[Show abstract][Hide abstract] ABSTRACT: Reproductive success stems from a finely regulated balance between follicular maturation and atresia, in which the role of carbohydrate structure is poorly understood. Herein, we describe for the first time a fraction of purified recombinant human Follicle Stimulating Hormone (FSH) that is capable of bringing about the cell death of granulosa cells and preventing follicular maturation in a rat model. Further analysis by mass spectrometry revealed the presence of the lectin Concanavalin-A (Con-A) within this fraction of recombinant FSH. Using both the fractionated FSH and Con-A, the observed cell death was predominantly located to the granulosa cells. Ex-vivo culture of rat follicles demonstrated that follicle degeneration occurred and resulted in the release of a denuded and deteriorated oocyte. Moreover, in vivo experiments confirmed an increase in atresia and a corresponding reduction confined to follicle in early antral stage. As a mechanism of action, Con-A reduces ovarian proliferation, Von Willenbrand staining and angiogenesis. Based on the observation that Con-A may induce granulosa cell death followed by follicle death, our results further demonstrate that follicular carbohydrate moiety is changing under the influence of FSH, which may allow a carbohydrate-binding lectin to increase granulosa cell death. The physiological consequences of circulating lectin-like molecules remain to be determined; however, our results suggest a potential exploitation of carbohydrate binding in fertility and ovarian cancer treatment. This work may shed light on a key role of carbohydrates in the still obscure physiological process of follicular selection and atresia.
[Show abstract][Hide abstract] ABSTRACT: Cathepsin D deficiency is a fatal neurodegenerative disease characterized by extreme loss of neurons and myelin. Our previous studies have demonstrated that structural and functional alterations in synapses are central to the disease pathogenesis. Therefore, we took a systematic approach to examine the synaptic proteome in cathepsin D knock-out mice, where the synaptic pathology resembles that of human patients. We applied quantitative mass spectrometry analysis on synaptosomal fractions isolated from cathepsin D knock-out and control mice at the age of 24days. From the approximately 600 identified proteins, 43 were present in different amounts (P<0.05, measured in triple biological replicates) in cathepsin D knock-out mice compared to controls. We connected and bridged these 43 proteins using protein interaction data, and overlaid the network with brain specific gene expression information. Subsequently, we superimposed the network with Gene Ontology, pathway, phenotype and disease involvement, allowing construction of a dynamic, disease-protein centered network and prediction of functional modules. The measured changes in the protein levels, as well as some of the bioinformatically predicted ones, were confirmed by quantitative Western blotting or qualitative immunohistochemistry. This combined approach indicated alterations in distinct cellular entities, previously not associated with the disease, and including microtubule associated cytoskeleton and cell projection organization. Cell spreading and wound healing assays confirmed strongly compromised spatial orientation, associated with changes in distribution of focal adhesions and integrin assembly, in cathepsin D deficient cells. These changes might contribute to commencement of synaptic alterations and neuronal degeneration observed in cathepsin D deficiency.
Neurobiology of Disease 10/2012; · 5.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Identification of protein targets for microRNAs (miRNAs) is a significant challenge due to the complexity of miRNA-mediated regulation. We have previously demonstrated that miR-193b targets estrogen receptor-α (ERα) and inhibits estrogen-induced growth of breast cancer cells. Here, we applied a high-throughput strategy using quantitative iTRAQ (isobaric tag for relative and absolute quantitation) reagents to identify other target proteins regulated by miR-193b in breast cancer cells. iTRAQ analysis of pre-miR-193b transfected MCF-7 cells resulted in identification of 743 unique proteins, of which 39 were down-regulated and 44 up-regulated as compared with negative control transfected cells. Computationally predicted targets of miR-193b were highly enriched (sevenfold) among the proteins whose level of expression decreased after miR-193b transfection. Only a minority of these (13%) showed similar effect at the mRNA level illustrating the importance of post-transcriptional regulation. The most significantly repressed proteins were selected for validation experiments. These data confirmed 14-3-3ζ (YWHAZ), serine hydroxyl transferase (SHMT2), and aldo-keto reductase family 1, member C2 (AKR1C2) as direct, previously uncharacterized, targets of miR-193b. Functional RNAi assays demonstrated that specific combinations of knockdowns of these target genes by siRNAs inhibited growth of MCF-7 cells, mimicking the effects of the miR-193b overexpression. Interestingly, the data imply that besides targeting ERα, the miR-193b effects include suppression of the local production of estrogens and other steroid hormones mediated by the AKR1C2 gene, thus provoking two separate molecular mechanisms inhibiting steroid-dependent growth of breast cancer cells. In conclusion, we present here a proteomic screen to identify targets of miR-193b, and a systems biological approach to mimic its effects at the level of cellular phenotypes. This led to the identification of multiple genes whose combinatorial knock-down likely mediates the strong anti-cancer effects observed for miR-193b in breast cancer cells.
[Show abstract][Hide abstract] ABSTRACT: Ataxin-3, the protein involved in Machado-Joseph disease, is able to bind ubiquitylated substrates and act as a deubiquitylating enzyme in vitro, and it has been involved in the modulation of protein degradation by the ubiquitin-proteasome pathway. C. elegans and mouse ataxin-3 knockout models are viable and without any obvious phenotype in a basal condition however their phenotype in stress situations has never been described.Considering the role of ataxin-3 in the protein degradation pathway, we analyzed the effects of heat shock, a known protein homeostasis stressor, in C. elegans ataxin-3 (ATX-3) knockout animals. We found that ATX-3 mutants have an exacerbated stress response and survive significantly better than wild type animals when subjected to a noxious heat shock stimulus. This increased thermotolerance of mutants was further enhanced by pre-exposure to a mild heat shock. At a molecular level, ATX-3 mutants have a distinct transcriptomic and proteomic profile with several molecular chaperones abnormally up-regulated during heat shock and recovery, consistent with the observed resistance phenotype.The improved thermotolerance in ATX-3 mutants is independent of heat shock factor 1, the maestro of the heat shock response, but fully dependent on DAF-16, a critical stress responsive transcription factor involved in longevity and stress resistance. We also show that the increased thermotolerance of ATX-3 mutants is mainly due to HSP-16.2, C12C8.1 and F44E5.5 given that the knockdown of these heat shock proteins using RNA interference causes the phenotype to revert. This report suggests that the absence of ATX-3 activates the DAF-16 pathway leading to an overexpression of molecular chaperones, which yields knockout animals with an improved capacity for dealing with deleterious stimuli.
PLoS ONE 01/2011; 6(4):e18512. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cathepsin D (CTSD) deficiencies are fatal neurological diseases that in human infants and in sheep are characterized by extreme loss of neurons and myelin. To date, similar morphological evidence for myelin disruption in CTSD knockout mice has not been reported. Here, we show that CTSD deficiency leads to pronounced myelin changes in the murine brain: myelin-related proteolipid protein and myelin basic protein were both markedly reduced at postnatal day 24, and the amount of lipids characteristically high in myelin (e.g. plasmalogen-derived alkenyl chains and glycosphingolipid-derived 20- and 24-carbon acyl chains) were significantly lowered compared with controls. These changes were accompanied by ultrastructural alterations of myelin, including significant thinning of myelin sheaths. Furthermore, in CTSD knockout brains there was a pronounced accumulation of cholesteryl esters and abnormal levels of proteins related to cholesterol transport, with an increased content of apolipoprotein E and a reduced content of ATP-binding cassette transporter A1. These results provide evidence for dysmyelination and altered trafficking of cholesterol in brains of CTSD knockout mice, and warrant further studies on the role of lipid metabolism in the pathogenesis of CTSD deficiencies.
Journal of Neurochemistry 10/2009; 112(1):193-203. · 3.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ataxin-3 is the protein involved in Machado-Joseph disease, a neurodegenerative disorder caused by a polyglutamine expansion. Ataxin-3 binds ubiquitylated proteins and acts as a deubiquitylating enzyme in vitro. It was previously proposed that ataxin-3, along with the VCP/p97 protein, escorts ubiquitylated substrates for proteasomal degradation, although other players of this escort complex were not identified yet. In this work, we show that the Caenorhabditis elegans ataxin-3 protein (ATX-3) interacts with both VCP/p97 worm homologs, CDC-48.1 and CDC-48.2 and we map the interaction domains. We describe a motility defect in both ATX-3 and CDC-48.1 mutants and, in addition, we identify a new protein interactor, UBXN-5, potentially an adaptor of the CDC-48-ATX-3 escort complex. CDC-48 binds to both ATX-3 and UBXN-5 in a non-competitive manner, suggesting the formation of a trimolecular complex. Both CDC-48 and ATX-3, but not UBXN-5, were able to bind K-48 polyubiquitin chains, the standard signal for proteasomal degradation. Additionally, we describe several common interactors of ATX-3 and UBXN-5, some of which can be in vivo targets of this complex.
Biochemical and Biophysical Research Communications 07/2009; 386(4):575-81. · 2.41 Impact Factor