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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., (© 2024. The Author(s).)

Although antipsychotics are routinely used in the treatment of schizophrenia for the last decades, their precise mechanism of action is still unclear. In this study, we investigated changes in the PC12 cells' proteome under the influence of clozapine, risperidone, and haloperidol to identify protein pathways regulated by antipsychotics. Analysis of the protein profiles in two time points: after 12 and 24 h of incubation with drugs revealed significant alterations in 510 proteins. Further canonical pathway analysis revealed an inhibition of ciliary trophic factor signaling after treatment with haloperidol and showed a decrease in acute phase response signaling in the risperidone group. Interestingly, all tested drugs have caused changes in PC12 proteome which correspond to inhibition of cytokines: tumor necrosis factor (TNF) and transforming growth factor beta 1 (TGF-β1). We also found that the 12-h incubation with clozapine caused up-regulation of protein kinase A signaling and translation machinery. After 24 h of treatment with clozapine, the inhibition of the actin cytoskeleton signaling and Rho proteins signaling was revealed. The obtained results suggest that the mammalian target of rapamycin complex 1 (mTORC1) and 2 (mTORC2) play a central role in the signal transduction of clozapine.

Microglia and astrocytes, two types of glial cells are known to be important targets for antidepressant drugs. Here we used a comprehensive proteomic analysis to examine the effect of imipramine on rat primary mixed glial culture. The two-dimensional differential gel electrophoresis method allowed us to identify 62 proteins that were altered by imipramine. Functional analysis revealed that imipramine influenced the level of proteins involved in oxidative stress; in particular, it elevated the level of glutathione transferases. Imipramine upregulated proteins related to glycolysis but down-regulated many mitochondrial proteins including enzymes involved in oxidative phosphorylation. Mitochondrial dysfunction, especially decrease of mitochondrial membrane potential can be counted as a side effect triggered by imipramine. Imipramine induced lowering of chaperone level and alterations suggesting impaired protein synthesis could be associated with increased apoptosis. One of the most pronounced effect of imipramine is the reduction of vimentin level, this protein is engaged in majority of biological processes which were found to be affected by imipramine. Many imipramine regulated proteins, including chaperones, cathepsins and annexins are involved in immune responses. Additionally, imipramine influenced proteins associated with phagocytosis and cell migration. Overall these findings indicate that imipramine produces complex effect on glial cells, primarily on microglia and suggest their transition towards a more quiescent, metabolically less demanding phenotype., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: The cell nucleus is a highly dynamic subcellular compartment performing crucial processes for functioning and administration of the cell. Proteomic analysis of isolated nuclear fraction enables in-depth insight into these processes leading to better understanding of physiological and pathological state of the brain. There is no universal method for nuclear proteome investigation and each biological material needs individual optimization. An additional difficulty is the large amount of nucleic acids, which impairs isoelectrofocusing of proteins and shotgun mass spectrometry analysis of complex peptide samples., New Method: We performed the first comprehensive optimization of intact nuclei isolation from mouse brain in combination with nuclear protein purification prior to two-dimensional gel electrophoresis (2DE) and gel-free proteomic analysis., Results: Application of sonication, digestion with nuclease and protein precipitation with acetone allowed to obtain high quality 2DE gels. Shotgun mass spectrometry analysis of isolated proteins proved an enrichment in nuclear proteins. The 66.4% of 265 identified proteins had assigned nucleus localization in UniProt database. Gene Ontology analysis using DAVID platform revealed the highest fold enrichment in spliceosome (24.5), nuclear periphery (12.4) and nuclear matrix (11.3)., Comparison With Existing Methods: The proposed procedure is tailored to mouse brain tissue nuclear subproteome investigation. The quality of isolated nuclei, the effectiveness of the protein purification, efficiency of protein recovery after precipitation and overall method reproducibility was taken into detailed consideration., Conclusions: The elaborated procedure could be further applied for in-depth proteomic analysis of molecular processes occurring in the mouse brain nucleus., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Unlabelled: Intrinsically disordered proteins (IDPs) are biologically active and crucial for cell function although they do not possess defined three-dimensional architecture. IDPs are especially prevalent in eukaryotic proteomes, and large-scale experiments have shown that many IDPs are nuclear proteins. Bioinformatic analyses have also demonstrated that the vast majority of transcription factors contain extended regions of intrinsic disorder. In the current study, we isolated and functionally analyzed IDPs expressed in the nuclei of HEK293 human cells. According to the results of MS analysis followed by subsequent analysis with the bioinformatic tools IUPred and RAPID (regression-based accurate predictor of intrinsic disorder), a heat-treatment method was able to enrich the nuclear lysate in IDPs. For approximately 85% of the proteins obtained, IUPred predicted a sequence of 30 or more consecutive disordered residues (DRs), and for approximately 83% of the proteins RAPID reported a content of at least 25% DRs (compared to ~66% and 49%, respectively, for the nuclear lysate). Gene Ontology analysis in terms of molecular function revealed that the obtained fraction was generally enriched in proteins involved in the process of transcription and especially in transcription factors. We also showed experimentally that IDPs are overrepresented in the cell nucleus., Significance: Intrinsically disordered proteins (IDPs) are crucial cellular molecules and are especially numerous in eukaryotes. In particular, IDPs act as signaling and regulatory proteins, and impairment in their functioning may lead to serious diseases. Large-scale bioinformatic studies of IDPs have provided essential knowledge about this group of proteins. However, experimental data reflect the actual situation in living cells. Our study is the first large-scale proteomic analysis of nuclear IDPs. We showed experimentally that IDPs are overrepresented in the nucleus in comparison to the whole cell. Analysis of molecular function indicated that the nuclear intrinsically disordered proteome (IDP-ome) is enriched in proteins involved in transcription regulation and especially in transcription factors. The IDP isolation method from human cell nuclei presented in this article could be further applied in differential proteomic studies., (Copyright © 2015 Elsevier B.V. All rights reserved.)