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.

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.

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.

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.

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.

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.

PD-L1 Overexpression, SWI/SNF Complex Deregulation, and Profound Transcriptomic Changes Characterize Cancer-Dependent Exhaustion of Persistently Activated CD4+ T Cells.

Growing tumors avoid recognition and destruction by the immune system. During continuous stimulation of tumor-infiltrating lymphocytes (TILs) by tumors, TILs become functionally exhausted; thus, they become unable to kill tumor cells and to produce certain cytokines and lose their ability to proliferate. This collectively results in the immune escape of cancer cells. Here, we show that breast cancer cells expressing PD-L1 can accelerate exhaustion of persistently activated human effector CD4+ T cells, manifesting in high PD-1 and PD-L1 expression level son T cell surfaces, decreased glucose metabolism genes, strong downregulation of SWI/SNF chromatin remodeling complex subunits, and p21 cell cycle inhibitor upregulation. This results in inhibition of T cell proliferation and reduction of T cell numbers. The RNAseq analysis on exhausted CD4+ T cells indicated strong overexpression of IDO1 and genes encoding pro-inflammatory cytokines and chemokines. Some interleukins were also detected in media from CD4+ T cells co-cultured with cancer cells. The PD-L1 overexpression was also observed in CD4+ T cells after co-cultivation with other cell lines overexpressing PD-L1, which suggested the existence of a general mechanism of CD4+ T cell exhaustion induced by cancer cells. The ChIP analysis on the PD-L1 promoter region indicated that the BRM recruitment in control CD4+ T cells was replaced by BRG1 and EZH2 in CD4+ T cells strongly exhausted by cancer cells. These findings suggest that epi-drugs such as EZH2 inhibitors may be used as immunomodulators in cancer treatment.

Anticancer agents found in environment affect Daphnia at population, individual and molecular levels.

Pharmaceuticals are used in medical treatment on a large scale and as a waste contaminate freshwater ecosystems. Growing amount of so-called civilization diseases, such as different type of cancer, significantly contribute to this form of pollution. The aim of the present study was to determine how the exposure to chemotherapeutics: cyclophosphamide (CP) and cisplatin (CDDP), at detected in environment concentrations, influence proteome profile, life history and population parameters of naturally setting surface waters Daphnia pulex and Daphnia pulicaria. The parameters important for crustaceans, survivorship and population growth rate, were importantly decreased by CDDP treatment but not influenced by CP. On the contrary, the individual growth rate was affected only by CP and exclusively in the case of D. pulicaria. In both clones treated with CP or CDDP, decreased number of eggs was observed. Interestingly, Daphnia males were less sensitive to tested chemotherapeutic than females. Proteome profile revealed that tested anticancer pharmaceuticals modified expression of some proteins involved in Daphnia metabolism. Moreover, males exposed to CDDP showed increased level of enzymes participating in DNA repair. Summing up, the contaminating environment chemotherapeutics reduced fitness of naturally occurring Daphnia species. In consequence this may affect functioning of the aquatic food webs.

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.

Hypoxic 3D in vitro culture models reveal distinct resistance processes to TKIs in renal cancer cells.

BACKGROUND: The aim of this study is to determine the effect of hypoxia on axitinib and sorafenib-treated renal cell carcinoma (RCC) cells. Hypoxia is a crucial factor influencing transcription process via protein modulation, which was shown i.e. in pancreatic cancer. Until now, hypoxia has been defined as associated with poorer outcome and inducing chemotherapy resistance in solid tumors. The unique phenomenon of pseudo-hypoxia connected with vhl mutation was observed in clear-cell, but not in papillary RCC, and the treatment of this subtype of cancer is still challenging. Despite the introduction of new antiangiogenic targeted therapies (inter alia tyrosine kinase inhibitors, TKIs), patients still develop both primary and acquired resistance. Overcoming resistance to TKIs, also in papillary RCC, may be possible by finding significantly modified protein expression. To do this, hypoxic 3D in vitro models must be developed to mimic both molecular pathways typical for low oxygen tension and cell-cell dynamics in tumor-like spatial structures. RESULTS: Clear-cell and papillary renal cell carcinoma (cc and pRCC) cell lines were used in the study to determine the impact of hypoxia on primary drug resistance phenomenon previously observed in papillary, but not in ccRCC. Resistance was confirmed in monolayer culture and in 3D models in soft agar and suspension culture. Human papillary kidney cancer stem-like cells (HKCSCs) cultured in hypoxia developed resistance to sorafenib, while when cultured in normoxia resistance to axitinib has developed. Flow cytometry revealed that hypoxia decreased proliferation rates in all investigated RCC cells. In HKCSCs, there was an increase of quiescent cells (Ki67-) and percentage of cells arrested in S phase. It also appeared that map2k1 and eif4b protein expression is altered in papillary RCC resistant to tested drugs at different oxygen tensions. Also, HKCSCs did not express vegfr-1, braf nor c-kit, TKIs target receptors, which were present in ccRCC cells sensitive to TKI treatment. CONCLUSIONS: The results confirm that low oxygen tension affects RCC cells. Hypoxia facilitates induction of sorafenib resistance in pRCC and induces map2k1 overexpression, while normoxic axitinib-resistant cells up-regulated eif4b. Further studies may determine if map2k1 or eif4b proteins play a role in pRCC resistance to TKIs. It is also of interest to establish if other than vegfr-1, braf, c-kit receptors can serve as potential molecular targets for more effective anti-RCC strategies.

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.

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.

Dimeric peroxiredoxins are druggable targets in human Burkitt lymphoma.

Burkitt lymphoma is a fast-growing tumor derived from germinal center B cells. It is mainly treated with aggressive chemotherapy, therefore novel therapeutic approaches are needed due to treatment toxicity and developing resistance. Disturbance of red-ox homeostasis has recently emerged as an efficient antitumor strategy. Peroxiredoxins (PRDXs) are thioredoxin-family antioxidant enzymes that scavenge cellular peroxides and contribute to red-ox homeostasis. PRDXs are robustly expressed in various malignancies and critically involved in cell proliferation, differentiation and apoptosis. To elucidate potential role of PRDXs in lymphoma, we studied their expression level in B cell-derived primary lymphoma cells as well as in cell lines. We found that PRDX1 and PRDX2 are upregulated in tumor B cells as compared with normal counterparts. Concomitant knockdown of PRDX1 and PRDX2 significantly attenuated the growth rate of lymphoma cells. Furthermore, in human Burkitt lymphoma cell lines, we isolated dimeric 2-cysteine peroxiredoxins as targets for SK053, a novel thiol-specific small-molecule peptidomimetic with antitumor activity. We observed that treatment of lymphoma cells with SK053 triggers formation of covalent PRDX dimers, accumulation of intracellular reactive oxygen species, phosphorylation of ERK1/2 and AKT and leads to cell cycle arrest and apoptosis. Based on site-directed mutagenesis and modeling studies, we propose a mechanism of SK053-mediated PRDX crosslinking, involving double thioalkylation of active site cysteine residues. Altogether, our results suggest that peroxiredoxins are novel therapeutic targets in Burkitt lymphoma and provide the basis for new approaches to the treatment of this disease.

Proteomic characterization of biogenesis and functions of matrix vesicles released from mineralizing human osteoblast-like cells.

Matrix vesicles (MVs), released by budding from apical microvilli of osteoblasts during bone formation and development, are involved in the initiation of mineralization by promoting the formation of hydroxyapatite in their lumen. To gain additional insights into MV biogenesis and functions, MVs and apical microvilli were co-isolated from mineralizing osteoblast-like Saos-2 cells and their proteomes were characterized using LC-ESI-MS/MS and compared. In total, 282 MV and 451 microvillar proteins were identified. Of those, 262 were common in both preparations, confirming that MVs originate from apical microvilli. The occurrence of vesicular trafficking molecules (e.g. Rab proteins) and of the on-site protein synthetic machinery suggests that cell polarization and apical targeting are required for the incorporation of specific lipids and proteins at the site of MV formation. MV release from microvilli may be driven by actions of actin-severing proteins (gelsolin, cofilin 1) and contractile motor proteins (myosins). In addition to the already known proteins involved in MV-mediated mineralization, new MV residents were detected, such as inorganic pyrophosphatase 1, SLC4A7 sodium bicarbonate cotransporter or sphingomyelin phosphodiesterase 3, providing additional insights into MV functions.

Proteins recruited by SH3 domains of Ruk/CIN85 adaptor identified by LC-MS/MS.

BACKGROUND: Ruk/CIN85 is a mammalian adaptor molecule with three SH3 domains. Using its SH3 domains Ruk/CIN85 can cluster multiple proteins and protein complexes, and, consequently, facilitates organisation of elaborate protein interaction networks with diverse regulatory roles. Previous research linked Ruk/CIN85 with the regulation of vesicle-mediated transport and cancer cell invasiveness. Despite the recent findings, precise molecular functions of Ruk/CIN85 in these processes remain largely elusive and further research is hampered by a lack of complete lists of its partner proteins. RESULTS: In the present study we employed a LC-MS/MS-based experimental pipeline to identify a considerable number (over 100) of proteins recruited by the SH3 domains of Ruk/CIN85 in vitro. Most of these identifications are novel Ruk/CIN85 interaction candidates. The identified proteins have diverse molecular architectures and can interact with other proteins, as well as with lipids and nucleic acids. Some of the identified proteins possess enzymatic activities. Functional profiling analyses and literature mining demonstrate that many of the proteins recruited by the SH3 domains of Ruk/CIN85 identified in this work were involved in the regulation of membranes and cytoskeletal structures necessary for vesicle-mediated transport and cancer cell invasiveness. Several groups of the proteins were also associated with few other cellular processes not previously related to Ruk/CIN85, most prominently with cell division. CONCLUSION: Obtained data support the notion that Ruk/CIN85 regulates vesicle-mediated transport and cancer cell invasiveness through the assembly of multimeric protein complexes governing coordinated remodelling of membranes and underlying cytoskeletal structures, and imply its important roles in formation of coated vesicles and biogenesis of invadopodia. In addition, this study points to potential involvement of Ruk/CIN85 in other cellular processes, chiefly in cell division.

An integrated LC-ESI-MS platform for quantitation of serum peptide ladders. Application for colon carcinoma study.

Mounting evidence indicates that MS analysis of the human blood peptidome allows to distinguish between cancer and non-cancer samples, giving promise for a new MS-based diagnostic tool. However, several aspects of already published work have been criticized and demand for more methodical approach has been formulated. Motivated by this we undertook a systematic study of the plasma and serum peptidome using an integrated ESI-LC-MS-based platform, equipped with new data analysis tools for relative and absolute peptide quantitation. We used a high resolution LC-ESI-MS to analyze well-separated MS signals corresponding to peptides, and measured the variability of >1000 peptide signal amplitudes across a set of plasma and serum samples from healthy individuals. By spiking serum samples with known amounts of isotopically labeled versions of a selected set of peptides we measured the variability of their absolute concentration in this sample set and demonstrated a strong influence of clotting time on the concentration of these peptides in serum. Finally, we used this new LC-ESI-MS analytical platform for the differential analysis of healthy versus colon cancer serum samples and found that it was possible to distinguish the two groups with 89.8% sensitivity and 94.6% specificity.