Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies with limited survival rate. Roles for peptidylarginine deiminases (PADs) have been studied in relation to a range of cancers with roles in epigenetic regulation (including histone modification and microRNA regulation), cancer invasion, and extracellular vesicle (EV) release. Hitherto though, knowledge on PADs in PDAC is limited. In the current study, two PDAC cell lines (Panc-1 and MiaPaCa-2) were treated with pan-PAD inhibitor Cl-amidine as well as PAD2, PAD3, and PAD4 isozyme-specific inhibitors. Effects were assessed on changes in EV signatures, including EV microRNA cargo (miR-21, miR-126, and miR-221), on changes in cellular protein expression relevant for pancreatic cancer progression and invasion (moesin), for mitochondrial housekeeping (prohibitin, PHB), and gene regulation (deiminated histone H3, citH3). The two pancreatic cancer cell lines were found to predominantly express PAD2 and PAD3, which were furthermore expressed at higher levels in Panc-1, compared with MiaPaCa-2 cells. PAD2 isozyme-specific inhibitor had the strongest effects on reducing Panc-1 cell invasion capability, which was accompanied by an increase in moesin expression, which in pancreatic cancer is found to be reduced and associated with pancreatic cancer aggressiveness. Some reduction, but not significant, was also found on PHB levels while effects on histone H3 deimination were variable. EV signatures were modulated in response to PAD inhibitor treatment, with the strongest effects observed for PAD2 inhibitor, followed by PAD3 inhibitor, showing significant reduction in pro-oncogenic EV microRNA cargo (miR-21, miR-221) and increase in anti-oncogenic microRNA cargo (miR-126). While PAD2 inhibitor, followed by PAD3 inhibitor, had most effects on reducing cancer cell invasion, elevating moesin expression, and modulating EV signatures, PAD4 inhibitor had negligible effects and pan-PAD inhibitor Cl-amidine was also less effective. Compared with MiaPaCa-2 cells, stronger modulatory effects for the PAD inhibitors were observed in Panc-1 cells, which importantly also showed strong response to PAD3 inhibitor, correlating with previous observations that Panc-1 cells display neuronal/stem-like properties. Our findings report novel PAD isozyme regulatory roles in PDAC, highlighting roles for PAD isozyme-specific treatment, depending on cancer type and cancer subtypes, including in PDAC.

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The purple sea urchin (Strongylocentrotus purpuratus) is a marine invertebrate of the class Echinoidea that serves as an important research model for developmental biology, cell biology, and immunology, as well as for understanding regenerative responses and ageing. Peptidylarginine deiminases (PADs) are calcium-dependent enzymes that mediate post-translational protein deimination/citrullination. These alterations affect protein function and may also play roles in protein moonlighting. Extracellular vesicles (EVs) are membrane-bound vesicles that are released from cells as a means of cellular communication. Their cargo includes a range of protein and RNA molecules. EVs can be isolated from many body fluids and are therefore used as biomarkers in physiological and pathological responses. This study assessed EVs present in the coelomic fluid of the purple sea urchin (Strongylocentrotus purpuratus), and identified both total protein cargo as well as the deiminated protein cargo. Deiminated proteins in coelomic fluid EVs were compared with the total deiminated proteins identified in coelomic fluid to assess putative differences in deiminated protein targets. Functional protein network analysis for deiminated proteins revealed pathways for immune, metabolic, and gene regulatory functions within both total coelomic fluid and EVs. Key KEGG and GO pathways for total EV protein cargo furthermore showed some overlap with deimination-enriched pathways. The findings presented in this study add to current understanding of how post-translational deimination may shape immunity across the phylogeny tree, including possibly via PAD activity from microbiota symbionts. Furthermore, this study provides a platform for research on EVs as biomarkers in sea urchin models.

The reindeer (caribou) Rangifer tarandus is a Cervidae in the order Artiodactyla. Reindeer are sedentary and migratory populations with circumpolar distribution in the Arctic, Northern Europe, Siberia and North America. Reindeer are an important wild and domesticated species, and have developed various adaptive strategies to extreme environments. Importantly, deer have also been identified to be putative zoonotic carriers, including for parasites, prions and coronavirus. Therefore, novel insights into immune-related markers are of considerable interest. Peptidylarginine deiminases (PADs) are a phylogenetically conserved enzyme family which causes post-translational protein deimination by converting arginine into citrulline in target proteins. This affects protein function in health and disease. Extracellular vesicles (EVs) participate in cellular communication, in physiological and pathological processes, via transfer of cargo material, and their release is partly regulated by PADs. This study assessed deiminated protein and EV profile signatures in plasma from sixteen healthy wild female reindeer, collected in Iceland during screening for parasites and chronic wasting disease. Reindeer plasma EV profiles showed a poly-dispersed distribution from 30 to 400 nm and were positive for phylogenetically conserved EV-specific markers. Deiminated proteins were isolated from whole plasma and plasma EVs, identified by proteomic analysis and protein interaction networks assessed by KEGG and GO analysis. This revealed a large number of deimination-enriched pathways for immunity and metabolism, with some differences between whole plasma and EVs. While shared KEGG pathways for whole plasma and plasma EVs included complement and coagulation pathways, KEGG pathways specific for EVs were for protein digestion and absorption, platelet activation, amoebiasis, the AGE–RAGE signaling pathway in diabetic complications, ECM receptor interaction, the relaxin signaling pathway and the estrogen signaling pathway. KEGG pathways specific for whole plasma were pertussis, ferroptosis, SLE, thyroid hormone synthesis, phagosome, Staphylococcus aureus infection, vitamin digestion and absorption, and prion disease. Further differences were also found between molecular function and biological processes GO pathways when comparing functional STRING networks for deiminated proteins in EVs, compared with deiminated proteins in whole plasma. This study highlights deiminated proteins and EVs as candidate biomarkers for reindeer health and may provide information on regulation of immune pathways in physiological and pathological processes, including neurodegenerative (prion) disease and zoonosis.

Peptidylarginine deiminases (PADs) and extracellular vesicles (EVs) may be indicative biomarkers of physiological and pathological status and adaptive responses, including to diseases and disorders of the central nervous system (CNS) and related to hypoxia. While these markers have been studied in hypoxia-intolerant mammals, in vivo investigations in hypoxia-tolerant species are lacking. Naked mole-rats (NMR) are among the most hypoxia-tolerant mammals and are thus a good model organism for understanding natural and beneficial adaptations to hypoxia. Thus, we aimed to reveal CNS related roles for PADs in hypoxia tolerance and identify whether circulating EV signatures may reveal a fingerprint for adaptive whole-body hypoxia responses in this species. We found that following in vivo acute hypoxia, NMR: (1) plasma-EVs were remodelled, (2) whole proteome EV cargo contained more protein hits (including citrullinated proteins) and a higher number of associated KEGG pathways relating to the total proteome of plasma-EVs Also, (3) brains had a trend for elevation in PAD1, PAD3 and PAD6 protein expression, while PAD2 and PAD4 were reduced, while (4) the brain citrullinome had a considerable increase in deiminated protein hits with hypoxia (1222 vs. 852 hits in normoxia). Our findings indicate that circulating EV signatures are modified and proteomic content is reduced in hypoxic conditions in naked mole-rats, including the circulating EV citrullinome, while the brain citrullinome is elevated and modulated in response to hypoxia. This was further reflected in elevation of some PADs in the brain tissue following acute hypoxia treatment. These findings indicate a possible selective role for PAD-isozymes in hypoxia response and tolerance.