Post Translational Modifications

Lampreys are a jawless vertebrate species belonging to an ancient vertebrate lineage that diverged from a common ancestor with humans ~500 million years ago. The sea lamprey (Petromyzon marinus) has a filter feeding ammocoete larval stage that metamorphoses into a parasitic adult, feeding both on teleost and elasmobranch fish. Lampreys are a valuable comparative model species for vertebrate immunity and physiology due to their unique phylogenetic position, unusual adaptive immune system, and physiological adaptions such as tolerance to salinity changes and urea. Peptidylarginine deiminases (PADs) are a phylogenetically conserved enzyme family which catalyses posttranslational deimination/citrullination in target proteins, enabling proteins to gain new functions (moonlighting). The identification of deiminated protein targets in species across phylogeny may provide novel insights into post-translational regulation of physiological and pathophysiological processes. Extracellular vesicles (EVs) are membrane vesicles released from cells that carry cargos of small molecules and proteins for cellular communication, involved in both normal and pathological processes. The current study identified deimination signatures in proteins of both total plasma and plasma-EVs in sea lamprey and furthermore reports the first characterisation of plasma-EVs in lamprey. EVs were poly-dispersed in the size range of 40-500 nm, similar to what is observed in other taxa, positive for CD63 and Flotillin-1. Plasma-EV morphology was confirmed by transmission electron microscopy. Assessment of deimination/citrullination signatures in lamprey plasma and plasma-EVs, revealed 72 deimination target proteins involved in immunity, metabolism and gene regulation in whole plasma, and 37 target proteins in EVs, whereof 24 were shared targets. Furthermore, the presence of deiminated histone H3, indicative of gene-regulatory mechanisms and also a marker of neutrophil extracellular trap formation (NETosis), was confirmed in lamprey plasma. Functional protein network analysis revealed some differences in KEGG and GO pathways of deiminated proteins in whole plasma compared with plasma-EVs. For example, while common STRING network clusters in plasma and plasma-EVs included Peptide chain elongation, Viral mRNA translation, Glycolysis and gluconeogenesis, STRING network clusters specific for EVs only included: Cellular response to heat stress, Muscle protein and striated muscle thin filament, Nucleosome, Protein processing in endoplasmic reticulum, Nucleosome and histone deacetylase complex. STRING network clusters specific for plasma were: Adipokinetic hormone receptor activity, Fibrinogen alpha/beta chain family, peptidase S1A, Glutathione synthesis and recycling-arginine, Fructose 1,6-bisphosphate metabolic process, Carbon metabolism and lactate dehydrogenase activity, Post-translational protein phosphorylation, Regulation of insulin-like growth factor transport and clotting cascade. Overall, for the EV citrullinome, five STRING network clusters, 10 KEGG pathways, 15 molecular GO pathways and 29 Reactome pathways were identified, compared with nine STRING network clusters, six KEGG pathways, two Molecular GO pathways and one Reactome pathway specific for whole plasma; while further pathways were shared. The reported findings indicate that major pathways relevant for immunity and metabolism are targets of deimination in lamprey plasma and plasma-EVs, with some differences, and may help elucidating roles for the conserved PAD enzyme family in regulation of immune and metabolic function throughout phylogeny.

One of the fundamental challenges in designing drug molecule against a disease target or protein is to predict binding affinity between target and drug or small molecule. In this review, our focus will be on advancement in the field of protein-small molecule interaction. This review has been divided into four major sections. In the first section, we will cover software developed for protein structure prediction. This will include prediction of binding pockets and post-translation modifications in proteins. In the second section, we will discuss software packages developed for predicting small-molecule interacting residues in a protein. Advances in the field of docking particularly advancement in the knowledge-based force fields will be discussed in the third part of the review. This section will also cover the method developed for predicting affinity between protein and drug molecules. The fourth section of the review will describe miscellaneous techniques used for designing drug molecules, like pharmacophore modelling. Our major emphasis in this review will be on computational tools that are available free for academic use.

Abstract

Embryonic development is the process by which multicellular organisms build their bodies. Hox genes are among the most relevant regulators of this process. Depending on when and where they are expressed, Hox genes set the segmental identity along the anterior-posterior (A-P) axis of all bilaterian animals. In vertebrate species the axial skeleton is one of the structures requiring patterning input from this family of genes. Hox genes encode for transcription factors characterized by the presence of the homeodomain, a protein motif mediating the binding of the protein to their target DNA. Although most Hox proteins bind rather similar DNA sequences, they typically have very specific functional activities. Relevant to this work is the role of the Hox10 genes in the axial skeleton, which specify lumbar vertebrae as a consequence of their intrinsic rib-blocking activity, a patterning property not shared by any other Hox gene. In most cases, functional specificity of Hox proteins is determined by interaction with particular cofactors, which typically bind parts of the Hox protein outside of their homeodomain. In this study, we identified 4 small linear motifs (C1, C2, C3, C4) in the N-terminal arm of Hox10 proteins, highly conserved both among members of the Hox10 group and throughout evolution. We tested their functional significance by generating Hoxa10 variants containing several deletions and mutations in these small motifs and measured their capability to elicit rib-inhibiting activity in a transgenic assay in mouse embryos. We identified two regions (C1 and C3) that reduced Hoxa10 activity to a certain extent when mutated. Moreover, we showed that these two small motifs are sufficient for conferring rib-inhibiting activity to a Hox protein, as when they were inserted into Hoxb9, which does not have intrinsic rib-blocking properties, the chimeric proteins gained this activity in our transgenic assay. In a complementary study, we identified potential protein partners of Hoxa10, using two yeast two-hybrid (Y2H) screens with a E11.5 cDNA library from MatchMaker ClonTech system. One of the screens, made with the whole Hoxa10 protein as a bait, resulted in the identification of Itm2a, Myl6, Arhgap, AdpRH, Plk1, eIF4a and Rnf138 as potential binding partners for Hoxa10. A second screen used the C1 motif of Hoxa10 as bait due to its importance for rib-removal function. In this screen we identified Tle4, Smad4, Cdc20 and Guanine Nucleotide BP as potential interactors of C1. The interaction between Hoxa10 and Smad4 through C1 was further validated in a mammalian system.

Proteogenomics provide opportunities for proteomic validation of gene structures, genomic alterations and functional relevance of novel findings obtained from genomic data analysis. However, for effective proteogenomic data integration, an extensive proteome profiling, approaching the gene coverage of genomics data, is critical. Here we developed a multi-stage database search method for comprehensive proteomics data analysis to complement whole transcriptome sequencing data. The method utilizes two complementary database search engines, MS-GF+ and MODa/MODi, in tandem. The MS/MS data were first subjected to MS-GF+ database search (1st stage search) and the unidentified MS/MS data from the 1st stage search were subsequently analyzed with the combined use of MODa and MODi (2nd stage search), tools for blind and unrestrictive modification search, respectively. When combined with mPE-MMR, a tool for accurate and extensive precursor masses assignments to MS/MS data, the multi-stage method exhibited a significant increase in identified peptides, modified peptides, mutated peptides, identified proteins and coding genes, compared to a conventional single-stage method. With the increased coverage of proteome profile, the genomics and proteomics data obtained from the same gastric tumor tissue were effectively integrated as evidenced by proBAMsuite analysis results, which showed abundant examples of peptides uniquely mapped to genomic locations as well as increased coverages of exon-exon junctions and coding regions with the multi-stage search method.

Biomarkers are decision-making tools at the basis of clinical diagnostics and essential for guiding therapeutic treatments. In this context, autoimmune diseases represent a class of disorders that need early diagnosis and steady monitoring. These diseases are usually associated with humoral or cell-mediated immune reactions against one or more of the body's own constituents. Autoantibodies fluctuating in biological fluids can be used as disease biomarkers and they can be, thus, detected by diagnostic immunoassays using native autoantigens. However, it is now accepted that post-translational modifications may affect the immunogenicity of self-protein antigens, triggering an autoimmune response and creating neo-antigens. In this case, post-translationally modified peptides represent a more valuable tool with respect to isolated or recombinant proteins. In fact, synthetic peptides can be specifically modified to mimic neo-antigens and to selectively detect autoantibodies as disease biomarkers. A ‘chemical reverse approach’ to select synthetic peptides, bearing specific post-translational modifications, able to fishing out autoantibodies from patients' biological fluids, can be successfully applied for the development of specific in vitro diagnostic/prognostic assays of autoimmune diseases. Herein, we report the successful application of this approach to the identification of biomarkers in different autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.

Abstract Proteogenomics provide opportunities for proteomic validation of gene structures, genomic alterations and functional relevance of novel findings obtained from genomic data analysis. However, for effective proteogenomic data integration, an extensive proteome profiling, approaching the gene coverage of genomics data, is critical. Here we developed a multi-stage database search method for comprehensive proteomics data analysis to complement whole transcriptome sequencing data. The method utilizes two complementary database search engines, MS-GF+ and MODa/MODi, in tandem. The MS/MS data were first subjected to MS-GF+ database search (1st stage search) and the unidentified MS/MS data from the 1st stage search were subsequently analyzed with the combined use of MODa and MODi (2nd stage search), tools for blind and unrestrictive modification search, respectively. When combined with m PE-MMR, a tool for accurate and extensive precursor masses assignments to MS/MS data, the multi-stage method exhibited a significant increase in identified peptides, modified peptides, mutated peptides, identified proteins and coding genes, compared to a conventional single-stage method. With the increased coverage of proteome profile, the genomics and proteomics data obtained from the same gastric tumor tissue were effectively integrated as evidenced by proBAMsuite analysis results, which showed abundant examples of peptides uniquely mapped to genomic locations as well as increased coverages of exon-exon junctions and coding regions with the multi-stage search method.

In humans and other eukaryotes, histone post-translational modifications (hPTMs) play an essential role in the epigenetic control of gene expression. In trypanosomatid parasites, conversely, gene regulation occurs mainly at the post-transcriptional level. However, our group has recently shown that hPTMs are abundant and varied in Trypanosoma cruzi, the etiological agent of Chagas Disease, signaling for possible conserved epigenetic functions. Here, we applied an optimized mass spectrometry-based proteomic workflow to provide a high-confidence comprehensive map of hPTMs, distributed in all canonical, variant and linker histones of T. cruzi. Our work expands the number of known T. cruzi hPTMs by almost 2-fold, representing the largest dataset of hPTMs available to any trypanosomatid to date, and can be used as a basis for functional studies on the dynamic regulation of chromatin by epigenetic mechanisms and the selection of candidates for the development of epigenetic drugs against tr...

The future potential of multiomic based treatments which can be delivered to cellular and molecular targets by nanomedicine-systems shall be taken from lab to clinic by the implementation of Translational Medicine for tailoring personalized, and precision medicine approaches.  Nanodelivery systems may transport therapeutic molecular agents to the targeted tissues, cells, and molecules by circumventing biological milieu interactions and avoiding RES elimination enhancing therapeutic index, while reducing and eliminating systemic toxicity. This way, we can circumvent in the future the traditional-medicine approach of one size fits all, and proceed to the implementation of stratified or precision-medicine, and personalized-medicine for each CVD patient based on multiomic-evidence improving screening, diagnosis, prevention, prognosis, monitoring and mainly treatment of currently incurable CVDs.

Peptidylarginine deiminases (PADs) are phylogenetically conserved calcium-dependent enzymes which post-translationally convert arginine into citrulline in target proteins in an irreversible manner, causing functional and structural changes in target proteins. Protein deimination causes generation of neo-epitopes, affects gene regulation and also allows for protein moonlighting. Furthermore, PADs have been found to be a phylogenetically conserved regulator for extracellular vesicle (EVs) release. EVs are found in most body fluids and participate in cellular communication via transfer of cargo proteins and genetic material. In this study, post-translationally deiminated proteins in serum and serum-EVs are described for the first time in camelids, using the llama (Lama glama L. 1758) as a model animal. We report a poly-dispersed population of llama serum EVs, positive for phylogenetically conserved EV-specific markers and characterised by TEM. In serum, 103 deiminated proteins were overall identified, including key immune and metabolic mediators including complement components, immunoglobulin-based nanobodies, adiponectin and heat shock proteins. In serum, 60 deiminated proteins were identified that were not in EVs, and 25 deiminated proteins were found to be unique to EVs, with 43 shared deiminated protein hits between both serum and EVs. Deiminated histone H3, a marker of neutrophil extracellular trap formation, was also detected in llama serum. PAD homologues were identified in llama serum by Western blotting, via cross reaction with human PAD antibodies, and detected at an expected 70 kDa size. This is the first report of deiminated proteins in serum and EVs of a camelid species, highlighting a hitherto unrecognized post-translational modification in key immune and metabolic proteins in camelids, which may be translatable to and inform a range of human metabolic and inflammatory pathologies.

Aging is one of the chief biomedical problems of the 21st century. After decades of basic research on biogerontology
(the science of aging), the aging process still remains an enigma. Although hundreds of "theories" on aging have
been formulated and many fundamental insights about age-related changes and genetic as well as environmental
interventions that change the pace of aging have been discovered, the actual why and how we age remain enigmatic.
In the post-genomic era there is an exponential increase in data. As a consequence it is a challenge to utilize all
information based on it and derive meaningful knowledge about biological phenomena. No individual scientist, no
group, nor consortium is capable of keeping up within their own field and are overwhelmed by the explosion of data
increase. Machine learning applied on biological data has the potential to solve this and cause a paradigm shift from
hypothesis-driven research (which predominates biological research including biogerontology) to data-driven
research.
This dissertation addresses this problem. In particular it proposes and executes the use of machine learning on
current existing data to predict drivers of aging (and therefore helps to distinguish causes from consequences),
interventions to counteract aging, and specific hypotheses to fill in research gaps that require experimental
validation.
The objective of this project is therefore to build computational models that are based on data relevant to the
phenomenon of aging and to predict as many of its aspects and dimensions as possible (thus elucidate their relations
to each other). For converting between and sorting within dimensions which are relevant to aging, different machine
learning models are evaluated. Ones models are built, it can be determined how much they can explain different
aspects of aging. Those models will also be capable of specifying which features are most relevant for prediction (in
both classification or regression). It is possible to train models that incorporate age-related changes based on
transcriptomic, proteomic, metabolomic, epigenomic as well as morphological data and their combinations. Machine
learning is further used to convert between and within them.
This work focuses on three types of predictors. Subsequently, discoveries are made with the statistical and learning
algorithms. The first model (lifespan predictor) is trained on predicting the lifespan based on genotype, environment
and combinations thereof. It is useful for predicting lifespan extending interventions on the population level. The
second model (age predictor) is trained on predicting the age given features measured on individuals. This is useful
for identifying biomarkers of aging and to determine the effects of interventions on the level of individuals. The third
model predicts functions/regulations of biological entities in regard to the aging process based on heterogeneous data
such as ontologies and diverse omics including time-series gene expression profiles (which can be visualized as
plots), and linked data. It is used to understand the role of genes and proteins as well as perhaps other entities such as
small molecules including lipids and other metabolites. Functions of proteins, which are still unknown, especially
those involved in yeast lipid metabolism and its regulation, can be predicted.
For this purpose we use primarily yeast as model organism as well as data on humans. Other biomedical model
organisms might be added if found beneficial.
The novel aspects of this research are for instance that 1) aging is investigated systematically in an unbiased
data-driven approach, 2) lifespan is predicted as continuous values, 3) age is predicted by combining multiple omics
data, 4) functions and regulations of biological entities like genes are predicted with high confidence from
heterogeneous data sources.
This thesis discovered that genetics is the most important feature of lifespan determination. Phenotypic features
related to lipid and membranes such as vacuolar morphology and autophagy activity are important for lifespan
determination according to the best performing models. A age predictor based on transcriptomics and proteomics can
highly accurately determine the age. It selected features are associated with both translation and lipid metabolism.
Among the top selected features are transcripts of genes when deleted exhibit abnormal vacuolar morphology as well
as targets of Opi1. Opi1 itself and its regulators were found to be differentially regulated post-transcriptional or
post-translational. Lastly, a function predictor for genes was created that achieved exceptional accuracy of
classifying aging genes. It learned for instance that piecemeal autophagy of the nucleus is strongly predictive for
aging-suppressor genes while cytoplasmic translation is strongly predictive for gerontogenes.
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This review point out several aspects regarding the mitogen-activated protein kinase (MAPK)/extracellular-regulated kinase (Erk) network, which are still pending issues in the understanding how this pathway integrate information to drive cell fates. Focusing on the role of Erk during cell cycle, it has to be underlined that Erk downstream effectors, which are required for mitosis progression and contribute to aneuploidy during tumorigenesis, remain to be determined. In addition to the identity of the terminal enzymes or effectors of Erk, it has to be stressed that the dynamic nature of the Erk signal is itself a key factor in cell phenotype decisions. Development of biophotonics strategies for monitoring the Erk network at the spatiotemporal level in living cells, as well as computational and hypothesis-driven approaches, are called to unravel the principles by which signaling networks create biochemical and biological specificities. Finally, Erk dynamics might also be impacted by other post-translational modification than phosphorylation, such as O-GlcNAcylation. J. Cell. Biochem. 109: 850–857, 2010. © 2010 Wiley-Liss, Inc.

A frameshift mutation in the transcript of the ubiquitin-B gene leads to a C-terminally extended ubiquitin (Ub), UBB(+1). UBB(+1) has been considered to inhibit proteasomes and as such to be the underlying cause for toxic protein buildup correlated with certain neuropathological conditions. We demonstrate that expression of extended Ub variants leads to accumulation of heterogeneously linked polyubiquitin conjugates, indicating a pervasive effect on Ub-dependent turnover. 20S proteasomes selectively proteolyzed Ub extensions, yet no evidence for inhibition of 26S holoenzymes was found. However, among susceptible targets for inhibition was Ubp6, the primary enzyme responsible for disassembly of Lys48 linkages at 26S proteasomes. Processing of Lys48 and Lys63 linkages by other deubiquitinating enzymes (DUBs) was also inhibited. Disruption of Ub-dependent degradation by extended Ub variants may therefore be attributed to their inhibitory effect on select DUBs, thus shifting research ef...

The Signal Transducer and Activator of Transcription (STAT) family of transcription factors is involved in inflammation, immunity, development, cancer, and response to injury, among other biological phenomena. Canonical STAT signaling is often represented as a 3-step pathway involving the sequential activation of a membrane receptor, an intermediate kinase and a STAT transcription factor. The rate-limiting phosphorylation at a highly conserved C-terminal tyrosine residue determines nuclear translocation and transcriptional activity of STATs. This apparent simplicity is actually misleading and can hardly explain the pleiotropic nature of STATs, the existence of various non-canonical STAT pathways, or the key role of the N-terminal domain in STAT functions. More than 80 post-translational modifications (PTMs) have been identified for STAT3, but their functions remain barely understood. Here, we provide a brief but comprehensive overview of these underexplored PTMs and their role on STAT3 canonical and non-canonical functions. A less tyrosine-centric point of view may be required to advance our understanding of STAT signaling.

Simple Summary: Alveolates are a major group of free living and parasitic organisms; some of which are serious pathogens of animals and humans. Apicomplexans and chromerids are two phyla belonging to the alveolates. Apicomplexans are obligate intracellular parasites; that cannot complete their life cycle without exploiting a suitable host. Chromerids are mostly photoautotrophs as they can obtain energy from sunlight; and are considered ancestors of the apicomplexans. The pathogenicity and life cycle strategies differ significantly between parasitic alveolates; with some causing major losses in host populations while others seem harmless to the host. As the life cycles of some are still poorly understood, a better understanding of the factors which can affect the parasitic alveolates' life cycles and survival is of great importance and may aid in new biomarker discovery. This study assessed new mechanisms relating to changes in protein structure and function (so-called "deimination" or "citrullination") in two key parasites-an apicomplexan and a chromerid-to assess the pathways affected by this protein modification. Our findings point to novel regulatory mechanisms in these parasites' lifecycles via protein deimination and may provide novel insights into their adaptability to different environments and hosts as well as host-pathogen coevolution Abstract: The alveolates (Superphylum Alveolata) comprise a group of primarily single-celled eukaryotes that have adopted extremely diverse modes of nutrition, such as predation, photoau-totrophy and parasitism. The alveolates consists of several major phyla including the apicom-plexans, 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 cy-toplasmic 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 Citation: Kristmundsson, Á.; Erlingsdóttir, Á.; Lange, S. Peptidylarginine Deiminase (PAD) and Post-Translational Protein Deimination-Novel Insights into Alveolata Metabolism, Epigenetic Regulation and Host-Pathogen Interactions.

Pre-mRNA splicing, the removal of noncoding intron sequences from the pre-mRNA, is a
critical reaction in eukaryotic gene expression. Pre-mRNA splicing is carried out by a remarkable macromolecular machine, the spliceosome, which undergoes dynamic rearrangements of its RNA and protein components to assemble its catalytic center. While significant progress has been made in describing the ‘‘moving parts’’ of this machine, the mechanisms by which spliceosomal proteins mediate the ordered rearrangements within the spliceosome remain elusive. Here we explore recent evidence from proteomics studies revealing extensive post-translational modification
of splicing factors. While the functional significance of most of these modifications remains
to be characterized, we describe recent studies in which the roles of specific post-translational modifications of splicing factors have been characterized.

S-nitrosation of cysteine plays an important role in storage and transport of NO, a key signaling molecule in vivo. An approach to detect this modification in the bare, charged amino acid is presented, based on IR multiple photon dissociation (IRMPD) spectroscopy. Protonated and deprotonated S-nitrosocysteine ions, [SNOCys+H]+ and [SNOCys-H]-, have been obtained by electrospray ionization and assayed for IR activity in either the 1000-1900 cm-1 or the 3000-3600 cm-1 wavenumber range. The so-obtained IRMPD spectra display characteristic features ascribed to the presence of the S-nitrosation motif, which are missing in the corresponding IRMPD spectra of the native cysteine ions, [Cys+H]+ and [Cys-H]-. In particular, the NO stretching mode is unambiguously identified by the red shift observed for the 15N-labelled species. The interpretation of the IRMPD spectra is supported by density functional theory calculations of the optimized geometries, relative energies and IR spectra of [SNOCys+H]+ and [SNOCys-H]-. Both sampled ions comprise a thermally averaged population of conformers contributing to the experimental IRMPD spectrum. This notion is supported by the agreement between the convoluted IR spectrum of the several conformers, and the recorded IRMPD spectrum. The gathered evidence points to a characteristic NO stretching mode that emerges as a pronounced feature at 1460-1490 cm-1  in the IRMPD spectrum of [SNOCys-H]-, namely in a region where [Cys-H]- displays no IRMPD activity. Conversely, the NO stretching vibration of [SNOCys+H]+ is enclosed in a wide absorption including the C=O stretching mode at 1780 cm-1. The [SNOCys-H]- negative ions are thus a promising benchmark in a search for S-nitrosation features using IRMPD spectroscopy.

Ahead of display, a non-original layer was observed on the surface of a fragment of a wall painting by Ambrogio Lorenzetti (active 1319, died 1348/9). FTIR analysis suggested proteinaceous content. Mass spectrometry was used to better characterise this layer and revealed two protein components: sheep and cow glue and chicken and duck egg white. Analysis of post-translational modifications detected several photo-oxidation products, which suggest that the egg experienced prolonged exposure to UV light and was likely applied long before the glue layer. Additionally, glycation products detected may indicate naturally occurring glycoprotein degradation or reaction with a carbohydrate material such as starch, identified by ATR-FTIR in a cross-section of a sample taken from the painting. Palaeoproteomics is shown to provide detailed characterisation of organic layers associated with mural paintings and therefore aids reconstruction of the conservation history of these objects.

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.

Despite the mechanisms for endogenous nitroxyl (HNO) production and action being incompletely understood, pharmacological donors show broad therapeutic promise and are in clinical trials. Mass spectrometry and site-directed mutagenesis showed that chemically distinct HNO donors 1-nitrosocyclohexyl acetate or Angeli's salt induced disulfides within cGMP-dependent protein kinase I-alpha (PKGIα), an interdisulfide between Cys42 of the two identical subunits of the kinase and a previously unobserved intradisulfide between Cys117 and Cys195 in the high affinity cGMP-binding site. Kinase activity was monitored in cells transfected with wildtype (WT), Cys42Ser or Cys117/195Ser PKGIα that cannot form the inter- or intradisulfide, respectively. HNO enhanced WT kinase activity, an effect significantly attenuated in inter- or intradisulfide-deficient PKGIα. To investigate whether the intradisulfide modulates cGMP binding, real-time imaging was performed in vascular smooth muscle cells expressing a FRET-biosensor comprising the cGMP-binding sites of PKGIα. HNO induced FRET changes similar to those elicited by an increase of cGMP, suggesting that intradisulfide formation is associated with activation of PKGIα. Intradisulfide formation in PKGIα correlated with enhanced HNO-mediated vasorelaxation in mesenteric arteries in vitro and arteriolar dilation in vivo in mice. HNO induces intradisulfide formation in PKGIα, inducing the same effect as cGMP binding, namely kinase activation and thus vasorelaxation.