Hana Kovarova

Mapping of the secretome of primary isolates of mammalian cells, stem cells and derived cell lines.

Within a mammalian organism, the interaction among cells both at short and long distances is mediated by soluble factors released by cells into the extracellular environment. The secreted proteins may involve extracellular matrix proteins, proteinases, growth factors, protein hormones, immunoregulatory cytokines, chemokines or other bioactive molecules that have a direct impact on target cell phenotype. Stem cells of mesenchymal, adipose, neural and embryonic origin, fibroblast feeder cells as well as primary isolates of astrocytes, endothelial and muscle cells have recently become targets of intensive secretome profiling with the search for proteins regulating cell survival, proliferation, differentiation or inflammatory response. Recent advances and challenges of the stem cell and primary cell secretome analysis together with the most relevant results are discussed in this review.

Protein Patterns of Pig Oocytes During In Vitro Maturation

Abstract In vitro maturation (IVM) of fully grown mammalian oocytes is characterized by initial germinal vesicle (GV) breakdown and rearrangement of microtubule network during the first meiosis (MI), followed by extrusion of the first polar body and block of the oocytes in metaphase of the second meiosis (MII). Only fully matured oocytes are capable of undergoing fertilization and the initiation of zygotic development. These observations are mostly based on morphological evaluation; however, the molecular events responsible for these processes are not known. In this study, we have launched the analysis of pig oocytes during in vitro maturation using a proteomics approach. First, oocyte proteins have been separated by two-dimensional gel electrophoresis and identified by mass spectrometry. Remarkably, several proteins, including peroxiredoxins, ubiquitin carboxyl-terminal hydrolase isozyme L1, and spermine synthase, are even more abundant than actin, usually the most abundant protein in somatic cells. Furthermore, we have initiated comparative analysis of the oocytes at different stages of maturation to characterize candidate proteins, which are differentially expressed during in vitro maturation. To date, we have identified antiquitin (D7A1), the member of aldehyde dehydrogenase family7 that has been significantly increased in MI and MII stages compared with GV oocytes. To our knowledge, this is the first pig oocyte proteome available so far that may be used as a reference map. The proteins that are differentially regulated during IVM may present potential biomarkers of oocyte maturation and quality. It is a useful inventory toward a deeper understanding of the mechanisms underlying reproduction and development.

Proteome Mining of Human Follicular Fluid Reveals a Crucial Role of Complement Cascade and Key Biological Pathways in Women Undergoing in Vitro Fertilization

In vitro fertilization (IVF) is fraught with problems and currently proteomics approaches are being tried out to examine the microenvironment of the follicle in order to assess biological and immunological parameters that may affect its development. Additionally, better understanding of reproductive process may help increase IVF birth rate per embryo transfer and at the same time avoid spontaneous miscarriages or life threatening conditions such as ovarian hyperstimulation syndrome. The primary aim of this study was to search for specific differences in protein composition of human follicular fluid (HFF) and plasma in order to identify proteins that accumulate or are absent in HFF. Depletion of abundant proteins combined with multidimensional protein fractionation allowed the study of middle- and lower-abundance proteins. Paired comparison study examining HFF with plasma/serum from women undergoing successful IVF revealed important differences in the protein composition which may improve our knowledge of the follicular microenvironment and its biological role. This study showed involvement of innate immune function of complement cascade in HFF. Complement inhibition and the presence of C-terminal fragment of perlecan suggested possible links to angiogenesis which is a vital process in folliculogenesis and placental development. Differences in proteins associated with blood coagulation were also found in the follicular milieu. Several specific proteins were observed, many of which have not yet been associated with follicle/oocyte maturation. These proteins together with their regulatory pathways may play a vital role in the reproductive process.

Antibody Microarray Analyses of Signal Transduction Protein Expression and Phosphorylation during Porcine Oocyte Maturation

Kinex antibody microarray analyses was used to investigate the regulation of 188 protein kinases, 24 protein phosphatases, and 170 other regulatory proteins during meiotic maturation of immature germinal vesicle (GV+) pig oocytes to maturing oocytes that had completed meiosis I (MI), and fully mature oocytes arrested at metaphase of meiosis II (MII). Increases in apparent protein levels of protein kinases accounted for most of the detected changes during the GV to MI transition, whereas reduced protein kinase levels and increased protein phosphorylation characterized the MI to MII transition. During the MI to MII period, many of the MI-associated increased levels of the proteins and phosphosites were completely or partially reversed. The regulation of these proteins were also examined in parallel during the meiotic maturation of bovine, frog, and sea star oocytes with the Kinex antibody microarray. Western blotting analyses confirmed altered expression levels of Bub1A, IRAK4, MST2, PP4C, and Rsk2, and the phosphorylation site changes in the kinases Erk5 (T218 + Y220), FAK (S722), GSK3-beta (Y216), MEK1 (S217 + S221) and PKR1 (T451), and nucleophosmin/B23 (S4) during pig oocyte maturation.

Proteomic analysis of porcine oocytes during in vitro maturation reveals essential role for the ubiquitin C-terminal hydrolase-L1

In this study, we performed proteomic analysis of porcine oocytes during in vitro maturation. Comparison of oocytes at the initial and final stages of meiotic division characterized candidate proteins that were differentially synthesized during in vitro maturation. While the biosynthesis of many of these proteins was significantly decreased, we found four proteins with increased biosynthetic rate, which are supposed to play an essential role in meiosis. Among them, the ubiquitin C-terminal hydrolase-L1 (UCH-L1) was identified by mass spectrometry. To study the regulatory role of UCH-L1 in the process of meiosis in pig model, we used a specific inhibitor of this enzyme, marked C30, belonging to the class of isatin O-acyl oximes. When germinal vesicle (GV) stage cumulus-enclosed oocytes were treated with C30, GV breakdown was inhibited after 28 h of culture, and most of the oocytes were arrested at the first meiosis after 44 h. The block of metaphase I-anaphase transition was not completely reversible. In addition, the inhibition of UCH-L1 resulted in elevated histone H1 kinase activity, corresponding to cyclin-dependent kinase(CDK1)-cyclin B1 complex, and a low level of monoubiquitin. These results supported the hypothesis that UCH-L1 might play a role in metaphase I-anaphase transition by regulating ubiquitin-dependent proteasome mechanisms. In summary, a proteomic approach coupled with protein verification study revealed an essential role of UCH-L1 in the completion of the first meiosis and its transition to anaphase.

Development of ovarian hyperstimulation syndrome: interrogation of key proteins and biological processes in human follicular fluid of women undergoing in vitro fertilization

Ovarian hyperstimulation syndrome (OHSS) is an iatrogenic complication and potentially life-threatening condition resulting from excessive ovarian stimulation during assisted reproductive technologies. Our aim was to identify candidate proteins in follicular fluid (FF) using various proteomic approaches which may help to identify patients at risk of OHSS. We analysed the proteome alterations in FF from patients suffering from severe forms of OHSS (OHSS+) compared with a control group of women without or with only mild signs of OHSS (OHSS-). The 12 abundant proteins of FF were removed using an immunoaffinity system. Pools of remaining depleted proteins were applied to the two-dimensional (2D) electrophoresis and 2D liquid chromatography and proteins in differentially expressed protein spots/fractions were identified by mass spectrometry. Among a total of 19 candidate proteins differentially expressed (P< 0.05) between OHSS+ and OHSS- FF samples, three proteins, namely ceruloplasmin, complement C3 and kininogen-1, were found using both 2D techniques. Computer modelling highlighted the important role of kininogen-1 as an anchor for mediated interactions with other identified proteins including ferritin light chain and ceruloplasmin, hepatocyte growth factor-like protein, as well as complement C3 and gelsolin, thus linking various biological processes including inflammation and angiogenesis, iron transport and storage, blood coagulation, innate immunity, cell adhesion and actin filament polymerization. The delineation of such processes may allow the development of informed corrective therapeutic intervention in patients at risk of OHSS and a set of key proteins of the FF may be helpful as potential biomarkers for monitoring IVF therapy.

Challenges in cancer research and multifaceted approaches for cancer biomarker quest

Recent advances in cancer biology have subsequently led to the development of new molecularly targeted anti‐cancer agents that can effectively hit cancer‐related proteins and pathways. Despite better insight into genomic aberrations and diversity of cancer phenotypes, it is apparent that proteomics too deserves attention in cancer research. Currently, a wide range of proteomic technologies are being used in quest for new cancer biomarkers with effective use. These, together with newer technologies such as multiplex assays could significantly contribute to the discovery and development of selective and specific cancer biomarkers with diagnostic or prognostic values for monitoring the disease state. This review attempts to illustrate recent advances in the field of cancer biomarkers and multifaceted approaches undertaken in combating cancer.

Proteomics of neural stem cells

The isolation of neural stem cells from fetal and adult mammalian CNS and the demonstration of functional neurogenesis in adult CNS have offered perspectives for treatment of many devastating hereditary and acquired neurological diseases. Due to this enormous potential, neural stem cells are a subject of extensive molecular profiling studies with a search for new markers and regulatory pathways governing their self-renewal as opposed to differentiation. Several in-depth proteomic studies have been conducted on primary or immortalized cultures of neural stem cells and neural progenitor cells, and yet more remains to be done. Additionally, neurons and glial cells have been obtained from embryonic stem cells and mesenchymal stem cells, and proteins associated with the differentiation process have been characterized to a certain degree with a view to further investigations. This review summarizes recent findings relevant to the proteomics of neural stem cells and discusses major proteins significantly regulated during neural stem cell differentiation with a view to their future use in cell-based regenerative and reparative therapy.

Protein signaling pathways in differentiation of neural stem cells

Neural stem cells (NSC) capable of differentiating into neurons, astrocytes and oligodendrocytes are a promising source of cells for the treatment of central nervous system diseases. Access to signaling proteins present in such cells in low copies and with specific regulatory functions has been very restrictive until now as judged by classical proteomic approaches and limitations due to scarcity of stem cell populations. Hence, we utilized the Kinex™ Antibody Microarray analysis where profiles of the proliferating porcine NSC and differentiated counterparts were compared and selected changes were verified by immunoblotting. Differentiated neural cells exhibited an increased level of RafB proto‐oncogene‐encoded protein‐serine kinase, MAP kinase protein‐serine kinase 3, heme oxygenase 2 (HO2) and protein phosphatase 4 catalytical subunit. On the other hand, relatively high level of G protein‐coupled receptor‐serine kinase 2 and enhanced phosphorylations of αB‐crystallin (S45), protein‐serine kinase C gamma (T655), protein‐serine kinase D (PKCμ; S738+S742) together with eukaryotic translation initiation factor 2 alpha (eIF2α) (S51) raised intriguing questions as regards their potential functionality within stem cells. In‐depth study of HO2 and phospho‐S45 αB‐crystallin confirmed expression profiles and intense cytoplasmic localization of HO2 in neurons but a weaker signal in glial cells. Phospho‐S45 αB‐crystallin was localized in nuclei of differentiated neural cells. Computer simulation of possible interaction network connecting regulated proteins, exposed additional relationships including direct interactions of HO2 with amyloid precursor protein or huntingtin‐associated protein 1.

Cancer cell resistance to aurora kinase inhibitors: identification of novel targets for cancer therapy.

Drug resistance is the major obstacle to successful cancer therapy. Our study focuses on resistance to Aurora kinase inhibitors tested as anti-cancer drugs in clinical trials. We have used 2D electrophoresis in the pH ranges of 4-7 and 6-11 followed by protein identification using MALDI-TOF/TOF to compare the protein composition of HCT116 colon cancer cells either sensitive to CYC116 and ZM447439 inhibitors or resistant toward these drugs. The analysis also included p53(+/+) and p53(-/-) phenotypes of HCT116 cells. Our findings demonstrate that platelet-activating factor acetylhydrolase and GTP-binding nuclear protein Ran contribute to the development of resistance to ZM447439 only where resistance is related to p53. On the other hand, serine hydroxymethyltransferase was found to promote the tumor growth in cells resistant to CYC116 without the influence of p53. Computer modeling of interaction networks highlighted a direct link of the p53-independent mechanism of resistance to CYC116 with autophagy. Importantly, serine hydroxymethyltransferase, serpin B5, and calretinin represent the target proteins that may help overcome resistance in combination therapies. In addition, serpin B5 and calretinin appear to be candidate biomarkers that may be accessible in patients for monitoring of cancer therapy with ease.