bluuebunny/crossref_metadata_2025 huggingface dataset:
13C-NMR study of the inhibition of δ-chymotrypsin by a tripeptide-glyoxal inhibitor
Authors: DJURDJEVIC-PAHL Aleksandra , HEWAGE Chandralal , MALTHOUSE J. Paul G.
Published: August 2015

A new inhibitor, Z-Ala-Pro-Phe-glyoxal (where Z is benzyloxycarbonyl),has been synthesized and shown to be a competitive inhibitor of δ-chymotrypsin, with a Ki of 25±8nM at pH7.0 and 25°C. Z-Ala-Pro-[1-13C]Phe-glyoxal and Z-Ala-Pro-[2-13C]Phe-glyoxal have been synthesized, and 13C-NMR has been used to determine how they interact with δ-chymotrypsin. Using Z-Ala-Pro-[2-13C]Phe-glyoxal we have detected a signal at 100.7p.p.m. which we assign to the tetrahedral adduct formed between the hydroxy group of Ser-195 and the 13C-enriched keto-carbon of the inhibitor. This signal is in a pH-dependent slow exchange with a signal at 107.6p.p.m. which depends on a pKa of ∼ 4.5, which we assign to oxyanion formation. Thus we are the first to detect an oxyanion pKa in a reversible chymotrypsin—inhibitor complex. A smaller titration shift of 100.7p.p.m. to 103.9p.p.m. with a pKa of ∼ 5.3 is also detected due to a rapid exchange process. This pKa is also detected with the Z-Ala-Pro-[1-13C]Phe-glyoxal inhibitor and gives a larger titration shift of 91.4p.p.m. to 97.3p.p.m., which we assign to the ionization of the hydrated aldehyde hydroxy groups of the enzyme-bound inhibitor. Protonation of the oxyanion in the oxyanion hole decreases the binding efficiency of the inhibitor. From this decrease in binding efficiency we estimate that oxyanion binding in the oxyanion hole reduces the oxyanion pKa by 1.3 pKa units. We calculate that the pKas of the oxyanions of the hemiketal and hydrated aldehyde moieties of the glyoxal inhibitor are both lowered by 6.4–6.9 pKa units on binding to chymotrypsin. Therefore we conclude that oxyanion binding in the oxyanion hole has only a minor role in decreasing the oxyanion pKa. We also investigate how the inhibitor breaks down at alkaline pH, and how it breaks down at neutral pH in the presence of chymotrypsin.
Transcriptional regulation of the human manganese superoxide dismutase gene: the role of specificity protein 1 (Sp1) and activating protein-2 (AP-2)
Authors: XU Yong , PORNTADAVITY Sureerut , ST CLAIR Daret K.
Published: August 2015

Manganese superoxide dismutase (MnSOD) plays an important role in regulating cellular redox conditions. Expression of MnSOD has been shown to protect against damage by oxidative stress and to suppress the malignant phenotype of human cancer cells. We have previously cloned the human MnSOD (SOD2) gene and analysed its 5′ proximal promoter, which has been characterized by a lack of a TATA or CAAT box and the presence of multiple GC boxes. To define further the molecular mechanisms for the regulation of MnSOD expression, multiple transcription factor-binding motifs containing overlapping specificity protein 1 (Sp1)- and activator protein (AP)-2-binding sites were identified by DNase I footprinting analysis. Functional studies in three cell lines with different levels of Sp1 and AP-2 proteins suggested that the cellular levels of these proteins may differentially regulate transcription via GC-binding motifs in the human SOD2 promoter. Co-transfection of an Sp1 expression vector resulted in an increase in the transcription of the promoter-driven reporter gene. In contrast, co-transfection of the AP-2 expression vector caused a decrease in transcription. Direct mutagenesis analysis of Sp1- and AP-2-binding sites showed that Sp1 is essential for transcription of the human SOD2 gene, whereas AP-2 plays a negative role in the transcription. Immunoprecipitation of Sp1 and AP-2 proteins demonstrated that Sp1 interacts with AP-2 in vivo. Two-hybrid analysis revealed that interaction between Sp1 and AP-2 plays both a positive and negative role in the transcription of the reporter gene in vivo. Taken together, our data indicate that AP-2 down-regulates transcription of the human SOD2 gene via its interaction with Sp1 within the promoter region. These findings, coupled with our previous observation that several cancer cell lines have mutations in the promoter region of the human MnSOD gene, which lead to an increase in an AP-2-binding site and a decrease in the promoter activity, signal the importance of understanding the promoter structure and the regulation of the human SOD2 gene by Sp1 and AP-2.
Interaction of syncollin with GP-2, the major membrane protein of pancreatic zymogen granules, and association with lipid microdomains
Authors: KALUS Ina , HODEL Alois , KOCH Annett , KLEENE Ralf , EDWARDSON J. Michael , SCHRADER Michael
Published: August 2015

Syncollin, a novel pancreatic zymogen granule protein, is present on the luminal side of the granule membrane. To address the function of syncollin, we searched for putative binding partners. Cross-linking experiments with purified syncollin, and granule content and membrane proteins revealed a direct interaction between syncollin and GP-2, a major glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein. An interaction was also observed when cross-linking was performed with recombinant GP-2. In addition, syncollin could be cross-linked to itself, supporting the suggestion that it exists as a homo-oligomer. Cleavage of the GPI anchor of GP-2 by treatment of granule membranes with phosphatidylinositol-specific phospholipase C had no effect on the membrane attachment of syncollin, indicating that it is not mediated exclusively via an interaction with GP-2. Syncollin was found to be associated with detergent-insoluble cholesterol/glycolipid-enriched complexes. These complexes floated to the lighter fractions of sucrose-density gradients and also contained GP-2, the lectin ZG16p, sulphated matrix proteoglycans and the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) syntaxin 3 and synaptobrevin 2. Our results indicate that membrane-associated syncollin is a component of lipid rafts, where it interacts both with GP-2 and membrane lipids. We suggest that the syncollin—GP-2 complex might play a role in signal transduction across the granule membrane.
Biochemical analysis of DnaA protein with mutations in both Arg328 and Lys372
Authors: MAKISE Masaki , MIMA Shinji , KOTERASAWA Motohiro , TSUCHIYA Tomofusa , MIZUSHIMA Tohru
Published: August 2015

The DnaA protein is the initiator of chromosomal DNA replication in Escherichia coli. Acidic phospholipids decrease its affinity for adenine nucleotides, and re-activate the ADP-bound form to the ATP-bound form. We have previously reported that two mutant forms, DnaAR328E and DnaAK372E, have decreased affinity for cardiolipin (CL). In the present study, we constructed a mutant DnaA protein, DnaA435, with both R328E and K372E, and compared its biochemical characteristics with those of DnaAR328E and DnaAK372E. DnaA435 could bind to oriC DNA, but did not bind ATP or ADP. In DnaA435, compared with DnaAR328E and DnaAK372E, CL caused less inhibition of oriC DNA binding, suggesting that amino acids R328 and K372 are involved in the interaction of DnaA with acidic phospholipids. DnaA435 could initiate DNA synthesis on oriC both in vivo and in vitro. Based on these results, we propose that ATP activates DnaA protein by changing its higher order structure around R328 and K372.
The mechanism of aggrecan release from cartilage differs with tissue origin and the agent used to stimulate catabolism
Authors: SZTROLOVICS Robert , WHITE Robert J. , ROUGHLEY Peter J. , MORT John S.
Published: August 2015

The mechanisms of aggrecan degradation in adult human articular, adult bovine nasal and fetal bovine epiphyseal cartilage in response to either interleukin-1β (IL-1β) or retinoic acid were compared using an explant culture system. Bovine nasal cartilage cultured with either IL-1β or retinoic acid exhibited significant release of glycosaminoglycan (GAG). For both factors, aggrecan proteolysis occurred predominantly at the ‘aggrecanase’ site, with no evidence for the action of matrix metalloproteinases, and resulted in the appearance of the corresponding G1 fragment in tissue extracts and in culture media. In human cartilage, little effect of IL-1β was seen, but abundant release of GAG occurred in the presence of retinoic acid, with evidence of aggrecanase action. Treatment of fetal epiphyseal cartilage with retinoic acid resulted in significant GAG release, whereas treatment with IL-1β did not. In the retinoic acid-treated tissue, however, no evidence for the cleavage of aggrecan in the interglobular region was apparent. Thus, in the fetal system, agents in addition to aggrecanase and matrix metalloproteinases appear to be active. Taken together, these data demonstrate that the pathways utilized for aggrecan catabolism may vary between different cartilages for a given stimulatory agent, and that, for a given tissue, different factors may elicit aggrecan release via different pathways.
Identification and characterization of four novel phosphorylation sites (Ser31, Ser325, Thr336 and Thr366) on LKB1/STK11, the protein kinase mutated in Peutz–Jeghers cancer syndrome
Authors: SAPKOTA Gopal P. , BOUDEAU Jérôme , DEAK Maria , KIELOCH Agnieszka , MORRICE Nick , ALESSI Dario R.
Published: August 2015

Peutz—Jeghers syndrome is an inherited cancer syndrome, which results in a greatly increased risk of developing tumours in those affected. The causative gene encodes a nuclear-localized protein kinase, termed LKB1, which is predicted to function as a tumour suppressor. The mechanism by which LKB1 is regulated in cells is not known, and nor have any of its physiological substrates been identified. Recent studies have demonstrated that LKB1 is phosphorylated in cells. As a first step towards identifying the roles that phosphorylation of LKB1 play, we have mapped the residues that are phosphorylated in human embryonic kidney (HEK)-293 cells, as well as the major in vitro autophosphorylation sites. We demonstrate that LKB1 expressed in HEK-293 cells, in addition to being phosphorylated at Ser431, a previously characterized phosphorylation site, is also phosphorylated at Ser31, Ser325 and Thr366. Incubation of wild-type LKB1, but not a catalytically inactive mutant, with manganese-ATP in vitro resulted in the phosphorylation of LKB1 at Thr336 as well as at Thr366. We were unable to detect autophosphorylation at Thr189, a site previously claimed to be an LKB1 autophosphorylation site. A catalytically inactive mutant of LKB1 was phosphorylated at Ser31 and Ser325 in HEK-293 cells to the same extent as the wild-type enzyme, indicating that LKB1 does not phosphorylate itself at these residues. We show that phosphorylation of LKB1 does not directly affect its nuclear localization or its catalytic activity in vitro, but that its phosphorylation at Thr336, and perhaps to a lesser extent at Thr366, inhibits LKB1 from suppressing cell growth.
Bisphenol A diglycidyl ether induces apoptosis in tumour cells independently of peroxisome proliferator-activated receptor-γ, in caspase-dependent and -independent manners
Authors: FEHLBERG Sebastian , TRAUTWEIN Stefan , GÖKE Alexandra , GÖKE Rüdiger
Published: August 2015

Peroxisome proliferator-activated receptors (PPARs) are nuclear transcription factors which are involved in many biological processes, such as regulation of cell differentiation, lipid metabolism, inflammation and cell death. PPARs consist of three families, PPAR-α, PPAR-δ and PPAR-γ. Bisphenol A diglycidyl ether (BADGE) has been described as a pure antagonist of PPAR-γ. However, recent data also revealed PPAR-γ-agonistic activities of BADGE. Here we show that BADGE kills transformed cells by apoptosis and promotes the cytotoxic effects of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and indomethacin. The cytotoxic effect of BADGE does not require PPAR-γ expression and is mediated in caspase-dependent and caspase-independent manners.
Heat-shock protein 70 attenuates nitric oxide-induced apoptosis in RAW macrophages by preventing cytochrome c release
Authors: KLEIN Sabine D. , BRÜNE Bernhard
Published: August 2015

Heat-shock protein (Hsp) 70 is an inhibitor of apoptosis and has been shown to protect against nitric oxide-mediated toxicity. To gain mechanistic insights into the actions of Hsp70, we stably transfected RAW 264.7 mouse macrophages with the human Hsp70 gene and investigated critical steps in the progression towards cell demise. Incubation of control and Hsp70-transfected macrophages with S-nitrosoglutathione induced accumulation of the tumour suppressor p53, expression of p21WAF1/CIP1 (where WAF1 corresponds to wild-type p53-activated fragment 1 and CIP1 corresponds to cyclin-dependent kinase-interacting protein 1) and G1 cell-cycle arrest. However, cytochrome c translocation to the cytosol and activation of caspase 9 and caspase 3 were markedly reduced in Hsp70-overexpressing cells. In addition, changes in nuclear morphology, as determined by Hoechst staining, and the appearance of cells in the sub-G1 phase were diminished in Hsp70-overexpressing cells compared with controls. We conclude that, in macrophages, Hsp70 interferes with cytochrome c release from mitochondria and, thereby, prevents nitric oxide-induced apoptosis, but leaves p53 accumulation and interference in the cell cycle intact.
α-Tocopheryl succinate, an agent with in vivo anti-tumour activity, induces apoptosis by causing lysosomal instability
Authors: NEUZIL Jiri , ZHAO Ming , OSTERMANN Georg , STICHA Martin , GELLERT Nina , WEBER Christian , EATON John W. , BRUNK Ulf T.
Published: August 2015

Certain vitamin E analogues, such as α-tocopheryl succinate (α-TOS), exhibit in vivo anti-tumour activity and, in vitro, induce apoptosis of cultured tumour cells. In the present study we report that these effects may be explained, at least in part, by destabilization of lysosomal membranes. α-TOS, but not α-tocopheryl acetate or α-tocopherol (α-TOH), induced early lysosomal destabilization followed by apoptosis. Similar effects were observed with β-TOS, whereas β-TOH was inactive. Cathepsin D-deficient cells were more resistant to α-TOS than their normal counterparts, and featured delayed caspase activation. Possible detergent and lysosomotropic effects of α- and β-TOS were suggested by their haemolytic activity in an in vitro test and their release of β-galactosidase from isolated lysosomes, whereas the non-succinylated analogues were inactive. The pro-apoptotic activity of α-TOS was pH-dependent, being greater at lower pH, typical of the interstitium of solid tumours. These findings indicate that lysosomal destabilization may partially or fully explain the induction of apoptosis in cultured cells by α-TOS and the mechanism whereby this agent exerts in vivo anti-tumour effects.
Mono-ADP-ribosyltransferases in human monocytes: regulation by lipopolysaccharide
Authors: GRAHNERT Andreas , FRIEDRICH Maik , PFISTER Martin , HAAG Friedrich , KOCH-NOLTE Friedrich , HAUSCHILDT Sunna
Published: August 2015

ADP-ribosyltransferase activity was shown to be present on the surface of human monocytes. Incubating the cells in the presence of BSA leads to an increase in enzyme activity. The acceptor amino acid mainly responsible for the ADP-ribose bond was identified as a cysteine residue. An increase in ADP-ribosyltransferase activity was observed when cells were treated for 16h with bacterial lipopolysaccharide (LPS). Possible candidates for catalysing the reaction are mono-ADP-ribosyltransferases (ARTs). When measuring expression of the mRNA of ART1, 3, 4 and 5, only ART3 mRNA was detected in unstimulated monocytes. Upon stimulation for 16h with LPS, lipoteichoic acid or peptidoglycan, ART4 mRNA was found to be expressed. No ART4 signal appeared after a 4h exposure of the cells to LPS. Cell-surface proteins were labelled when incubating monocytes with [32P]NAD+. Their molecular masses were 29, 33, 43, 45, 60 and 82kDa. In response to LPS an additional protein of 31kDa was found to be labelled. The bound label was resistant to treatment with NH2OH but sensitive to HgCl2, characteristic of a cysteine-linked ADP-ribosylation.
The ratio between tetrahydrobiopterin and oxidized tetrahydrobiopterin analogues controls superoxide release from endothelial nitric oxide synthase: an EPR spin trapping study
Authors: VÁSQUEZ-VIVAR Jeannette , MARTÁSEK Pavel , WHITSETT Jennifer , JOSEPH Joy , KALYANARAMAN Balaraman
Published: August 2015

Augmentation of superoxide levels has been linked to impaired relaxation in hypertension, diabetes and hypercholesterolaemia. Purified endothelial nitric oxide synthase (eNOS) generates superoxide under limited availability of 5,6,7,8-tetrahydrobiopterin (BH4). Thus alterations in endothelial BH4 levels have been postulated to stimulate superoxide production from eNOS. This possibility was examined by determining the concentration-dependent effects of BH4, and its analogues, on superoxide formation by eNOS. Superoxide was quantified by EPR spin trapping, which is the only available technique to quantify superoxide from eNOS. Using 5-ethoxycarbonyl-5-methyl-pyrroline N-oxide, we show that only fully reduced BH4 diminished superoxide release from eNOS, with efficiency BH4>6-methyl-BH4>5-methyl-BH4. In contrast, partially oxidized BH4 analogues, 7,8-dihydrobiopterin (7,8-BH2) and sepiapterin had no effect. Neither l-arginine nor NG-nitro-l-arginine methyl ester (l-NAME) abolished superoxide formation. Together, BH4 and l-arginine stimulated ˙NO production at maximal rates of 148nmol/min per mg of protein. These results indicate that BH4 acts as a ‘redox switch’, decreasing superoxide release and enhancing ˙NO formation. This role was verified by adding 7,8-BH2 or sepiapterin to fully active eNOS. Both 7,8-BH2 and sepiapterin enhanced superoxide release while inhibiting ˙NO formation. Collectively, these results indicate that the ratio between oxidized and reduced BH4 metabolites tightly regulates superoxide formation from eNOS. The pathological significance of this scenario is discussed.
A cytochrome c mutant with high electron transfer and antioxidant activities but devoid of apoptogenic effect
Authors: ABDULLAEV Ziedulla Kh. , BODROVA Marina E. , CHERNYAK Boris V. , DOLGIKH Dmitry A. , KLUCK Ruth M. , PEREVERZEV Mikhail O. , ARSENIEV Alexander S. , EFREMOV Roman G. , KIRPICHNIKOV Mikhail P. , MOKHOVA Elena N. , NEWMEYER Donald D. , RODER Heinrich , SKULACHEV Vladimir P.
Published: August 2015

A cytochrome c mutant lacking apoptogenic function but competent in electron transfer and antioxidant activities has been constructed. To this end, mutant species of horse and yeast cytochromes c with substitutions in the N-terminal α-helix or position 72 were obtained. It was found that yeast cytochrome c was much less effective than the horse protein in activating respiration of rat liver mitoplasts deficient in endogenous cytochrome c as well as in inhibition of H2O2 production by the initial segment of the respiratory chain of intact rat heart mitochondria. The major role in the difference between the horse and yeast proteins was shown to be played by the amino acid residue in position 4 (glutamate in horse, and lysine in yeast; horse protein numbering). A mutant of the yeast cytochrome c containing K4E and some other ‘horse’ modifications in the N-terminal α-helix, proved to be (i) much more active in electron transfer and antioxidant activity than the wild-type yeast cytochrome c and (ii), like the yeast cytochrome c, inactive in caspase stimulation, even if added in 400-fold excess compared with the horse protein. Thus this mutant seems to be a good candidate for knock-in studies of the role of cytochrome c-mediated apoptosis, in contrast with the horse K72R, K72G, K72L and K72A mutant cytochromes that at low concentrations were less active in apoptosis than the wild-type, but were quite active when the concentrations were increased by a factor of 2–12.
Isoform-specific knockdown and expression of adaptor protein ShcA using small interfering RNA
Authors: KISIELOW Malgorzata , KLEINER Sandra , NAGASAWA Michiaki , FAISAL Amir , NAGAMINE Yoshikuni
Published: August 2015

Many eukaryotic genes are expressed as multiple isoforms through the differential utilization of transcription/translation initiation sites or alternative splicing. The conventional approach for studying individual isoforms in a clean background (i.e. without the influence of other isoforms) has been to express them in cells or whole organisms in which the target gene has been deleted; this is time-consuming. Recently an efficient post-transcriptional gene-silencing method has been reported that employs a small interfering double-stranded RNA (siRNA). On the basis of this method we report a rapid alternative approach for isoform-specific gene expression. We show how the adaptor protein ShcA can be suppressed and expressed in an isoform-specific manner in a human cell line. ShcA exists in three isoforms, namely p66, p52 and p46, which differ only in their N-terminal regions and are derived from two different transcripts, namely p66 and p52/p46 mRNAs. An siRNA with a sequence shared by the two transcripts suppressed all of them. However, another siRNA whose sequence was present only in p66 mRNA suppressed only the p66 isoform, suggesting that the siRNA signal did not propagate to other regions of the target mRNA. The expression of individual isoforms was achieved by first down-regulating all isoforms by the common siRNA and then transfecting with an expression vector for each isoform that harboured silent mutations at the site corresponding to the siRNA. This allowed functional analysis of individual ShcA isoforms and may be more generally applicable for studying genes encoding multiple proteins.
Evidence that zymogen granules do not function as an intracellular Ca2+ store for the generation of the Ca2+ signal in rat parotid acinar cells
Authors: NEZU Akihiro , TANIMURA Akihiko , MORITA Takao , IRIE Kazuharu , YAJIMA Toshihiko , TOJYO Yosuke
Published: August 2015

Rat parotid acinar cells lacking zymogen granules were obtained by inducing granule discharge with the β-adrenoceptor agonist isoproterenol. To assess whether zymogen granules are involved in the regulation of Ca2+ signalling as intracellular Ca2+ stores, changes in cytosolic free Ca2+ ion concentration ([Ca2+]i) were studied with imaging microscopy in fura-2-loaded parotid acinar cells lacking zymogen granules. The increase in [Ca2+]i induced by muscarinic receptor stimulation was initiated at the apical pole of the acinar cells, and rapidly spread as a Ca2+ wave towards the basolateral region. The magnitude of the [Ca2+]i response and the speed of the Ca2+ wave were essentially similar to those in control acinar cells containing zymogen granules. Western blot analysis of the inositol 1,4,5-trisphosphate receptor (IP3R) was performed on zymogen granule membranes and microsomes using anti-IP3R antibodies. The immunoreactivity of all three IP3Rs was clearly observed in the microsomal preparations. Although a weak band of IP3R type-2 was detected in the zymogen granule membranes, this band probably resulted from contamination by the endoplasmic reticulum (ER), because calnexin, a marker protein of the ER, was also detected in the same preparation. Furthermore, Western blotting and reverse transcriptase-PCR analysis failed to provide evidence for the expression of ryanodine receptors in rat parotid acinar cells, whereas expression was clearly detectable in rat skeletal muscle, heart and brain. These results suggest that zymogen granules do not have a critical role in Ca2+ signalling in rat parotid acinar cells.
Monitoring of exocytosis and endocytosis of insulin secretory granules in the pancreatic β-cell line MIN6 using pH-sensitive green fluorescent protein (pHluorin) and confocal laser microscopy
Authors: OHARA-IMAIZUMI Mica , NAKAMICHI Yoko , TANAKA Toshiaki , KATSUTA Hidenori , ISHIDA Hitoshi , NAGAMATSU Shinya
Published: August 2015

The dynamics of exocytosis/endocytosis of insulin secretory granules in pancreatic β-cells remains to be clarified. In the present study, we visualized and analysed the motion of insulin secretory granules in MIN6 cells using pH-sensitive green fluorescent protein (pHluorin) fused to either insulin or the vesicle membrane protein, phogrin. In order to monitor insulin exocytosis, pHluorin, which is brightly fluorescent at approximately pH7.4, but not at approximately pH5.0, was attached to the C-terminus of insulin. To monitor the motion of insulin secretory granules throughout exocytosis/endocytosis, pHluorin was inserted between the third and fourth amino acids after the identified signal-peptide cleavage site of rat phogrin cDNA. Using this method of cDNA construction, pHluorin was located in the vesicle lumen, which may enable discrimination of the unfused acidic secretory granules from the fused neutralized ones. In MIN6 cells expressing insulin—pHluorin, time-lapse confocal laser scanning microscopy (5 or 10s intervals) revealed the appearance of fluorescent spots by depolarization after stimulation with 50mM KCl and 22mM glucose. The number of these spots in the image at the indicated times was counted and found to be consistent with the results of insulin release measured by RIA during the time course. In MIN6 cells expressing phogrin—pHluorin, data showed that fluorescent spots appeared following high KCl stimulation and remained stationary for a while, moved on the plasma membrane and then disappeared. Thus we demonstrate the visualized motion of insulin granule exocytosis/endocytosis using the pH-sensitive marker, pHluorin.
Expression, purification and characterization of human glutamate dehydrogenase (GDH) allosteric regulatory mutations
Authors: FANG Jie , HSU Betty Y. L. , MacMULLEN Courtney M. , PONCZ Mortimer , SMITH Thomas J. , STANLEY Charles A.
Published: August 2015

Glutamate dehydrogenase (GDH) catalyses the reversible oxidative deamination of l-glutamate to 2-oxoglutarate in the mitochondrial matrix. In mammals, this enzyme is highly regulated by allosteric effectors. The major allosteric activator and inhibitor are ADP and GTP, respectively; allosteric activation by leucine may play an important role in amino acid-stimulated insulin secretion. The physiological significance of this regulation has been highlighted by the identification of children with an unusual hyperinsulinism/hyperammonaemia syndrome associated with dominant mutations in GDH that cause a loss in GTP inhibition. In order to determine the effects of these mutations on the function of the human GDH homohexamer, we studied the expression, purification and characterization of two of these regulatory mutations (H454Y, which affects the putative GTP-binding site, and S448P, which affects the antenna region) and a mutation designed to alter the putative binding site for ADP (R463A). The sensitivity to GTP inhibition was impaired markedly in the purified H454Y (ED50, 210μM) and S448P (ED50, 3.1μM) human GDH mutants compared with the wild-type human GDH (ED50, 42nM) or GDH isolated from heterozygous patient cells (ED50, 290 and 280nM, respectively). Sensitivity to ADP or leucine stimulation was unaffected by these mutations, confirming that they interfere specifically with the inhibitory GTP-binding site. Conversely, the R463A mutation completely eliminated ADP activation of human GDH, but had little effect on either GTP inhibition or leucine activation. The effects of these three mutations on ATP regulation indicated that this nucleotide inhibits human GDH through binding of its triphosphate tail to the GTP site and, at higher concentrations, activates the enzyme through binding of the nucleotide to the ADP site. These data confirm the assignment of the GTP and ADP allosteric regulatory sites on GDH based on X-ray crystallography and provide insight into the structural mechanisms involved in positive and negative allosteric control and in inter-subunit co-operativity of human GDH.
De novo-synthesized ceramide is involved in cannabinoid-induced apoptosis
Authors: PULGAR Teresa GÓMEZ del , VELASCO Guillermo , SÁNCHEZ Cristina , HARO Amador , GUZMÁN Manuel
Published: August 2015

Δ9-Tetrahydrocannabinol (THC) and other cannabinoids have been shown to induce apoptosis of glioma cells via ceramide generation. In the present study, we investigated the metabolic origin of the ceramide responsible for this cannabinoid-induced apoptosis by using two subclones of C6 glioma cells: C6.9, which is sensitive to THC-induced apoptosis; and C6.4, which is resistant to THC-induced apoptosis. Pharmacological inhibition of ceramide synthesis de novo, but not of neutral and acid sphingomyelinases, prevented THC-induced apoptosis in C6.9 cells. The activity of serine palmitoyltransferase (SPT), which catalyses the rate-limiting step of ceramide synthesis de novo, was remarkably enhanced by THC in C6.9 cells, but not in C6.4 cells. However, no major changes in SPT mRNA and protein levels were evident. Changes in SPT activity paralleled changes in ceramide levels. Pharmacological inhibition of ceramide synthesis de novo also prevented the stimulation of extracellular-signal-regulated kinase and the inhibition of protein kinase B triggered by cannabinoids. These findings show that de novo-synthesized ceramide is involved in cannabinoid-induced apoptosis of glioma cells.
Efficient identification of photolabelled amino acid residues by combining immunoaffinity purification with MS: revealing the semotiadil-binding site and its relevance to binding sites for myristates in domain III of human serum albumin
Authors: KAWAHARA Kohichi , KUNIYASU Akihiko , MASUDA Katsuyoshi , ISHIGURO Masaji , NAKAYAMA Hitoshi
Published: August 2015

To identify photoaffinity-labelled amino acid residue(s), we devised an effective method utilizing immunoaffinity purification of photolabelled fragments, followed by matrix-assisted laser-desorption ionization-time of flight (MALDI-TOF) MS and nanoelectrospray ionization tandem MS (nano-ESI-MS/MS) analysis. Human serum albumin (HSA) was photolabelled with an azidophenyl derivative of semotiadil, FNAK {(+)-(R)-3,4-dihydro-2-[5-methoxy-2-[3-[N-methyl-N-[2-(3-azidophenoxy)-ethyl]amino]propoxyl]phenyl]-4-methyl-2H-1,4-benzothiazin-3-(4H)-one}, since HSA is a major binding protein for semotiadil in serum. After lysyl endopeptidase digestion, photolabelled HSA fragments were adsorbed selectively on to Sepharose beads on which an anti-semotiadil antibody was immobilized, and fractions were eluted quantitatively by 50% acetonitrile/10mM HCl. MALDI-TOF MS analysis of the eluted fraction showed that it contained two photolabelled fragments of m/z 2557.54 (major) and 1322.44 (minor), corresponding to Lys-414—Lys-432 and Ala-539—Lys-545, respectively. Further nano-ESI-MS/MS analysis revealed that Lys-414 was the photolabelled amino acid residue in fragment 414–432 and Lys-541 was a likely candidate in fragment 539–545. Based on the photolabelling results, we constructed a three-dimensional model of the FNAK—HSA complex, revealing that FNAK resides in a pocket that overlaps considerably with myristate (Myr)-binding sites, Myr-3 and −4, by comparison with crystallographic data of HSA—Myr complexes described in Curry, Mandelkow, Brick and Franks (1998) Nat. Struct. Biol. 5, 827–835. Moreover, addition of Myr increased photo-incorporation into Lys-414, whereas incorporation into Lys-541 decreased under conditions of [Myr]/[HSA]<1. Further addition of Myr, however, uniformly decreased photo-incorporation into both Lys residues. These results indicate that FNAK labelling can also be used to monitor Myr binding in domain III. An interpretation for the concomitant local conformational change of HSA is provided.
Molecular dissection of membrane-transport proteins: mass spectrometry and sequence determination of the galactose–H+ symport protein, GalP, of Escherichia coli and quantitative assay of the incorporation of [ring-2-13C]histidine and 15NH3
Authors: VENTER Henrietta , ASHCROFT Alison E. , KEEN Jeffrey N. , HENDERSON Peter J.F. , HERBERT Richard B.
Published: August 2015

The molecular mass of the galactose—H+ symport protein GalP, as its histidine-tagged derivative GalP(His)6, has been determined by electrospray MS (ESI-MS) with an error of <0.02%. One methionine residue, predicted to be present from the DNA sequence, was deduced to be absent. This is a significant advance on the estimation of the molecular masses of membrane-transport proteins by SDS/PAGE, where there is a consistent under-estimation of the true molecular mass due to anomalous electrophoretic migration. Addition of a size-exclusion chromatography step after Ni2+-nitrilotriacetate affinity purification was essential to obtain GalP(His)6 suitable for ESI-MS. Controlled trypsin, trypsin+chymotrypsin and CNBr digestion of the protein yielded peptide fragments suitable for ESI-MS and tandem MS analysis, and accurate mass determination of the derived fragments resulted in identification of 82% of the GalP(His)6 protein. Tandem MS analysis of selected peptides then afforded 49% of the actual amino acid sequence of the protein; the absence of the N-terminal methionine was confirmed. Matrix-assisted laser-desorption ionization MS allowed identification of one peptide that was not detected by ESI-MS. All the protein/peptide mass and sequence determinations were in accord with the predictions of amino acid sequence deduced from the DNA sequence of the galP gene. [ring-2-13C]Histidine was incorporated into GalP(His)6in vivo, and ESI-MS analysis enabled the measurement of a high (80%) and specific incorporation of label into the histidine residues in the protein. MS could also be used to confirm the labelling of the protein by 15NH3 (93% enrichment) and [19F]tryptophan (83% enrichment). Such MS measurements will serve in the future analysis of the structures of membrane-transport proteins by NMR, and of their topology by indirect techniques.
Transcriptional regulation of human 3′-phosphoadenosine 5′-phosphosulphate synthase 2
Authors: SHIMIZU Chikara , FUDA Hirotoshi , LEE Young C. , STROTT Charles A.
Published: August 2015

Sulphonation is a fundamental process that is essential for normal growth and development as well as maintenance of the internal milieu. The universal sulphonate donor molecule essential for all sulphoconjugation reactions is adenosine 3′-phosphate 5′-phosphosulphate (PAPS), which is produced from ATP and inorganic sulphate by the action of bifunctional PAPS synthase. There are two isozymes encoded by genes located on chromosome 4 (PAPS synthase 1) and chromosome 10 (PAPS synthase 2). The promoter for PAPS synthase 2 contains neither a TATAAA nor a CCAAT box, although a consensus initiator motif is present. Three human cell lines were used to examine promoter activity after transfection with various lengths of the 5′-flanking region of the PAPS synthase 2 gene fused to a reporter gene. Proximal promoter activity was located between bp −84 and bp −124 upstream of the purported transcription start site. This region contains two GC/GT boxes that are essential for full promoter activity, as indicated by deletion analysis and supported further by mutagenesis. A nuclear extract of SW13 cells, which highly express PAPS synthase 2, contained proteins that bound to probes possessing promoter-specific GC/GT boxes. Furthermore, the presence of specificity protein (Sp) 1, Sp2 and Sp3 proteins in the nuclear extract was confirmed by supershift analysis. Co-transfection experiments using SL2 cells yielded additional support for the involvement of Sp1 in transcriptional regulation of the PAPS synthase 2 gene; the involvement of Sp2 and/or Sp3 remains to be clarified further.
Cloning and expression of two novel pig liver and kidney fatty acid hydroxylases [cytochrome P450 (CYP)4A24 and CYP4A25]
Authors: LUNDELL Kerstin
Published: August 2015

A new member of the cytochrome P450 (CYP) 4A subfamily (CYP4A21) was recently cloned by PCR from pig liver [Lundell, Hansson, and Wikvall (2001) J. Biol. Chem. 276, 9606–9612]. This enzyme does not catalyse ω- or (ω-1)-hydroxylation of lauric acid, the model substrate for CYP4A enzymes. Instead, CYP4A21 participates in bile acid biosynthesis in the pig. Extensive studies, primarily conducted to verify the aberrant amino acids found in CYP4A21 within a normally conserved CYP4A motif, revealed that besides CYP4A21 two additional sequences were co-amplified by PCR. These two sequences (designated CYP4A24 and CYP4A25), generated from both pig liver and kidney, were characterized by restriction-enzyme analysis and were subsequently cloned. The deduced amino acid sequences of CYP4A24 and CYP4A25 share extensive sequence identity (97%). Both enzymes, expressed in yeast cells, exhibit ω-and (ω-1)-hydroxylase activities towards lauric acid and palmitic acid. The positions of the variable regions between CYP4A24 and CYP4A25, which are confined to β-sheets 1 and 4, indicate a possible difference in substrate specificity or regioselectivity. The porcine CYP4A21, CYP4A24 and CYP4A25 enzymes, with an overall identity of 94%, have probably evolved from a common ancestral gene, perhaps in conjunction with species-specific habits.
Expression in mammalian cell cultures reveals interdependent, but distinct, functions for Star and Rhomboid proteins in the processing of the Drosophila transforming-growth-factor-α homologue Spitz
Authors: PASCALL John C. , LUCK Jane E. , BROWN Kenneth D.
Published: August 2015

We report here distinct interdependent functions for two proteins, Star and Rhomboid, that are key determinants of the epidermal-growth-factor (EGF)-receptor signalling pathway in Drosophila. When we expressed the Drosophila EGF-receptor ligand Spitz in mammalian cells, the protein failed to traffic to the plasma membrane, as assessed by either cell-surface protein biotinylation or immunocytochemical staining. However, when we co-expressed Star with Spitz, trafficking of Spitz to the cell surface could be demonstrated. Only when we co-expressed Spitz, Star and Rhomboid could the release of soluble Spitz protein into the medium be shown. Taken together, our results indicate that Star is required for the intracellular trafficking of Spitz, and that Rhomboid is essential for the release of soluble Spitz protein from cells.
Activation of rhodopsin kinase
Authors: McCARTHY Nina E.M. , AKHTAR Muhammad
Published: August 2015

The present study confirms our original assertion that peptides corresponding to the C-terminal sequence of rhodopsin are phosphorylated by rhodopsin kinase (RK), but only in the presence of photo-activated rhodopsin [Rho∗, which is functionally equivalent to metarhodopsin II (Meta II)]. Under optimized conditions, the extent of peptide phosphorylation reached up to 60% that of Rho∗. Rho∗ phosphorylation began to plateau within 15min of the initiation of photolysis, whereas the peptide phosphorylation continued linearly for >60min. This lack of co-ordination in the phosphorylation of the physiological (Rho∗) and synthetic (peptide) substrates necessitated the present study, which showed that RK is activated for peptide phosphorylation not only by Meta II but also by Meta III, as well as by the phosphorylated derivatives of these species, but not by opsin. These results led to the conclusion that all the derivatives of opsin, which contain the Schiff base linkage with the all-trans-retinylidene moiety, retain the ability to activate RK.
Characterization of a putative α-mannosyltransferase involved in phosphatidylinositol trimannoside biosynthesis in Mycobacterium tuberculosis
Authors: KREMER Laurent , GURCHA Sudagar S. , BIFANI Pablo , HITCHEN Paul G. , BAULARD Alain , MORRIS Howard R. , DELL Anne , BRENNAN Patrick J. , BESRA Gurdyal S.
Published: August 2015

Phosphatidyl-myo-inositol mannosides (PIMs), lipomannan (LM) and lipoarabinomannan (LAM) are an important class of bacterial factors termed modulins that are found in tuberculosis and leprosy. Although their structures are well established, little is known with respect to the molecular aspects of the biosynthetic machinery involved in the synthesis of these glycolipids. On the basis of sequence similarity to other glycosyltransferases and our previous studies defining an α-mannosyltransferase from Mycobacterium tuberculosis, named PimB [Schaeffer, Khoo, Besra, Chatterjee, Brennan, Belisle and Inamine (1999) J. Biol. Chem. 274, 31625–31631], which catalysed the formation of triacyl (Ac3)-PIM2 (i.e. the dimannoside), we have identified a related gene from M. tuberculosis CDC1551, now designated pimC. The use of a cell-free assay containing GDP-[14C]mannose, amphomycin and membranes from Myobacterium smegmatis overexpressing PimC led to the synthesis of a new alkali-labile PIM product. Fast-atom-bombardment MS established the identity of the new enzymically synthesized product as Ac3PIM3 (i.e. the trimannoside). The results indicate that pimC encodes an α-mannosyltransferase involved in Ac3PIM3 biosynthesis. However, inactivation of pimC in Myobacterium bovis Bacille Calmette—Guérin (BCG) did not affect the production of higher PIMs, LM and LAM when compared with wild-type M. bovis BCG, suggesting the existence of redundant gene(s) or an alternate pathway that may compensate for this PimC deficiency. Further analyses, which compared the distribution of pimC in a panel of M. tuberculosis strains, revealed that pimC was present in only 22% of the clinical isolates examined.
Association of thrombospondin-1 with the actin cytoskeleton of human thrombin-activated platelets through an αIIbβ3- or CD36-independent mechanism
Authors: SAUMET Anne , de JESUS Nando , LEGRAND Chantal , DUBERNARD Véronique
Published: August 2015

Thrombospondin-1 (TSP-1) is an adhesive glycoprotein which, when secreted from α-granules of activated platelets, can bind to the cell surface and participate in platelet aggregate formation. In this study, we show that thrombin activation leads to the rapid and specific association of a large amount of secreted α-granular TSP-1 with the actin cytoskeleton. This cytoskeletal association of TSP-1 was correlated with platelet secretion, but not aggregation, and was inhibited by cytochalasin D, an inhibitor of actin polymerization. Association of TSP-1 with the actin cytoskeleton was mediated by membrane receptors, as shown by using MAII, a TSP-1-specific monoclonal antibody that inhibited both TSP-1 surface binding to activated platelets and cytoskeletal association. TSP-1 and its potential membrane receptors, e.g. αIIbβ3 integrin, CD36 and CD47, concomitantly associated with the actin cytoskeleton. However, studies on platelets from a patient with type I Glanzmann's thrombasthenia lacking αIIbβ3 and another with barely detectable CD36 showed normal TSP-1 surface expression and association with the actin cytoskeleton. Likewise, no involvement of CD47 in TSP-1 association with the actin cytoskeleton could be inferred from experiments with control platelets using the function-blocking anti-CD47 antibody B6H12. Finally, assembly of signalling complexes, as observed through translocation of tyrosine-phosphorylated proteins and kinases to the actin cytoskeleton, was found to occur in concert with cytoskeletal association of TSP-1, in control platelets as well as in thrombasthenic and CD36-deficient platelets. Our results imply a role for the actin cytoskeleton in the membrane-surface expression process of TSP-1 molecules and suggest a possible coupling of TSP-1 receptors to signalling events occurring independently of αIIbβ3 or CD36. These results provide new insights into the link between surface-bound TSP-1 and the contractile actin microfilament system which may promote platelet aggregate cohesion.
Sensitive and real-time determination of H2O2 release from intact peroxisomes
Authors: MUELLER Sebastian , WEBER Angelika , FRITZ Reiner , MÜTZE Sabine , ROST Daniel , WALCZAK Henning , VÖLKL Alfred , STREMMEL Wolfgang
Published: August 2015

Peroxisomes are essential and ubiquitous cell organelles having a key role in mammalian lipid and oxygen metabolism. The presence of flavine oxidases makes them an important intracellular source of H2O2: an obligate product of peroxisomal redox reactions and a key reactive oxygen species. Peroxisomes proliferate in response to external signals triggered by peroxisome-proliferator-activated receptor signalling pathways. Peroxisome-derived oxidative stress as a consequence of this proliferation is increasingly recognized to participate in pathologies ranging from carcinogenesis in rodents to alcoholic and non-alcoholic steatosis hepatitis in humans. To date, no sensitive approach exists to record H2O2 turnover of peroxisomes in real time. Here, we introduce a sensitive chemiluminescence method that allows the monitoring of H2O2 generation and degradation in real time in suspensions of intact peroxisomes. Importantly, removal, as well as release of, H2O2 can be assessed at nanomolar, non-toxic concentrations in the same sample. Owing to the kinetic properties of catalase and oxidases, H2O2 forms fast steady-state concentrations in the presence of various peroxisomal substrates. Substrate screening suggests that urate, glycolate and activated fatty acids are the most important sources for H2O2 in rodents. Kinetic studies imply further that peroxisomes contribute significantly to the β-oxidation of medium-chain fatty acids, in addition to their essential role in the breakdown of long and very long ones. These observations establish a direct quantitative release of H2O2 from intact peroxisomes. The experimental approach offers new possibilities for functionally studying H2O2 metabolism, substrate transport and turnover in peroxisomes of eukaryotic cells.
A novel TATA-box-binding factor from the silk glands of the mulberry silkworm, Bombyx mori
Authors: SRINIVASAN Lakshmi , GOPINATHAN Karumathil P.
Published: August 2015

The presence of one or more TATATAA motifs in the flanking sequences of individual members of a multi-gene AGly1 family from the mulberry silkworm, Bombyx mori, negatively modulated the transcription of the gene copies. Characterization of proteins from posterior silk gland nuclear extracts, binding to the TATATAA motif, identified a novel 43kD protein, designated here as P43 TATA-box-binding factor (TBF). The protein was purified to homogeneity. P43 TBF binding was highly sequence-specific and showed a 100-fold-higher affinity for binding than the TATA-box-binding protein (TBP). The protein also showed binding to the TATAAA sequence of the actin5C promoter. P43 TBF inhibited transcription of all the tRNA genes examined, as well as RNA polymerase II transcription from the actin5C promoter. The amino acid sequence of eleven peptides generated from P43 TBF did not share homology with proteins that bind the TATA box, such as TBP, TRF (TBP-related factor) or TLFs (TBP-like factors) reported from other sources. Inhibition of transcription of tRNA genes by P43 TBF could not be reversed by TBP. The inhibitory effect appeared to be exerted through sequestration of the associated transcription factors.
Stimulation of erythroleukaemia cell differentiation by extracellular high-mobility group-box protein 1 is independent of the receptor for advanced glycation end-products
Authors: SPARATORE Bianca , PEDRAZZI Marco , PASSALACQUA Mario , GAGGERO Deborah , PATRONE Mauro , PONTREMOLI Sandro , MELLONI Edon
Published: August 2015

In several cell types the binding of extracellular high-mobility group-box protein 1 (HMGB1) with the receptor for advanced glycation end-products (RAGE) induces cytoskeletal reorganization and cell motility. To establish whether RAGE is also involved in murine erythroleukaemia (MEL) cell differentiation stimulated by HMGB1, we have demonstrated that these cells express a 51kDa protein identified as RAGE, and then we have produced stable transfectants overexpressing wild-type (wt) RAGE or a dominant negative (dn) RAGE mutant lacking the cytoplasmic domain to analyse the differentiation process in these cells. Several experimental findings indicated that RAGE was not involved in the MEL cell differentiation programme. This was also supported by the identical stimulatory effect exerted by HMGB1 on both wt- or dn-RAGE transfectants. We have also observed that HMGB1 binds a 65kDa protein on the surface of MEL cells, supporting the hypothesis that alternative targets of HMGB1 are expressed on the MEL cell membrane and may be involved as mediators of its signalling.