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  • 1
    Keywords: SACCHAROMYCES-CEREVISIAE ; BINDING ; ESCHERICHIA-COLI ; OLIGOMERIZATION ; REDUCTASE ; thioredoxin ; PEROXIDASE-ACTIVITY ; SWISS-MODEL ; NMR SOLUTION STRUCTURE ; 1-CYS PEROXIREDOXIN
    Abstract: BACKGROUND: Peroxiredoxins are important heterogeneous thiol-dependent hydroperoxidases with a variety of isoforms and enzymatic mechanisms. A special subclass of glutaredoxin/glutathione-dependent peroxiredoxins has been discovered in bacteria and eukaryotes during the last decade, but the exact enzymatic mechanisms of these enzymes remain to be unraveled. METHODS: We performed a comprehensive analysis of the enzyme kinetics and redox states of one of these glutaredoxin/glutathione-dependent peroxiredoxins, the antioxidant protein from the malaria parasite Plasmodium falciparum, using steady-state kinetic measurements, site-directed mutagenesis, redox mobility shift assays, gel filtration, and mass spectrometry. RESULTS: P. falciparum antioxidant protein requires not only glutaredoxin but also glutathione as a true substrate for the reduction of hydroperoxides. One peroxiredoxin cysteine residue and one glutaredoxin cysteine residue are sufficient for catalysis, however, additional cysteine residues of both proteins result in alternative redox states and conformations in vitro with implications for redox regulation. Our data furthermore point to a glutathione-dependent peroxiredoxin activation and a negative subunit cooperativity. CONCLUSIONS: The investigated glutaredoxin/glutathione/peroxiredoxin system provides numerous new insights into the mechanism and redox regulation of peroxiredoxins. GENERAL SIGNIFICANCE: As a member of the special subclass of glutaredoxin/glutathione-dependent peroxiredoxins, the P. falciparum antioxidant protein could become a reference protein for peroxiredoxin catalysis and regulation.
    Type of Publication: Journal article published
    PubMed ID: 23624334
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  • 2
    Keywords: GENE-EXPRESSION ; BREAST-CANCER ; CRYSTAL-STRUCTURE ; BRAIN-TUMORS ; MOLECULAR-MECHANISMS ; COORDINATION-COMPLEXES ; HUMAN GLUTATHIONE-REDUCTASE ; THIOREDOXIN REDUCTASE INHIBITION ; PHOSPHINE GOLD(I) COMPLEXES ; ANTICANCER THERAPEUTICS
    Abstract: Glioblastoma, an aggressive brain tumor, has a poor prognosis and a high risk of recurrence. An improved chemotherapeutic approach is required to complement radiation therapy. Gold(I) complexes bearing phosphole ligands are promising agents in the treatment of cancer and disturb the redox balance and proliferation of cancer cells by inhibiting disulfide reductases. Here, we report on the antitumor properties of the gold(I) complex 1-phenyl-bis(2-pyridyl)phosphole gold chloride thio-beta-d-glucose tetraacetate (GoPI-sugar), which exhibits antiproliferative effects on human (NCH82, NCH89) and rat (C6) glioma cell lines. Compared to carmustine (BCNU), an established nitrosourea compound for the treatment of glioblastomas that inhibits the proliferation of these glioma cell lines with an IC50 of 430muM, GoPI-sugar is more effective by two orders of magnitude. Moreover, GoPI-sugar inhibits malignant glioma growth in vivo in a C6 glioma rat model and significantly reduces tumor volume while being well tolerated. Both the gold(I) chloro- and thiosugar-substituted phospholes interact with DNA albeit more weakly for the latter. Furthermore, GoPI-sugar irreversibly and potently inhibits thioredoxin reductase (IC50 4.3nM) and human glutathione reductase (IC50 88.5nM). However, treatment with GoPI-sugar did not significantly alter redox parameters in the brain tissue of treated animals. This might be due to compensatory upregulation of redox-related enzymes but might also indicate that the antiproliferative effects of GoPI-sugar in vivo are rather based on DNA interaction and inhibition of topoisomerase I than on the disturbance of redox equilibrium. Since GoPI-sugar is highly effective against glioblastomas and well tolerated, it represents a most promising lead for drug development. This article is part of a Special Issue entitled: Thiol-Based Redox Processes.
    Type of Publication: Journal article published
    PubMed ID: 24440405
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  • 3
    Keywords: IN-VIVO ; DRUG-DELIVERY ; PHASE-II TRIAL ; BREAST-CANCER CELLS ; IRON-OXIDE NANOPARTICLES ; gold nanoparticles ; POLYMERIC NANOPARTICLES ; SERUM-ALBUMIN NANOPARTICLES ; PLASMID-LOADED NANOPARTICLES ; PEP-PCL NANOPARTICLES
    Abstract: Radiation therapy is one of the most commonly used non-surgical interventions in tumor treatment and is often combined with other modalities to enhance its efficacy. Despite recent advances in radiation oncology, treatment responses, however, vary considerably between individual patients. A variety of approaches have been developed to enhance radiation response or to counteract resistance to ionizing radiation. Among them, a relatively novel class of radiation sensitizers comprises nanoparticles (NPs) which are highly efficient and selective systems in the nanometer range. NPs can either encapsulate radiation sensitizing agents, thereby protecting them from degradation, or sensitize cancer cells to ionizing radiation via their physicochemical properties, e.g. high Z number. Moreover, they can be chemically modified for active molecular targeting and the imaging of tumors. In this review we will focus on recent developments in nanotechnology, different classes and modifications of NPs and their radiation sensitizing properties.
    Type of Publication: Journal article published
    PubMed ID: 26142869
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  • 4
    Keywords: IN-VITRO ; PROSTATE-CANCER ; TANDEM MASS-SPECTROMETRY ; tumor microenvironment ; SERINE-PROTEASE ; Caveolin-1 ; STATISTICAL-MODEL ; EPITHELIAL CANCERS ; INTERFACE ZONE ; SPFH DOMAIN
    Abstract: Fibroblast activation protein alpha (FAPalpha) is a cell surface protease expressed by cancer-associated fibroblasts in the microenvironment of most solid tumors. As there is increasing evidence for proteases having non-catalytic functions, we determined the FAPalpha interactome in cancer-associated fibroblasts using the quantitative immunoprecipitation combined with knockdown (QUICK) method. Complex formation with adenosin deaminase, erlin-2, stomatin, prohibitin, Thy-1 membrane glycoprotein, and caveolin-1 was further validated by immunoblotting. Co-immunoprecipitation (co-IP) of the known stoichiometric FAPalpha binding partner dipeptidyl-peptidase IV (DPPIV) corroborated the proteomic strategy. Reverse co-IPs validated the FAPalpha interaction with caveolin-1, erlin-2, and stomatin while co-IP upon RNA-interference mediated knock-down of DPPIV excluded adenosin deaminase as a direct FAPalpha interaction partner. Many newly identified FAPalpha interaction partners localize to lipid rafts, including caveolin-1, a widely-used marker for lipid raft localization. We hypothesized that this indicates a recruitment of FAPalpha to lipid raft structures. In density gradient centrifugation, FAPalpha co-fractionates with caveolin-1. Immunofluorescence optical sectioning microscopy of FAPalpha and lipid raft markers further corroborates recruitment of FAPalpha to lipid rafts and invadopodia. FAPalpha is therefore an integral component of stromal lipid rafts in solid tumors. In essence, we provide one of the first interactome analyses of a cell surface protease and translate these results into novel biological aspects of a marker protein for cancer-associated fibroblasts.
    Type of Publication: Journal article published
    PubMed ID: 26209915
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  • 5
    Keywords: SPECTRA ; PROTEIN ; PROTEINS ; EFFICIENCY ; MOLECULES ; COMPLEX ; COMPLEXES ; DNA ; MECHANISM ; BINDING ; MOLECULE ; virus ; inactivation ; PCR ; DAMAGE ; CAPSID PROTEIN ; DNA-DAMAGE ; POLYMERASE-CHAIN-REACTION ; NETHERLANDS ; BLOOD COMPONENTS ; CHAIN-REACTION ; ENVELOPED VIRUSES ; GLYCOCONJUGATED PORPHYRINS ; METHYLENE-BLUE ; PHOTODYNAMIC INACTIVATION ; photodynamic virus inactivation ; Q-BETA ; RED-CELLS ; ROSE-BENGAL ; SECONDARY STRUCTURE ; STRUCTURAL-CHANGES ; T7 phage ; tetraphenyl porphyrin
    Abstract: We investigated the efficiency and the mechanism of action of a tetraphenyl porphyrin derivative in its photoreaction with T7 phage as surrogate of non-enveloped DNA viruses. TPFP was able to sensitize the photoinactivation of T7 phage in spite of the lack of its binding to the nucleoprotein complex. The efficiency of TPFP photosensitization was limited by the aggregation and by the photobleaching of porphyrin molecules. Addition of sodium azide or 1,3-dimethyl-2-thiourea (DMTU) to the reaction mixture moderated T7 inactivation, however, neither of them inhibited T7 inactivation completely. This result suggests that both Type I and Type 11 reaction play a role in the virus inactivation. Optical melting studies revealed structural changes in the protein part but not in the DNA of the photochemically treated nucleoprotein complex. Polymerase chain reaction (PCR) also failed to demonstrate any DNA damage. Circular dichroism (CD) spectra of photosensitized nucleoprotein complex indicated changes in the secondary structure of both the DNA and proteins. We suggest that damages in the protein capsid and/or loosening of protein-DNA interaction can be responsible for the photodynamic inactivation of T7 phage. The alterations in DNA secondary structure might be the result of photochemical damage in phage capsid proteins. (C) 2003 Elsevier B.V. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 14642821
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  • 6
    Keywords: CELLS ; EXPRESSION ; PATHWAYS ; METABOLISM ; ACTIVATION ; MEMBRANE ; CERAMIDE ; HEPATOCYTE APOPTOSIS ; NONALCOHOLIC STEATOHEPATITIS ; FATTY LIVER-DISEASE
    Abstract: In the pathogenesis of nonalcoholic fatty liver disease, accumulation of lipids in hepatocytes and hepatocyte apoptosis are strongly implicated in disease progression from the potentially reversible condition of steatosis to severe acute and chronic liver injury. Acyl-CoA synthetase 5, a member of the ACSL gene family that catalyzes the activation of long-chain fatty acids for lipid biosynthesis, is the only ACSL isoform that is both, located on mitochondria and functionally involved in enterocyte apoptosis. In this study, the regulation of human ACSL5 in hepatocellular fatty acid degeneration and its involvement in hepatocyte apoptosis was investigated using models of in vitro and in vivo steatosis as well as plasmid-mediated stable gene transfer and RNAi-mediated gene silencing. ACSL5 mRNA and protein were strongly increased by uptake of dietary derived fatty acids in primary human hepatocytes, HepG2 cells and human steatotic liver. Over-expression of ACSL5 decreased HepG2 cell viability and increased susceptibility to TRAIL- and TNF alpha-, but not FAS- induced apoptosis, whereas knock down of ACSL5 reduced apoptosis susceptibility. High ACSL5 activity resulted in enhanced caspase-3/7 activity, but was not accompanied by up-regulation of death receptors, DR4, DR5 or TNF-R1. This study gives evidence that hepatocyte steatosis is associated with ACSL5 up-regulation resulting in increased susceptibility to hepatic cell death. We propose that ACSL5 could play a role in promoting fatty acid-induced lipoapoptosis in hepatocytes as important mechanism in fatty liver-related disorders.
    Type of Publication: Journal article published
    PubMed ID: 20470896
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  • 7
    Keywords: FACTOR RECEPTOR ; ACTIVATION ; DOMAIN ; BINDING ; BOVINE PAPILLOMAVIRUS ; TRANSFORMATION ; EPITHELIAL-CELL LINE ; ONCOPROTEIN ; GOLGI ; CIRCULAR-DICHROISM
    Abstract: The product of the E5 oncogene in human papillomaviruses (HPVs) participates in cellular transformation. The sequences of E5 from high-risk HPV types are closely related, and the ability to transform is thought to be associated with their structure. Structural determination by standard biophysical methods has proved impossible due to the extreme hydrophobicity of the gene product. We have achieved limited solubility by dividing the sequence into three, structurally distinct domains. Synthetic peptides corresponding to these domains have been examined using circular dichroism (CD) spectroscopy, a method that can detect secondary structure elements in highly dilute protein solutions. Using data on the secondary structure content of these domains under different conditions and in systematic combination to detect constructive domain interactions, a model of HPV E5 structure and position in the membrane is proposed that is consistent with what is known of the larger family of leucine-rich repeat (LRR) proteins to which it belongs.
    Type of Publication: Journal article published
    PubMed ID: 12429498
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  • 8
  • 9
    Keywords: MESSENGER-RNA ; FATTY-ACIDS ; ARACHIDONIC-ACID ; MOLECULAR-CLONING ; MOUSE SKIN CARCINOGENESIS ; CDNA CLONING ; RECESSIVE CONGENITAL ICHTHYOSIS ; PLATELET-TYPE 12-LIPOXYGENASE ; FUNCTIONAL TUMOR-SUPPRESSOR ; 15-LIPOXYGENASE-2 EXPRESSION
    Abstract: Lipoxygenases (LOX) are key enzymes in the biosynthesis of a variety of highly active oxylipins which act as signaling molecules involved in the regulation of many biological processes. LOX are also able to oxidize complex lipids and modify membrane structures leading to structural changes that play a role in the maturation and terminal differentiation of various cell types. The mammalian skin represents a tissue with highly abundant and diverse LOX metabolism. Individual LOX isozymes are thought to play a role in the modulation of epithelial proliferation and/or differentiation as well as in inflammation, wound healing, inflammatory skin diseases and cancer. Emerging evidence indicates a structural function of a particular LOX pathway in the maintenance of skin permeability barrier. Loss-of-function mutations in the LOX genes ALOX12B and ALOXE3 have been found to represent the second most common cause of autosomal recessive congenital ichthyosis and targeted disruption of the corresponding LOX genes in mice resulted in neonatal death due to a severely impaired permeability barrier function. Recent data indicate that LOX action in barrier function can be traced back to the oxygenation of linoleate-containing ceramides which constitutes an important step in the formation of the corneocyte lipid envelope. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.
    Type of Publication: Journal article published
    PubMed ID: 23954555
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  • 10
    Abstract: Brite adipocytes recently discovered in humans are of considerable importance in energy expenditure by converting energy excess into heat. This property could be useful in the treatment of obesity, and nutritional aspects are relevant to this important issue. Using hMADS cells as a human cell model which undergoes a white to a brite adipocyte conversion, we had shown previously that arachidonic acid, the major metabolite of the essential nutrient Omega6-linoleic acid, plays a major role in this process. Its metabolites PGE2 and PGF2 alpha inhibit this process via a calcium-dependent pathway, whereas in contrast carbaprostacyclin (cPGI2), a stable analog of prostacyclin, activates white to brite adipocyte conversion. Herein, we show that cPGI2 generates via its cognate cell-surface receptor IP-R, a cyclic AMP-signaling pathway involving PKA activity which in turn induces the expression of UCP1. In addition, cPGI2 activates the pathway of nuclear receptors of the PPAR family, i.e. PPARalpha and PPARgamma, which act separately from IP-R to up-regulate the expression of key genes involved in the function of brite adipocytes. Thus dual pathways are playing in concert for the occurrence of a browning process of human white adipocytes. These results make prostacyclin analogs as a new class of interesting molecules to treat obesity and associated diseases.
    Type of Publication: Journal article published
    PubMed ID: 26775637
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