Search

ABSTRACT Mycotoxin zearalenone (ZEN) is a secondary metabolite produced by some Fusarium species that contaminate a large variety of grains and feedstuffs worldwide. ZEN has been associated with a wide variety of adverse health effects including hepatotoxic, hematologic, immunotoxic and genotoxic. In order to better understand the mechanism of ZEN toxicity, a proteomic approach was applied to characterize cellular responses of hepatocarcinoma cells (HepG2) to ZEN exposure. Protein extracts from cultured HepG2 cells treated with 100 µ m ZEN for 8 h, as well as extracts from control cells. The screening method applied to compare the proteome was based on the stable isotope approach of isobaric tagging for relative and absolute quantification (iTRAQ). This study identified 982 proteins, among which peptides and their corresponding proteins were identified and quantified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Ingenuity pathways analysis software was then used to determine the biological functions and canonical pathways associated with the ZEN-responsive proteins. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Background: Mutations in the Planar Cell Polarity (PCP) core gene Vangl2 cause the most severe neural tube defects (NTD) in mice and humans. Genetic studies show that the Vangl2 gene genetically interacts with a close homologue Vangl1. How precisely Vangl2 and Vangl1 proteins interact and crosstalk has remained a difficult issue to address, with the main obstacle being the accurate discrimination of the two proteins, which share close sequence homology. Experimental evidence previously presented has been sparse and addressed with ectopically expressed proteins or with antibodies unable to biochemically discriminate Vangl1 from Vangl2, therefore giving rise to unclear results. Methodology and Main Findings: A highly specific monoclonal anti-Vangl2 antibody was generated and rigorously tested on both recombinant and extracted Vangl2 using surface plasmon resonance (SPR) analysis, western blot, and immunoprecipitation experiments. This antibody efficiently affinity-purified Vangl2 from cell lysates and allowed the unambiguous identification of endogenous Vangl2 by proteomic analysis. Vangl1 was also present in Vangl2 immunoprecipitates, establishing the first biochemical evidence for the existence of Vangl2/Vangl1 heterodimers at an endogenous level. Epitope-tagged Vangl2 and Vangl1 confirmed that both proteins interact and colocalize at the plasma membrane. The Vangl2 antibody is able to acutely assess differential expression levels of Vangl2 protein in culture cell lines, as corroborated with gene expression analysis. We characterised Vangl2 expression in the cochlea of homozygous and heterozygous Lp mutant mice bearing a point mutation within the C-terminal Vangl2 region that leads to profound PCP defects. Our antibody could detect much lower levels of Vangl2Lp protein in mutant mice compared to the wild type mice. Conclusion: Our results provide an in-depth biochemical characterisation of the interaction observed between Vangl paralogues. [ABSTRACT FROM AUTHOR]

Proinsulin is a key autoantigen in type I diabetes. Evidence in the mouse has underscored the importance of the insulin B chain region in autoimmunity to pancreatic beta cells. In man, a majority of proteasome cleavage sites are predicted by proteasome cleavage algorithms within this region. To study CD8+ T cell responses to the insulin B chain and adjacent C peptide, we selected 8- to 11-mer peptides according to proteasome cleavage patterns obtained by digestion of two peptides covering proinsulin residues 28 to 64. We studied their binding to purified HLA class I molecules and their recognition by T cells from diabetic patients. Peripheral blood mononuclear cells from 17 of 19 recent-onset and 12 of 13 long-standing type I diabetic patients produced IFN-y in response to proinsulin peptides as shown by using an ELISPOT assay. In most patients, the response was against several class I-restricted pep- tides. Nine peptides were recognized within the proinsulin region covering residues 34 to 61. Four yielded a high frequency of recognition in HLA-Al and -B8 patients. Three peptides located in the proinsulin region 41-51 were shown to bind several HLA molecules and to be recognized in a high percentage of diabetic patients. [ABSTRACT FROM AUTHOR]

To elucidate the molecular mechanisms of the transendothelial migration of leukocytes, we attempted to identify the cellular proteins capable of interaction with the cytoplasmic domain of the intercellular adhesion molecule-1 (ICAM-1) in a rat brain microvessel endothelial cell line (RBE4 cells). A 27-amino-acid synthetic peptide, corresponding to the cytoplasmic domain of rat ICAM-1, was covalently linked to a Sepharose matrix. Upon affinity chromatography of RBE4 cell cytosol, several ICAM-1-interacting proteins were specifically eluted by the soluble peptide. Two of these proteins have been identified by microsequencing as the cytoskeletal protein β-tubulin and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GraP-DH). Experiments carried out with purified GraP-DH or CNBr fragments of GraP-DH indicated that binding to the ICAM-1 matrix was mediated by the C-terminal domain of GraP-DH, containing the binding site of the cofactor NAD+, and that NAD+ could compete with this binding. Using a series of ICAM-1 C-terminal truncated peptides, we could demonstrate that (a) the nitric-oxide-induced covalent linkage of NAD+ to GraP-DH was impaired by these peptides, (b) the glycolytic activity of GraP-DH was drastically inhibited by a truncated peptide containing the 15 C-terminal residues, (c) nitric oxide appeared to prevent this inhibition. Together, our results demonstrate that GraP-DH specifically associates with the isolated ICAM-1 cytoplasmic domain. Since GraP-DH is known as a microtubule bundling protein, these findings suggest that, in a cellular environment, GraP-DH may behave as an adaptor molecule by linking ICAM-1 to the microtubule network. The role of nitric oxide in the modulation of this interaction deserves further investigation. [ABSTRACT FROM AUTHOR]

In duck erythroblasts, two major populations of untranslated messenger (m) RNP can be separated by sucrose gradient centrifugation in low ionic strength. One of these contains globin mRNA associated to protein factors, among them the prosomes. The other, sedimenting in the 35S zone, contains non-globin mRNA. From this ‘35S’ mRNP, a new RNP particle called the prosome-like particle was isolated and characterized [Akhayat, O., Infante, A. A., Infante, D., Martins de Sa, C., Grossi de Sa, M.-F. & Scherrer, K. (1987) Eur. J. Biochem. 170, 23–33]. The PLP is a multimer of a protein of Mr 21000, and contains small RNA species. The particle is tightly associated with repressed mRNA and inhibits in vitro protein synthesis, We show here that the protein of Mr 21 000, constituting the prosome-like particle, is apoferritin. Different approaches confirm the RNP character of this particle and provide evidence that some of its RNA species are tRNA. The hypothesis is discussed as to whether (apo-)ferritin might serve other functions in addition to iron storage. [ABSTRACT FROM AUTHOR]

The activity of the Src family protein-tyrosine kinase p56lck is regulated by phosphorylation and dephosphorylation of two critical tyrosine residues Tyr394 and Tyr505. Tyr394 is autophosphorylated after p56lck activation, whereas phosphorylation of Tyr505 is believed to be due to p56lck which negatively modulates p56lck activity. To determine whether Tyr505 could be autophosphorylated, we used the prokaryotic glutathione S-transferase expression system to express wild-type Lck, the mutants [Y394F]Lck and [Y505F]Lck, a kinase-deficient p56lck with a mutation of the A-TP-binding site [K273E]Lck and a double mutant [Y394F, Y505F]Lck. We studied the kinase activities and the patterns of autophosphorylation for tyrosine residues in these mutants and wild-type Lck both in vivo and in vitro. Wild-type Lck. [Y505F]Lck and [Y394F]Lck were phosphorylated on tyrosine. Both the kinasedeficient mutant[K273E]Lck and the double mutant [Y394F. Y505F]Lck did not react with monoclonal anti-phosphotyrosine antibody [anti-Y(P) mAb], thus providing evidence that (a) the bacterial strains used lacked intrinsic protein-tyrosine kinase activities, and therefore tyrosine phosphorylations of wild-type Lck, [Y505F]Lck and [Y394F]Lck are due to autophosphorylation occurring in vivo in bacteria, and (b) that p56lck can only be autophosphorylated on two tyrosine residues, namely Tyr394 and Tyr505. Phosphopeptide mapping analysis confirmed that p56lck can undergo autophos-, phorylation tm these two tyrosine residues. We propose that autophosphorylation at Tyr505 of p56lck may represent an accessory mechanism for the down-regulation of the tyrosine kinase activity of p56lck. [ABSTRACT FROM AUTHOR]

Three monoclonal antibodies (mAb) directed against the regulatory domain of the protein kinase Cγ (PKCγ); 15G4, 5A2 and 36G9, were shown to display distract properties with respect to PKCγ kinase activity [Cazaubon. S., Marais, R., Parker, P. & Strosberg, A.D. (1989) Ear, J. Biochem. 182, 401 -406]. The mAb 5A2 and 36G9, which act as potent inhibitors of the cofactor-dependent kinase activity, can no longer bind PKCγ in the presence of phosphatidylserine and phosphatidylserine/phorbol ester, respectively; 15G4 binding is not influenced by effectors. Due to this functional relationship between the inhibitory mAb- and cofactor-binding sites, we sought to localize the mAb epitopes with respect to the functional sites of PKCγ. For this purpose, several deletions were introduced at the 5' end of the PKCγ cDNA and the mutant proteins were expressed in Escherichia coli. The determination of the immunoreactivity of the deleted PKCγ proteins shows that the amino acid residues essential to the binding of 5A2 and 36G9 are directly adjacent to the second cysteine-rich motif: these are contained in the sequences at positions 151- 163 and 164-197. respectively. In addition, various deletions around the C1 region of the regulatory domain allowed the identification of the second cysteine-rich motif as a functional binding site for phorbol dibutyrate. These deletion studies thus demonstrate that the epitopes recognized by the inhibitory mAbs 5A2 and 36G9 are distinct from the cofactor-binding sites. This suggests that the binding of phosphatidylserine and phorbol ester reduce conformational changes in the regulatory domain of PKC, which are thus responsible for the loss of the 5A2 and 36G9 immunoreactivity of the native protein. In this conformational state, PKCγ conserves its ability to interact with the non-inhibitory mAb 15G4. By using synthetic peptides, the 15G4 epitope was localized to the sequence 297 - 310 in the V3 variable region. This indicates that the flexibility of the V3 region, which delimits the C-terminus of the regulatory domain, may not be necessary for the allosteric activation of PKC. In view of these results, we propose that PKC activation by its cofactors results in intramolecular changes which allow the enzyme to bind exogenous substrates. [ABSTRACT FROM AUTHOR]

Drug development in oncology is highly challenging, with less than 5% success rate in clinical trials. This alarming figure points out the need to study in more details the multiple biological effects of drugs in specific contexts. Indeed, the comprehensive assessment of drug poly-pharmacology can provide insights into their therapeutic and adverse effects, to optimize their utilization and maximize the success rate of clinical trials. Recent technological advances have made possible in-depth investigation of drug poly-pharmacology. This review first highlights high-throughput methodologies that have been used to unveil new mechanisms of action of existing drugs. Then, we discuss how emerging chemo-proteomics strategies allow effectively dissecting the poly-pharmacology of drugs in an unsupervised manner. • Poor target validation and lack of biomarkers lead to high rate of drug attrition. • Compound-centric and functional genomic screens can identify cancer vulnerabilities. • Chemo-proteomics can unveil the poly-pharmacology of drugs. [ABSTRACT FROM AUTHOR]

First described in the milkweed bug Oncopeltus fasciatus, planar cell polarity (PCP) is a developmental process essential for embryogenesis and development of polarized structures in Metazoans. This signaling pathway involves a set of evolutionarily conserved genes encoding transmembrane (Vangl, Frizzled, Celsr) and cytoplasmic (Prickle, Dishevelled) molecules. Vangl2 is of major importance in embryonic development as illustrated by its pivotal role during neural tube closure in human, mouse, Xenopus, and zebrafish embryos. Here, we report on the molecular and functional characterization of a Vangl2 isoform, Vangl2-Long, containing an N-terminal extension of about 50 aa, which arises from an alternative nearcognate AUA translation initiation site, lying upstream of the conventional start codon. While missing in Vangl1 paralogs and in all invertebrates, including Drosophila, this N-terminal extension is conserved in all vertebrate Vangl2 sequences. We show that Vangl2-Long belongs to a multimeric complex with Vangl1 and Vangl2. Using morpholino oligonucleotides to specifically knockdown Vangl2-Long in Xenopus, we found that this isoform is functional and required for embryo extension and neural tube closure. Furthermore, both Vangl2 and Vangl2-Long must be correctly expressed for the polarized distribution of the PCP molecules Pk2 and Dvl1 and for centriole rotational polarity in ciliated epidermal cells. Altogether, our study suggests that Vangl2-Long significantly contributes to the pool of Vangl2 molecules present at the plasma membrane to maintain PCP in vertebrate tissues. [ABSTRACT FROM AUTHOR]

Background: Individual functional modifications shape the ability of wildlife populations to cope with anthropogenic environmental changes. But instead of adaptive response, human-altered environments can generate a succession of deleterious functional changes leading to the extinction of the population. To study how persistent anthropogenic changes impacted local species' population status, we characterised population structure, genetic diversity and individual response of gene expression in the tree frog Hyla orientalis along a gradient of radioactive contamination around the Chernobyl nuclear power plant. Results: We detected lower effective population size in populations most exposed to ionizing radiation in the Chernobyl Exclusion Zone that is not compensated by migrations from surrounding areas. We also highlighted a decreased body condition of frogs living in the most contaminated area, a distinctive transcriptomics signature and stop-gained mutations in genes involved in energy metabolism. While the association with dose will remain correlational until further experiments, a body of evidence suggests the direct or indirect involvement of radiation exposure in these changes. Conclusions: Despite ongoing migration and lower total dose rates absorbed than at the time of the accident, our results demonstrate that Hyla orientalis specimens living in the Chernobyl Exclusion Zone are still undergoing deleterious changes, emphasizing the long-term impacts of the nuclear disaster. [ABSTRACT FROM AUTHOR]

Proteomic, cellular and biochemical analysis of the stress protein NUPR1 reveals that it binds to PARP1 into the nucleus and inhibits PARP1 activity in vitro. Mutations on residues Ala33 or Thr68 of NUPR1 or treatment with its inhibitor ZZW-115 inhibits this effect. PARylation induced by 5-fluorouracil (5-FU) treatment is strongly enhanced by ZZW-115 and associated with a decrease of NAD+/NADH ratio and rescued by the PARP inhibitor olaparib. Cell death induced by ZZW-115 treatment of pancreas cancer-derived cells is rescued by olaparib and improved with PARG inhibitor PDD00017273. The mitochondrial catastrophe induced by ZZW-115 treatment or by genetic inactivation of NUPR1 is associated to a hyperPARylation of the mitochondria, disorganization of the mitochondrial network, mitochondrial membrane potential decrease, and with increase of superoxide production, intracellular level of reactive oxygen species (ROS) and cytosolic levels of Ca2+. These features are rescued by olaparib or NAD+ precursor nicotinamide mononucleotide in a dose-dependent manner and partially by antioxidants treatments. In conclusion, inactivation of NUPR1 induces a hyperPARylation, which in turn, induces a mitochondrial catastrophe and consequently a cell death through a non-canonical Parthanatos, since apoptosis inducing-factor (AIF) is not translocated out of the mitochondria. NUPR1 binds to PARP1 and inhibits PARP1 activity in pancreatic cancer cells, with the NUPR1 inhibitor ZZW-115 abolishing the protective effect of NUPR1, inducing strong hyperPARylation and cell death by mitochondrial dysfunction. [ABSTRACT FROM AUTHOR]

Pancreatic ductal adenocarcinoma (PDA) tumor cells are deprived of oxygen and nutrients and therefore must adapt their metabolism to ensure proliferation. In some physiological states, cells rely on ketone bodies to satisfy their metabolic needs, especially during nutrient stress. Here, we show that PDA cells can activate ketone body metabolism and that β‐hydroxybutyrate (βOHB) is an alternative cell‐intrinsic or systemic fuel that can promote PDA growth and progression. PDA cells activate enzymes required for ketogenesis, utilizing various nutrients as carbon sources for ketone body formation. By assessing metabolic gene expression from spontaneously arising PDA tumors in mice, we find HMG‐CoA lyase (HMGCL), involved in ketogenesis, to be among the most deregulated metabolic enzymes in PDA compared to normal pancreas. In vitro depletion of HMGCL impedes migration, tumor cell invasiveness, and anchorage‐independent tumor sphere compaction. Moreover, disrupting HMGCL drastically decreases PDA tumor growth in vivo, while βOHB stimulates metastatic dissemination to the liver. These findings suggest that βOHB increases PDA aggressiveness and identify HMGCL and ketogenesis as metabolic targets for limiting PDA progression. Synopsis: The molecular mechanisms underlying metabolic flexibility of solid tumor cells exposed to oxygen deprivation remain poorly understood. This study identifies the ketogenic enzyme HMG‐CoA lyase (HMGCL) and its derivative β‐hydroxybutyrate (βOHB) as essential drivers of pancreatic cancer, highlighting ketone body metabolism as a regulatory nexus in metastasis. Systemic βOHB is metabolized by pancreatic adenocarcinoma (PDA) cells, fueling the TCA cycle.HMGCL and βOHB promote PDA tumor aggressiveness and metastatic dissemination.Ketone body metabolic enzymes are upregulated in PDA.PDA cells de novo generate ketone bodies from various carbon sources.Depletion of HMGCL limits oncogenic properties of pancreatic tumor cells in vivo, which is rescued by exogenous βOHB. [ABSTRACT FROM AUTHOR]

Metastatic progression is the leading cause of mortality in breast cancer. Invasive tumor cells develop invadopodia to travel through basement membranes and the interstitial matrix. Substantial efforts have been made to characterize invadopodia molecular composition. However, their full molecular identity is still missing due to the difficulty in isolating them. To fill this gap, we developed a non-hypothesis driven proteomic approach based on the BioID proximity biotinylation technology, using the invadopodia-specific protein Tks5α fused to the promiscuous biotin ligase BirA* as bait. In invasive breast cancer cells, Tks5α fusion concentrated to invadopodia and selectively biotinylated invadopodia components, in contrast to a fusion which lacked the membrane-targeting PX domain (Tks5β). Biotinylated proteins were isolated by affinity capture and identified by mass spectrometry. We identified known invadopodia components, revealing the pertinence of our strategy. Furthermore, we observed that Tks5 newly identified close neighbors belonged to a biologically relevant network centered on actin cytoskeleton organization. Analysis of Tks5β interactome demonstrated that some partners bound Tks5 before its recruitment to invadopodia. Thus, the present strategy allowed us to identify novel Tks5 partners that were not identified by traditional approaches and could help get a more comprehensive picture of invadopodia molecular landscape. [ABSTRACT FROM AUTHOR]

Exosomes, extracellular vesicles (EVs) of endosomal origin, emerge as master regulators of cell-to-cell signaling in physiology and disease. Exosomes are highly enriched in tetraspanins (TSPNs) and syndecans (SDCs), the latter occurring mainly in proteolytically cleaved form, as membrane-spanning C-terminal fragments of the proteins. While both protein families are membrane scaffolds appreciated for their role in exosome formation, composition, and activity, we currently ignore whether these work together to control exosome biology. Here we show that TSPN6, a poorly characterized tetraspanin, acts as a negative regulator of exosome release, supporting the lysosomal degradation of SDC4 and syntenin. We demonstrate that TSPN6 tightly associates with SDC4, the SDC4- TSPN6 association dictating the association of TSPN6 with syntenin and the TSPN6-dependent lysosomal degradation of SDC4-syntenin. TSPN6 also inhibits the shedding of the SDC4 ectodomain, mimicking the effects of matrix metalloproteinase inhibitors. Taken together, our data identify TSPN6 as a regulator of the trafficking and processing of SDC4 and highlight an important physical and functional interconnection between these membrane scaffolds for the production of exosomes. These findings clarify our understanding of the molecular determinants governing EV formation and have potentially broad impact for EV-related biomedicine. [ABSTRACT FROM AUTHOR]