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Peptidylarginine Deiminase (PAD) and Post-Translational Protein Deimination-Novel Insights into Alveolata Metabolism, Epigenetic Regulation and Host-Pathogen Interactions.

ABSTRACT: The alveolates (Superphylum Alveolata) comprise a group of primarily single-celled eukaryotes that have adopted extremely diverse modes of nutrition, such as predation, photoautotrophy and parasitism. The alveolates consists of several major phyla including the apicomplexans, a large group of unicellular, spore forming obligate intracellular parasites, and chromerids, which are believed to be the phototrophic ancestors of the parasitic apicomplexans. Molecular pathways involved in Alveolata host-pathogen interactions, epigenetic regulation and metabolism in parasite development remain to be fully understood. Peptidylarginine deiminases (PADs) are a phylogenetically conserved enzyme family which causes post-translational protein deimination, affecting protein function through the conversion of arginine to citrulline in a wide range of target proteins, contributing to protein moonlighting in physiological and pathological processes. The identification of deiminated protein targets in alveolate parasites may therefore provide novel insight into pathogen survival and host-pathogen interactions. The current study assessed PAD homologues and deiminated protein profiles of two alveolate parasites, Piridium sociabile (Chromerida) and Merocystis kathae (Apicomplexa). Histological analysis verified strong cytoplasmic PAD expression in both Alveolates, detected deiminated proteins in nuclear and cytoplasmic compartments of the alveolate parasites and verified the presence of citrullinated histone H3 in Alveolata nucleus, indicating roles in epigenetic regulation. Histone H3 citrullination was also found significantly elevated in the host tissue, indicative of neutrophil extracellular trap formation, a host-defence mechanism against a range of pathogens, particularly those that are too large for phagocytosis. Proteomic analysis of deiminated proteins from both Alveolata identified GO and KEGG pathways strongly relating to metabolic and genetic regulation, with some species-specific differences between the apicomplexan and the chromerid. Our findings provide novel insights into roles for the conserved PAD/ADI enzyme family in the regulation of metabolic and epigenetic pathways in alveolate parasites, possibly also relating to their life cycle and host-pathogen interactions.

SUBMITTER: Kristmundsson A

PROVIDER: S-EPMC7996758 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundWhile mammalian cortical granules are important in fertilization, their biochemical composition and functions are not fully understood. We previously showed that the ABL2 antibody, made against zona free mouse blastocysts, binds to a 75-kDa cortical granule protein (p75) present in a subpopulation of mouse cortical granules. The purpose of this study was to identify and characterize p75, examine its distribution in unfertilized oocytes and preimplantation embryos, and investigate its biological role in fertilization.ResultsTo identify p75, the protein was immunoprecipitated from ovarian lysates with the ABL2 antibody and analyzed by tandem mass spectrometry (MS/MS). A partial amino acid sequence (VLIGGSFY) was obtained, searched against the NCBI nonredundant database using two independent programs, and matched to mouse peptidylarginine deiminase (PAD). When PAD antibody was used to probe western blots of p75, the antibody detected a single protein band with a molecular weight of 75 kDa, confirming our mass spectrometric identification of p75. Immunohistochemistry demonstrated that PAD was present in the cortical granules of unfertilized oocytes and was released from activated and in vivo fertilized oocytes. After its release, PAD was observed in the perivitelline space, and some PAD remained associated with the oolemma and blastomeres' plasma membranes as a peripheral membrane protein until the blastocyst stage of development. In vitro treatment of 2-cell embryos with the ABL2 antibody or a PAD specific antibody retarded preimplantation development, suggesting that cortical granule PAD plays a role after its release in preimplantation cleavage and early embryonic development.ConclusionOur data showed that PAD is present in the cortical granules of mouse oocytes, is released extracellularly during the cortical reaction, and remains associated with the blastomeres' surfaces as a peripheral membrane protein until the blastocyst stage of development. Our in vitro study supports the idea that extracellular PAD functions in preimplantation development.

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Peptidylarginine Deiminase (PAD) and Post-Translational Protein Deimination-Novel Insights into Alveolata Metabolism, Epigenetic Regulation and Host-Pathogen Interactions.

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