bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of liver plasma membrane transporters in gallstone-susceptible and gallstone-resistant mice
Authors: MÜLLER Oliver , SCHALLA Carmen , SCHEIBNER Jürgen , STANGE Eduard F. , FUCHS Michael
Published: August 2015

We tested the hypothesis that differential expression of liver plasma membrane transporters might account for variations in biliary lipid secretion rates between gallstone-susceptible C57L/J and gallstone-resistant AKR/J mice. Plasma membrane fractions and total RNA isolated from livers of mice fed with a control or lithogenic (15% fat/1.25% cholesterol/0.5% cholic acid) diet were used for measurements of steady-state gene expression of hepatobiliary transport systems for bile salts (Ntcp1/Slc10a1, Oatp1/Slc21a1 and Bsep/Abcb11), phospholipids (Mdr2/Abcb4), organic anions (Mrp2/Abcc2) and organic cations (Oct1/Slc22a1). Irrespective of the diet, the steady-state gene expression of hepatobiliary transporters did not differ significantly between the two strains. Despite a higher basal bile flow and bile-salt secretion in C57L mice, Mrp2 (Abcc2) and Bsep (Abcb11) expression did not differ between the two strains. Elevated biliary phospholipid secretion in response to the lithogenic diet was linked to increased Mdr2 (Abcb4) protein expression, whereas the induction of Oct1 (Slc22a1) might reflect an enhanced uptake of choline for augmented phospholipid synthesis. In response to the lithogenic diet, Bsep (Abcb11) protein expression was up-regulated only marginally and bile salt secretion did not increase. The down-regulation of Ntcp1 (Slc10a1) protein expression might protect hepatocytes from high intracellular bile-salt loads. We conclude that variations in protein function rather than in the gene expression of liver plasma membrane transporters might account for variations in biliary lipid secretion rates. Our findings support the concept that the formation of lithogenic bile is caused by the hypersecretion of bile salts as a result of augmented availability of canalicular membrane cholesterol, possibly amplified by bile-salt—phospholipid uncoupling due to the increased bile flow.
Structural and functional characterization of the human and mouse fibulin-1 gene promoters: role of Sp1 and Sp3
Authors: CASTOLDI Mirco , CHU Mon-Li
Published: August 2015

Fibulin-1 is a multifunctional extracellular protein involved in diverse biological processes including cardiovascular development, haemostasis and cancer. To investigate the transcriptional regulation of the gene encoding fibulin-1 we cloned and analysed about 4.0kb of the 5′-flanking regions of both the human and mouse fibulin-1 genes. The human and mouse fibulin-1 promoters share little sequence similarity except for a short region of approx. 150–170bp immediately upstream of the translation start site. The conserved region contains a TATA-like sequence (ATAATT) and multiple consensus binding sites for Sp1 and activator protein 2 (AP-2). That the short conserved region in each gene confers basal promoter activity is demonstrated by transient transfections of promoter deletion constructs for both the human and mouse genes into cells that express fibulin-1 constitutively. Co-transfections of promoter constructs with expression plasmids for Sp1, Sp3 and Sp4 into Drosophila SL2 cells indicate that Sp1 and Sp3 are essential for transcriptional activation and that these two factors act synergistically. Electrophoretic mobility-shift assays show that Sp1 and Sp3, but not AP-2, bind to the basal promoter of the human fibulin-1 gene. The results demonstrate the functional importance of Sp1 and Sp3 in regulating the expression of the fibulin-1 gene.
Induction of glutathione synthesis by oxidized low-density lipoprotein and 1-palmitoyl-2-arachidonyl phosphatidylcholine: protection against quinone-mediated oxidative stress
Authors: MOELLERING Douglas R. , LEVONEN Anna-Liisa , GO Young-Mi , PATEL Rakesh P. , DICKINSON Dale A. , FORMAN Henry Jay , DARLEY-USMAR Victor M.
Published: August 2015

Exposure of endothelial cells to oxidized low-density lipoprotein (oxLDL) leads to diverse cellular effects, including induction of the intracellular antioxidant GSH. It is not known whether lipid-or protein-derived oxidation products cause GSH induction and whether this involves increased activity of the key enzyme in its synthesis, glutamate—cysteine ligase (GCL). Furthermore, the effect of oxLDL exposure on the cell's ability to combat oxidative stress has not been previously examined. In the present study we found that, in bovine aortic endothelial cells, LDL or 1-palmitoyl-2-arachidonyl phosphatidylcholine oxidized by different reactive oxygen and nitrogen species induced GSH synthesis. However, prevention of GSH synthesis during exposure to oxLDL caused extensive cell death. The mediator causing GSH induction was shown to be a polar lipid and resulted in the increased activity of GCL as well as increased protein levels of the regulatory subunit of GCL. Pretreatment with both oxLDL and the polar lipid subfraction of the oxLDL protected cells against the toxicity of 2,3-dimethoxynaphthoquinone (DMNQ), a superoxide- and H2O2-forming compound. The potential of a low level of lipid peroxidation products to initiate cytoprotective pathways are discussed.
A dominant-negative isoform lacking exons 11 and 12 of the human hypoxia-inducible factor-1α gene
Authors: CHUN Yang-Sook , CHOI Eunjoo , KIM Tae-You , KIM Myung-Suk , PARK Jong-Wan
Published: August 2015

Hypoxia-inducible factor-1α (HIF-1α), a member of the transcription family characterized by a basic helix-loop-helix (bHLH) domain and a PAS domain, regulates the transcription of hypoxia-inducible genes involved in erythropoiesis, vascular remodelling and glucose/energy metabolism. It contains bHLH/PAS domains in the N-terminal half, and a nuclear localization signal (NLS) and two transactivation domains (TADs) in the C-terminal half. It also has an oxygen-dependent degradation (ODD) domain, which is required to degrade HIF-1α protein by the ubiquitin—proteasome pathway. In this study, we identified a new alternatively spliced variant of human HIF-1α mRNA, which lacked both exons 11 and 12, producing a frame shift and giving a shorter form of HIF-1α. In the corresponding protein, a part of the ODD domain, both TADs and the C-terminal NLS motif were missing. Expression of endogenous HIF-1α variant protein was identified using immunoprecipitation and immunoblotting methods. The expressed HIF-1α variant exhibited neither the activity of transactivation nor hypoxia-induced nuclear translocation. In contrast with HIF-1α, the variant was strikingly stable in normoxic conditions and not up-regulated to such an extent by hypoxia, cobalt ions or desferrioxamine. It was also demonstrated that the HIF-1α variant competed with endogenous HIF-1α and suppressed HIF-1 activity, resulting in the down-regulation of mRNA expression of hypoxia-inducible genes. The association of the variant and arylhydrocarbon receptor nuclear translocator in the cytoplasm may be related to the inhibition of HIF-1 activity. It is assumed that this isoform preserves the balance between aerobic and anaerobic metabolism by counteracting the overaction of HIF-1α.
The distinct methods by which manganese and iron regulate the Nramp transporters in yeast
Authors: PORTNOY Matthew E. , JENSEN Laran T. , CULOTTA Valeria Cizewski
Published: August 2015

The bakers yeast Saccharomyces cerevisiae expresses three Smf metal transport proteins that are differentially regulated by metal ions. Smf1p and Smf2p are regulated at the post-translational level by manganese, whereas Smf3p is regulated by iron through a mechanism that, up until now, was unknown. Through promoter and protein-domain swapping experiments, we now demonstrate that the manganese regulation of Smf1p involves an internal protein-coding region that is separate from the N-terminal domain of this transporter. By comparison, iron regulation of Smf3p involves the upstream non-coding region of the gene. Using SMF3—lacZ reporter constructs, we identified two distinct regions of the SMF3 promoter that contribute to iron regulation: (1) approx. nt −435 to −350 that contain dual consensus recognition sites for the Aft1 iron transcription factor; and (2) nt −348 to −247 that do not contain obvious Aft1 binding sites. The −348 to −247 region by itself can confer strong iron regulation to the heterologous CYC1 core promoter, and therefore harbours a putative upstream activating sequence for iron. Iron regulation of SMF3 was dramatically reduced, but not completely eliminated, in strains lacking both the AFT1 and AFT2 iron regulatory factors. Together with the promoter mapping studies, these results suggest that both Aft-dependent and Aft-independent pathways may contribute to iron regulation of SMF3.
Mass-spectrometric analysis of agonist-induced retinoic acid receptor γ conformational change
Authors: PETERSON Valerie J. , BAROFSKY Elisabeth , DEINZER Max L. , DAWSON Marcia I. , FENG Kai-Chia , ZHANG Xiao-kun , MADDURU Machender R. , LEID Mark
Published: August 2015

Apo and holo forms of retinoic acid receptors, and other nuclear receptors, display differential sensitivity to proteolytic digestion that likely reflects the distinct conformational states of the free and liganded forms of the receptor. We have developed a method for rapid peptide mapping of holo-retinoic acid receptor γ that utilizes matrix-assisted laser-desorption—ionization time-of-flight MS to identify peptide fragments that are derived from the partially proteolysed holo-receptor. The peptide maps of retinoic acid receptor γ bound by four different agonists were identical, suggesting that all four ligands induced a similar conformational change within the ligand-binding domain of the receptor. In all cases, this agonist-induced conformational change promoted the direct association of retinoic acid receptor γ with the transcriptional co-activator p300 and inhibited interaction of the receptor with the nuclear receptor co-repressor. SR11253, a compound previously reported to exert mixed retinoic acid receptor γ agonist/antagonist activities in cultured cells, was found to bind directly to, but only weakly altered the protease-sensitivity of, the receptor and failed to promote interaction of the receptor with p300 or induce dissociation of receptor—nuclear receptor co-repressor complexes. This technique should be generally applicable to other members of the nuclear receptor superfamily that undergo an induced structural alteration upon agonist or antagonist binding, DNA binding and/or protein—protein interaction.
The death domain of Rab3 guanine nucleotide exchange protein in GDP/GTP exchange activity in living cells
Authors: COPPOLA Thierry , PERRET-MENOUD Véronique , GATTESCO Sonia , MAGNIN Sarah , POMBO Isabel , BLANK Ulrich , REGAZZI Romano
Published: August 2015

Rab3 GTPases regulate exocytosis of neurons, endocrine and exocrine cells. In the present paper, we report a system to measure the guanine nucleotide status of Rab3 proteins in living cells. The assay is based on the ability of the Rab3 interacting molecule RIM to extract selectively the GTP-bound form of Rab3. Using this system, we found that approx. 20% of wild-type Rab3A, -B, -C or -D transfected in the insulin-secreting cell line HIT-T15 is in the GTP-bound conformation. The pool of activated Rab3 is decreased under conditions that stimulate exocytosis or by co-expression of the Rab3 GTPase-activating protein. In contrast, co-expression of Mss4 or Rab3-GEP (guanine nucleotide exchange protein) increases by approx. 3-fold the GTP-bound pool of Rab3 isoforms. Rab3-GEP is very similar to MADD, a death domain-containing protein that associates with the type 1 tumour necrosis factor receptor. We observed that the death domain of Rab3-GEP is involved in intramolecular interactions and that deletions or mutations that affect this domain of the protein impair the nucleotide exchange activity towards Rab3. We propose that the death domain of Rab3-GEP acts as a molecular switch and co-ordinates multiple functions of the protein by exchanging its binding partners.
Ecdysone-inducible expression of oncogenic Ha-Ras in NIH 3T3 cells leads to transient nuclear localization of activated extracellular signal-regulated kinase regulated by mitogen-activated protein kinase phosphatase-1
Authors: PLOWS David , BRIASSOULI Paraskevi , OWEN Carolyn , ZOUMPOURLIS Vassilis , GARRETT Michelle D. , PINTZAS Alexander
Published: August 2015

The Ras family of GTP-binding proteins are key transducers of extracellular signals, particularly through the mitogen-activated protein kinase (MAPK) pathway. Constitutively active forms of Ras are found in a variety of tumours, suggesting an important role for this pathway in cancer. Here we report that initial cellular exposure to oncogenic Ras chronically activated the MAPK pathway in the cytoplasm, but transiently activated the same pathway in the nucleus. Nuclear-activated extracellular signal-regulated kinase (ERK) was rapidly dephosphorylated, with consequent short-term activation of the Elk-1 transcription factor and expression of the c-fos gene. Additional experiments suggested that the regulatory mechanism involved requires the calcium-dependent protein phosphotyrosine phosphatase MAPK phosphatase-1 (MKP-1). This is the first report on the ability of Ras, in the absence of growth factors, to transiently activate the MAPK pathway in the nucleus and show an involvement of MKP-1 in nuclear ERK2 regulation. In addition we show that transient activation of the MAPK pathway is sufficient to drive chronic cell-cycle progression. We conclude that, whereas the MAPK pathway is necessary to initiate cellular proliferation and transformation, the transient nature of the MAPK pathway activation suggests the involvement of additional signalling pathway(s) regulated by Ras.
Molecular interactions between desmosomal cadherins
Authors: SYED Shabih-e-Hassnain , TRINNAMAN Brian , MARTIN Stephen , MAJOR Sarah , HUTCHINSON Jon , MAGEE Anthony I.
Published: August 2015

Desmocollins (Dscs) and desmogleins (Dsgs) are cell-adhesion molecules involved in the formation of desmosome cell—cell junctions and share structural similarities to classical cadherins such as E-cadherin. In order to identify and provide quantitative information on the types of protein—protein interactions displayed by the type 2 isoforms and investigate the role of Ca2+ in this process, we have developed an Escherichia coli expression system to generate recombinant proteins containing the first two extracellular domains, namely Dsg2(1-2) and Dsc2(1-2). Analytical ultracentrifugation, chemical cross-linking, CD, fluorescence and BIAcore have been used to provide the first direct evidence of Ca2+ binding to desmosomal cadherins. These studies suggest that Dsc2(1-2) not only exhibits homophilic interactions in solution, but can also form heterophilic interactions with Dsg2(1-2). The latter, on the other hand, shows much weaker homophilic association. Our results further demonstrate that heterophilic interactions are Ca2+-dependent, whereas the Ca2+-dependence of homophilic association is less clear. Our data indicate that the functional properties of Dsc2(1-2) are more similar to those of classical cadherins, consistent with the observation that Dsc shares a higher level of sequence homology with classical cadherins than does Dsg. In addition to corroborating the conclusions of previously reported transfection studies which suggest the formation of lateral heterodimers and homodimers, our results also provide direct quantitative information on the strength of these interactions which are essential for understanding the adhesion mechanism.
Purification, molecular cloning and heterologous expression of a glutathione S-transferase involved in insecticide resistance from the rice brown planthopper, Nilaparvata lugens
Authors: VONTAS John G. , SMALL Graham J. , NIKOU Dimitra C. , RANSON Hilary , HEMINGWAY Janet
Published: August 2015

A novel glutathione S-transferase (GST)-based pyrethroid resistance mechanism was recently identified in Nilaparvata lugens [Vontas, Small and Hemingway (2001) Biochem. J. 357, 65–72]. To determine the nature of GSTs involved in conferring this resistance, the GSTs from resistant and susceptible strains of N. lugens were partially purified by anion exchange and affinity chromatography. The majority of peroxidase activity, previously correlated with resistance, was confined to the fraction that bound to the affinity column, which was considerably elevated in the resistant insects. A cDNA clone encoding a GST (nlgst1-1) —the first reported GST sequence from Hemiptera with up to 54% deduced amino-acid identity with other insect class I GSTs—was isolated from a pyrethroid-resistant strain. Northern analysis showed that nlgst1-1 was overexpressed in resistant insects. nlgst1-1 was expressed in Escherichia coli, purified and characterized. The ability of the recombinant protein to bind to the S-hexylglutathione affinity matrix, its substrate specificities and its immunological properties confirmed that this GST was one from the elevated subset of N. lugens GSTs. Peroxidase activity of the recombinant nlgst1-1 indicated that it had a role in resistance, through detoxification of lipid peroxidation products induced by pyrethroids. Southern analysis of genomic DNA from the resistant and susceptible strains indicated that GST-based insecticide resistance may be associated with gene amplification in N. lugens.
The unfolded protein response in a dolichyl phosphate mannose-deficient Chinese hamster ovary cell line points out the key role of a demannosylation step in the quality-control mechanism of N-glycoproteins
Authors: FOULQUIER François , HARDUIN-LEPERS Anne , DUVET Sandrine , MARCHAL Ingrid , MIR Anne Marie , DELANNOY Philippe , CHIRAT Frédéric , CACAN René
Published: August 2015

The CHO (Chinese hamster ovary) glycosylation mutant cell line, B3F7, transfers the truncated glycan Glc3Man5GlcNAc2 on to nascent proteins. After deglucosylation, the resulting Man5GlcNAc2 glycan is subjected to two reciprocal enzymic processes: the action of an endoplasmic-reticulum (ER) kifunensine-sensitive α1,2-mannosidase activity to yield a Man4GlcNAc2 glycan, and the reglucosylation involved in the quality-control system which ensures that only correctly folded glycoproteins leave the ER. We show that the recombinant secreted alkaline phosphatase (SeAP) produced in stably transfected B3F7 cells, is co-immunoprecipitated with the GRP78 (glucose-regulated protein 78), a protein marker of the unfolded protein response (UPR). The level of GRP78 transcription has been evaluated by reverse transcription-PCR (RT-PCR) and we demonstrate that B3F7 cells present a constitutively higher level of UPR in the absence of inductors, compared with Pro−5 cells. Interestingly, a decrease was observed in the UPR and an increase in SeAP secretion in the kifunensine-treated B3F7 cells. Altogether, these data highlight the relationships between the glycan structure, the quality control system and the UPR. Moreover, they support the idea that a specific demannosylation step is a key event of the glycoprotein quality control in B3F7 cells.
Activation of c-Jun N-terminal kinase promotes survival of cardiac myocytes after oxidative stress
Authors: DOUGHERTY Christopher J. , KUBASIAK Lori A. , PRENTICE Howard , ANDREKA Peter , BISHOPRIC Nanette H. , WEBSTER Keith A.
Published: August 2015

Reperfusion injury occurs when ischaemic tissue is reperfused. It involves the generation and release of reactive oxygen that activates numerous signalling pathways and initiates cell death. Exposure of isolated cardiac myocytes to chronic hypoxia followed by reoxygenation results in the early activation of c-Jun N-terminal kinase (JNK) and death by apoptosis of approx. 30% of the myocytes. Although JNK activation has been described in a number of models of ischaemia/reperfusion, the contribution of JNK activation to cell fate has not been established. Here we report that the activation of JNK by reoxygenation correlates with myocyte survival. Transfection of myocytes with JNK pathway interfering plasmid vectors or infection with adenoviral vectors support the hypothesis that JNK is protective. Transfection or infection with JNK inhibitory mutants increased the rates of apoptosis by almost 2-fold compared with control cultures grown aerobically or subjected to hypoxia and reoxygenation. Caspase 9 activity, measured by LEHD cleavage, increased > 3-fold during reoxygenation and this activity was enhanced significantly at all times in cultures infected with dominant negative JNK adenovirus. Hypoxia—reoxygenation mediated a biphasic (2.6- and 2.9-fold) activation of p38 mitogen-activated protein kinase, as well as a small increase of tumour necrosis factor α (TNFα) secretion, but treatments with the p38 MAPK-specific inhibitor SB203580 or saturating levels of a TNFα-1 blocking antibody provided only partial protection against apoptosis. The results suggest that JNK activation is protective and that the pathway is largely independent of p38 MAPK or secreted TNFα.
Impaired chromosome partitioning and synchronization of DNA replication initiation in an insertional mutant in the Vibrio harveyi cgtA gene coding for a common GTP-binding protein
Authors: SŁOMIṄSKA Monika , KONOPA Grażyna , WĘGRZYN Grzegorz , CZYŻ Agata
Published: August 2015

The Vibrio harveyi cgtA gene product belongs to a subfamily of small GTP-binding proteins, called Obg-like proteins. Members of this subfamily are present in diverse organisms ranging from bacteria to humans. On the other hand, the functions of these proteins in the regulation of cellular processes are largely unknown. Genes coding for these proteins are essential in almost all bacteria investigated thus far. However, a viable V. harveyi insertional mutant in the cgtA gene was described recently. Therefore, this mutant gives a unique opportunity to study functions of a member of the subfamily of Obg-like proteins. Here we demonstrate that the mutant cells often form long filaments with expanded, non-partitioned or rarely partitioned chromosomes. Such a phenotype suggests impairment of the mechanism of chromosome partition. Flow cytometric studies revealed that synchronization of chromosome replication initiation is also significantly disturbed in the cgtA mutant. Moreover, in contrast to wild-type V. harveyi, inhibition of chromosome replication and/or of cell division in the mutant bacteria caused significant increase in the number of large cells, suggesting that the cgtA gene product may be involved in the coupling of cell growth to chromosome replication and cell division. These results indicate that CgtA, an Obg-like GTP-binding protein, plays an important role in the regulation of chromosomal functions.
The β-appendages of the four adaptor-protein (AP) complexes: structure and binding properties, and identification of sorting nexin 9 as an accessory protein to AP-2
Authors: LUNDMARK Richard , CARLSSON Sven R.
Published: August 2015

Adaptor protein (AP) complexes are essential components for the formation of coated vesicles and the recognition of cargo proteins for intracellular transport. Each AP complex exposes two appendage domains with that function to bind regulatory accessory proteins in the cytosol. Secondary structure predictions, sequence alignments and CD spectroscopy were used to relate the β-appendages of all human AP complexes to the previously published crystal structure of AP-2. The results suggested that the β-appendages of AP-1, AP-2 and AP-3 have similar structures, consisting of two subdomains, whereas that of AP-4 lacks the inner subdomain. Pull-down and overlay assays showed partial overlap in the binding specificities of the β-appendages of AP-1 and AP-2, whereas the corresponding domain of AP-3 displayed a unique binding pattern. That AP-4 may have a truncated, non-functional domain was indicated by its apparent inability to bind any proteins from cytosol. Of several novel β-appendage-binding proteins detected, one that had affinity exclusively for AP-2 was identified as sorting nexin 9 (SNX9). SNX9, which contains a phox and an Src homology 3 domain, was found in large complexes and was at least partially associated with AP-2 in the cytosol. SNX9 may function to assist AP-2 in its role at the plasma membrane.
Mechanism of the regulation of type IB phosphoinositide 3OH-kinase byG-protein βγ subunits
Authors: KRUGMANN Sonja , COOPER Matthew A. , WILLIAMS Dudley H. , HAWKINS Phillip T. , STEPHENS Len R.
Published: August 2015

Type IB phosphoinositide 3OH-kinase (PI3K) is activated by G-protein βγ subunits (Gβγs). The enzyme is soluble and largely cytosolic in vivo. Its substrate, PtdIns(4,5)P2, and the Gβγs are localized at the plasma membrane. We have addressed the mechanism by which Gβγs regulate the PI3K using an in vitro approach. We used sedimentation assays and surface plasmon resonance to determine association of type IB PI3K with lipid monolayers and vesicles of varying compositions, some of which had Gβγs incorporated. Association and dissociation rate constants were determined. Our results indicated that in an assay situation in vitro the majority of PI3K will be associated with lipid vesicles, irrespective of the presence or absence of Gβγs. In line with this, a constitutively active membrane-targeted PI3K construct could still be activated substantially by Gβγs in vitro. We conclude that Gβγs activate type IB PI3K by a mechanism other than translocation to the plasma membrane.
Solute carrier 11a1 (Slc11a1; formerly Nramp1) regulates metabolism and release of iron acquired by phagocytic, but not transferrin-receptor-mediated, iron uptake
Authors: MULERO Victoriano , SEARLE Susan , BLACKWELL Jenefer M. , BROCK Jeremy H.
Published: August 2015

Solute carrier 11a1 (Slc11a1; formerly Nramp1; where Nramp stands for natural-resistance-associated macrophage protein) is a proton/bivalent cation antiporter that localizes to late endosomes/lysosomes and controls resistance to pathogens. In the present study the role of Slc11a1 in iron turnover is examined in macrophages transfected with Slc11a1Gly169 (wild-type) or Slc11a1Asp169 (mutant = functional null) alleles. Following direct acquisition of transferrin (Tf)-bound iron via the Tf receptor, iron uptake and release was equivalent in wild-type and mutant macrophages and was not influenced by interferon-γ/lipopolysaccharide activation. Following phagocytosis of [59Fe]Tf—anti-Tf immune complexes, iron uptake was equivalent and up-regulated similarly with activation, but intracellular distribution was markedly different. In wild-type macrophages most iron was in the soluble (60%) rather than insoluble (12%) fraction, with 28% ferritin (Ft)-bound. With activation, the soluble component increased to 82% at the expense of Ft-bound iron (< 5%). In mutant macrophages, 40–50% of iron was in insoluble form, 50–60% was soluble and < 5% was Ft-bound. Western-blot analysis confirmed failure of mutant macrophages to degrade complexes 24h after phagocytic uptake. Confocal microscopy showed that complexes were within lysosome-associated membrane protein 1-positive vesicles in wild-type and mutant macrophages at 30min and 24h, implying failure in the degradative process in mature phagosomes in mutant macrophages. NO-mediated iron release was 2.4-fold higher in activated wild-type macrophages compared with mutant macrophages. Overall, our data suggest that iron acquired by phagocytosis and degradation is retained within the phagosomal compartment in wild-type macrophages, and that NO triggers iron release by direct secretion of phagosomal contents rather than via the cytoplasm.
Glutamic acid-65 is an essential residue for catalysis in Proteus mirabilis glutathione S-transferase B1-1
Authors: ALLOCATI Nerino , MASULLI Michele , CASALONE Enrico , SANTUCCI Silvia , FAVALORO Bartolo , PARKER Michael W. , DI ILIO Carmine
Published: August 2015

The functional role of three conserved amino acid residues in Proteus mirabilis glutathione S-transferase B1-1 (PmGST B1-1) has been investigated by site-directed mutagenesis. Crystallographic analyses indicated that Glu65, Ser103 and Glu104 are in hydrogen-bonding distance of the N-terminal amino group of the γ-glutamyl moiety of the co-substrate, GSH. Glu65 was mutated to either aspartic acid or leucine, and Ser103 and Glu104 were both mutated to alanine. Glu65 mutants (Glu65→Asp and Glu65→Leu) lost all enzyme activity, and a drastic decrease in catalytic efficiency was observed for Ser103→Ala and Glu104→Ala mutants toward both 1-chloro-2,4-dinitrobenzene and GSH. On the other hand, all mutants displayed similar intrinsic fluorescence, CD spectra and thermal stability, indicating that the mutations did not affect the structural integrity of the enzyme. Taken together, these results indicate that Ser103 and Glu104 are significantly involved in the interaction with GSH at the active site of PmGST B1-1, whereas Glu65 is crucial for catalysis.
Promoter characterization of the novel human matrix metalloproteinase-26 gene: regulation by the T-cell factor-4 implies specific expression of the gene in cancer cells of epithelial origin
Authors: MARCHENKO George N. , MARCHENKO Natalia D. , LENG Jay , STRONGIN Alex Y.
Published: August 2015

A novel matrix metalloproteinase-26 (MMP-26) is known to be specifically expressed in epithelial carcinomas. To facilitate studies of MMP-26 transcriptional regulation, we have cloned and characterized a 1kb 5′-flanking region of the human MMP-26 gene. Altogether, our findings indicate that the MMP-26 promoter has distinctive structural and functional features among MMP genes. An unusual polyadenylation site proximal to the transcription-factor-binding sites protects transcription of the MMP-26 gene from the upstream promoters and represents a part of the stringent transcriptional regulation of the gene. The MMP-26 gene has a consensus TATA-box and one transcriptional start site located 60 and 35 nucleotides upstream of the translational start site, respectively. The MMP-26 promoter was able to drive luciferase expression in human A549 lung carcinoma, HT1080 fibrosarcoma and HEK293 embryonic kidney cells. The basal transcription efficiency of the MMP-26 promoter is relatively low, thereby explaining the minute expression of the gene in most cells and tissues. When compared with other MMP genes, the MMP-26 promoter contains binding sites for a few transcription factors. Sequential deletion and mutation analysis, and electrophoretic mobility-shift assay have identified the T-cell factor-4 (Tcf-4) motif and the activator protein-1 site as the major regulatory elements of the MMP-26 promoter. Since previous studies have established that the Tcf-4 transcription factor is subjected exclusively to regulation through the β-catenin/E(epithelial)-cadherin pathway, this implies the specific expression of MMP-26 in cancer cells of epithelial origin.
Thermodynamics of the ligandin function of human class Alpha glutathione transferase A1-1: energetics of organic anion ligand binding
Authors: SAYED Yasien , HORNBY Judith A.T. , LOPEZ Marimar , DIRR Heini
Published: August 2015

In addition to their catalytic functions, cytosolic glutathioneS-transferases (GSTs) are a major reserve of high-capacity binding proteins for a large variety of physiological and exogenous non-substrate compounds. This ligandin function has implicated GSTs in numerous ligand-uptake, -transport and -storage processes. The binding of non-substrate ligands to GSTs can inhibit catalysis. In the present study, the energetics of the binding of the non-substrate ligand 8-anilino-1-naphthalene sulphonate (ANS) to wild-type human class Alpha GST with two type-1 subunits (hGSTA1-1) and its ΔPhe-222 deletion mutant were studied by isothermal titration calorimetry. The stoichiometry of binding to both proteins is one ANS molecule per GST subunit with a greater affinity for the wild-type (Kd=65μM) than for the ΔPhe-222 mutant (Kd=105μM). ANS binding to the wild-type protein is enthalpically driven and it is characterized by a large negative heat-capacity change, ΔCp. The negative ΔCp value for ANS binding indicates a specific interface with a significant hydrophobic component in the protein—ligand complex. The negatively charged sulphonate group of the anionic ligand is apparently not a major determinant of its binding. Phe-222 contributes to the binding affinity for ANS and the hydrophobicity of the binding site.
Species specificity of O-linked carbohydrate chains of the oviducal mucins in amphibians: structural analysis of neutral oligosaccharide alditols released by reductive β-elimination from the egg-jelly coats of Rana clamitans
Authors: DELPLACE Florence , MAES Emmanuel , LEMOINE Jérôme , STRECKER Gérard
Published: August 2015

The extracellular matrix (the so-called jelly coat) surrounding amphibian eggs mainly comprises highly O-glycosylated proteins. These oviducal mucins have an important role in the fertilization process, and their carbohydrate chains are remarkably species-specific. Alkaline reductive treatment of the jelly-coat material of the frog Rana clamitans led to the release of oligosaccharide alditols. The neutral oligosaccharide alditols were fractionated and purified by successive chromatographic techniques. The structures of 27 of them, ranging from three to sixteen monosaccharides, were established by a combination of NMR spectroscopy, methylation analyses and matrix-assisted laser-desorption ionization—time of flight MS. Typically, some of the neutral compounds appeared to possess the core structure: Gal(β1–3)[GlcNAc(β1–6)]Gal(β1–3)[GlcNAc(β1–6)]GalNAc-ol (where GalNAc-ol represents N-acetylgalactosaminitol). Moreover, a novel type of chain termination, characterized by an unusual sequence {Fuc(α1–2)Gal(α1–3)Gal(α1–4)Gal(β1–3/4)} was observed. Indeed, the most complex representative structure of this series was found to be: Fuc(α1–2)Gal(α1–3)Gal(α1–4)Gal(β1–3)[Fuc(α1–2)Gal(α1–3)Gal(α1–4)Gal(β1–4)GlcNAc(β1–6)]Gal(β1–3)[Fuc(α1–2)Gal(α1–3)Gal(α1–4)Gal(β1–4)GlcNAc(β1–6)]GalNAc-ol.
Alteration of pore properties of Escherichia coli OmpF induced by mutation of key residues in anti-loop 3 region
Authors: BREDIN Jérôme , SAINT Nathalie , MALLÉA Monique , DÉ Emmanuelle , MOLLE Gérard , PAGÈS Jean-Marie , SIMONET Valérie
Published: August 2015

The Escherichia coli OmpF pore is governed by an internal constriction consisting of the negatively charged loop 3 folded into the lumen and the positively charged barrel wall located on the opposite side across the pore, ‘anti-loop 3'. To investigate the role of anti-loop 3 in solute diffusion, four site-directed mutations, K16A, K16D, R132A and R132D, were introduced into this eyelet region. The mutant porins were expressed efficiently and inserted into the outer membrane, and the thermal stabilities of the resulting trimers were determined. Diffusion of cefepime, a recently developed cephalosporin, was analysed in vivo. In vitro studies were performed on purified porins reconstituted in planar lipid bilayers to measure conductance, selectivity and voltage closure, as well as in liposomes for patch-clamp and sugar-swelling assays. All substitutions modified the ion-channel parameters, and minor conformational changes in the OmpF eyelet region were predicted from modelling studies. Our data show that Lys-16, and to a lesser extent Arg-132, are involved in voltage-gating and pore selectivity via their side-chain charges. Substitution K16D, which causes a severe decrease in critical voltage (Vc), may generate a channel susceptible to membrane potential, which perturbs cefepime diffusion. These results suggest that the Lys-16 residue plays an important role in the process of diffusion through the OmpF lumen.
Enzymic degradation of plasma arginine using arginine deiminase inhibits nitric oxide production and protects mice from the lethal effects of tumour necrosis factor α and endotoxin
Authors: THOMAS J. Brandon , HOLTSBERG Frederick W. , ENSOR C. Mark , BOMALASKI John S. , CLARK Mike A.
Published: August 2015

Septic shock is mediated in part by nitric oxide (NO) and tumour necrosis factor α (TNFα). NO is synthesized primarily from extracellular arginine. We tested the ability of an arginine-degrading enzyme to inhibit NO production in mice and to protect mice from the hypotension and lethality that occur after the administration of TNFα or endotoxin. Treatment of BALB/c mice with arginine deiminase (ADI) formulated with succinimidyl succinimide polyethylene glycol of Mr 20000 (ADI-SS PEG20000) eliminated all measurable plasma arginine (from normal levels of ∼155μM arginine to 2μM). In addition, ADI-SS PEG20000 also inhibited the production of NO, as quantified by plasma nitrate+nitrite. Treatment of mice with TNFα or endotoxin resulted in a dose-dependent increase in NO production and lethality. Pretreatment of mice with ADI-SS PEG20000 resulted in increased resistance to the lethal effects of TNFα and endotoxin. These observations are consistent with NO production resulting, to some extent, from the metabolism of extracellular arginine. The toxic effects of TNFα and endotoxin may be partially inhibited by enzymic degradation of plasma arginine by ADI-SS PEG20000. Interestingly, pretreatment with ADI-SS PEG20000 did not inhibit the anti-tumour activity of TNFα in vitro or in vivo. This treatment may allow greater amounts of TNFα, as well as other cytokines, to be administered while abrogating side effects such as hypotension and death.
Interaction of the 1α,25-dihydroxyvitamin D3 receptor at the distal promoter region of the bone-specific osteocalcin gene requires nucleosomal remodelling
Authors: PAREDES Roberto , GUTIÉRREZ José , GUTIERREZ Soraya , ALLISON Lizabeth , PUCHI Marcia , IMSCHENETZKY Maria , van WIJNEN Andre , LIAN Jane , STEIN Gary , STEIN Janet , MONTECINO Martin
Published: August 2015

1α,25-Dihydroxyvitamin D3-mediated transcriptional control of the bone-specific osteocalcin (OC) gene requires the integration of regulatory signals at the vitamin D-responsive element (VDRE) and flanking tissue-specific sequences. The 1α,25-dihydroxyvitamin D3 receptor (VDR) is a member of the nuclear receptor superfamily and forms a heterodimeric complex with the receptor for 9-cis retinoic acid (RXR) that binds to the VDRE sequence. We have demonstrated previously that changes in chromatin structure at the VDRE region of the rat OC gene promoter accompany transcriptional enhancement in vivo, suggesting a requirement for chromatin remodelling. Here we show that the VDRE in the distal region of the OC gene promoter is refractory to binding of the VDR—RXR complex when organized in a nucleosomal context. Addition of the ligand 1α,25-dihydroxyvitamin D3 or the presence of other transcription factors, such as YY1 and Runx/Cbfa (core-binding factor α), which also bind to sequences partially overlapping or near the VDRE, is not sufficient to render the VDRE accessible. Thus the VDR—RXR, unlike other steroid receptors, such as glucocorticoid receptor, progesterone receptor and thyroid receptor, is unable to bind its target sequence within a nucleosomal context. Taken together these results demonstrate that nucleosomal remodelling is required for in vivo occupancy of binding sites in the distal region of the OC gene promoter by the regulatory factors responsible for 1α,25-dihydroxyvitamin D3-dependent enhancement of transcription.
A continuous-wave electron–nuclear double resonance (X-band) study of the Cu2+ sites of particulate methane mono-oxygenase of Methylococcus capsulatus (strain M) in membrane and pure dopamine β-mono-oxygenase of the adrenal medulla
Authors: KATTERLE Bettina , GVOZDEV Rudolf I. , ABUDU Ntei , LJONES Torbjørn , ANDERSSON K. Kristoffer
Published: August 2015

All methanotrophic bacteria express a membrane-bound (particulate) methane mono-oxygenase (pMMO). In the present study, we have investigated pMMO in membrane fragments from Methylococcus capsulatus (strain M). pMMO contains a typical type-2 Cu2+ centre with the following EPR parameters: gz 2.24, gx,y 2.06, ACuz 19.0mT and ACux,y 1.0mT. Simulation of the Cu2+ spectrum yielded a best match by using four equivalent nitrogens (AN = 1.5mT, 42MHz). Incubation with ferricyanide neither changed nor increased the amount of EPR-active Cu2+, in contrast with other reports. The EPR visible copper seems not to be part of any cluster, as judged from the microwave power saturation behaviour. Continuous-wave electron—nuclear double resonance (CW ENDOR; 9.4GHz, 5–20K) experiments at g⊥ of the Cu(II) spectrum show a weak coupling to protons with an AH of 2.9MHz that corresponds to a distance of 3.8Å (1Å≡0.1nm), assuming that it is a purely dipolar coupling. Incubation in 2H2O leads to a significant decrease in these 1H-ENDOR intensities, showing that these protons are exchangeable. This result strongly suggests that the EPR visible copper site of pMMO is accessible to solvent, which was confirmed by the chelation of the Cu2+ by diethyldithiocarbamic acid. The 1H and 14N hyperfine coupling constants confirm a histidine ligation of the EPR visible copper site in pMMO. The hyperfine structure in the ENDOR or EPR spectra of pMMO is not influenced by the inhibitors azide, cyanide or ammonia, indicating that they do not bind to the EPR visible copper. We compared pMMO with the type-2 Cu2+ enzyme, dopamine β-mono-oxygenase (DβM). For DβM, it is assumed that the copper site is solvent-accessible. CW ENDOR shows similar weakly coupled and 2H2O-exchangeable protons (2.9MHz), as observed in pMMO, as well as the strongly coupled nitrogens (40MHz) from the co-ordinating N of the histidines in DβM. In conclusion, the resting EPR visible Cu in pMMO is not part of a trinuclear cluster, as has been suggested previously.
Cell-specific activity of neprilysin 2 isoforms and enzymic specificity compared with neprilysin
Authors: ROSE Christiane , VOISIN Stéphanie , GROS Claude , SCHWARTZ Jean-Charles , OUIMET Tanja
Published: August 2015

Neprilysin (NEP) 2 is a recently cloned glycoprotein displaying a high degree of sequence identity with neprilysin (EC 3.4.24.11), the prototypical member of the M13 subfamily of metalloproteases. Whereas NEP is involved in the metabolism of several bioactive peptides by plasma membranes of various cells, the enzymic properties and physiological functions of NEP2 are unknown. Here we characterize the cell-expression modalities and enzymic specificity of two alternatively spliced isoforms of NEP2 in Chinese hamster ovary and AtT20 cells. In the two cell lines, both isoforms are type II glycoproteins inserted in the endoplasmic reticulum as inactive precursors. Maturation detected by Western-blot analysis of glycosidase digests was cell-specific and more efficient in the endocrine cell line. The enzymic activity of both isoforms semi-purified from AtT20 cells reveals comparable specificities in terms of model substrates, pH optima and inhibitory patterns. NEP2 activity was compared with that of NEP regarding potencies of transition-state inhibitors, modes of hydrolysis, maximal hydrolysis rates and apparent affinities of bioactive peptides. Although all transition-state inhibitors of NEP inhibited NEP2 activity, albeit with different potencies, and many peptides were cleaved at the same amide bond by both peptidases, differences could be observed, i.e. in the hydrolysis of gonadotropin-releasing hormone and cholecystokinin, which occurred at different sites and more efficiently in the case of NEP2. Differences in cleavage of bioactive peptides, in cell-trafficking patterns and in tissue distribution indicate that NEP and NEP2 play distinct physiological roles in spite of their high degree of sequence identity.
Oxidized lipid accumulates in the presence of α-tocopherol in atherosclerosis
Authors: UPSTON Joanne M. , TERENTIS Andrew C. , MORRIS Kathryn , KEANEY John F. , STOCKER Roland
Published: August 2015

Oxidative modification of low-density lipoproteins in the arterial wall is a key feature of atherogenesis and widely believed to cause and/or accelerate lesion development. Linked to this is the expectation that vascular antioxidants are depleted during oxidation invivo. However, whether α-tocopherol (vitamin E), an important lipid-soluble antioxidant, is depleted early in atherogenesis and can prevent lipid peroxidation invivo is unresolved. To address this we examined the content of specific configurational isomers (cis/trans) of lipid hydro(pero)xides in lesions, which represent the major non-enzymic oxidation products, as formation and accumulation of cis/trans isomers is influenced by α-tocopherol in studies invitro. Concordant with our previous findings that large amounts of oxidized lipid co-exist with relatively normal α-tocopherol levels in human lesions, we now show that cis/trans isomers predominate over other products in human carotid and aortic lesions and in lesion lipoproteins. Further, dietary vitamin E supplementation of rabbits after arterial injury significantly increases both the aortic levels of α-tocopherol and the overall content of cis/trans isomers. These data are fully consistent with α-tocopherol acting as a hydrogen donor during lipid oxidation in vivo and suggest that α-tocopherol does not prevent lipoprotein lipid oxidation in the diseased vessel wall.
Turnover of matrix proteins in mammalian mitochondria
Authors: HUTH Walter , ROLLE Stefan , WUNDERLICH Ilona
Published: August 2015

In cultured hepatocytes the turnover of several mitochondrial matrix proteins (e.g. acetyl-CoA acetyltransferase) appears to be initiated by CoA-mediated, sequential transformation into CoA-modified forms. This modification favours the notion that intramitochondrial degradation by a matrix-resident ATP-dependent protease may be preceded by a specific modification by CoA. In a mitochondrial matrix fraction the MgATP-dependent decrease in anti-CoA immunoreactivity coincided with both a decrease in the anti-protein immunoreactivity of acetyl-CoA acetyltransferase and/or of 3-ketoacyl-CoA thiolase, and with the appearance of proteolytic fragments. A closer analysis of the degradation pattern revealed, however, a breakdown of the unmodified acetyl-CoA acetyltransferase and of its CoA-modified form, A1, whereas the form that is more highly modified by CoA, A2, proved to be inaccessible towards an ATP-dependent protease. In mammalian mitochondrial matrix, proteins can be degraded selectively by a matrix-resident ATP-dependent protease. The process of CoA modification results finally in the protection of matrix proteins from degradation. In cultured hepatocytes, leupeptin, an inhibitor of lysosomal proteases, did not affect the steady-state level of the mitochondrial matrix protein acetyl-CoA acetyltransferase. However, leupeptin mediated a specific accumulation of mitochondrial matrix proteins in the cytosolic fractions of hepatocytes cultured over a 24h period. The levels of acetyl-CoA acetyltransferase, 3-ketoacyl-CoA thiolase and glutamate dehydrogenase proteins increased 1.9-, 2.0- and 2.2-fold respectively. Their status as mature, oligomeric, but enzymically inactive enzymes strongly suggests that they originate from a leakage of autophagosomes, a constituent of the non-selective autophagic/lysosomal pathway for degradation of whole mitochondria.