bluuebunny/crossref_metadata_2025 huggingface dataset:
Probing of conformational changes in human O6-alkylguanine-DNA alkyl transferase protein in its alkylated and DNA-bound states by limited proteolysis
Authors: KANUGULA Sreenivas , GOODTZOVA Karina , PEGG E. Anthony
Published: August 2015

Human O6-alkylguanine-DNA alkyl transferase (hAGT) is a DNA repair protein that protects cells from alkylation damage by transferring an alkyl group from the O6-position of guanine to a cysteine residue in the active site (-PCHR-) of the protein. The structure of the hAGT protein (23 kDa) has been probed by limited proteolysis with trypsin and Glu-C endoproteases and analysis of the polypeptide fragments by SDS/PAGE. The native hAGT protein had limited accessibility to digestion with trypsin and Glu-C in spite of a number of potential cleavage sites. Initial cleavage by trypsin occurred at residue Lys-193 to give a 21 kDa polypeptide fragment, and this polypeptide underwent further cleavage at residues Arg-128 and Lys-165. These trypsin-cleavage sites became more accessible to digestion in the presence of double-stranded DNA (dsDNA), indicating that hAGT undergoes a change in its conformation on binding to DNA. However, the trypsin cutting site at the Arg-128 position was less available for digestion in the presence of single-stranded DNA (ssDNA), suggesting that the hAGT protein has a different conformation when bound to ssDNA compared with dsDNA. When protease digestion was carried out on wild-type protein, preincubated with the low-molecular-mass pseudosubstrate O6-benzylguanine, increased susceptibility to proteases was observed. A mutant C145A hAGT protein, which cannot repair O6-alkylguanine because the Cys-145 acceptor site in the active site of the protein is changed to Ala, showed identical trypsin cleavage to the wild type, but its digestion was not affected by O6-benzylguanine. These results suggest that alkylation of hAGT leads to an altered conformation. The acquisition of increased susceptibility to proteases upon DNA binding and alkylation demonstrates that hAGT undergoes considerable conformational changes in its structure upon binding to DNA and after repair of alkylation damage.
DNA recognition by quinoxaline antibiotics: use of base-modified DNA molecules to investigate determinants of sequence-specific binding of triostin A and TANDEM
Authors: BAILLY Christian , WARING J. Michael
Published: August 2015

The methodology of DNAase I footprinting has been adapted to investigate the sequence-specific binding of two quinoxaline drugs to DNA fragments containing natural and modified bases. In order to help comprehend the molecular origin of selectivity in the bis-intercalation of triostin A and TANDEM at CpG and TpA sites respectively, we have specifically examined the effect of the 2-amino group of guanine on their sequence specificity by using DNA in which that group has been either removed from guanine, added to adenine or both. Previous studies suggested that the recognition of particular nucleotide sequences by these drugs might be dependent upon the placement of the purine 2-amino group, serving as a positive or a negative effector for triostin A and TANDEM respectively. However, the footprinting data reported here indicate that this is not entirely correct, since they show that the 2-amino group of guanine is important for the binding of triostin A to DNA but has absolutely no influence on the interaction of TANDEM with TpA steps. Apparently the binding of triostin A to CpG sites is primarily due to hydrogen bonding interaction between the cyclic peptide of the antibiotic and the 2-amino group of guanine residues, whereas the selective binding of TANDEM to TpA sites is not hydrogen-bond driven and probably originates mainly from steric and/or hydrophobic interactions, perhaps involving indirect recognition of a suitable minor groove structure.
Transcriptional regulation of MHC class I gene expression in rat oligodendrocytes
Authors: MAVRIA Georgia , HALL T. Kersten , JONES A. Richard , BLAIR G. Eric
Published: August 2015

MHC class I molecules are normally expressed at very low levels in the brain and their up-regulation in response to cytokines and viral infections has been associated with a number of neurological disorders. Here we demonstrate that the down-regulation of surface class I molecules in differentiated primary rat oligodendrocytes was accompanied by reduced steady-state levels of class I heavy-chain mRNA. Transient expression assays were performed in oligodendrocytes and fibroblasts, using a mouse H-2Kb class I promoter chloramphenicol acetyltransferase plasmid termed pH2KCAT (which contained 5ʹ-flanking sequences from -2033 to +5 bp of the H-2Kb gene relative to the transcriptional start site at +1 bp). These assays showed that H-2Kb promoter activity was reduced in oligodendrocytes but not in class I-expressing fibroblasts. H-2Kb promoter activity was up-regulated in oligodendrocytes co-transfected with a plasmid expression vector encoding the transcriptional activator tax of human T-cell leukaemia virus type I, showing that down-regulation of promoter activity was reversible. Deletion mutant analysis of the H-2Kb promoter revealed the presence of negative regulatory elements that were functional in oligodendrocytes at -1.61 to -1.07 kb and -242 to -190 bp. Deletion of sequences in pH2KCAT encompassing the downstream element totally abolished promoter activity in both oligodendrocytes and fibroblasts, whereas a deletion within the upstream negative regulatory element increased promoter activity specifically in oligodendrocytes. The upstream negative regulatory element also down-regulated a linked heterologous herpes simplex virus thymidine kinase promoter in oligodendrocytes, but not in fibroblasts. Gel retardation assays using overlapping DNA probes that spanned the entire -1.61 to -1.07 kb region revealed the presence of a number of DNA-binding activities that were present in oligodendrocyte, but not in fibroblast nuclear extracts.
Tyrosine kinase-dependent modulation of 3-hydroxy-3-methylglutaryl-CoA reductase in human breast adenocarcinoma SKBR-3 cells
Authors: ASSLAN Raghda , PRADINES Anne , FAVRE Gilles , GAILLARD Francis LE
Published: August 2015

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase is the major rate-limiting enzyme in sterol and non-sterol isoprenoid synthesis. Isoprenoids are involved in the mechanisms of cell proliferation and transformation leading notably to crucial post-translational maturation of small G-proteins of the Ras superfamily. HMG-CoA reductase is among the most highly regulated enzymes. It is controlled by several feedback regulation mechanisms induced by sterol and non-sterol metabolites. The present results show that tyrosine kinase activity is also involved in the regulation of HMG-CoA reductase activity in the human breast cancer cell line SKBR-3. Incubation of SKBR-3 cells with the tyrosine kinase inhibitor, herbimycin A, induces a concentration-dependent reduction of HMG-CoA reductase activity with an IC50 of 80 nM. The inhibition of HMG-CoA reductase activity by herbimycin A is also time-dependent. A similar effect of herbimycin A was obtained on the steady-state level of the HMG-CoA reductase protein. The effect of herbimycin A is probably specific as it abolished the stimulation of reductase activity by epidermal growth factor. To elucidate the molecular basis of the inhibition of HMG-CoA reductase activity and protein level by herbimycin A, we performed experiments to study the metabolic turnover of this enzyme using [35S]methionine and [35]cysteine. Herbimycin A (1 μM) did not have any significant effect on the rate of HMG-CoA reductase protein degradation but did affect its rate of synthesis and mRNA levels. The decrease in protein synthesis rate correlates with the lower reductase protein level but is more pronounced than the decrease in mRNA levels. Taken together, the results reveal a novel pathway of regulation of HMG-CoA reductase expression and activity by cellular tyrosine kinase activities.
Regulation of expression of human intestinal bile acid-binding protein in Caco-2 cells
Authors: KANDA Tatsuo , FOUCAND Laurent , NAKAMURA Yuichi , NIOT Isabelle , BESNARD Philippe , FUJITA Michiyo , SAKAI Yasuo , HATAKEYAMA Katsuyoshi , ONO Teruo , FUJII Hiroshi
Published: August 2015

Molecular mechanisms of the bile acid active transport system in the ileal enterocytes remain unknown. We examined whether bile acids affect human enterocyte gene expression of intestinal bile acid-binding protein (I-BABP), a component of this transport system. Differentiated Caco-2 cells were incubated in the presence of human bile, bile acids or other lipids. The level of I-BABP expression was evaluated by Northern and Western blot analyses. A 24 h incubation of Caco-2 cells in a medium containing either bile or bile acids resulted in a remarkable 7.5-fold increase in the I-BABP mRNA level over the control level. Neither cholesterol, palmitic acid, phosphatidylcholine nor cholestyramine treated bile showed any difference in I-BABP mRNA expression from the control. Bile acid treatment increased the level of I-BABP mRNA in Caco-2 cells in a time- and dose-dependent manner. Western blot analysis showed that this induction led to increase in cytosolic I-BABP. Chenodeoxycholic acid and deoxycholic acid showed greater induction effects than other hydrophilic bile acids, including their own glycine conjugates. Pretreatment by actinomycin D or cycloheximide completely inhibited the up-regulation of I-BABP expression by bile acid. Bile acids, especially lipophilic bile acids, increase the I-BABP expression in Caco-2-cells, suggesting that luminal bile acids play an important role in regulating the I-BABP gene expression.
The molecular basis for UV vision in birds: spectral characteristics, cDNA sequence and retinal localization of the UV-sensitive visual pigment of the budgerigar (Melopsittacus undulatus)
Authors: WILKIE E. Susan , VISSERS Peter M. A. M. , DAS Debipriya , DeGRIP J. Willem , BOWMAKER K. James , HUNT M. David
Published: August 2015

Microspectrophotometric (msp) studies have shown that the colour-vision system of many bird species is based on four pigments with absorption peaks in the red, green, blue and UV regions of the spectrum. The existence of a fourth pigment (UV) is the major difference between the trichromacy of humans and the tetrachromacy of such birds, and recent studies have shown that it may play a determining role in such diverse aspects of behaviour as mate selection and detection of food. Avian visual pigments are composed of an opsin protein covalently bound via a Schiff-base linkage to the chromophore 11-cis-retinal. Here we report the cDNA sequence of a UV opsin isolated from an avian species, Melopsittacus undulatus (budgerigar or small parakeet). This sequence has been expressed using the recombinant baculovirus system; the pigment generated from the expressed protein on addition of 11-cis-retinal yielded an absorption spectrum typical of a UV photopigment, with λmax 365±3 nm. This is the first UV opsin from an avian species to be sequenced and expressed in a heterologous system. In situ hybridization of this sequence to budgerigar retinas selectively labelled a sub-set of UV cones, representing approx. 9% of the total cone population, that are distributed in a semi-regular pattern across the entire retina.
The effects of free [Ca2+] on the cytosolic face of the inositol (1,4,5)-trisphosphate receptor at the single channel level
Authors: THROWER C. Edwin , LEA J. A. Edward , DAWSON P. Alan
Published: August 2015

Cytosolic free Ca2+ has been shown to have both activating and inhibitory effects upon the inositol (1,4,5) trisphosphate receptor (InsP3R) during intracellular Ca2+ release. The effects of cytosolic free Ca2+ on the InsP3R have already been monitored using cerebellar microsomes (containing InsP3R) incorporated into planar lipid bilayers [Bezprozvanny, Watras and Ehrlich (1991) Nature (London) 351, 751-754]. In these experiments the open probability of the channel exhibited a ‘bell-shaped Ca2+ dependence’. However, this has only been seen when the receptor is in the presence of its native membrane (e.g. microsomal vesicles). Using solubilized, purified InsP3R incorporated into planar lipid bilayers using the ‘tip-dip’ technique, investigations were carried out to see if the same effect was seen in the absence of the native membrane. Channel activity was observed in the presence of 4 μM InsP3 and 200 nM free Ca2+. Mean single channel current was 2.69 pA and more than one population of lifetimes was observed. Two populations had mean open times of approx. 9 and 97 ms. Upon increasing the free [Ca2+] to 2 μM, the mean single channel current decreased slightly to 2.39 pA, and the lifetimes increased to 30 and 230 ms. Elevation of free [Ca2+] to 4 μM resulted in a further decrease in mean single channel current to 1.97 pA as well as a decrease in lifetime to approx. 8 and 194 ms. At 10 μM free [Ca2+] no channel activity was observed. Thus, with purified receptor in artificial bilayers, free [Ca2+] on the cytosolic face of the receptor has major effects on channel behaviour, particularly on channel closure, although inhibition of channel activity is not seen until very high free [Ca2+] is reached.
Double-stranded-RNA-activated protein kinase (PKR) regulates Ca2+ stores in Xenopus oocytes
Authors: THOMAS David , KIM Hak Yong , MORGAN Roseline , HANLEY R. Michael
Published: August 2015

Expression of the double-stranded-RNA-dependent protein kinase (PKR) in Xenopus oocytes attenuated Ca2+ entry-dependent membrane currents activated by depletion of Ca2+ stores, whereas expression of a dominant-negative PKR mutant had the opposite effect. These results appeared to be due to perturbation of releasable Ca2+ stores, and not actions of PKR on protein synthesis. PKR may thus have novel protein substrates and cellular functions in Ca2+ storage and signalling.
Identification of a novel murine glutathione S-transferase class mu gene
Authors: de BRUIN Wieke C. C. , te MORSCHE René H. M. , WAGENMANS J. M. Muriel , ALFERINK C. Jeroen , TOWNSEND J. Alan , WIERINGA Bé , PETERS H. M. Wilbert
Published: August 2015

Screening of a genomic mouse DNA library with a glutathione S-transferase class mu cDNA probe resulted in the identification of mGSTM5, a novel member of the murine glutathione S-transferase class mu gene family. Here we present the sequence of the promoter region, the exon-intron organization of the gene and the isolation and characterization of its complete cDNA. Conceptual translation of the cDNA sequence revealed that several amino acid positions have been changed in ‘invariant’ mu class signature sequences in mGSTM5. Reverse transcriptase polymerase chain reaction using gene specific primers revealed that mGSTM5 is uniquely expressed in mouse liver, stomach and small intestine.
Pseudo-enzymatic S-acylation of a myristoylated Yes protein tyrosine kinase peptide in vitro may reflect non-enzymatic S-acylation in vivo
Authors: BAÑÓ M. Carmen , JACKSON S. Caroline , MAGEE I. Anthony
Published: August 2015

Covalent attachment of a variety of lipid groups to proteins is now recognized as a major group of post-translational modifications. S-acylation of proteins at cysteine residues is the only modification considered dynamic and thus has the potential for regulating protein function and/or localization. The activities that catalyse reversible S-acylation have not been well characterized and it is not clear whether both the acylation and the deacylation steps are regulated, since in principle it would be sufficient to control only one of them. Both apparently enzymatic and non-enzymatic S-acylation of proteins have previously been reported. Here we show that a synthetic myristoylated c-Yes protein tyrosine kinase undecapeptide undergoes spontaneous S-acylation in vitro when using a long chain acyl-CoA as acyl donor in the absence of any protein. The S-acylation was dependent on myristoylation of the substrate, the length of the incubation period, temperature and substrate concentration. When COS cell fractions were added to the S-acylation reaction no additional peptide:S-acyltransferase activity was detected. These results are consistent with the possibility that membrane-associated proteins may undergo S-acylation in vivo by non-enzymatic transfer of acyl groups from acyl-CoA. In this case, the S-acylation-deacylation process could be controlled by a regulated depalmitoylation mechanism.
Identification of a new endogenous platelet-activating factor-like molecule in gingival crevicular fluid
Authors: ANTONOPOULOU Smaragdi , DEMOPOULOS A. Constantinos , ARGYROPOULOS Dimitris , BALTAS George , KOTSIFAKI Helen , DIAMANTI-KIPIOTI Anthoula
Published: August 2015

Periodontal disease is an inflammatory disease and the major cause of tooth loss in adults. Bacteria and their products are the causative agents of this disease. Endogenous molecules mediate the inflammatory process and play a major role in its amplification and perpetuation as well as in the ensuing tissue destruction. The relationship between platelet-activating factor (PAF) and periodontal disease has not so far been examined thoroughly. We have isolated a phospholipid molecule with PAF-like activity from gingival crevicular fluid. This molecule, purified on HPLC, causes washed platelet aggregation with EC50 value 0.1 μM, based on phosphorus determination. It acts through PAF-receptors and is inactivated by PAF-acetylhydrolase. In addition, this phospholipid presents biological activity towards human platelets. The combination of the results obtained from the chemical and enzymic treatments, the biological assays as well as results from the electrospray analysis, leads to the conclusion that this phospholipid is a hydroxyl-PAF analogue with relative molecular mass 703. This PAF-like molecule may be implicated in periodontal disease.
Comparative biochemical and pharmacological characterization of the mouse 5HT5A 5-hydroxytryptamine receptor and the human β2-adrenergic receptor produced in the methylotrophic yeast Pichia pastoris
Authors: WEISS H. Markus , HAASE Winfried , MICHEL Hartmut , REILÄNDER Helmut
Published: August 2015

Over the last few years, Pichia pastoris has been developed into a powerful expression system for a multitude of foreign genes. Here, we demonstrate that the P. pastoris expression system has similar power to the baculovirus expression system in high-level production of two G-protein-coupled receptors, the mouse 5HT5A 5-hydroxtryptamine receptor and the human β2-adrenergic receptor. Different expression plasmids were constructed in which the cDNAs of the two receptors were cloned under the transcriptional control of the highly inducible promoter of the P. pastoris alcohol oxidase 1 (AOX1) gene. In three expression plasmids, the receptors were fused to the Saccharomyces cerevisiae α-factor prepropeptide and also to the c-myc tag or the FLAG tag to permit immunological detection of the receptors. After transformation into P. pastoris strains KM71 and SMD 1163, recombinant clones were selected and tested for the production of the 5HT5A receptor and the β2-adrenergic receptor by radioligand binding using [N-methyl-3H]lysergic acid diethylamide and [5,7-3H](-)CGP-12177 respectively. The production level of the 5HT5A receptor was improved by a factor of three by fusion with the α-factor prepropeptide. Also, the higher gene dosage resulting from multiple insertions of the expression cassette led to an improvement in production by a factor of two for both receptors. The addition of the adrenergic antagonist alprenolol to the culture medium had a positive effect on the number of specific binding sites detectable in clones producing the β2-adrenergic receptor. For the 5HT5A receptor the addition of yohimbine resulted in a similar but smaller effect. Binding assays revealed that approx. 25 pmol of β2-adrenergic receptor and approx. 40 pmol of 5HT5A receptor per mg of membrane protein in crude membrane preparations were produced. The pharmacological profiles for the heterologously produced receptors, estimated by ligand-displacement analysis using certain adrenergic and serotoninergic agonists and antagonists, were comparable with those reported for the receptors expressed in mammalian systems. Immunoblot analysis of the 5HT5A receptor revealed an apparent molecular mass about 20 kDa higher than expected from the amino acid sequence. Here, the Kex2 endopeptidase failed to process the α-factor leader correctly. Blocking glycosylation in vivo by tunicamycin or in vitro deglycosylation of membranes by endoglycosidase H resulted in correct processing. In contrast, the β2-adrenergic receptor fusion to the α-factor leader was correctly processed by the internal Kex2 endopeptidase. The Kex2-processed β2-adrenergic receptor was not glycosylated. In conclusion, the high-level production of the two receptors in P. pastoris will allow their purification in quantities sufficient for subsequent biophysical and structural studies.
Bradykinin-induced amyloid precursor protein secretion: a protein kinase C-independent mechanism that is not altered in fibroblasts from patients with sporadic Alzheimer's disease
Authors: RACCHI Marco , IANNA Paola , BINETTI Giuliano , TRABUCCHI Marco , GOVONI Stefano
Published: August 2015

We treated human skin fibroblasts with bradykinin (BK) and observed a concentration-dependent increase in the release of soluble amyloid precursor protein (sAPP). The estimated EC50 for the observed effect is 2.8 nM, which is of the same order of magnitude as the reported Kd of BK binding in human skin fibroblasts. The effect of BK on sAPP secretion appears to be dependent on interaction of the ligand with the B2 type of BK receptors but independent of activation of protein kinase C. We also show that sAPP release after BK treatment in fibroblasts from patients with sporadic Alzheimer's disease is not different from control cells and is paralleled by equivalent levels of inositol trisphosphate production. A discussion of the differences from previously published work focuses on the possible divergent alterations in transduction systems in fibroblasts from patients with familial and sporadic Alzheimer's disease. Our results are the first example of receptor-mediated sAPP release in human skin fibroblasts and the first demonstration of the co-existence of protein kinase C-dependent and -independent mechanisms in these cells.
Molecular cloning of membrane cofactor protein (MCP; CD46) on B95a cell, an Epstein–Barr virus-transformed marmoset B cell line: B95a-MCP is susceptible to infection by the CAM, but not the Nagahata strain of the measles virus
Authors: MURAKAMI Yusuke , SEYA Tsukasa , KURITA Mitsue , FUKUI Aya , UEDA Shigeharu , NAGASAWA Shigeharu
Published: August 2015

Measles virus (MV) infects not only human beings but also some simian species. The MV receptor on Vero cells (a cell line established from African Green monkey kidney cells) and human cells has been shown to be the membrane cofactor protein MCP/CD46, which is an inhibitor of autologous complement (C) activation. B95a, an Epstein-Barr virus (EBV)-transformed marmoset B cell line, is a simian cell line used for MV selection and is much more susceptible to MV than Vero cells. In the present study, we isolated cDNAs encoding MCP homologues from B95a cDNA library and assessed whether B95a-MCP is responsible for the high susceptibility of B95a to MV. The deduced amino acid sequence of the cDNA of B95a-MCP was 76% identical to that of human-MCP, and the recombinant B95a-MCP exerts C inhibitor activity. Although CAM, a vaccine strain of MV, infected Chinese hamster ovary (CHO) cells expressing B95a-MCP, Nagahata strain, a wild type of MV, failed to infect the CHO transfectants, suggesting that additional membrane molecules of B95a are responsible for the high susceptibility of B95a to the Nagahata strain.
Evolution of alanine:glyoxylate aminotransferase intracellular targeting: structural and functional analysis of the guinea pig gene
Authors: BIRDSEY Graeme M. , DANPURE Christopher J.
Published: August 2015

The distribution of alanine:glyoxylate aminotransferase 1 (AGT) within liver cells has changed many times during mammalian evolution. Depending on the particular species, AGT can be found in mitochondria or peroxisomes, or mitochondria and peroxisomes. In some cases significant cytosolic AGT is also present. In the livers of most rodents, AGT has what is thought to be the more ‘ancestral ’ distribution (i.e. mitochondrial and peroxisomal). However, AGT is distributed very differently in the guinea pig, being peroxisomal and cytosolic. In this study, we have attempted to determine the molecular basis for the loss of mitochondrial AGT targeting and the apparent inefficiency of peroxisomal targeting of AGT in the guinea pig. Our results show that the former is owing to the evolutionary loss of the more 5´ of two potential transcription and translation initiation sites, resulting in the loss of the ancestral N-terminal mitochondrial targeting sequence from the open reading frame. Guinea pig AGT is targeted to peroxisomes via the peroxisomal targeting sequence type 1 (PTS1) peroxisomal import machinery, even though its C-terminal tripeptide, HRL, deviates from the standard consensus PTS1 motif. Although HRL appears to target AGT to peroxisomes less efficiently than the classical PTS1 SKL, the main reason for the low efficiency of AGT peroxisomal targeting in guinea pig cells (compared with cells from other species) lies not with guinea pig AGT but with some other, as yet undefined, part of the guinea pig peroxisomal import machinery.
Regulation of mitochondrial biogenesis in brown adipose tissue: nuclear respiratory factor-2/GA-binding protein is responsible for the transcriptional regulation of the gene for the mitochondrial ATP synthase β subunit
Authors: VILLENA Josep A. , VIÑAS Octavi , MAMPEL Teresa , IGLESIAS Roser , GIRALT Marta , VILLARROYA Francesc
Published: August 2015

The regulation of transcription of the gene for the β subunit of the FoF1 ATP synthase (ATPsynβ) in brown adipose tissue has been studied as a model to determine the molecular mechanisms for mitochondrial biogenesis associated with brown adipocyte differentiation. The expression of the ATPsynβ mRNA is induced during the brown adipocyte differentiation that occurs during murine prenatal development or when brown adipocytes differentiate in culture. This induction occurs in parallel with enhanced gene expression for other nuclear and mitochondrially-encoded components of the respiratory chain/oxidative phosphorylation system (OXPHOS). Transient transfection assays indicated that the expression of the ATPsynβ gene promoter is higher in differentiated HIB-1B brown adipocytes than in non-differentiated HIB-1B cells. A major transcriptional regulatory site was identified between nt -306 and -266 in the ATPsynβ promoter. This element has a higher enhancer capacity in differentiated brown adipocyte HIB-1B cells than in non-differentiated cells. Electrophoretic shift analysis indicated that Sp1and nuclear respiratory factor-2/GA-binding protein (NRF2/GABP) were the main nuclear proteins present in brown adipose tissue that bind this site. Double-point mutant analysis indicated a major role for the NRF2/GABP site in the enhancer capacity of this element in brown fat cells. It is proposed that NRF2/GABP plays a pivotal role in the co-ordinated enhancement of OXPHOS gene expression associated with mitochondrial biogenesis in brown adipocyte differentiation.
Binding of α-melanocyte-stimulating hormone to its G-protein-coupled receptor on B-lymphocytes activates the Jak/STAT pathway
Authors: BUGGY Joseph J.
Published: August 2015

α-Melanocyte-stimulating hormone (α-MSH) is a 13-amino-acid peptide with a variety of physiological effects, including the stimulation of melanocyte proliferation and melanogenesis, temperature control, control of prolactin release and the modulation of cytokine action in the immune system. There are five known subtypes of G-protein-coupled receptors, which bind with different affinities to α-MSH. This paper provides evidence that Ba/F3 pro-B-lymphocyte cells express the gene for the melanocortin 5 (MC5) receptor and specifically bind α-MSH. Western-blot analysis reveals that α-MSH binding stimulates Janus kinase 2 (JAK2) and signal transducers and activators of transcription (STAT1) tyrosine phosphorylation in both Ba/F3 cells and human cultured IM-9 lymphocytes. α-MSH is further revealed to activate JAK2 in mouse L-cells stably expressing the human MC5 receptor. Finally, α-MSH binding is shown to result in an enhancement of cellular proliferation. These findings identify a new protein tyrosine kinase pathway in the action of α-MSH, and suggest that α-MSH plays an important role in B-lymphocyte function via the activation of the same intracellular phosphorylation pathway used by cytokines and growth factors.
Purification and characterization of a new cystatin inhibitor from Taiwan cobra (Naja naja atra) venom
Authors: BRILLARD-BOURDET Michèle , NGUYÊN Vinh , FERRER-DI MARTINO Michèle , GAUTHIER Francis , MOREAU Thierry
Published: August 2015

Cobra cystatin, a new cysteine-proteinase inhibitor of the cystatin superfamily, was isolated from the venom of the Taiwan cobra (Naja naja atra) by affinity chromatography on S-carboxymethylpapain–Sepharose and reverse-phase chromatography. The venom contained two forms of the inhibitor, one of 11870 Da and the other of 12095 Da, as determined by MS, and pI values of 6.2 and 6.1. Cobra cystatin strongly inhibits cysteine proteinases of the papain family, but not calpain. Papain, cathepsin L, cathepsin B and cathepsin S are inhibited with Ki values of 0.19, 0.1, 2.5 and 1.2 nM respectively. The amino acid sequence of cobra cystatin shows that it is a Type 2 cystatin. The amino acid sequence is 73% identical with that of the cystatin in African-puff-adder (Bitis arietans) venom, with which it shares a unique six-residue insertion in a region opposite the reactive inhibitory site. Cobra cystatin is 25–42% identical with other Type 2 cystatins, the most closely related being the recently described human cystatin M, which also has a similar five-residue insertion starting at position 76 (chicken cystatin numbering). A molecular phylogenetic tree of 16 representative members of Family 2 cystatins was constructed by parsimony analysis; it suggests that snake cystatins, together with Tachypleus tridentatus (Japanese horseshoe crab) cystatin and human cystatin M, form a new subfamily within cystatin Family 2.
Expression of v-src in mammary epithelial cells induces transcription via STAT3
Authors: SMITH Paul D. , CROMPTON Mark R.
Published: August 2015

Transgenic mouse models of mammary tumorigenesis and analyses of human breast tumour samples have indicated a role for Src proteins in the tumorigenic process. The downstream effectors of Src function in mammary epithelial cells are less well understood. STAT proteins constitute a family of transcription factors whose activation by cytokine and non-cytokine receptors leads to tyrosine phosphorylation, dimerization and translocation from the cytoplasm to the nucleus. In the nucleus they activate the transcription of specific genes by binding to consensus DNA elements. STATs 1 and 3 can be activated by both cytokine and non-cytokine receptors, and bind as homodimers or heterodimers to viral simian sarcoma virus (sis)-inducible elements such as that found in the c-fospromoter. Here we report that one of the downstream effectors of Src function in mammary epithelial cells is STAT3. We demonstrate that v-srcexpression in mammary epithelial cells induces Tyr-705 phosphorylation, nuclear translocation and DNA binding of STAT3. Furthermore, we demonstrate that v-srccan induce STAT3-dependent transcription. These observations are the first direct evidence that v-srccan regulate transcription through the activation of STAT proteins, and add a further level of complexity to the understanding of the mode of action of v-src.
Inactivating peptide of the Shaker B potassium channel: conformational preferences inferred from studies on simple model systems
Authors: ENCINAR José A. , FERNÁNDEZ Asia M. , GIL-MARTÍN Emilio , GAVILANES Francisco , ALBAR Juan P. , FERRAGUT José A. , GONZÁLEZ-ROS José M.
Published: August 2015

Previous studies on the interaction between the inactivating peptide of the Shaker B K+ channel (ShB peptide, H2N-MAAVAGLYGLGEDRQHRKKQ) and anionic phospholipid vesicles, used as model targets, have shown that the ShB peptide: (i) binds to the vesicle surface with high affinity; (ii) readily adopts a strongly hydrogen-bonded β-structure; and (iii) becomes inserted into the hydrophobic bilayer. We now report fluorescence studies showing that the vesicle-inserted ShB peptide is in a monomeric form and, therefore, the observed β-structure must be intramolecularly hydrogen-bonded to produce a β-hairpin conformation. Also, additional freeze–fracture and accessibility-to-trypsin studies, which aimed to estimate how deeply and in which orientation the folded monomeric peptide inserts into the model target, have allowed us to build structural models for the target-inserted peptide. In such models, the peptide has been folded near G6 to configure a long β-hairpin modelled to produce an internal cancellation of net charges in the stretch comprising amino acids 1–16. As to the positively charged C-terminal portion of the ShB peptide (RKKQ), this has been modelled to be in parallel with the anionic membrane surface to facilitate electrostatic interactions. Since the negatively charged surface and the hydrophobic domains in the model vesicle target may partly imitate those present at the inactivation ‘entrance ’ in the channel protein [Kukuljan, M., Labarca, P. and Latorre, R. (1995) Am. J. Physiol. Cell Physiol. 268, C535–C556], we believe that the structural models postulated here for the vesicle-inserted peptide could help to understand how the ShB peptide associates with the channel during inactivation and why mutations at specific sites in the ShB peptide sequence, such as that in the ShB-L7E peptide, result in non-inactivating peptide variants.
Activation of S6 kinase in human neutrophils by calcium pyrophosphate dihydrate crystals: protein kinase C-dependent and phosphatidylinositol-3-kinase-independent pathways
Authors: TUDAN Christopher , JACKSON John K. , CHARLTON Lorin , PELECH Steven L. , SAHL Bill , BURT Helen M.
Published: August 2015

Phosphatidylinositol 3-kinase (PI 3-kinase) has been shown previously to be a central enzyme in crystal-induced neutrophil activation. Since activation of the 70 kDa S6 kinase (p70S6K) has been shown to be dependent on PI 3-kinase activation in mammalian cells, and since the former is a key enzyme in the transmission of signals to the cell nucleus, activation of p70S6K was investigated in crystal-stimulated neutrophils. Cytosolic fractions from calcium pyrophosphate dihydrate (CPPD)-crystal-activated neutrophils were separated by Mono Q chromatography and analysed for phosphotransferase activity using a range of substrates and probed by Western analysis using antibodies to p70S6K and mitogen-activated protein kinase (MAP kinase). CPPD crystals induced a robust, transient activation (peak activity at 2 min) of p70S6K that was fully inhibited by pretreatment with rapamycin. This is the first report of the activation of p70S6K in neutrophil signal transduction pathways induced by an agonist. This crystal-induced activation of p70S6K could also be inhibited by a protein kinase C (PKC) inhibitor (Compound 3), but not by the PI 3-kinase inhibitor wortmannin. CPPD crystals also activated the ERK1 and ERK2 forms of MAP kinase (wortmannin insensitive), PKC (Compound 3 sensitive) and protein kinase B (wortmannin sensitive) in neutrophils. These data suggest that activation of p70S6K may proceed through a PI 3-kinase- and protein kinase B-independent but PKC-dependent pathway in crystal-activated neutrophils.
Synergistic transcriptional activation of the mouse urokinase plasminogen activator (uPA) gene and of its enhancer activator protein 1 (AP1) site by cAMP and retinoic acid
Authors: MIRA-Y-LOPEZ Rafael , JARAMILLO Silvia , JING Yongkui
Published: August 2015

We have investigated the mechanism whereby all-trans retinoic acid (tRA) potentiates the 8-bromo-cAMP (8-BrcAMP)-dependent transcription of the urokinase plasminogen activator (uPA) gene in SC115 mouse mammary carcinoma cells. Photoaffinity labelling experiments showed that tRA did not alter the cellular content of cAMP-dependent protein kinase regulatory subunits I and II. In agreement with this, nuclear run-on analysis in the presence of the translational inhibitor puromycin demonstrated that the effect of 8-BrcAMP and its potentiation by tRA were independent of protein synthesis. A transiently transfected 6.6 kb uPA 5´-flanking region-chloramphenicol acetyltransferase (CAT) fusion gene mimicked the response of the endogenous uPA gene. Thus 1 mM 8-BrcAMP induced a 100–200% increase in CAT content, 100 nM tRA had no effect and 100 nM tRA+1 mM 8-BrcAMP induced a 300–500% increase in cells co-transfected with tRA receptor and/or 9-cis-RA receptor. Analysis of 5´-deleted constructs showed that the tRA effect required at least two cis regions: -2657 to -2186, encompassing the 100 bp uPA enhancer, and -709 to -324, which exhibited silencing activity. Neither region contained a tRA-response element-like motif. Because tRA receptor and 9-cis-RA receptor interact with activator protein 1 (AP1), we tested whether tRA regulated the uPA enhancer AP1 site in the presence of 8-BrcAMP. We found that a dimer of this site fused to a minimal uPA–CAT fusion gene was responsive to 1 mM 8-BrcAMP (100% CAT increase), not responsive to 100 nM tRA, and synergistically responsive to 100 nM tRA+1 mM 8-BrcAMP (240% CAT increase) in cells co-transfected with Fos and Jun. Synergistic activation of the same construct and of the 6.6 kb uPA–CAT fusion gene was also obtained using tRA and 100 nM PMA. We conclude that multiple cis elements, probably including the uPA enhancer AP1 site, mediate the tRA potentiation of uPA transcription.
Release of the glycosylphosphatidylinositol-anchored enzyme ecto-5′-nucleotidase by phospholipase C: catalytic activation and modulation by the lipid bilayer
Authors: LEHTO Marty T. , SHAROM Frances J.
Published: August 2015

Many hydrolytic enzymes are attached to the extracellular face of the plasma membrane of eukaryotic cells by a glycosylphosphatidylinositol (GPI) anchor. Little is currently known about the consequences for enzyme function of anchor cleavage by phosphatidylinositol-specific phospholipase C. We have examined this question for the GPI-anchored protein 5´-nucleotidase (5´-ribonucleotide phosphohydrolase; EC 3.1.3.5), both in the native lymphocyte plasma membrane, and following purification and reconstitution into defined lipid bilayer vesicles, using Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC). Membrane-bound, detergent-solubilized and cleaved 5´-nucleotidase all obeyed Michaelis–Menten kinetics, with a Km for 5´-AMP in the range 11–16 µM. The GPI anchor was removed from essentially all 5´-nucleotidase molecules, indicating that there is no phospholipase-resistant pool of enzyme. However, the phospholipase was much less efficient at cleaving the GPI anchor when 5´-nucleotidase was present in detergent solution, dimyristoyl phosphatidylcholine, egg phosphatidylethanolamine and sphingomyelin, compared with the native plasma membrane, egg phosphatidylcholine and a sphingolipid/cholesterol-rich mixture. Lipid molecular properties and bilayer packing may affect the ability of PI-PLC to gain access to the GPI anchor. Catalytic activation, characterized by an increase in Vmax, was observed following PI-PLC cleavage of reconstituted 5´-nucleotidase from vesicles of several different lipids. The highest degree of activation was noted for 5´-nucleotidase in egg phosphatidylethanolamine. An increase in Vmax was also noted for a sphingolipid/cholesterol-rich mixture, the native plasma membrane and egg phosphatidylcholine, whereas vesicles of sphingomyelin and dimyristoyl phosphatidylcholine showed little activation. Km generally remained unchanged following cleavage, except in the case of the sphingolipid/cholesterol-rich mixture. Insertion of the GPI anchor into a lipid bilayer appears to reduce the catalytic efficiency of 5´-nucleotidase, possibly via a conformational change in the enzyme, and activity is restored on release from the membrane.
Enzymic, cysteine-specific ADP-ribosylation in bovine liver mitochondria
Authors: JORCKE Dierk , ZIEGLER Mathias , HERRERO-YRAOLA Andrés , SCHWEIGER Manfred
Published: August 2015

NAD+ glycohydrolase (NADase) and non-enzymic ADP-ribosylation have been thought to be involved in the regulation of mitochondrial Ca2+ fluxes. In this study it was found that several conditions (5 mM nicotinamide, 5 mM 3-aminobenzamide, 2 mM EDTA, 1 mM ATP, 10 mM dithiothreitol) known to strongly inhibit the NADase decreased ADP-ribosylation in bovine liver mitochondrial membranes with [32P]NAD+ as substrate to only a limited extent, if at all. The reaction led to the specific modification of two proteins with apparent molecular masses of approx. 26 and 53 kDa. An excess of added free ADP-ribose diminished the incorporation of label from [32P]NAD+ only slightly. Dithiothreitol inactivated the NADase, whereas ADP-ribosylation was unaffected. At low concentrations (25 µM) ADP-ribosylation was efficient with NAD+, but not ADP-ribose, as substrate. Under these conditions mitochondrial ADP-ribosylation seems to occur as an enzymic reaction rather than a non-enzymic transfer of ADP-ribose previously liberated from NAD+ by NAD+ glycohydrolase. The chemical stability of the protein-ADP-ribose bonds in the mitochondrial membranes indicated that cysteine residues are the predominant acceptors. Moreover, yeast aldehyde dehydrogenase, known to be a substrate for thiol-associated ADP-ribosylation, was efficiently ADP-ribosylated by using the mitochondrial activity and NAD+ as substrate. The modification of a cysteine residue in the aldehyde dehydrogenase was verified by the observation that pretreatment of this acceptor protein with N-ethylmaleimide substantially decreased its modification. It is therefore concluded that bovine liver mitochondria contain a cysteine-specific ADP-ribosyltransferase.
Enzyme-mediated cytosine deamination by the bacterial methyltransferase M.MspI
Authors: ZINGG Jean-Marc , SHEN Jiang-Cheng , JONES Peter A.
Published: August 2015

Most prokaryotic (cytosine-5)-DNA methyltransferases increase the frequency of deamination at the cytosine targeted for methylation in vitro in the absence of the cofactor S-adenosylmethionine (AdoMet) or the reaction product S-adenosylhomocysteine (AdoHcy). We show here that, under the same in vitro conditions, the prokaryotic methyltransferase, M.MspI (from Moraxella sp.), causes very few cytosine deaminations, suggesting a mechanism in which M.MspI may avoid enzyme-mediated cytosine deamination. Two analogues of AdoMet, sinefungin and 5´-amino-5´-deoxyadenosine, greatly increased the frequency of cytosine deamination mediated by M.MspI presumably by introducing a proton-donating amino group into the catalytic centre, thus facilitating the formation of an unstable enzyme–dihydrocytosine intermediate and hydrolytic deamination. Interestingly, two naturally occurring analogues, adenosine and 5´-methylthio-5´-deoxyadenosine, which do not contain a proton-donating amino group, also weakly increased the deamination frequency by M.MspI, even in the presence of AdoMet or AdoHcy. These analogues may trigger a conformational change in the enzyme without completely inhibiting the access of solvent water to the catalytic centre, thus allowing hydrolytic deamination of the enzyme–dihydrocytosine intermediate. Under normal physiological conditions the enzymes M.HpaII (from Haemophilus parainfluenzae), M.HhaI (from Haemophilus hemolytica) and M.MspI all increased the in vivo deamination frequency at the target cytosines with comparable efficiency.
Quantitative assessment of comparative potencies of cholesterol-crystal-promoting factors: relation to mechanistic characterization
Authors: NISHIOKA Tomoji , TAZUMA Susumu , YAMASHITA Gunji , KAJIYAMA Goro
Published: August 2015

The crystallization of cholesterol is affected by various factors in bile. The present study evaluated the relative importance of cholesterol-nucleation-promoting factors and partially characterized the mechanisms of their action. Model biles with an identical relative composition of cholesterol, egg-yolk phosphatidylcholine and taurocholate, except for replacing phosphatidylcholine (5–20%) with dilinoleoyl-phosphatidylcholine or taurocholate (10–30%) with taurodeoxycholate. Cholesterol crystallization was quantitatively assessed spectrophotometrically and morphologically estimated by the laser-scattering diffraction analyser and video-enhanced microscopy in the absence and presence of concanavalin A-binding glycoprotein isolated from human bile. In a series of experiments, lipid distribution among particulate species was determined after isolation by FPLC. In all experiments, cholesterol crystallization was dose-dependently enhanced with a rank order of: concanavalin A-binding glycoprotein > dilinoleoyl-phosphatidylcholine > taurodeoxycholate. No morphological alteration was evident for vesicles and crystals, but the cholesterol/phospholipid ratio in vesicles was increased significantly by replacement with dilinoleoyl-phosphatidylcholine and excess cholesterol. A high proportion of relatively hydrophilic phosphatidylcholine species such as dilinoleoyl-phosphatidylcholine and excess cholesterol in bile cause a redistribution of cholesterol to increase a vesicular cholesterol/phospholipid ratio, eventually promoting cholesterol crystallization, whereas concanavalin A-binding glycoprotein acts via differing mechanisms.
Isolation and characterization of the human aldehyde oxidase gene: conservation of intron/exon boundaries with the xanthine oxidoreductase gene indicates a common origin
Authors: TERAO Mineko , KUROSAKI Mami , DEMONTIS Silvia , ZANOTTA Stefania , GARATTINI Enrico
Published: August 2015

Aldehyde oxidase (AO) is a molybdo-flavo enzyme involved in the metabolism of various endogenous and exogenous N-heterocyclic compounds of pharmacological and toxicological importance. The enzyme is the product of a gene which is implicated in the aetio-pathogenesis of familial recessive amyotrophic lateral sclerosis. Here, we report the cloning and structural characterization of the human AO gene. AO is a single copy gene approximately 85 kb long with 35 transcribed exons. The transcription-initiation site and the sequence of the 5´-flanking region, containing several putative regulatory elements, were determined. The 5´-flanking region contains a functional promoter, as assessed by appropriate reporter constructs in transient transfection experiments. Comparison of the AO gene structure shows conservation of the position and type of exon/intron junctions relative to those observed in the gene coding for another molybdo-flavoprotein, i.e. xanthine oxidoreductase (XOR). As the two genes code for proteins with a high level of amino acid identity, our results strongly suggest that the AO and XOR genetic loci arose as the consequence of a duplication event. Southern blot analysis conducted on genomic DNA from various animal species with specific cDNA probes indicates that the AO gene is less conserved than the XOR gene during evolution.