Classical Substrates Research Articles

Pepsin is nitrated in the rat stomach, acquiring antiulcerogenic activity: A novel interaction between dietary nitrate and gut proteins

Dietary nitrate is reduced to nitrite and nitric oxide (•NO) in the gut, producing reactive species able to nitrate proteins and lipids. We investigated intragastric production of •NO and nitrating agents in vivo by examining selective nitration of pepsinogen and pepsin. We further addressed the functional impact of nitration on peptic activity by evaluating the progression of secretagogue-induced ulcers. Pepsinogen nitration was assessed in healthy and diclofenac-induced ulcerated rat stomachs. Both groups were fed nitrite or water by oral gavage. Protein nitration was studied by immunofluorescence and immunoprecipitation. In parallel experiments, pentagastrin was administered to rats and nitrite was then instilled intragastrically. •NO levels were measured before and after nitrite administration by chemiluminescence. Macroscopic damage was assessed and nitrated pepsin was examined in the margin of ulcers. Protein nitration was detected physiologically in the stomach of healthy animals. Nitrite had a dual effect on intragastric nitration: overall nitration was decreased under physiological conditions but enhanced by acute inflammation. Pepsin and pepsinogen were also nitrated via a nitrite-dependent pathway. Nitration of both pepsin and its zymogen led to decreased peptic activity in response to classical substrates (e.g., collagen). Under conditions of acute ulceration, nitrite-dependent pepsin nitration prevented the development of gastric ulcers. Dietary nitrite generates nitrating agents in the stomach in vivo, markedly decreasing peptic activity. Under inflammatory and ulcerogenic conditions pepsin nitration attenuates the progression of gastric ulceration. These results suggest that dietary nitrite-dependent nitration of pepsin may have a novel antiulcerogenic effect in vivo.

Is Oxidized Thioredoxin a Major Trigger for Cysteine Oxidation? Clues from a Redox Proteomics Approach

Cysteine oxidation mediates oxidative stress toxicity and signaling. It has been long proposed that the thioredoxin (Trx) system, which consists of Trx and thioredoxin reductase (Trr), is not only involved in recycling classical Trx substrates, such as ribonucleotide reductase, but it also regulates general cytoplasmic thiol homeostasis. To investigate such a role, we have performed a proteome-wide analysis of cells expressing or not the two components of the Trx system. We have compared the reversibly oxidized thiol proteomes of wild-type Schizosaccharomyces pombe cells with mutants lacking Trx or Trr. Specific Trx substrates are reversibly-oxidized in both strain backgrounds; however, in the absence of Trr, Trx can weakly recycle its substrates at the expense of an alternative electron donor. A massive thiol oxidation occurs only in cells lacking Trr, with 30% of all cysteine-containing peptides being reversibly oxidized; this oxidized cysteine proteome depends on the presence of Trxs. Our observations lead to the hypothesis that, in the absence of its reductase, the natural electron donor Trx becomes a powerful oxidant and triggers general thiol oxidation.

Transforming a drug/H + antiporter into a polyamine importer by a single mutation

EmrE, a multidrug antiporter from Escherichia coli, has presented biochemists with unusual surprises. Here we describe the transformation of EmrE, a drug/H(+) antiporter to a polyamine importer by a single mutation. Antibiotic resistance in microorganisms may arise by mutations at certain chromosomal loci. To investigate this phenomenon, we used directed evolution of EmrE to assess the rate of development of novel specificities in existing multidrug transporters. Strikingly, when a library of random mutants of EmrE was screened for resistance to two major antibacterial drugs--norfloxacin, a fluoroquinolone, and erythromycin, a macrolide--proteins with single mutations were found capable of conferring resistance. The mutation conferring erythromycin resistance resulted from substitution of a fully conserved and essential tryptophan residue to glycine, and, as expected, this protein lost its ability to recognize and transport the classical EmrE substrates. However, this protein functions now as an electrochemical potential driven importer of a new set of substrates: aliphatic polyamines. This mutant provides a unique paradigm to understand the function and evolution of distinct modes of transport.

PatA and PatB Form a Functional Heterodimeric ABC Multidrug Efflux Transporter Responsible for the Resistance of Streptococcus pneumoniae to Fluoroquinolones

All bacterial multidrug ABC transporters have been shown to work as either homodimers or heterodimers. Two possibly linked genes, patA and patB from Streptococcus pneumococcus, that encode half-ABC transporters have been shown previously to be involved in fluoroquinolone resistance. We showed that the ΔpatA, ΔpatB, or ΔpatA/ΔpatB mutant strains were more sensitive to unstructurally related compounds, i.e., ethidium bromide or fluoroquinolones, than the wild-type R6 strain. Inside-out vesicles prepared from Escherichia coli expressing PatA and/or PatB transported Hoechst 33342, a classical substrate of multidrug transporters, only when both PatA and PatB were coexpressed. This transport was inhibited either by orthovanadate or by reserpine, and mutation of the conserved Walker A lysine residue of either PatA or PatB fully abrogated Hoechst 33342 transport. PatA, PatB, and the PatA/PatB heterodimer were purified from detergent-solubilized E. coli membrane preparations. Protein dimers were identified in all cases, albeit in different proportions. In contrast to the PatA/PatB heterodimers, homodimers of PatA or PatB failed to show a vanadate-sensitive ATPase activity. Thus, PatA and PatB need to interact together to make a functional drug efflux transporter, and they work only as heterodimers.

Flavone potently stimulates an apical transporter for flavonoids in human intestinal Caco‐2 cells

Based on the studies suggesting that active transport mechanisms contribute to the absorption of flavonoids into human intestinal Caco-2 cells, we here used the structurally similar fluorescent rhodamine 123 to test a possible influence of flavonoids on its uptake. Rhodamine absorption displayed saturation kinetics with a K(m) of 1.1 μM and a pH-optimum of 8.5 and was stimulated by flavone four-fold in its V(max) . Ring C of the other 16 flavonoids tested turned out to be of special importance in order to act as potent inhibitors for rhodamine transport, with a positive charge there, as present in the anthocyanidins, or a 2,3 double bond together with an aromatic ring fused to position 2, as present in flavones and flavonols, being essential structural requirements. Flavone-stimulated rhodamine uptake was unaffected by classical substrates of organic cation transporters or inhibitors of adenosine triphosphate (ATP)-dependent efflux pumps. Also, inhibitors of mitogen-activated protein kinases or tyrosine kinases did not influence the transport, whose stimulation, however, was essentially dependent on the simultaneous presence of flavone. The existence of a flavone-activated apical flavonoid transporter in Caco-2 cells was finally associated with the potently diminished transepithelial apical to basolateral fluxes of (14) C-kaempferol in the presence of competing unlabeled flavonoid substrates. In conclusion, flavone activates an as yet unidentified transporter for flavonoids in the apical membrane of Caco-2 cells.

Abstract 259: Sam68 Regulates Endothelin-Converting Enzyme-1b Expression and Blood Pressure Homeostasis

We have previously reported that E2F2, a transcription factor known for its role in cell cycle, regulates the expression of endothelin-converting enzyme-1b (ECE-1b) in endothelial cells and contributes to the maintenance of vascular contractility and blood pressure (BP). However, the molecular mechanisms underlying this novel, cell-cycle independent function of E2F2 remain largely elusive. In this study, we profiled E2F2 interactome in the nucleus of human umbilical vein endothelial cells by utilizing a proteomic approach. We identified that Sam68, a classic RNA-binding protein and Src kinase substrate, acts as an E2F2-interacting protein; co-immunoprecipitation analyses confirmed that both endogenous and ectopically-expressed Sam68 interact with E2F2. Overexpression of Sam68 repressed whereas knockdown of Sam68 increased E2F2-induced ECE-1b promoter activity and mRNA expression. Chromatin immunoprecipitation assays further confirmed that E2F2 and Sam68 co-localize on ECE-1b promoter, indicating that ECE-1b is a direct transcriptional target of E2F2 and Sam68. In vivo, Sam68 knockout (KO) mice displayed a significantly lowered BP as compared to WT littermates (tail-cuff measurements of mean BP, KO vs. WT: 104 ± 6 vs. 123 ± 7 mmHg, n=25 males of age 4-6 months, P<0.05). Together, our studies have revealed a previously unknown function of Sam68 as a transcriptional co-repressor of E2F2 and a critical regulator of BP homeostasis.

Cyanamide-mediated Inhibition of N-acetyltransferase 1

Cyanamide has been used for decades for medical intentions in the treatment of alcoholism and for agricultural purposes as a plant growth regulator and bud-breaking agent. Its therapeutic effect is mediated by reversible inhibition of aldehyde dehydrogenase and it was reported to be metabolized in vivo mainly via coenzyme A dependent N-acetylation by N-acetyltransferases. Although described to be a substrate for N-acetyltransferases (NATs), cyanamide has a different molecular structure to arylamines and hydrazines, the preferred substrates for N-acetyltransferases. Therefore, a more detailed investigation of its interrelations with N-acetyltransferases was performed. We analyzed the impact of cyanamide on NAT1 activities of human monocytes (monocytic THP-1 cells) using the classical substrate p-aminobenzoic acid. We found that a 24h treatment with physiologically relevant concentrations of cyanamide decreased the NAT1 activity significantly. Based on this observation we performed additional experiments using recombinant human NAT1 and NAT2 to achieve further insights. In detail a significant dose- and time-dependent inhibition of NAT1 activity was observed for 100 and 1000μM cyanamide using recombinant human NAT1*4. However, cyanamide did not inhibit recombinant NAT2*4. Experiments testing cyanamide as substrate did not provide evidence that cyanamide is metabolized via coenzyme A dependent N-acetylation in vitro by human NAT1 or NAT2, THP-1 or human liver cytosol. Therefore we can conclude that the observed enzyme inhibition (around 50% and 25% after treatment with 0.5 and 0.25mM CA, respectively) is not based on substrate-dependent down-regulation of NAT1. Further mechanistic and kinetic studies indicated that cyanamide reacts with the active site cysteine residue of NAT1, leading to its rapid inhibition (significant inhibition after 30min and 2h for 1000 and 100μM CA, respectively). Addition of the reduction agent dithiothreitol (DTT) did not modify the effect, indicating that oxidative processes that can be reversed by 5mM DTT are not likely involved in the inhibition. Taken together our results show that cyanamide is able to inhibit NAT1 most likely via interaction with the active site cysteine residue. Thereby cyanamide might modulate NAT1 dependent detoxification and activation of arylamines.

Substrate-inhibitory analysis of monoamine oxidase from hepatopancreas of the octopus Bathypolypus arcticus

Study of the substrate-inhibitory specificity of mitochondrial monoamine oxidase (MAO) of hepatopancreas of the octopus Bathypolypus arcticus revealed distinctive peculiarities of catalytic properties of this enzyme. The studied enzyme, on one hand, like the classic MAO of homoiothermal animals, is able to deaminate tyramine, serotonin, benzylamine, tryptamine, beta-phenylethylamine, while, on the other hand, deaminates histamine and does not deaminate putrescine--classic substrates of diamine oxidase (DAO). Results of the substrate-inhibitory analysis with use of chlorgiline and deprenyl are indirect proofs of the existence in the octopus hepatopancreas of one molecular MAO form. Semicarbazide and pyronine G turned out to be weak irreversible inhibitors, four derivatives of acridine--irreversible inhibitors of the intermediate effectiveness with respect to the octopus hepatopancreas MAO; specificity of action of inhibitors at deamination of different substrates was equal.

Evaluation of Inhibition Selectivity for Human Cytochrome P450 2A Enzymes

Cytochrome P450 (P450) enzymes are mixed-function oxidases that catalyze the metabolism of xenobiotics and endogenous biochemicals. Selective inhibitors are needed to accurately distinguish the contributions of individual P450 enzymes in the metabolism of drugs and the activation of procarcinogens in human tissues, but very frequently these enzymes have substantial overlapping selectivity. We evaluated a chemically diverse set of nine previously identified CYP2A6 inhibitors to determine which are able to discriminate between human CYP2A enzymes CYP2A6 and the 94%-identical CYP2A13 enzyme. Inhibitor binding to recombinant purified enzyme was evaluated, and affinities were determined. K(i) values were determined for inhibition of p-nitrophenol 2-hydroxylation, a reaction accomplished by CYP2A13 and CYP2A6 with more similar catalytic efficiencies (k(cat)/K(m) 0.19 and 0.12 μM⁻¹ · min⁻¹, respectively) than hydroxylation of the classic substrate coumarin (0.11 and 0.53 μM⁻¹ · min⁻¹, respectively). Of the nine compounds assayed, only tranylcypromine and (R)-(+)-menthofuran had a greater than 10-fold preference for CYP2A6 inhibition versus CYP2A13 inhibition. Most compounds evaluated [tryptamine, 4-dimethylaminobenzaldehyde, phenethyl isothiocyanate, β-nicotyrine, (S)-nicotine, and pilocarpine] demonstrated only moderate or no preference for inhibition of one CYP2A enzyme over the other. However, 8-methoxypsoralen has a 6-fold lower K(i) for CYP2A13 than for CYP2A6. This information is useful to inform reinterpretation of previous data with these inhibitors and to guide future studies seeking to determine which human CYP2A enzyme is responsible for the in vivo metabolism of compounds in human tissues expressing both enzymes.

Reactions and properties of the dinuclear molybdenum complex [(η5-C5H3)2(CMe2)(SiMe2)]Mo2(CO)6

Reactions of the title complex (1), which features the rigid doubly-bridged biscyclopentadienyl ligand and the unusually long Mo–Mo bond in the structure, with a variety of classic substrates are explored. Complex 1 reacts with I2 in CH2Cl2 to give the expected Mo–Mo cleaved product [(η5-C5H3)2(CMe2)(SiMe2)]Mo2(CO)6I2 (2A) with a Mo–Mo distance of 5.329Å. While the same reaction in benzene forms the product 2B, which has the same structural formula as 2A but a different conformation, in which the Mo–Mo distance is 6.442Å. Reaction of 1 with Ph2E2 (E=S, Se) in refluxing toluene or under UV irradiation yields products with the carbonyls completely removed, [(η5-C5H3)2(CMe2)(SiMe2)]Mo2(EPh)2(μ-EPh)2 (3a–b). Reaction of 1 with [Et2NC(S)S]2 results in the di-substituted product [(η5-C5H3)2(CMe2)(SiMe2)]Mo2(CO)4(Et2NC(S)S)2 (4). Reaction of 1 with C2Ph2 affords the acetylene-bridged derivative [(η5-C5H3)2(CMe2)(SiMe2)]Mo2(CO)4(μ-C2Ph2) (5). Reaction of 1 with an excess of PPh3 in refluxing toluene only provides the mono-substituted derivative [(η5-C5H3)2(CMe2)(SiMe2)]Mo2(CO)5(PPh3) (6), while the reaction of 1 with excess P(OPh)3 under the same conditions produces not only the mono-substituted derivative [(η5-C5H3)2(CMe2)(SiMe2)]Mo2(CO)5(P(OPh)3) (7), but also the di-substituted derivative [(η5-C5H3)2(CMe2)(SiMe2)]Mo2(CO)4(P(OPh)3)2 (8). The reactivity and properties of the doubly-bridged molybdenum complex 1 and the corresponding non-bridged and singly-bridged analogs are compared. The molecular structures of 2A, 2B, 3a, 5 and 8 have been determined by single crystal X-ray diffraction.

Cortactin Tyrosine Phosphorylation Promotes Its Deacetylation and Inhibits Cell Spreading

BackgroundCortactin is a classical Src kinase substrate that participates in actin cytoskeletal dynamics by activating the Arp2/3 complex and interacting with other regulatory proteins, including FAK. Cortactin has various domains that may contribute to the assembly of different protein platforms to achieve process specificity. Though the protein is known to be regulated by post-translational modifications such as phosphorylation and acetylation, how tyrosine phosphorylation regulates cortactin activity is poorly understood. Since the basal level of tyrosine phosphorylation is low, this question must be studied using stimulated cell cultures, which are physiologically relevant but unreliable and difficult to work with. In fact, their unreliability may be the cause of some contradictory findings about the dynamics of tyrosine phosphorylation of cortactin in different processes.Methodology/Principal FindingsIn the present study, we try to overcome these problems by using a Functional Interaction Trap (FIT) system, which involves cotransfecting cells with a kinase (Src) and a target protein (cortactin), both of which are fused to complementary leucine-zipper domains. The FIT system allowed us to control precisely the tyrosine phosphorylation of cortactin and explore its relationship with cortactin acetylation.Conclusions/SignificanceUsing this system, we provide definitive evidence that a competition exists between acetylation and tyrosine phosphorylation of cortactin and that phosphorylation inhibits cell spreading. We confirmed the results from the FIT system by examining endogenous cortactin in different cell types. Furthermore, we demonstrate that cell spreading promotes the association of cortactin and FAK and that tyrosine phosphorylation of cortactin disrupts this interaction, which may explain how it inhibits cell spreading.

Ku-band bandpass filters using novel micromachined substrate integrated waveguide structure with embedded silicon vias in benzocyclobutene dielectrics

In this paper, Ku-band bandpass filters (BPFs) based on a novel micromachined substrate integrated waveguide (SIW) platform with square complementary split-ring resonators (CSRRs) have been demonstrated. The proposed SIW platform adopts embedded metal-coated silicon vias in benzocyclobutene (BCB) dielectric substrate filled into a silicon trench. Unlike conventional SIW BPFs using classical microwave substrates, the proposed BPFs can be integrated with silicon-based circuits or RF MEMS devices since the fabrication process is fully compatible with the silicon process while providing low-loss properties of the waveguide. Three types of BPFs using CPW and microstrip feeding configurations have been designed and fabricated. The microstrip-fed BPF using the same CSRRs shows an improved insertion loss of 1.39dB at 13.8GHz with reduced device size compared to the CPW-fed BPF which has an insertion loss of 2.27dB at 14.1GHz. The microstrip-fed BPF with different CSRR configurations designed to improve shape factor shows an attenuation factor of 124dB/GHz, with an insertion loss of 2.3dB at 21.2GHz.

Well-redispersed ceria nanoparticles: Promising peroxidase mimetics for H2O2 and glucose detection

Well-redispersed ceria nanoparticles (CeO2 NPs) were synthesized by a simple hydrothermal method. The prepared CeO2 NPs exhibited excellent catalytic activity towards classical peroxidase substrate 3,3,5,5-tetramethylbiphenyl dihydrochloride (TMB·2HCl) in the presence of H2O2, based on which a colorimetric method that is highly sensitive and selective was developed for glucose detection. The composition, structure, morphology and peroxidase-like catalytic activity of CeO2 NPs are investigated in detail by using X-ray diffraction (XRD), transmission electron microscopy (TEM), high resolution transmission electron microscopy (HRTEM), selected area electron diffraction (SAED), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectrometer (FT-IR), thermal analysis (TG) and UV-vis absorption spectroscopy. According to this method, the detection of H2O2 and glucose are in linear range from 6.0 × 10−7 to 1.5 × 10−6 mol L−1 and 6.6 × 10−6 to 1.3 × 10−4 mol L−1, with the detection limit down to 5.0 × 10−7 mol L−1 H2O2 and 3.0 × 10−6 mol L−1 glucose, respectively. Further, this simple, cheap, highly sensitive and selective colorimetric method for glucose detection was successfully applied for the determination of glucose in human serum samples.

Characterization of ppGalNAc-T18, a member of the vertebrate-specific Y subfamily of UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferases †

The first step of mucin-type O-glycosylation is catalyzed by members of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T; EC 2.4.1.41) family. Each member of this family has unique substrate specificity and expression profiles. In this report, we describe a new subfamily of ppGalNAc-Ts, designated the Y subfamily. The Y subfamily consists of four members, ppGalNAc-T8, -T9, -T17 and -T18, in which the conserved YDX(5)WGGENXE sequence in the Gal/GalNAc-T motif of ppGalNAc-Ts is mutated to LDX(5)YGGENXE. Phylogenetic analysis revealed that the Y subfamily members only exist in vertebrates. All four Y subfamily members lack in vitro GalNAc-transferase activity toward classical substrates possibly because of the UDP-GalNAc-binding pocket mutants. However, ppGalNAc-T18, the newly identified defining member, was localized in the endoplasmic reticulum rather than the Golgi apparatus in lung carcinoma cells. The knockdown of ppGalNAc-T18 altered cell morphology, proliferation potential and changed cell O-glycosylation. ppGalNAc-T18 can also modulate the in vitro GalNAc-transferase activity of ppGalNAc-T2 and -T10, suggesting that it may be a chaperone-like protein. These findings suggest that the new Y subfamily of ppGalNAc-Ts plays an important role in protein glycosylation; characterizing their functions will provide new insight into the role of ppGalNAc-Ts.

Ficolin-B marks apoptotic and necrotic cells

Ficolins are a group of proteins consisting of a fibrinogen-like and a collagen-like domain. They play a role in innate immunity by activating the complement system via the lectin pathway upon binding to carbohydrate patterns on pathogens. Two types of ficolins have been identified in mice, ficolin A and ficolin B (FcnB). We show in this article that recombinant FcnB binds to late apoptotic cells and to apoptotic bodies as well as to necrotic cells but not to early apoptotic cells. This binding was calcium-dependent and could be competitively inhibited by acetylated BSA, a classical binding substrate of FcnB. In addition, DNA inhibited binding of FcnB to apoptotic and necrotic cells, indicating that DNA exposed by dying cells could also be a ligand for FcnB. Thus, FcnB may play a role in the removal of damaged host cells and maintenance of tissue homeostasis.

Important considerations for effective gas sensors based on metal oxide nanoneedles films

Gas sensor devices based on either polycrystalline or nanoneedle tungsten oxide were deposited in situ on classical ceramic substrates via AACVD, and subsequently functionalized with gold nanoparticles via sputtering. The sensing properties of the films were tested to a wide range of analytes, revealing high responses to ethanol, hydrogen, and nitrogen dioxide, at low operating temperatures (≤250 °C), and a lack of response to carbon monoxide, ammonia, and hydrogen sulfide. In addition, the sensing responses to hydrogen and nitrogen dioxide, at 100 °C and 150 °C, were improved by using gold functionalized structures. Modest differences of the sensor response magnitude in polycrystalline, and nanoneedle films were observed, suggesting the need of special substrate platforms for effective application of nanostructured films in gas sensors devices.

A heme peroxidase of the ascomyceteous lichen Leptogium saturninum oxidizes high-redox potential substrates

Lichens belonging to the order Peltigerales display strong activity of multi-copper oxidases (e.g. tyrosinase) as well as heme-containing peroxidases. The lichen peroxidase was purified to homogeneity from the thallus of Leptogium saturninum ( LsaPOX) by fast protein liquid chromatography and then partially characterized. The oligomeric protein occurs as both 79 kDa dimeric and 42 kDa monomeric forms, and displayed broad substrate specificity. In addition to an ability to oxidize classic peroxidase substrates (e.g. 2,6-dimethoxyphenol), the enzyme could convert recalcitrant compounds such as synthetic dyes (e.g. Azure B and Reactive Blue 5), 4-nitrophenol and non-phenolic methoxylated aromatics (e.g. veratryl alcohol). Comparing LsaPOX with a basidiomycete dye-decolorizing (DyP)-type peroxidase from Auricularia auricula-judae showed that the lichen enzyme has a high-redox potential, with oxidation capabilities ranging between those of known plant and fungal peroxidases. Internal peptide fragments show homology (up to 60%) with putative proteins from free-living ascomycetes (e.g. Penicillium marneffei and Neosartorya fischeri), but not to sequences of algal or cyanobacterial peptides or to known fungal, bacterial or plant peroxidases. LsaPOX is the first heme peroxidase purified from an ascomyceteous lichen that may help the organism to successfully exploit the extreme micro-environments in which they often grow.

ABCB1/MDR1 contributes to the anticancer drug-resistant phenotype of IPH-926 human lobular breast cancer cells

Contribution of the ABCB1/MDR1/P-glycoprotein drug transporter to breast cancer resistance has been controversial. One issue is that ABCB1-dependent drug-resistance has primarily been investigated in mammary epithelial cell models technically manipulated to overexpress ABCB1, either by gene transfer using appropriate expression vectors or by chronic anticancer drug-selection. However, an unmodified human breast cancer cell line with an endogenous overexpression of ABCB1 has not been described thus far. Using Affymetrix microarray analyses, we identified an endogenous overexpression of several tumor-biologically relevant transcripts including ABCB1, BCAR4, CCL28, SCGB2A2 and PIP in IPH-926, an anticancer drug-resistant human lobular breast cancer cell line derived from a chemo-refractory mammary carcinoma patient. In a panel of twenty breast cancer cell lines examined, overexpression of ABCB1 mRNA and protein was exclusively detected in IPH-926. This was further validated using chronically in vitro drug-selected KB-V-1 cells as a widely used reference model to accurately define an ABCB1 overexpression. IPH-926 and KB-V-1 displayed a similar overexpression of ABCB1. Flow cytometric analyses showed that IPH-926 but not ABCB1-negative breast cancer cells extruded the anticancer agent doxorubicin, a classical substrate of the ABCB1 drug transporter. PSC-833 (valspodar), a selective ABCB1 inhibitor, blocked this efflux, restored apoptotic PARP cleavage and increased doxorubicin sensitivity in IPH-926 and KB-V-1. To our knowledge, IPH-926 represents the first human breast cancer cell line with a genuine, endogenous overexpression of ABCB1. IPH-926 provides evidence that ABCB1 can occasionally cause anticancer drug-resistance in breast cancer patients and offers a new tool for the evaluation of compounds to overcome drug-resistance.

Catalytic characteristics of monoamine oxidase of whitefish Coregonus lavaretus ludoga

Study of substrate-inhibitory specificity of liver mitochondrial monoamine oxidase (MAO) of sexually mature individuals of the whitefish Coregonus lavaretus ludoga P. from the Ladoga Lake has revealed distinguished peculiarities of catalytical properties of this enzyme. The studied MAO, on one hand, like the classical enzyme of homoiothermal animals, is able to deaminate tyramine, serotonin, benzylamine, tryptamine, and beta-phenylalanine, but, on the other hand, to deaminate histamine, the classic substrate of diamine oxidase. The found equal activity and sorptional ability of the enzyme toward six studied substrates including histamine, as well as results of the substrate-inhibitory analysis with use of specific inhibitors--deprenyl and chlorgilin--indicate homogeneity of the enzyme. The detected for the first time among the fish MAO wide substrate specificity and an unusually low sensitivity to both studied acetylene inhibitors does not allow ascribing unanimously the studied enzyme to the known MAO forms of organs and tissues of homoiothermal organisms. Apparently, the revealed enzyme form of poikilothermal organism is not the true MAO, but performs a large amine oxidase function.

Structural and functional basis for RNA cleavage by Ire1

BackgroundThe unfolded protein response (UPR) controls the protein folding capacity of the endoplasmic reticulum (ER). Central to this signaling pathway is the ER-resident bifunctional transmembrane kinase/endoribonuclease Ire1. The endoribonuclease (RNase) domain of Ire1 initiates a non-conventional mRNA splicing reaction, leading to the production of a transcription factor that controls UPR target genes. The mRNA splicing reaction is an obligatory step of Ire1 signaling, yet its mechanism has remained poorly understood due to the absence of substrate-bound crystal structures of Ire1, the lack of structural similarity between Ire1 and other RNases, and a scarcity of quantitative enzymological data. Here, we experimentally define the active site of Ire1 RNase and quantitatively evaluate the contribution of the key active site residues to catalysis.ResultsThis analysis and two new crystal structures suggest that Ire1 RNase uses histidine H1061 and tyrosine Y1043 as the general acid-general base pair contributing ≥ 7.6 kcal/mol and 1.4 kcal/mol to transition state stabilization, respectively, and asparagine N1057 and arginine R1056 for coordination of the scissile phosphate. Investigation of the stem-loop recognition revealed that additionally to the stem-loops derived from the classic Ire1 substrates HAC1 and Xbp1 mRNA, Ire1 can site-specifically and rapidly cleave anticodon stem-loop (ASL) of unmodified tRNAPhe, extending known substrate specificity of Ire1 RNase.ConclusionsOur data define the catalytic center of Ire1 RNase and suggest a mechanism of RNA cleavage: each RNase monomer apparently contains a separate catalytic apparatus for RNA cleavage, whereas two RNase subunits contribute to RNA stem-loop docking. Conservation of the key residues among Ire1 homologues suggests that the mechanism elucidated here for yeast Ire1 applies to Ire1 in metazoan cells, and to the only known Ire1 homologue RNase L.