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.

Post-Translational Protein Deimination Signatures in Serum and Serum-Extracellular Vesicles of Bos taurus Reveal Immune, Anti-Pathogenic, Anti-Viral, Metabolic and Cancer-Related Pathways for Deimination

“…The presence of deiminated histones H2A, H2B, H3 and H4 was identified in serum-EVs only, but not in whole bovine serum. Interestingly, a similar EV-mediated export of deiminated histones, as observed in cow serum-EVs in the current study, was recently identified in the naked mole-rat, an animal with unusual immunological and metabolic traits [98], as well as in alligator, also an animal with unusual antimicrobial, including anti-viral responses [93]. Deiminated histone H3 is commonly used as an indicator for neutrophil extracellular trap formation (NETosis) [31,244], which has been related to PAD4 and implicated in anti-viral responses in cattle respiratory syncytial virus disease [42], in response to parasitic infections in cattle [245][246][247] as well as in response to certain antibiotics [248].…”

“…It must be noted that neither anti-human PAD1 nor PAD6 antibodies were tested against bovine sera in the current study. The anti-human antibodies have previously been shown to cross-react with PADs from diverse taxa, with PAD2, which is considered the phylogenetically most conserved isoform, predominantly cross-reacting best with other species, including bony and cartilaginous fish, birds, mole-rat, alligator, camelids, cetaceans and pinnipeds [15][16][17]92,93,[96][97][98][99].…”

“…C3 is the central component of the complement cascade and has furthermore been implicated in tissue remodeling during teleost ontogeny [121][122][123][124] as well as in regeneration [125]. A range of complement proteins have recently been identified by our group to be deiminated in a range of taxa throughout the phylogenetic tree including teleost and cartilaginous fish, camelids, cetaceans, pinnipeds, camelids and peglagic seabirds [16,17,[91][92][93][94][95][96]98,99]. The bovine complement system [126] has been widely studied, including in immune defences against bovine anaplasmosis [127], in relation to heat-stress in dairy cows [128], susceptibility to bacterial infection relating to copper-deficiency [129].…”

“…In the cow, adiponectin is a known glycoprotein and has been assessed in bovine tissues as well as body fluids [179] and amongst other been identified as a pro-survival signal in ER stress in the mammary gland [180]. Adiponectin has recently been identified as a deimination candidate in several taxa with unusual metabolism, including camelids, the naked mole-rat, orca and alligator [92,93,96,98]. A range of apolipoproteins was furthermore identified to be deiminated in the current study in cow serum and serum-EVs.…”

“…Furthermore, HIF-1 has recently been identified as a genomic marker differing between cattle breeds, including highland-adapted cattle [241]. Interestingly, enrichment in deiminated proteins in KEGG HIF-1 signalling pathways has previously been identified in EVs only, in some species with unusual immune and metabolic function including the naked mole-rat and deep-diving whales, both of which are animal models of hypoxia-tolerance and cancer-resistance [96,98]. Furthermore, deimination of HIF-1 regulation were recently identified in aggressive brain cancer (glioblastoma) cells [100].…”