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The stiffening or softening of cancers observed in nanoindentation experiments has been recognized as a marker of cancer-related changes. In bladder cancers, continuous stretching/destretching is observed due to its functionality, indicating that shear forces dominate the mechanical response of these cells. Thus, nanoindentation and microrheological measurements conducted in parallel allow for a fully reliable mechanomarker of cancer progression. Here, bladder cancer cell lines, i.e., non-malignant cell cancer of the ureter (HCV29), bladder carcinoma (HT1376), and transitional cell carcinoma (T24), were studied. Nanoindentation and microrheological experiments were conducted on individual cells, cell monolayers, and spheroids that were formed using non-adherent surface plates. The results show that nanoindentation experiments can only differentiatebetween non-malignant HCV29 (stiffer) and cancerous HT1376 and T24 (softer) cells. Applying microrheology recognizes the type of grade 3 bladder cancers (carcinoma HT1376 or transitional cell carcinoma T24 cells). Weshowed that actin filaments are a vital element defining the rheological properties of spheroids. Differences inmechanical properties of cell monolayers could be associated with thick actin bundles and intercellular connections,with some extracellular matrix (ECM) contributing to the stiffening of such monolayers. Our findings demonstrate that a complete image of how cancer cells respond to mechanical stress (compressive and shear forces) can only be obtained after microrheological measurements using the transition frequency separating elastic and viscous regimes as a non-labeled biomarker of bladder cancer progression.

Cancer progression is associated with changes in cell mechanical and rheological properties that could be probed by atomic force microscopy (AFM). In this study, we applied AFM to measure elastic (by compressing the cells) and viscoelastic (by applying shear stress) properties of bladder cancers in relation to their culture morphology (in single cells, cell monolayers, and spheroids). Three different cell lines, HCV29 (non-malignant cell cancer of ureter), HT1376 (grade III bladder carcinoma), T24 (grade IV transitional cell carcinoma), were investigated. Nanoindentation measurements only differentiate between non-malignant and cancer cells, but it is difficult to distinguish between specific bladder cancers. By applying microrheological measurements, we confirm that non-malignant cells are more rigid than cancer cells but more importantly, it was possible to differentiate between two cancerous cell lines, regardless of the culture conditions. As each of them is characterized by a distinct actin filament network inside the cell, we showed that actin filaments are a key element in defining the rheological properties of spheroids originating from cells having thick actin bundles. Our results showed that HCV29 cells are more rigid than the studied cancer cells, indicating that normal cells are resistant to compressive and shear forces. Therefore, we conclude that cell mechanical and rheological properties may serve as a biophysical marker to distinguish normal and cancer cells of different malignancies.

Protein content of extracellular vesicles (EVs) can modulate different processes during carcinogenesis. Novel proteomic strategies have been applied several times to profile proteins present in exosomes released by urothelial bladder cancer (UBC) cells. However, similar studies have not been conducted so far on another population of EVs, i.e., ectosomes. In the present study we used a shotgun nanoLC–MS/MS proteomic approach to investigate the protein content of ectosomes released in vitro by T-24 UBC cells and HCV-29 normal ureter epithelial cells. In addition, cancer-promoting effects exerted by UBC-derived ectosomes on non-invasive cells in terms of cell proliferation and migratory properties were assessed. In total, 1158 proteins were identified in T-24-derived ectosomes, while HCV-29-derived ectosomes contained a lower number of 259 identified proteins. Qualitative analysis revealed 938 proteins present uniquely in T-24-derived ectosomes, suggesting their potential applications in bladder cancer management as diagnostic and prognostic biomarkers. In addition, T-24-derived ectosomes increased proliferation and motility of recipient cells, likely due to the ectosomal transfer of the identified cancer-promoting molecules. The present study provided a focused identification of biologically relevant proteins in UBC-derived ectosomes, confirming their role in UBC development and progression, and their applicability for further biomarker-oriented studies in preclinical or clinical settings.

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. Supplementary Information The online version contains supplementary material available at 10.1007/s11064-021-03348-4.

Tetrameric transthyretin (TTR) transports thyroid hormones and retinol in plasma and cerebrospinal fluid and performs protective functions under stress conditions. Ageing and mutations result in TTR destabilisation and the formation of the amyloid deposits that dysregulate Ca2+ homeostasis. Our aim was to determine whether Ca2+ affects the structural stability of TTR. We show, using multiple techniques, that Ca2+ does not induce prevalent TTR dissociation and/or oligomerisation. However, in the presence of Ca2+, TTR exhibits altered conformational flexibility and different interactions with the solvent molecules. These structural changes lead to the formation of the sub-populations of non-native TTR conformers and to the destabilisation of the structure of TTR. Moreover, the sub-population of TTR molecules undergoes fragmentation that is augmented by Ca2+. We postulate that Ca2+ constitutes the structural and functional switch between the native and non-native forms of TTR, and therefore tip the balance towards age-dependent pathological calcification.

Kti12 and PSTK are closely related and highly similar proteins implicated in different aspects of tRNA metabolism. Kti12 has been identified as an essential regulatory factor of the Elongator complex, involved in the modification of uridine bases in eukaryotic tRNAs. PSTK phosphorylates the tRNASec-bound amino acid serine, which is required to synthesize selenocysteine. Kti12 and PSTK have previously been studied independently in various organisms, but only appear simultaneously in some animalia, including humans. As Kti12- and PSTK-related pathways are clinically relevant, it is of prime importance to understand their biological functions and mutual relationship in humans. Here, we use different tRNA substrates to directly compare the enzymatic activities of purified human KTI12 and human PSTK proteins. Our complementary Co-IP and BioID2 approaches in human cells confirm that Elongator is the main interaction partner of KTI12 but additionally indicate potential links to proteins involved in vesicular transport, RNA metabolism and deubiquitination. Moreover, we identify and validate a yet uncharacterized interaction between PSTK and γ-taxilin. Foremost, we demonstrate that human KTI12 and PSTK do not share interactors or influence their respective biological functions. Our data provide a comprehensive analysis of the regulatory networks controlling the activity of the human Elongator complex and selenocysteine biosynthesis.

Intrinsically disordered proteins (IDPs) play crucial roles in cell functioning, although they do not possess defined three-dimensional architecture. They are highly abundant in the cell nucleus, and the vast majority of transcription factors (TFs) contain extended regions of intrinsic disorder. IDPs do not respond to denaturing conditions in a standard manner, and this can be used for their separation from structured proteins. Here we describe a protocol for the isolation and characterization of nuclear IDPs in which heat treatment is used for enrichment of IDPs in samples. The whole workflow comprises the following steps: nuclei isolation from HEK293 (human embryonic kidney) cells, protein extraction, enrichment of IDPs, sample preparation for mass spectrometric analysis, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, in silico assessment of protein disorder, and Gene Ontology analysis.

Rhabdomyosarcoma (RMS) is a predominant soft tissue tumor in children and adolescents. For high-grade RMS with metastatic involvement, the 3-year overall survival rate is only 25 to 30%. Thus, understanding the regulatory mechanisms involved in promoting the metastasis of RMS is important. Here, we demonstrate for the first time that the SNAIL transcription factor regulates the metastatic behavior of RMS both in vitro and in vivo. SNAIL upregulates the protein expression of EZRIN and AKT, known to promote metastatic behavior, by direct interaction with their promoters. Our data suggest that SNAIL promotes RMS cell motility, invasion and chemotaxis towards the prometastatic factors: HGF and SDF-1 by regulating RHO, AKT and GSK3β activity. In addition, miRNA transcriptome analysis revealed that SNAIL-miRNA axis regulates processes associated with actin cytoskeleton reorganization. Our data show a novel role of SNAIL in regulating RMS cell metastasis that may also be important in other mesenchymal tumor types and clearly suggests SNAIL as a promising new target for future RMS therapies.

Cutaneous melanoma (CM) is an aggressive type of skin cancer for which effective biomarkers are still needed. Recently, the protein content of extracellular vesicles (ectosomes and exosomes) became increasingly investigated in terms of its functional role in CM and as a source of novel biomarkers, however, the data concerning the proteome of CM-derived ectosomes is very limited. We used the shotgun nanoLC&ndash, MS/MS approach to the profile protein content of ectosomes from primary (WM115, WM793) and metastatic (WM266-4, WM1205Lu) CM cell lines. Additionally, the effect exerted by CM ectosomes on recipient cells was assessed in terms of cell proliferation (Alamar Blue assay) and migratory properties (wound healing assay). All cell lines secreted heterogeneous populations of ectosomes enriched in the common set of proteins. A total of 1507 unique proteins were identified, with many of them involved in cancer cell proliferation, migration, escape from apoptosis, epithelial&ndash, mesenchymal transition and angiogenesis. Isolated ectosomes increased proliferation and motility of recipient cells, likely due to the ectosomal transfer of different cancer-promoting molecules. Taken together, these results confirm the significant role of ectosomes in several biological processes leading to CM development and progression, and might be used as a starting point for further studies exploring their diagnostic and prognostic potential.

Intrinsically disordered proteins (IDPs) play crucial roles in cell functioning, although they do not possess defined three-dimensional architecture. They are highly abundant in the cell nucleus, and the vast majority of transcription factors (TFs) contain extended regions of intrinsic disorder. IDPs do not respond to denaturing conditions in a standard manner, and this can be used for their separation from structured proteins. Here we describe a protocol for the isolation and characterization of nuclear IDPs in which heat treatment is used for enrichment of IDPs in samples. The whole workflow comprises the following steps: nuclei isolation from HEK293 (human embryonic kidney) cells, protein extraction, enrichment of IDPs, sample preparation for mass spectrometric analysis, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, in silico assessment of protein disorder, and Gene Ontology analysis.

A lipid-binding protein apolipophorin III (apoLp-III), an exchangeable component of lipophorin particles, is involved in lipid transport and immune response in insects. In Galleria mellonella, apoLp-III binding to high-density lipophorins and formation of low-density lipophorin complexes upon immune challenge was reported. However, an unanswered question remains whether apoLp-III could form different complexes in a pathogen-dependent manner. Here we report on pathogen- and time-dependent alterations in the level of apoLp-III free and lipophorin-bound form that occur in the hemolymph and hemocytes shortly after immunization of G. mellonella larvae with different pathogens, i.e. Gram-negative bacterium Escherichia coli, Gram-positive bacterium Micrococcus luteus, yeast-like fungus Candida albicans, and filamentous fungus Fusarium oxysporum. These changes were accompanied by differently persistent re-localization of apoLp-III in the hemocytes. The apoLp-III-interacting proteins were recovered from immune hemolymph by affinity chromatography on a Sepharose bed with immobilized anti-apoLp-III antibodies. ApoLp-I, apoLp-II, hexamerin, and arylphorin were identified as main components that bound to apoLp-III; the N-terminal amino acid sequences of G. mellonella apoLp-I and apoLp-II were determined for the first time. In the recovered complexes, the pathogen-dependent differences in the content of individual apolipophorins were detected. Apolipophorins may thus be postulated as signaling molecules responding in an immunogen-dependent manner in early steps of G. mellonella immune response.

Using cyto-morphological analysis of somatic embryogenesis (SE) in the tree fern Cyathea delgadii as a guide, we performed a comparative proteomic analysis in stipe explants undergoing direct SE. Plant material was cultured on hormone-free medium supplemented with 2% sucrose. Phenol extracted proteins were separated using two-dimensional gel electrophoresis (2-DE) and mass spectrometry was performed for protein identification. A total number of 114 differentially regulated proteins was identified during early SE, i.e. when the first cell divisions started and several-cell pro-embryos were formed. Proteins were assigned to seven functional categories: carbohydrate metabolism, protein metabolism, cell organization, defense and stress responses, amino acid metabolism, purine metabolism, and fatty acid metabolism. Carbohydrate and protein metabolism were found to be the most sensitive SE functions with the greatest number of alterations in the intensity of spots in gel. Differences, especially in non-enzymatic and structural protein abundance, are indicative for cell organization, including cytoskeleton rearrangement and changes in cell wall components. The highest induced changes concern those enzymes related to fatty acid metabolism. Global analysis of the proteome reveals several proteins that can represent markers for the first 16 days of SE induction and expression in fern. The findings of this research improve the understanding of molecular processes involved in direct SE in C. delgadii .

Light-dependent protochlorophyllide oxidoreductase (POR, E.C. 1.3.1.33) is a plant enzyme that directly needs light to conduct a biochemical reaction. In the present paper we confirmed that POR forms large oligomers in solution before binding of substrates. We carried out the research using different techniques: cross-linking, native gel electrophoresis and FRET measurements. Mass spectrometry analysis of the cross-link products provided the first structural data about the organisation of the oligomer of POR. The results indicated that the catalytic motifs of the adjacent subunits become close to each other upon binding of substrates. Moreover, we identified two mutations of POR that disturbed its oligomerisation properties: Δ85–88 and Δ240–270. Additionally, a complete loss of the catalytic activity was observed for the following mutations: Δ189–194, Δ240–270, Δ318–331 and Δ392–393.

Bisphenol A (BPA) is commonly present in plastics used for food storage and preservation. The release of BPA from these products results in a permanent human exposition to BPA; however, the quality and quantity of BPA adverse effects remain a matter of controversy. The common presence of BPA in the human environment and the controversies concerning the relations of human exposition to BPA and cancer incidence justify the research on the interactions between BPA and pro-metastatic signaling in cancer cells. Here, we describe a novel BPA-reactive signaling axis that induces the epithelial-mesenchymal transition (EMT) in lung adenocarcinoma A549 cells. BPA exerted negligible effects on their properties in a wide range of concentrations (10 nM - 100 nM), whereas it considerably induced A549 invasiveness at high concentrations (10 μM). The BPA-induced EMT was illustrated by morphologic changes, E/N-cadherin switch and vimentin/Snail-1/connexin(Cx)43 up-regulation in A549 populations. It was followed by enhancement of A549 drug-resistance. Corresponding effects of BPA were observed in prostate cancer cell populations. Concomitantly, we observed increased levels and perinuclear accumulation of estrogen-related receptor gamma (ERRγ) in BPA-treated cells, its interactions with Cx43/Snail-1, and the corresponding effects of phenol red on A549 cells. Collectively, these data identify a novel, pro-metastatic Snail-1/Cx43/ERRγ signaling pathway. Its reactivity to BPA underlies the induction of cancer cells' invasiveness in the presence of high BPA concentrations in vitro. Thus, the chronic exposition of cancer cells to extrinsic and intrinsic BPA should be considered as a potential obstacle in a cancer therapy.

Metronomic agents reduce the effective doses and adverse effects of cytostatics in cancer chemotherapy. Therefore, they can enhance the treatment efficiency of drug-resistant cancers. Cytostatic and anti-angiogenic effects of fenofibrate (FF) suggest that it can be used for the metronomic chemotherapy of drug-resistant prostate tumors. To estimate the effect of FF on the drug-resistance of prostate cancer cells, we compared the reactions of na&iuml, ve and drug-resistant cells to the combined treatment with docetaxel (DCX)/mitoxantrone (MTX) and FF. FF sensitized drug-resistant DU145 and PC3 cells to DCX and MTX, as illustrated by their reduced viability and invasive potential observed in the presence of DCX/MTX and FF. The synergy of the cytostatic activities of both agents was accompanied by the inactivation of P-gp-dependent efflux, dysfunction of the microtubular system, and induction of polyploidy in DCX-resistant cells. Chemical inhibition of PPAR&alpha, and reactive oxygen species (ROS)-dependent pathways by GW6471 and N-acetyl-L-cysteine, respectively, had no effect on cell sensitivity to combined DCX/FF treatment. Instead, we observed the signs of adenosine triphosphate (ATP) deficit and autophagy in DCX/FF-treated drug-resistant cells. Furthermore, the cells that had been permanently propagated under DCX- and DCX/FF-induced stress did not acquire DCX/FF-resistance. Instead, relatively slow proliferation of DCX-resistant cells was efficiently inhibited by FF. Collectively, our observations show that FF reduces the effective doses of DCX by interfering with the drug resistance and energy metabolism of prostate cancer cells. Concomitantly, it impairs the chemotherapy-induced microevolution and expansion of DCX/FF-resistant cells. Therefore, FF can be applied as a metronomic agent to enhance the efficiency of palliative chemotherapy of prostate cancer.

Background Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively eliminates tumor cells. However, the short biological half-life of this molecule limits its potential use in the clinic. Our aim was to construct a recombinant strain of nonpathogenic Lactococcus lactis bacteria as a vector for effective and prolonged human TRAIL production. Herein, we examined the expression and secretion conditions leading to the production of biologically active protein in vitro. Results The human soluble TRAIL-cDNA (hsTRAIL-cDNA) with optimized codons was designed to fit the codon usage pattern (codon bias) of the L. lactis host. This cDNA construct was synthesized and cloned in lactococcal plasmid secretion vector pNZ8124 under the control of the nisin-induced PnisA promoter. The pNZ8124-hsTRAIL plasmid vector was transformed into the L. lactis NZ9000 host strain cells by electroporation. Secretion of the protein occurred at the neutral pH during induction, with optimized concentration of the inducer and presence of serine proteases inhibitor. Using Western blotting and amino acid sequencing method we found that TRAIL was secreted in two forms, as visualized by the presence of two distinct molecular size bands, both deprived of the usp45 protein, the bacterial signal peptide. By the use of MTS assay we were able to prove that hsTRAIL present in supernatant from L. lactis (hsTRAIL+) broth culture was cytotoxic to human HCT116 colon cancer cells but not to normal human fibroblasts. Flow cytometry analysis revealed TRAIL-induced apoptosis of cancer cells. Conclusions We designed recombinant L. lactis bacteria, which efficiently produce biologically active, anti-tumorigenic human TRAIL in vitro. Further studies in tumor-bearing NOD-SCID mice will reveal whether the TRAIL-secreting L. lactis bacteria can be used as a safe carrier of this protein, capable of inducing effective elimination of human colon cancer cells in vivo.

Accumulating evidence suggests that six proteases encoded in the spl operon of a dangerous human pathogen, Staphylococcus aureus, may play a role in virulence. Interestingly, SplA, B, D, and E have complementary substrate specificities while SplF remains to be characterized in this regard. Here, we describe the prerequisites of a heterologous expression system for active SplF protease and characterize the enzyme in terms of substrate specificity and its structural determinants. Substrate specificity of SplF is comprehensively profiled using combinatorial libraries of peptide substrates demonstrating strict preference for long aliphatic sidechains at the P1 subsite and significant selectivity for aromatic residues at P3. The crystal structure of SplF was provided at 1.7 Å resolution to define the structural basis of substrate specificity of SplF. The obtained results were compared and contrasted with the characteristics of other Spl proteases determined to date to conclude that the spl operon encodes a unique extracellular proteolytic system.

The complex network of anatomical connections of the nucleus accumbens (NAc) makes it an interface responsible for the selection and integration of cognitive and affective information to modulate appetitive or aversively motivated behaviour. There is evidence for NAc dysfunction in schizophrenia. NAc also seems to be important for antipsychotic drug action, but the biochemical characteristics of drug-induced alterations within NAc remain incompletely characterized. In this study, a comprehensive proteomic analysis was performed to describe the differences in the mechanisms of action of clozapine (CLO) and risperidone (RIS) in the rat NAc. Both antipsychotics influenced the level of microtubule-regulating proteins, i.e., stathmin, and proteins of the collapsin response mediator protein family (CRMPs), and only CLO affected NAD-dependent protein deacetylase sirtuin-2 and septin 6. Both antipsychotics induced changes in levels of other cytoskeleton-related proteins. CLO exclusively up-regulated proteins involved in neuroprotection, such as glutathione synthetase, heat-shock 70-kDa protein 8 and mitochondrial heat-shock protein 75. RIS tuned cell function by changing the pattern of post-translational modifications of some proteins: it down-regulated the phosphorylated forms of stathmin and dopamine and the cyclic AMP-regulated phosphoprotein (DARPP-32) isoform but up-regulated cyclin-dependent kinase 5 (Cdk5). RIS modulated the level and phosphorylation state of synaptic proteins: synapsin-2, synaptotagmin-1 and adaptor-related protein-2 (AP-2) complex.

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.

IgM is a multivalent antibody which evolved as a first line defense of adaptive immunity. It consists of heavy and light chains assembled into a complex oligomer. In mouse serum there are two forms of IgM, a full-length and a truncated one. The latter contains μ’ chain, which lacks a variable region. Although μ’ chain was discovered many years ago, its origin has not yet been elucidated. Our results indicate that μ’ chain is generated from a full-length heavy chain by non-enzymatic cleavage of the protein backbone. The cleavage occurred specifically after Asn209 and is prevented by mutating this residue into any other amino acid. The process requires the presence of other proteins, preferentially with an acidic isoelectric point, and is facilitated by neutral or alkaline pH. This unique characteristic of the investigated phenomenon distinguishes it from other, already described, Asn-dependent protein reactions. A single IgM molecule is able to bind up to 12 epitopes via its antigen binding fragments (Fabs). The cleavage at Asn209 generates truncated IgM molecules and free Fabs, resulting in a reduced IgM valence and probably affecting IgM functionality in vivo.

Acireductone dioxygenase (ARD) is an intriguing enzyme from the methionine salvage pathway that is capable of catalysing two different oxidation reactions with the same substrate depending on the type of the metal ion in the active site. To date, the structural information regarding the ARD–acireductone complex is limited and possible reaction mechanisms are still under debate. The results of joint experimental and computational studies undertaken to advance knowledge about ARD are reported. The crystal structure of an ARD from Homo sapiens was determined with selenomethionine. EPR spectroscopy suggested that binding acireductone triggers one protein residue to dissociate from Fe(2+), which allows NO (and presumably O(2)) to bind directly to the metal. Mössbauer spectroscopic data (interpreted with the aid of DFT calculations) was consistent with bidentate binding of acireductone to Fe(2+) and concomitant dissociation of His88 from the metal. Major features of Fe vibrational spectra obtained for the native enzyme and upon addition of acireductone were reproduced by QM/MM calculations for the proposed models. A computational (QM/MM) study of the reaction mechanisms suggests that Fe(2+) promotes O—O bond homolysis, which elicits cleavage of the C1—C2 bond of the substrate. Higher M(3+) /M(2+) redox potentials of other divalent metals do not support this pathway, and instead the reaction proceeds similarly to the key reaction step in the quercetin 2,3-dioxygenase mechanism.

Rationale: Extracellular vesicles (EVs) are tiny membrane-enclosed droplets released by cells through membrane budding or exocytosis. The myocardial reparative abilities of EVs derived from induced pluripotent stem cells (iPSCs) have not been directly compared with the source iPSCs. Objective: To examine whether iPSC-derived EVs can influence the biological functions of cardiac cells in vitro and to compare the safety and efficacy of iPSC-derived EVs (iPSC-EVs) and iPSCs for cardiac repair in vivo. Methods and Results: Murine iPSCs were generated, and EVs isolated from culture supernatants by sequential centrifugation. Atomic force microscopy, high-resolution flow cytometry, real-time quantitative RT-PCR, and mass spectrometry were used to characterize EV morphology and contents. iPSC-EVs were enriched in miRNAs and proteins with proangiogenic and cytoprotective properties. iPSC-EVs enhanced angiogenic, migratory, and antiapoptotic properties of murine cardiac endothelial cells in vitro. To compare the cardiac reparative capacities in vivo, vehicle, iPSCs, and iPSC-EVs were injected intramyocardially at 48 hours after a reperfused myocardial infarction in mice. Compared with vehicle-injected mice, both iPSC- and iPSC-EV–treated mice exhibited improved left ventricular function at 35 d after myocardial infarction, albeit iPSC-EVs rendered greater improvement. iPSC-EV injection also resulted in reduction in left ventricular mass and superior perfusion in the infarct zone. Both iPSCs and iPSC-EVs preserved viable myocardium in the infarct zone, whereas reduction in apoptosis was significant with iPSC-EVs. iPSC injection resulted in teratoma formation, whereas iPSC-EV injection was safe. Conclusions: iPSC-derived EVs impart cytoprotective properties to cardiac cells in vitro and induce superior cardiac repair in vivo with regard to left ventricular function, vascularization, and amelioration of apoptosis and hypertrophy. Because of their acellular nature, iPSC-EVs represent a safer alternative for potential therapeutic applications in patients with ischemic myocardial damage.

For over the last 50 years, the molecular mechanism of anti-psychotic drugs' action has been far from clear. While risperidone is very often used in clinical practice, the most efficient known anti-psychotic drug is clozapine (CLO). However, the biochemical background of CLO's action still remains elusive. In this study, we performed comparative proteomic analysis of rat cerebral cortex following chronic administration of these two drugs. We observed significant changes in the expression of cytoskeletal, synaptic, and regulatory proteins caused by both antipsychotics. Among other proteins, alterations in collapsin response mediator proteins, CRMP2 and CRMP4, were the most spectacular consequences of treatment with both drugs. Moreover, risperidone increased the level of proteins involved in cell proliferation such as fatty acid-binding protein-7 and translin-associated factor X. CLO significantly up-regulated the expression of visinin-like protein 1, neurocalcin δ and mitochondrial, stomatin-like protein 2, the calcium-binding proteins regulating calcium homeostasis, and the functioning of ion channels and receptors. Using two-dimensional differential electrophoresis, we demonstrate that chronic treatment the healthy rats with anti-psychotics, clozapine and risperidone, induce changes in expression of cytoskeletal, synaptic, and regulatory proteins in the cerebral cortex. While risperidone increases the level of proteins regulating cell proliferation, namely, fatty acid-binding protein-7 and translin-associated factor X, the clozapine significantly up-regulates calcium sensors, i.e., visinin-like protein 1 and neurocalcin δ. 2D DIGE, Differential in Gel Electrophoresis; Cy2, Cy3, and Cy5 are cyanine dyes.

Growing evidence indicates that intracellular signaling mediated by extracellular vesicles (EVs) released by stem cells plays a considerable role in triggering the regenerative program upon transplantation. EVs from umbilical cord mesenchymal stem cells (UC-MSC-EVs) have been shown to enhance tissue repair in animal models. However, translating such results into clinical practice requires optimized EV collection procedures devoid of animal-originating agents. Thus, in this study, we analyzed the influence of xeno-free expansion media on biological properties of UC-MSCs and UC-MSC-EVs for future applications in cardiac repair in humans. Our results show that proliferation, differentiation, phenotype stability, and cytokine secretion by UC-MSCs vary depending on the type of xeno-free media. Importantly, we found distinct molecular and functional properties of xeno-free UC-MSC-EVs including enhanced cardiomyogenic and angiogenic potential impacting on target cells, which may be explained by elevated concentration of several pro-cardiogenic and pro-angiogenic microRNA (miRNAs) present in the EVs. Our data also suggest predominantly low immunogenic capacity of certain xeno-free UC-MSC-EVs reflected by their inhibitory effect on proliferation of immune cells in vitro. Summarizing, conscious selection of cell culture conditions is required to harvest UC-MSC-EVs with the optimal desired properties including enhanced cardiac and angiogenic capacity, suitable for tissue regeneration. Key message Type of xeno-free media influences biological properties of UC-MSCs in vitro.Certain xeno-free media promote proliferation and differentiation ability of UC-MSCs.EVs collected from xeno-free cultures of UC-MSCs are biologically active.Xeno-free UC-MSC-EVs enhance cardiac and angiogenic potential of target cells.Type of xeno-free media determines immunomodulatory effects mediated by UC-MSC-EVs. Electronic supplementary material The online version of this article (doi:10.1007/s00109-016-1471-7) contains supplementary material, which is available to authorized users.

Sphingomyelin-binding proteins of the lysenin family were originally identified in earthworms belonging to the genus Eisenia comprised of at least two distinct species, E. andrei and E. fetida, until recently considered subspecies or morphotypes of E. foetida (sic). In the present study the presence of lysenin and lysenin-related protein 2 (LRP-2, known also as fetidin) was detected in coelomocytes retrieved from all investigated adult specimens of E. andrei, and E. fetida. They were accompanied by LRP-3 and LRP-1 in some specimens of E. andrei and E. fetida, respectively. Lysenins were not observed in a third composting lumbricid species, Dendrobaena veneta, which served as a convenient negative reference for techniques and procedures used in the study. The pore-forming potential of soluble and cellular fractions of coelomic fluid was studied towards sheep red blood cells and sphingomyelin-rich liposomes. After experimental depletion the potential was restored in parallel with restoration of chloragocyte-derived eleocytes in both E. andrei and E. fetida.

Cancer cells are known to release extracellular vesicles that often promote disease development and progression. The present study investigated the protein content and glycosylation pattern of ectosomes released in vitro by a human primary uveal melanoma Mel202 cell line. Ectosomes released by Mel202 cells were isolated from conditioned media using sequential centrifugation, and a nano-LC-MS/MS approach was used to determine their protein content. Subsequently, proteins from ectosomes, the whole cell extracts, and the membrane fractions were probed with a panel of lectins using Western blotting and flow cytometry to reveal characteristic glycan structures. As many as 2527 unique proteins were identified, and many of them are known to be involved in cancer cell proliferation and altered metabolism, tumor invasion, metastasis, or drug resistance. Lectin-based studies revealed a distinct glycosylation pattern between Mel202-derived ectosomes and the parental cell membranes. Selective enrichment of ectosomal proteins with bisected complex type N-glycans and &alpha, 2,6-linked sialic acids may be significant for ectosome formation and sequestration. Differences in the surface glycosylation of Mel202 cells and ectosomes supports recent findings that the budding of ectosomes occurs within strictly determined fragments of the plasma membrane, and thus ectosomes contain a unique protein and glycan composition.

The ultraspiracle protein (Usp), together with an ecdysone receptor (EcR) forms a heterodimeric ecdysteroid receptor complex, which controls metamorphosis in Drosophila melanogaster. Although the ecdysteroid receptor is considered to be a source of elements for ecdysteroid inducible gene switches in mammals, nothing is known about posttranslational modifications of the receptor constituents in mammalian cells. Up until now there has been no study about Usp sumoylation. Using Ubc9 fusion-directed sumoylation system, we identified Usp as a new target of SUMO1 and SUMO3 modification. Mutagenesis studies on the fragments of Usp indicated that sumoylation can occur alternatively on several defined Lys residues, i.e. three (Lys16, Lys20, Lys37) in A/B region, one (Lys424) in E region and one (Lys506) in F region. However, sumoylation of one Lys residue within A/B region prevents modification of other residues in this region. This was also observed for Lys residues in carboxyl-terminal fragment of Usp, i.e. comprising E and F regions. Mass spectrometry analysis of the full-length Usp indicated that the main SUMO attachment site is at Lys20. EcR, the heterodimerization partner of Usp, and muristerone A, the EcR ligand, do not influence sumoylation patterns of Usp. Another heterodimerization partner of Usp - HR38 fused with Ubc9 interacts with Usp in HEK293 cells and allows sumoylation of Usp independent of the direct fusion to Ubc9. Taken together, we propose that sumoylation of DmUsp can be an important factor in modulating its activity by changing molecular interactions.

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.

Candida tropicalis is one of the most frequent causes of serious disseminated candidiasis in human patients infected by non-albicans Candida species, but still relatively little is known about its virulence mechanisms. In our current study, the interactions between the cell surface of this species and a multifunctional human protein - high-molecular-mass kininogen (HK), an important component of the plasma contact system involved in the development of the inflammatory state - were characterized at the molecular level. The quick release of biologically active kinins from candidal cell wall-adsorbed HK was presented and the HK-binding ability was assigned to several cell wall-associated proteins. The predicted hyphally regulated cell wall protein (Hyr) and some housekeeping enzymes exposed at the cell surface (known as "moonlighting proteins") were found to be the major HK binders. Accordingly, after purification of selected proteins, the dissociation constants of the complexes of HK with Hyr, enolase, and phosphoglycerate mutase were determined using surface plasmon resonance measurements, yielding the values of 2.20 × 10(-7) M, 1.42 × 10(-7) M, and 5.81 × 10(-7) M, respectively. Therefore, in this work, for the first time, the interactions between C. tropicalis cell wall proteins and HK were characterized in molecular terms. Our findings may be useful for designing more effective prevention and treatment approaches against infections caused by this dangerous fungal pathogen.

Introduction: Stem cell-derived extracellular vesicles (EVs) are known to facilitate cell-to-cell communication. The potential of mesenchymal stem cell (MSC)-derived EVs for cardiac repair when compared with MSC whole cell injection remains unknown. Hypothesis: We hypothesized that therapy with MSC-EVs after a reperfused myocardial infarction (MI) will result in greater improvement in LV structure and function compared with injection of MSCs. Methods: EVs were collected by sequential centrifugation from culture supernatants of murine bone marrow MSCs. We used tunable resistive pulse sensing technology (TRPS), flow cytometry, real-time qRT-PCR and mass spectrometry to analyze MSC-EVs. Wild-type (C57BL/6) mice underwent a 30-min coronary occlusion followed by reperfusion, and 48 h later received intramyocardial injection of vehicle (PBS, n=14), MSCs (n=14), or MSC-EVs (n=14). Echocardiography was performed 4 d prior to MI (BSL) and at 48 h and 30 d after injection. Mice were sacrificed after 30 d. Results: MSC-EVs were noted to be small vesicles with average size of 233.0 ± 70.2 nm (Fig), and expressed MSC-specific markers, including CD29, CD90.2, CD105, Sca-1 and CD49e (Fig). MSC-EVs were enriched in mRNAs, miRNAs and proteins from donor MSCs; and carried transcripts of genes regulating cardiac/vascular differentiation, such as GATA-4, Nkx2.5, Tie-2, and GATA-2. In vivo, LV ejection fraction (EF) was significantly and similarly reduced in all groups at 48 h after MI (Fig). At 30 d after MI, mice injected with MSC-EVs exhibited significantly higher EF (Fig) and smaller LV end-diastolic volume compared with vehicle-treated and MSC-injected mice. Conclusions: We conclude that MSC-EVs represent natural nanocarriers capable of transferring bioactive contents and inducing cardiac repair following intramyocardial injection after MI in vivo. The use of MSC-EVs may potentially improve the outcomes of cell therapy in humans.

Introduction: Induced pluripotent stem cells (iPSCs) have recently emerged as a promising therapy for cardiac repair. Whether extracellular vesicles (EVs) derived from iPSCs can confer similar benefits remains unclear. Hypothesis: We hypothesized that injection of iPSC-derived EVs would produce at least similar improvement in cardiac structure and function compared with iPSCs. Methods: Murine iPSCs were generated using standard protocol and iPSC-EVs were harvested from culture supernatants by sequential centrifugation. Wild-type (C57BL/6) mice underwent a 30-min coronary occlusion followed by reperfusion, and 48 h later received intramyocardial injection of vehicle (PBS, n=14), iPSCs (n=15) or iPSC-EVs (n=13). Echocardiography was performed 4 d prior to coronary occlusion/reperfusion (BSL) and at 48 h and 30 d after injection. Mice were sacrificed at 30 d. Results: At 48 h after injection, LVEF was significantly and similarly reduced in all groups (Fig). At 30 d after MI, mice injected with iPSC-EVs exhibited significantly greater EF (Fig) compared with vehicle-treated mice. iPSC-EV-injected hearts also showed significantly smaller LVEDV compared with vehicle-treated mice, indicating improved remodeling. Although iPSCs-injected hearts also showed somewhat improved function and structure, several mice in this group grew tumors at myocardial injection sites. Conclusions: We conclude that intramyocardial injection of iPSC-EVs after a reperfused MI improves LV function and remodeling. The use of iPSC-EVs also averts cardiac tumor formation that was noted in mice after iPSC whole cell injection. These findings support therapeutic potential of iPSC-EVs in patients after MI.

Introduction: Extracellular vesicles (EVs) are tiny membrane-enclosed vesicles released by cells by membrane budding or exocytosis. The molecular profile of contents and regenerative potential of EVs derived from murine induced pluripotent stem cells (miPSCs) have not been investigated. Hypothesis: We hypothesized that miPSC-derived EVs carry bioactive substances, including mRNA and miRNA, which would influence the biology and fate of target cells. Methods: Murine iPSCs were generated using established protocols and EVs were isolated from culture supernatants by sequential centrifugation. Atomic force microscopy (AFM), flow cytometry, and real-time RT-PCR were used to characterize EV contents. Endothelial cells were exposed to miPSC-EVs in culture. Results: AFM and dynamic light scattering showed that miPSC-EVs are homogenous spherical vesicles smaller than 100 nm (Fig, A). High-sensitivity flow cytometry confirmed the presence of several iPSC- specific markers along with typical exosomal markers (CD9, CD63 and CD81) on miPSC-EVs. miPSC-EVs were enriched in mRNAs, miRNAs and proteins from donor iPSCs as shown by real-time RT-PCR and mass spectroscopy, respectively. Moreover, miPSC-EVs contained transcripts regulating pluripotency, self-renewal and differentiation, including Oct-4, Nanog, GATA-4 as well as miR290-295 cluster (Fig, B,C). Importantly, several miRNAs were found to be higher in EVs when compared with parental miPSCs (Fig, B,C). Endothelial cells treated with miPSC-EVs exhibited greater proliferative, metabolic and angiogenic activities, and were more resistant to apoptosis. Conclusions: Our data show, for the first time, that miPSC-derived EVs are natural nanocarriers capable of transferring bioactive contents to mature cells affecting target cell function as well as regenerative potential. We conclude that iPSC-EVs may represent safe therapeutic alternatives to whole cell-based therapy for cardiovascular repair.

International audience; This paper reports on the properties of bilayers composed of dioctadecyldimethylammonium bromide (DODAB) and oleic acid (OA) at various molar ratios. The mole fraction of OA, X(OA), was varied in the range of 0-1 and the total lipid content was constant and equal to 10 mM. The DODAB/OA dispersions were extruded at a temperature higher than that of the gel-liquid transition of DODAB. The morphology of bilayer structures formed in the dispersions was inspected using a cryogenic transmission electron microscopy (cryo-TEM) and a differential interference contrast microscopy (DIC). The observations revealed that the incorporation of OA into DODAB bilayer results in a decrease of the membrane curvature. Anisotropy measurements using 1,6-diphenylhexatriene (DPH) as a rotator probe demonstrated that the DODAB/OA membrane microviscosity decreased considerably for X(OA)>0.4. The thermal behavior of DODAB/OA membranes has been studied by differential scanning calorimetry (DSC). In the case of the systems in which X(OA)

In the present study, detailed information is presented on the hetero-dimerization of the serotonin 5-HT(2A) receptor and the dopamine D(2) receptor. Biophysical approaches (fluorescence spectroscopy as well as fluorescence lifetime microscopy) were used to determine the degree of fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent protein labeled receptor variants co-expressed in human embryonic kidney 293 cells (HEK293). Recorded data demonstrate the existence of energy transfer between the wild-type forms of 5-HT(2A)R and D(2)R, pointing toward the formation of hetero-5-HT(2A)R/D(2)R dimers and homo-5-HT(2A)R/5-HT(2A)R dimers. Moreover, the present study investigates the role of specific motifs (one containing adjacent arginine residues (217RRRRKR222) in the third intracellular loop (ic3) of D(2)R, and the other consisting of acidic glutamate residues (454EE455) in the C-tail of (5-HT(2A)R) in the formation of noncovalent complexes between these receptors. Our results suggest that these regions of 5-HT(2A)R and D(2)R may be involved in the interaction between these two proteins. On the other hand, the above-mentioned motifs do not play an important role in the homo-dimerization of these receptors. Furthermore, we estimated the influence of specific receptor ligands on the dimerization processes. Agonists (DOI and quinpirole) and antagonists (ketanserin and butaclamol) cause different effects on FRET efficiency depending on whether homo- or hetero-complexes are present. These data may have therapeutic implications, since (using the immunofluorescence double labeling protocols) the co-localization of these two receptors was demonstrated in the medial prefrontal cortex and pars reticulate of the substantia nigra of the rat brain.

The negative regulation of p53, a major human tumor suppressor, by Mdm2 and Mdmx is crucial for the survival of a cell, whereas its aberrant function is a common feature of cancer. Both Mdm proteins act through the spatial occlusion of the p53 transactivation (TA) domain and by the ubiquitination of p53, resulting in its degradation. Two p53 homologues, p63 and p73, have been described in humans. Unlike p53, these proteins regulate developmental processes rather than genome stability. Both p63 and p73 contain TA domains homologous to that of p53, but relatively little is known about their regulation by Mdm2 or Mdmx. Here, we present a detailed characterization of the interaction of Mdm2 and Mdmx with the TA domains of p63 and p73. Earlier reports of Mdm2 and Mdmx interactions with p73 are substantiated by the detailed quantitative characterization reported in this study. Most importantly, earlier contradictions concerning the presumed interaction of the Mdm proteins with p63 are convincingly resolved and for the first time, the affinities of these interactions are determined. Finally, the contribution of these findings to our understanding of the physiological role of these interactions is discussed.