In vitro methylation of the U7 snRNP subunits Lsm11 and SmE by the PRMT5/MEP50/pICln methylosome.

U7 snRNP is a multisubunit endonuclease required for 3' end processing of metazoan replication-dependent histone pre-mRNAs. In contrast to the spliceosomal snRNPs, U7 snRNP lacks the Sm subunits D1 and D2 and instead contains two related proteins, Lsm10 and Lsm11. The remaining five subunits of the U7 heptameric Sm ring, SmE, F, G, B, and D3, are shared with the spliceosomal snRNPs. The pathway that assembles the unique ring of U7 snRNP is unknown. Here, we show that a heterodimer of Lsm10 and Lsm11 tightly interacts with the methylosome, a complex of the arginine methyltransferase PRMT5, MEP50, and pICln known to methylate arginines in the carboxy-terminal regions of the Sm proteins B, D1, and D3 during the spliceosomal Sm ring assembly. Both biochemical and cryo-EM structural studies demonstrate that the interaction is mediated by PRMT5, which binds and methylates two arginine residues in the amino-terminal region of Lsm11. Surprisingly, PRMT5 also methylates an amino-terminal arginine in SmE, a subunit that does not undergo this type of modification during the biogenesis of the spliceosomal snRNPs. An intriguing possibility is that the unique methylation pattern of Lsm11 and SmE plays a vital role in the assembly of the U7 snRNP.

Comparative proteomic analysis of the ovarian fluid and eggs of Siberian sturgeon.

BACKGROUND: Sturgeon species are living fossils that exhibit unique reproductive characteristics, and elucidation of the molecular processes governing the formation and quality of sturgeon eggs is crucial. However, comprehensive data on the protein composition of sturgeon ovarian fluid (OF) and eggs and their functional significance are lacking. To address this knowledge gap, the aim of the present study was to conduct a comprehensive comparative proteomic analysis of Siberian sturgeon OF and eggs using liquid chromatography-mass spectrometry (LC-MS/MS). RESULTS: A total of 617 proteins were identified in OF, and 565 proteins were identified in eggs. A total of 772 proteins showed differential abundance. Among the differentially abundant proteins, 365 were more abundant in OFs, while 407 were more abundant in eggs. We identified 339 proteins unique to OFs and 287 proteins specific to eggs, and further investigated the top 10 most abundant proteins in each. The functional annotation of the OF proteins highlighted their predominant association with immune system processes, including the complement and coagulation cascade, neutrophil and leukocyte-mediated immunity, cholesterol metabolism, and regulation of the actin cytoskeleton. Analysis of egg proteins revealed enrichment in metabolic pathways, such as oxidative phosphorylation and fatty acid metabolism, and protein ubiquitination and translation. OF-specific proteins included extracellular matrix and secretory vesicles, and eggs were enriched in proteins localized to mitochondria and ribosome components. CONCLUSIONS: This study presents the first comprehensive characterization of the protein composition of sturgeon OF and eggs and elucidates their distinct functional roles. These findings advance our understanding of sturgeon reproduction, OF-egg signaling and the origin of OF proteins. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium with the dataset identifier PXD044168 to ensure accessibility for further research.

N-glycosylation acts as a switch for FGFR1 trafficking between the plasma membrane and nuclear envelope.

Fibroblast growth factor receptor 1 (FGFR1) is a heavily N-glycosylated cell surface receptor tyrosine kinase that transmits signals across the plasma membrane, in response to fibroblast growth factors (FGFs). Balanced FGF/FGFR1 signaling is crucial for the development and homeostasis of the human body, and aberrant FGFR1 is frequently observed in various cancers. In addition to its predominant localization to the plasma membrane, FGFR1 has also been detected inside cells, mainly in the nuclear lumen, where it modulates gene expression. However, the exact mechanism of FGFR1 nuclear transport is still unknown. In this study, we generated a glycosylation-free mutant of FGFR1, FGFR1.GF, and demonstrated that it is localized primarily to the nuclear envelope. We show that reintroducing N-glycans into the D3 domain cannot redirect FGFR1 to the plasma membrane or exclude the receptor from the nuclear envelope. Reestablishment of D2 domain N-glycans largely inhibits FGFR1 accumulation in the nuclear envelope, but the receptor continues to accumulate inside the cell, mainly in the ER. Only the simultaneous presence of N-glycans of the D2 and D3 domains of FGFR1 promotes efficient transport of FGFR1 to the plasma membrane. We demonstrate that while disturbed FGFR1 folding results in partial FGFR1 accumulation in the ER, impaired FGFR1 secretion drives FGFR1 trafficking to the nuclear envelope. Intracellular FGFR1.GF displays a high level of autoactivation, suggesting the presence of nuclear FGFR1 signaling, which is independent of FGF. Using mass spectrometry and proximity ligation assay, we identified novel binding partners of the nuclear envelope-localized FGFR1, providing insights into its cellular functions. Collectively, our data define N-glycosylation of FGFR1 as an important regulator of FGFR1 kinase activity and, most importantly, as a switchable signal for FGFR1 trafficking between the nuclear envelope and plasma membrane, which, due to spatial restrictions, shapes FGFR1 interactome and cellular function. Video Abstract.

Sugar Starvation Disrupts Lipid Breakdown by Inducing Autophagy in Embryonic Axes of Lupin (Lupinus spp.) Germinating Seeds.

Under nutrient deficiency or starvation conditions, the mobilization of storage compounds during seed germination is enhanced to primarily supply respiratory substrates and hence increase the potential of cell survival. Nevertheless, we found that, under sugar starvation conditions in isolated embryonic axes of white lupin (Lupinus albus L.) and Andean lupin (Lupinus mutabilis Sweet) cultured in vitro for 96 h, the disruption of lipid breakdown occurs, as was reflected in the higher lipid content in the sugar-starved (-S) than in the sucrose-fed (+S) axes. We postulate that pexophagy (autophagic degradation of the peroxisome-a key organelle in lipid catabolism) is one of the reasons for the disruption in lipid breakdown under starvation conditions. Evidence of pexophagy can be: (i) the higher transcript level of genes encoding proteins of pexophagy machinery, and (ii) the lower content of the peroxisome marker Pex14p and its increase caused by an autophagy inhibitor (concanamycin A) in -S axes in comparison to the +S axes. Additionally, based on ultrastructure observation, we documented that, under sugar starvation conditions lipophagy (autophagic degradation of whole lipid droplets) may also occur but this type of selective autophagy seems to be restricted under starvation conditions. Our results also show that autophagy occurs at the very early stages of plant growth and development, including the cells of embryonic seed organs, and allows cell survival under starvation conditions.

Proteomic analysis of carp seminal plasma provides insights into the immune response to bacterial infection of the male reproductive system.

Aeromonas salmonicida is recognized as a significant bacterial pathogen in ulcerative disease of cyprinid fish. However, the mechanism of immunity to these bacteria in common carp is still not well understood, especially the immune regulation in the gonad to bacterial infection. The aims of our study were to analyze changes in the seminal plasma proteome following A. salmonicida infection in carp males. The observed pathological changes in the tissue (liver, spleen, kidney and testis) morphology and upregulation of immune-related genes (tnfa2, il6a) confirmed the successful infection challenge. Using mass spectrometry-based label-free quantitative proteomics, we identified 1402 seminal plasma proteins, and 44 proteins (20 up- and 24 downregulated) were found to be differentially abundant between infected and control males. Most differentially abundant proteins were involved in the immune response mechanisms, such as acute phase response, complement activation and coagulation, inflammation, lipid metabolism, cell-cell and cell-matrix adhesion, creatine-phosphate biosynthesis and germ cell-Sertoli cell junction signaling. Bacterial infection also caused profound changes in expression of selected genes in the testis and hematopoietic organs, which contributed to changes in seminal proteins. The altered seminal proteins and bacterial proteins in seminal plasma may serve as valuable markers of infection in the testis.

Proteome-Wide Analysis of Protein Lysine N-Homocysteinylation in Saccharomyces cerevisiae.

Protein N-homocysteinylation by a homocysteine (Hcy) metabolite, Hcy-thiolactone, is an emerging post-translational modification (PTM) that occurs in all tested organisms and has been linked to human diseases. The yeast Saccharomyces cerevisiae is widely used as a model eukaryotic organism in biomedical research, including studies of protein PTMs. However, patterns of global protein N-homocysteinylation in yeast are not known. Here, we identified 68 in vivo and 197 in vitro N-homocysteinylation sites at protein lysine residues (N-Hcy-Lys). Some of the N-homocysteinylation sites overlap with other previously identified PTM sites. Protein N-homocysteinylation in vivo, induced by supplementation of yeast cultures with Hcy, which elevates Hcy-thiolactone levels, was accompanied by significant changes in the levels of 70 yeast proteins (38 up-regulated and 32 down-regulated) involved in the ribosomal structure, amino acid biosynthesis, and basic cellular pathways. Our study provides the first global survey of N-homocysteinylation and accompanying changes in the yeast proteome caused by elevated Hcy level. These findings suggest that protein N-homocysteinylation and dysregulation of cellular proteostasis may contribute to the toxicity of Hcy in yeast. Homologous proteins and N-homocysteinylation sites are likely to be involved in Hcy-related pathophysiology in humans and experimental animals. Data are available via ProteomeXchange with identifier PXD020821.

Palm Oil-Rich Diet Affects Murine Liver Proteome and S-Palmitoylome.

Palmitic acid (C16:0) is the most abundant saturated fatty acid in animals serving as a substrate in synthesis and ß-oxidation of other lipids, and in the modification of proteins called palmitoylation. The influence of dietary palmitic acid on protein S-palmitoylation remains largely unknown. In this study we performed high-throughput proteomic analyses of a membrane-enriched fraction of murine liver to examine the influence of a palm oil-rich diet (HPD) on S-palmitoylation of proteins. HPD feeding for 4 weeks led to an accumulation of C16:0 and C18:1 fatty acids in livers which disappeared after 12-week feeding, in contrast to an accumulation of C16:0 in peritoneal macrophages. Parallel proteomic studies revealed that HPD feeding induced a sequence of changes of the level and/or S-palmitoylation of diverse liver proteins involved in fatty acid, cholesterol and amino acid metabolism, hemostasis, and neutrophil degranulation. The HPD diet did not lead to liver damage, however, it caused progressing obesity, hypercholesterolemia and hyperglycemia. We conclude that the relatively mild negative impact of such diet on liver functioning can be attributed to a lower bioavailability of palm oil-derived C16:0 vs. that of C18:1 and the efficiency of mechanisms preventing liver injury, possibly including dynamic protein S-palmitoylation.

Interplay between DsbA1, DsbA2 and C8J_1298 Periplasmic Oxidoreductases of Campylobacter jejuni and Their Impact on Bacterial Physiology and Pathogenesis.

The bacterial proteins of the Dsb family catalyze the formation of disulfide bridges between cysteine residues that stabilize protein structures and ensure their proper functioning. Here, we report the detailed analysis of the Dsb pathway of Campylobacter jejuni. The oxidizing Dsb system of this pathogen is unique because it consists of two monomeric DsbAs (DsbA1 and DsbA2) and one dimeric bifunctional protein (C8J_1298). Previously, we showed that DsbA1 and C8J_1298 are redundant. Here, we unraveled the interaction between the two monomeric DsbAs by in vitro and in vivo experiments and by solving their structures and found that both monomeric DsbAs are dispensable proteins. Their structures confirmed that they are homologs of EcDsbL. The slight differences seen in the surface charge of the proteins do not affect the interaction with their redox partner. Comparative proteomics showed that several respiratory proteins, as well as periplasmic transport proteins, are targets of the Dsb system. Some of these, both donors and electron acceptors, are essential elements of the C. jejuni respiratory process under oxygen-limiting conditions in the host intestine. The data presented provide detailed information on the function of the C. jejuni Dsb system, identifying it as a potential target for novel antibacterial molecules.

HDAC6 inhibition upregulates CD20 levels and increases the efficacy of anti-CD20 monoclonal antibodies.

Downregulation of CD20, a molecular target for monoclonal antibodies (mAbs), is a clinical problem leading to decreased efficacy of anti-CD20-based therapeutic regimens. The epigenetic modulation of CD20 coding gene (MS4A1) has been proposed as a mechanism for the reduced therapeutic efficacy of anti-CD20 antibodies and confirmed with nonselective histone deacetylase inhibitors (HDACis). Because the use of pan-HDACis is associated with substantial adverse effects, the identification of particular HDAC isoforms involved in CD20 regulation seems to be of paramount importance. In this study, we demonstrate for the first time the role of HDAC6 in the regulation of CD20 levels. We show that inhibition of HDAC6 activity significantly increases CD20 levels in established B-cell tumor cell lines and primary malignant cells. Using pharmacologic and genetic approaches, we confirm that HDAC6 inhibition augments in vitro efficacy of anti-CD20 mAbs and improves survival of mice treated with rituximab. Mechanistically, we demonstrate that HDAC6 influences synthesis of CD20 protein independently of the regulation of MS4A1 transcription. We further demonstrate that translation of CD20 mRNA is significantly enhanced after HDAC6 inhibition, as shown by the increase of CD20 mRNA within the polysomal fraction, indicating a new role of HDAC6 in the posttranscriptional mechanism of CD20 regulation. Collectively, our findings suggest HDAC6 inhibition is a rational therapeutic strategy to be implemented in combination therapies with anti-CD20 monoclonal antibodies and open up novel avenues for the clinical use of HDAC6 inhibitors.

Thioloxidoreductase HP0231 of Helicobacter pylori impacts HopQ-dependent CagA translocation.

Thioloxidoreductase HP0231 of Helicobacter pylori plays essential roles in gastric colonization and related gastric pathology. Comparative proteomics and analysis of complexes between HP0231 and its protein substrates suggested that several Hop proteins are its targets. HP0231 is a dimeric oxidoreductase that functions in an oxidizing Dsb (disulfide bonds) pathway of H. pylori. H. pylori HopQ possesses six cysteine residues, which generate three consecutive disulfide bridges. Comparison of the redox state of HopQ in wild-type cells to that in hp0231-mutated cells clearly indicated that HopQ is a substrate of HP0231. HopQ binds CEACAM1, 3, 5 and 6 (carcinoembryonic antigen-related cell adhesion molecules). This interaction enables T4SS-mediated translocation of CagA into host cells and induces host signaling. Site directed mutagenesis of HopQ (changing cysteine residues into serine) and analysis of the functioning of HopQ variants showed that HP0231 influences the delivery of CagA into host cells, in part through its impact on HopQ redox state. Introduction of a C382S mutation into HopQ significantly affects its reaction with CEACAM receptors, which disturbs T4SS functioning and CagA delivery. An additional effect of HP0231 on other adhesins and their redox state, resulting in their functional impairment, cannot be excluded.

Inhibition of protein disulfide isomerase induces differentiation of acute myeloid leukemia cells.

A cute myeloid leukemia is a malignant disease of immature myeloid cells. Despite significant therapeutic effects of differentiation-inducing agents in some acute myeloid leukemia subtypes, the disease remains incurable in a large fraction of patients. Here we show that SK053, a thioredoxin inhibitor, induces differentiation and cell death of acute myeloid leukemia cells. Considering that thioredoxin knock-down with short hairpin RNA failed to exert antiproliferative effects in one of the acute myeloid leukemia cell lines, we used a biotin affinity probe-labeling approach to identify potential molecular targets for the effects of SK053. Mass spectrometry of proteins precipitated from acute myeloid leukemia cells incubated with biotinylated SK053 used as a bait revealed protein disulfide isomerase as a potential binding partner for the compound. Biochemical, enzymatic and functional assays using fluorescence lifetime imaging confirmed that SK053 binds to and inhibits the activity of protein disulfide isomerase. Protein disulfide isomerase knockdown with short hairpin RNA was associated with inhibition of cell growth, increased CCAAT enhancer-binding protein α levels, and induction of differentiation of HL-60 cells. Molecular dynamics simulation followed by the covalent docking indicated that SK053 binds to the fourth thioredoxin-like domain of protein disulfide isomerase. Differentiation of myeloid precursor cells requires the activity of CCAAT enhancer-binding protein α, the function of which is impaired in acute myeloid leukemia cells through various mechanisms, including translational block by protein disulfide isomerase. SK053 increased the levels of CCAAT enhancer-binding protein α and upregulated mRNA levels for differentiation-associated genes. Finally, SK053 decreased the survival of blasts and increased the percentage of cells expressing the maturation-associated CD11b marker in primary cells isolated from bone marrow or peripheral blood of patients with acute myeloid leukemia. Collectively, these results provide a proof-of-concept that protein disulfide isomerase inhibition has potential as a therapeutic strategy for the treatment of acute myeloid leukemia and for the development of small-molecule inhibitors of protein disulfide isomerase.

Nucleolar Enrichment of Brain Proteins with Critical Roles in Human Neurodevelopment.

To study nucleolar involvement in brain development, the nuclear and nucleolar proteomes from the rat cerebral cortex at postnatal day 7 were analyzed using LC-MS/iTRAQ methodology. Data of the analysis are available via ProteomeXchange with identifier PXD002188. Among 504 candidate nucleolar proteins, the overrepresented gene ontology terms included such cellular compartmentcategories as "nucleolus", "ribosome" and "chromatin". Consistent with such classification, the most overrepresented functional gene ontology terms were related to RNA metabolism/ribosomal biogenesis, translation, and chromatin organization. Sixteen putative nucleolar proteins were associated with neurodevelopmental phenotypes in humans. Microcephaly and/or cognitive impairment were the most common phenotypic manifestations. Although several such proteins have links to ribosomal biogenesis and/or genomic stability/chromatin structure (e.g. EMG1, RPL10, DKC1, EIF4A3, FLNA, SMC1, ATRX, MCM4, NSD1, LMNA, or CUL4B), others including ADAR, LARP7, GTF2I, or TCF4 have no such connections known. Although neither the Alazami syndrome-associated LARP7nor the Pitt-Hopkins syndrome-associated TCF4 were reported in nucleoli of non-neural cells, in neurons, their nucleolar localization was confirmed by immunostaining. In cultured rat hippocampal neurons, knockdown of LARP7 reduced both perikaryal ribosome content and general protein synthesis. Similar anti-ribosomal/anti-translation effects were observed after knockdown of the ribosomal biogenesis factor EMG1 whose deficiency underlies Bowen-Conradi syndrome. Finally, moderate reduction of ribosome content and general protein synthesis followed overexpression of two Pitt-Hopkins syndrome mutant variants of TCF4. Therefore, dysregulation of ribosomal biogenesis and/or other functions of the nucleolus may disrupt neurodevelopment resulting in such phenotypes as microcephaly and/or cognitive impairment.

N-Homocysteinylation impairs collagen cross-linking in cystathionine ß-synthase-deficient mice: a novel mechanism of connective tissue abnormalities.

Cystathionine ß-synthase (CBS) deficiency, a genetic disorder in homocysteine (Hcy) metabolism in humans, elevates plasma Hcy-thiolactone and leads to connective tissue abnormalities that affect the cardiovascular and skeletal systems. However, the underlying mechanism of these abnormalities is not understood. Hcy-thiolactone has the ability to form isopeptide bonds with protein lysine residues, which generates N-homocysteinylated protein. Because lysine residues are involved in collagen cross-linking, N-homocysteinylation of these lysines should impair cross-linking. Using a Tg-I278T Cbs-/- mouse model of hyperhomocysteinemia (HHcy) which replicates the connective tissue abnormalities observed in CBS-deficient patients, we found that N-Hcy-collagen was elevated in bone, tail, and heart of Cbs-/- mice, whereas pyridinoline cross-links were significantly reduced. Plasma deoxypyridinoline cross-link and cross-linked carboxyterminal telopeptide of type I collagen were also significantly reduced in the Cbs-/- mice. Lysine oxidase activity and mRNA level were not reduced by the Cbs-/- genotype. We also showed that collagen carries S-linked Hcy bound to the thiol of N-linked Hcy. In vitro experiments showed that Hcy-thiolactone modifies lysine residues in collagen type I α-1 chain. Residue K160, located in the nonhelical N-telopeptide region and involved in pyridinoline cross-link formation, was also N-homocysteinylated in vivo Taken together, our findings showed that N-homocysteinylation of collagen in Cbs-/- mice impairs its cross-linking. These findings explain, at least in part, connective tissue abnormalities observed in HHcy.-Perla-Kajan, J., Utyro, O., Rusek, M., Malinowska, A., Sitkiewicz, E., Jakubowski, H. N-Homocysteinylation impairs collagen cross-linking in cystathionine ß-synthase-deficient mice: a novel mechanism of connective tissue abnormalities.

Tandem mass tag-based proteomic analysis of granulosa and theca interna cells of the porcine ovarian follicle following in vitro treatment with vitamin D3 and insulin alone or in combination.

This study was performed to investigate the proteomic basis underlying the interaction between vitamin D3 (VD) and insulin (I) within ovarian follicle using the pig as a model. Porcine antral follicles were incubated in vitro for 12 h with VD alone and I alone or in combination (VD + I) or with no treatment as the control (C). In total, 7690 and 7467 proteins were identified in the granulosa and theca interna compartments, respectively. Comparative proteomic analysis revealed 97 differentially abundant proteins (DAPs) within the granulosa layer and 11 DAPs within the theca interna layer. In the granulosa compartment, VD affected proteome leading to the promotion of cell proliferation, whereas I influenced mainly proteins related to cellular adhesion. The VD + I treatment induced granulosa cell proliferation probably via the DAPs involved in DNA synthesis and the cell cycle regulation. In the theca interna layer, VD alone or in co-treatment with I affected DAPs associated with cholesterol transport and lipid and steroid metabolic processes that was further confirmed by diminished lipid droplet accumulation. SIGNIFICANCE: The application of quantitative proteomics demonstrated for the first time the complexity of VD and I interactions in porcine ovarian follicle, providing a framework for understanding the molecular mechanisms underlying their cross-talk. Although identified DAPs were related to crucial ovarian processes, including the granulosa cell proliferation and cholesterol transport in the theca interna layer, novel molecular pathways underlying these processes have been proposed. The identified unique proteins may serve as indicators of VD and I interactions in both follicle layers, and could be useful biomarkers of ovarian pathologies characterized by impaired VD and I levels, such as polycystic ovary syndrome.

Silencing of the PHLDA1 leads to global proteome changes and differentiation pathways of human neuroblastoma cells.

Neuroblastoma (NB) is the most common extracranial pediatric solid tumor originating from the abnormal development of cells of the sympathoadrenal lineage of the neural crest. Targeting GD2 ganglioside (GD2), a glycolipid expressed on neuroblastoma cells, with GD2 ganglioside-recognizing antibodies affects several pivotal signaling routes that drive or influence the malignant phenotype of the cells. Previously performed gene expression profiling helped us to identify the PHLDA1 (pleckstrin homology-like domain family A member 1) gene as the most upregulated gene in the IMR-32 human neuroblastoma cells treated with the mouse 14G2a monoclonal antibody. Mass spectrometry-based proteomic analyses were applied to better characterize a role of PHLDA1 protein in the response of neuroblastoma cells to chimeric ch14.18/CHO antibody. Additionally, global protein expression profile analysis in the IMR-32 cell line with PHLDA1 silencing revealed the increase in biological functions of mitochondria, accompanied by differentiation-like phenotype of the cells. Moreover, mass spectrometry analysis of the proteins co-immunoprecipitated using anti-PHLDA1-specific antibody, selected a group of possible PHLDA1 binding partners. Also, a more detailed analysis suggested that PHLDA1 interacts with the DCAF7/AUTS2 complex, a key component of neuronal differentiation in vitro. Importantly, our results indicate that PHLDA1 silencing enhances the EGF receptor signaling pathway and combinatory treatment of gefitinib and ch14.18/CHO antibodies might be beneficial for neuroblastoma patients. Data are available via ProteomeXchange with the identifier PXD044319.

Daphnia stress response to environmental concentrations of chloramphenicol-multi-omics approach.

Commonly used medicines, when discarded or improperly disposed of, are known to contaminate freshwater ecosystems. Pharmaceuticals can be toxic and mutagenic, and can modify freshwater organisms, even at environmentally relevant concentrations. Chloramphenicol (CAP) is an antibiotic banned in Europe. However, it is still found in surface waters around the world. The aim of this study was to evaluate the impact of chloramphenicol contamination in freshwater on the model organism Daphnia magna. Specific life history parameters, proteome, and host-associated microbiome of four D. magna clones were analyzed during a three-generation exposure to CAP at environmental concentrations (32 ng L-1). In the first generation, no statistically significant CAP effect at the individual level was detected. After three generations, exposed animals were smaller at first reproduction and on average produced fewer offspring. The differences in D. magna's life history after CAP treatment were in accordance with proteome changes. D. magna's response to CAP presence indicates the high stress that the tested organisms are under, e.g., male production, upregulation of ubiquitin-conjugating enzyme E2 and calcium-binding protein, and downregulation of glutathione transferase. The CAP-exposed D. magna proteome profile confirms that CAP, being reactive oxygen species (ROS)-inducing compounds, contributes to structural changes in mitochondria. Microbiome analysis showed a significant difference in the Shannon index between control and CAP-exposed animals, the latter having a more diverse microbiome. Multilevel analyses, together with long exposure in the laboratory imitating conditions in a polluted environment, allow us to obtain a more complete picture of the impact of CAP on D. magna.

Understanding functions of eEF1 translation elongation factors beyond translation. A proteomic approach.

Mammalian translation elongation factors eEF1A1 and eEF1A2 are 92% homologous isoforms whose mutually exclusive tissue-specific expression is regulated during development. The isoforms have similar translation functionality, but show differences in spatial organization and participation in various processes, such as oncogenesis and virus reproduction. The differences may be due to their ability to interact with isoform-specific partner proteins. We used the identified sets of eEF1A1 or eEF1A2 partner proteins to identify cell complexes and/or processes specific to one particular isoform. As a result, we found isoform-specific interactions reflecting the involvement of different eEF1A isoforms in different cellular processes, including actin-related, chromatin-remodeling, ribonuclease H2, adenylyl cyclase, and Cul3-RING ubiquitin ligase complexes as well as initiation of mitochondrial transcription. An essential by-product of our analysis is the elucidation of a number of cellular processes beyond protein biosynthesis, where both isoforms appear to participate such as large ribosomal subunit biogenesis, mRNA splicing, DNA mismatch repair, 26S proteasome activity, P-body and exosomes formation, protein targeting to the membrane. This information suggests that a relatively high content of eEF1A in the cell may be necessary not only to maintain efficient translation, but also to ensure its participation in various cellular processes, where some roles of eEF1A have not yet been described. We believe that the data presented here will be useful for deciphering new auxiliary functions of eEF1A and its isoforms, and provide a new look at the known non-canonical functions of this main component of the human translation-elongation machinery.

Visfatin impact on the proteome of porcine luteal cells during implantation.

Visfatin (VIS) is a hormone belonging to the adipokines' group secreted mainly by the adipose tissue. VIS plays a crucial role in the control of energy homeostasis, inflammation, cell differentiation, and angiogenesis. VIS expression was confirmed in the hypothalamic-pituitary-gonadal (HPG) axis structures, as well as in the uterus, placenta, and conceptuses. We hypothesised that VIS may affect the abundance of proteins involved in the regulation of key processes occurring in the corpus luteum (CL) during the implantation process in pigs. In the present study, we performed the high-throughput proteomic analysis (liquid chromatography with tandem mass spectrometry, LC-MS/MS) to examine the in vitro influence of VIS (100 ng/mL) on differentially regulated proteins (DRPs) in the porcine luteal cells (LCs) on days 15-16 of pregnancy (implantation period). We have identified 511 DRPs, 276 of them were up-regulated, and 235 down-regulated in the presence of VIS. Revealed DRPs were assigned to 162 gene ontology terms. Western blot analysis of five chosen DRPs, ADAM metallopeptidase with thrombospondin type 1 motif 1 (ADAMTS1), lanosterol 14-α demethylase (CYP51A1), inhibin subunit beta A (INHBA), notch receptor 3 (NOTCH3), and prostaglandin E synthase 2 (mPGES2) confirmed the veracity and accuracy of LC-MS/MS method. We indicated that VIS modulates the expression of proteins connected with the regulation of lipogenesis and cholesterologenesis, and, in consequence, may be involved in the synthesis of steroid hormones, as well as prostaglandins' metabolism. Moreover, we revealed that VIS affects the abundance of protein associated with ovarian cell proliferation, differentiation, and apoptosis, as well as CL new vessel formation and tissue remodelling. Our results suggest important roles for VIS in the regulation of ovarian functions during the peri-implantation period.

Proteome Profiling of the Dystrophic mdx Mice Diaphragm.

Mdx mice with a spontaneous mutation in exon 23 of the Dmd gene represent the most common model to investigate the pathophysiology of Duchenne muscular dystrophy (DMD). The disease, caused by the lack of functional dystrophin, is characterized by irreversible impairment of muscle functions, with the diaphragm affected earlier and more severely than other skeletal muscles. We applied a label-free (LF) method and the more thorough tandem mass tag (TMT)-based method to analyze differentially expressed proteins in the diaphragm of 6-week-old mdx mice. The comparison of both methods revealed 88 commonly changed proteins. A more in-depth analysis of the TMT-based method showed 953 significantly changed proteins, with 867 increased and 86 decreased in dystrophic animals (q-value < 0.05, fold-change threshold: 1.5). Consequently, several dysregulated processes were demonstrated, including the immune response, fibrosis, translation, and programmed cell death. Interestingly, in the dystrophic diaphragm, we found a significant decrease in the expression of enzymes generating hydrogen sulfide (H2S), suggesting that alterations in the metabolism of this gaseous mediator could modulate DMD progression, which could be a potential target for pharmacological intervention.

Characteristics of size-exclusion chromatography enriched porcine follicular fluid extracellular vesicles.

Extracellular vesicles (EVs) are membrane-bound nanoparticles that are released by different cell types and play a crucial role in the intercellular communication. They carry various biomolecular compounds such as DNA, RNA, proteins, and lipids. Given that EVs are a new element of the communication within the ovarian follicle, extensive research is needed to optimize method of their isolation. The aim of the study was to assess size-exclusion chromatography (SEC) as a tool for effective EVs isolation from porcine ovarian follicular fluid. The characterization of EVs was performed by nanoparticle tracking analysis, transmission electron microscopy, atomic force microscopy, mass spectrometry and Western blot. We determined EVs concentration, size distribution, zeta potential, morphology, purity, and marker proteins. Our results show that SEC is an effective method for isolation of EVs from porcine follicular fluid. They displayed predominantly exosome properties with sufficient purity and possibility for further functional analyses, including proteomics.