat plasma. Reconstruction of protein-protein interactions based on known and predicted interactions using STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) analysis. (A) Coloured nodes represent query proteins and first shell of interactors. (B) KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways relating to the identified proteins and reported in STRING are highlighted as follows: red = complement and coagulation cascade; dark green = Staphylococcus aureus infection; purple = pertussis; yellow = platelet activation; light blue = systemic lupus erythematosus (SLE); orange = prion diseases; dark blue = cholesterol metabolism; light green = vitamin digestion and absorption; dark red = ferroptosis; pink = drug metabolism. Coloured nodes represent query proteins and first shell of interactors; white nodes are second shell of interactors. Coloured lines indicate whether protein interactions are identified via known interactions (curated databases, experimentally determined), predicted interactions (gene neighbourhood, gene fusion, gene co-occurrence) or via text mining, co-expression or protein homology (see the colour key for connective lines included in the figure).

Figure 4

Protein-protein interaction networks of deiminated proteins identified in plasma-EVs of naked mole-rat. Reconstruction of protein-protein interactions based on known and predicted interactions using STRING analysis. (A) Coloured nodes represent query proteins and first shell of interactors. (B) KEGG pathways relating to the identified proteins and reported in STRING are highlighted as follows: red = complement and coagulation cascade; dark green = Staphylococcus aureus infection; purple = HIF-signalling pathway; yellow = platelet activation; light blue = systemic lupus erythematosus (SLE); orange = oestrogen signalling pathway; dark blue = cholesterol metabolism; light green = vitamin digestion and absorption; pink = glycolysis/gluconeogenesis. Coloured nodes represent query proteins and first shell of interactors, white nodes are second shell of interactors. Coloured lines indicate whether protein interactions are identified via known interactions (curated databases, experimentally determined), predicted interactions (gene neighbourhood, gene fusion, gene co-occurrence) or via text mining, co-expression or protein homology (see the colour key for connective lines included in the figure).

Figure 5

Extracellular vesicle profiling in naked mole-rat plasma. (A) Nanoparticle tracking analysis (NTA) shows a size distribution of EVs from naked mole-rat in the range of mainly 50 to 300 nm, with representative NTA profiles of EVs from 3 different animals (NTA-1, NTA-2, NTA-3). (B) Western blotting analysis confirms that naked mole-rat EVs are positive for the phylogenetically conserved EV-specific markers CD63 and Flot-1. (C) Transmission electron microscopy (TEM) analysis of naked mole-rat plasma-derived EVs shows typical EV morphology; a composite figure is shown and the scale bar (100 nm) applies for all images in the panel. (D) EV yield in plasma of 12 individual naked mole-rats is shown. (E) EV modal size in plasma of 12 individual naked mole-rats is presented.

Figure 6

MicroRNA (miR) analysis of three key miRs related to cancer, inflammation, metabolism and hypoxia, in naked mole-rat plasma-derived EVs. (A). The onco-related miR21 showed the highest relative expression of the three miRs tested, being 394-fold higher than miR155 and 153-fold higher than miR210, respectively (p < 0.0001; n = 3). (B). The relative expression of the hypoxia and metabolic related miR210 was 2.6–fold higher than the inflammatory miR155 (p = 0.0002; n = 3); (miR21, miR155 and miR210 expression is represented as red circles, orange squares and blue triangles, respectively).