bluuebunny/crossref_metadata_2025 huggingface dataset:
Biochemical characterization of the 2-ketoacid reductases encoded by ycdW and yiaE genes in Escherichia coli
Authors: NUÑEZ M. Felisa , PELLICER M. Teresa , BADIA Josefa , AGUILAR Juan , BALDOMA Laura
Published: August 2015

Glyoxylate is an important intermediate of the central microbial metabolism formed from acetate, allantoin or glycolate. Depending on the physiological conditions, glyoxylate is incorporated into the central metabolism by the combined actions of the activity of malate synthase and the D-glycerate pathway, or alternatively it can be reduced to glycolate by constitutive glyoxylate reductase activity. At present no information is available on this latter enzyme in Escherichia coli, although similar enzymes, classified as 2-hydroxyacid dehydrogenases, have been characterized in other organisms. A BLAST search using as the query sequence the hydroxypyruvate/glyoxylate reductase from Cucumis sativus identified as an orthologue the yiaE gene of E. coli encoding a ketoaldonate reductase. Use of this sequence in a subsequent BLAST search yielded the ycdW gene as a good candidate to encode glyoxylate reductase in this bacterium. Cloning and overexpression of the ycdW gene showed that its product displayed a high NADPH-linked glyoxylate reductase activity, and also catalysed the reduction of hydroxypyruvate with a lower efficiency. Disruption of the ycdW gene by a chloramphenicol acetyltransferase (‘CAT’) cassette did not totally abolish the glyoxylate reductase activity, indicating that another enzyme accomplished this function. The similarity with YiaE led us to test whether this protein was responsible for the remaining glyoxylate reductase activity. Purification of YcdW and YiaE proteins permitted their kinetic characterization and comparison. Analysis of the catalytic power (kcat/Km) disclosed a higher ratio of YcdW for glyoxylate and of YiaE for hydroxypyruvate.
Identification of the G13 (cAMP-response-element-binding protein-related protein) gene product related to activating transcription factor 6 as a transcriptional activator of the mammalian unfolded protein response
Authors: HAZE Kyosuke , OKADA Tetsuya , YOSHIDA Hiderou , YANAGI Hideki , YURA Takashi , NEGISHI Manabu , MORI Kazutoshi
Published: August 2015

Eukaryotic cells control the levels of molecular chaperones and folding enzymes in the endoplasmic reticulum (ER) by a transcriptional induction process termed the unfolded protein response (UPR). The mammalian UPR is mediated by the cis-acting ER stress response element consisting of 19nt (CCAATN9CCACG), the CCACG part of which is considered to provide specificity. We recently identified the basic leucine zipper (bZIP) protein ATF6 as a mammalian UPR-specific transcription factor; ATF6 is activated by ER stress-induced proteolysis and binds directly to CCACG. Here we report that eukaryotic cells express another bZIP protein closely related to ATF6 in both structure and function. This protein encoded by the G13 (cAMP response element binding protein-related protein) gene is constitutively synthesized as a type II transmembrane glycoprotein anchored in the ER membrane and processed into a soluble form upon ER stress as occurs with ATF6. The proteolytic processing of ATF6 and the G13 gene product is accompanied by their relocation from the ER to the nucleus; their basic regions seem to function as a nuclear localization signal. Overexpression of the soluble form of the G13 product constitutively activates the UPR, whereas overexpression of a mutant lacking the activation domain exhibits a strong dominant-negative effect. Furthermore, the soluble forms of ATF6 and the G13 gene product are unable to bind to several point mutants of the cis-acting ER stress response element in vitro that hardly respond to ER stress in vivo. We thus concluded that the two related bZIP proteins are crucial transcriptional regulators of the mammalian UPR, and propose calling the ATF6 gene product ATF6α and the G13 gene product ATF6β.
Characterization of a novel phosphatidylinositol 3-phosphate-binding protein containing two FYVE fingers in tandem that is targeted to the Golgi
Authors: CHEUNG Peter C. F. , TRINKLE-MULCAHY Laura , COHEN Philip , LUCOCQ John M.
Published: August 2015

We have identified a novel protein of predicted molecular mass 40kDa that contains two FYVE domains in tandem and has therefore been named TAFF1 (TAndem FYVE Fingers-1). The protein is expressed predominantly in heart and binds to PtdIns3P specifically, even though the FYVE domains in TAFF1 lacks the first Arg of the consensus sequence R(K/R)HHCR, critical for the PtdIns3P binding of other FYVE domains identified so far. The first Arg is replaced by a Thr and Ser in the N-terminal and C-terminal FYVE domains of TAFF1 respectively. Mutational analysis indicates that both FYVE domains are required for high affinity binding to PtdIns3P. Cell localization studies using a green fluorescent protein fusion show that TAFF1 is localized to the Golgi, and that the Golgi targeting sequence is located within the N-terminal 187 residues and not in either FYVE domain.

The sequence of TAFF1has been deposited with the GenBank®, EMBL, DDBJ and GSDB Nucleotide Sequence Databases under accession number AF311602.
Protein–coenzyme interactions in adenosylcobalamin-dependent glutamate mutase
Authors: HUHTA Marja S. , CHEN Hao-Ping , HEMANN Craig , HILLE C. Russ , MARSH E. Neil G.
Published: August 2015

Glutamate mutase catalyses an unusual isomerization involving free-radical intermediates that are generated by homolysis of the cobalt–carbon bond of the coenzyme adenosylcobalamin (coenzyme B12). A variety of techniques have been used to examine the interaction between the protein and adenosylcobalamin, and between the protein and the products of coenzyme homolysis, cob(II)alamin and 5′-deoxyadenosine. These include equilibrium gel filtration, isothermal titration calorimetry, and resonance Raman, UV-visible and EPR spectroscopies. The thermodynamics of adenosylcobalamin binding to the protein have been examined and appear to be entirely entropy-driven, with ∆S = 109 Jċmol-1ċK-1. The cobalt–carbon bond stretching frequency is unchanged upon coenzyme binding to the protein, arguing against a ground-state destabilization of the cobalt–carbon bond of adenosylcobalamin by the protein. However, reconstitution of the enzyme with cob(II)alamin and 5′-deoxyadenosine, the two stable intermediates formed subsequent to homolysis, results in the blue-shifting of two of the bands comprising the UV-visible spectrum of the corrin ring. The most plausible interpretation of this result is that an interaction between the protein, 5′-deoxyadenosine and cob(II)alamin introduces a distortion into the ring corrin that perturbs its electronic properties.
Pectate lyase 10A from Pseudomonas cellulosa is a modular enzyme containing a family 2a carbohydrate-binding module
Authors: BROWN Ian E. , MALLEN Marie H. , CHARNOCK Simon J. , DAVIES Gideon J. , BLACK Gary W.
Published: August 2015

Pectate lyase 10A (Pel10A) enzyme from Pseudomonas cellulosa is composed of 649 residues and has a molecular mass of 68.5kDa. Sequence analysis revealed that Pel10A contained a signal peptide and two serine-rich linker sequences that separate three modules. Sequence similarity was seen between the 9.2kDa N-terminal module of Pel10A and family 2a carbohydrate-binding modules (CBMs). This N-terminal module of Pel10A was shown to encode an independently functional module with affinity to crystalline cellulose. A high sequence identity of 66% was seen between the 14.2kDa central module of Pel10A and the functionally uncharacterized central modules of the xylan-degrading enzymes endoxylanase 10B, arabinofuranosidase 62C and esterase 1D, also from P. cellulosa. The 35.8kDa C-terminal module of Pel10A was shown to have 30 and 36% identities with the family 10 pectate lyases from Azospirillum irakense and an alkaliphilic strain of Bacillus sp. strain KSM-P15, respectively. This His-tagged C-terminal module of the Pel10A was shown to encode an independent catalytic module (Pel10Acm). Pel10Acm was shown to cleave pectate and pectin in an endo-fashion and to have optimal activity at pH10 and in the presence of 2mM Ca2+. Highest enzyme activity was detected at 62°C. Pel10Acm was shown to be most active against pectate (i.e. polygalacturonic acid) with progressively less activity against 31, 67 and 89% esterified citrus pectins. These data suggest that Pel10A has a preference for sequences of non-esterified galacturonic acid residues. Significantly, Pel10A and the P. cellulosa rhamnogalacturonan lyase 11A, in the accompanying article [McKie, Vincken, Voragen, van den Broek, Stimson and Gilbert (2001) Biochem. J. 355, 167–177], are the first CBM-containing pectinases described to date.
Effect of polyamine depletion on caspase activation: a study with spermine synthase-deficient cells
Authors: STEFANELLI Claudio , PIGNATTI Carla , TANTINI Benedetta , FATTORI Monia , STANIC Ivana , MACKINTOSH Caroline A. , FLAMIGNI Flavio , GUARNIERI Carlo , CALDARERA Claudio M. , PEGG Anthony E.
Published: August 2015

Activation of the caspase proteases represents a central point in apoptosis. The requirement for spermine for the processes leading to caspase activation has been studied in transformed embryonic fibroblasts obtained from gyro (Gy) mutant male mice. These cells lack spermine synthase activity and thus provide a valuable model to study the role of spermine in cell processes. Gy fibroblasts do not contain spermine and have a higher spermidine content. However, when compared with fibroblasts obtained from normal male littermates (N cells), Gy fibroblasts were observed to grow normally. The lack of spermine did not affect the expression of Bcl-2, and caspases 3 and 9 were activated by etoposide in both N and Gy cells, indicating that spermine is dispensable for caspase activation. Spermine deficiency did not significantly influence caspase activity in cells treated with etoposide, cycloheximide or staurosporine, but sensitized the cells to UV irradiation, which triggered significantly higher caspase activity in Gy cells compared with N cells. α-Difluoromethylornithine (DFMO), an inhibitor of polyamine synthesis that is able to deplete cells of putrescine and spermidine, but usually does not influence spermine content, was able to produce a more complete polyamine depletion in Gy cells. This depletion, which included spermine deficiency, dramatically increased caspase activation and cell death in Gy fibroblasts exposed to UV irradiation. On the other hand, in either N or Gy cells, DFMO treatment did not influence caspase activity triggered by staurosporine, but inhibited it when the inducers were cycloheximide or etoposide. In Gy cells depleted of polyamines by DFMO, polyamine replenishment with either spermidine or spermine was sufficient to restore caspase activity induced by etoposide, indicating that, in this model, polyamines have an interchangeable role in supporting caspase activation. Therefore, spermine is not required for such activation, and the effect and specificity of polyamine depletion on caspase activity may be very different, depending on the role of polyamines in the specific death pathways engaged by different stimuli. Some inducers of apoptosis, for example etoposide, absolutely require polyamines for caspase activation, yet the lack of polyamines, particularly spermine, strongly increases caspase activation when induced by UV irradiation.
Phosphorylation of murine double minute clone 2 (MDM2) protein at serine-267 by protein kinase CK2 in vitro and in cultured cells
Authors: HJERRILD Majbrit , MILNE Diane , DUMAZ Nicolas , HAY Trevor , ISSINGER Olaf-Georg , MEEK David
Published: August 2015

Murine double minute clone 2 oncoprotein (MDM2) is a key component in the regulation of the tumour suppressor p53. MDM2 mediates the ubiqutination of p53 in the capacity of an E3 ligase and targets p53 for rapid degradation by the proteosome. Stress signals which impinge on p53, leading to its activation, promote disruption of the p53-MDM2 complex, as in the case of ionizing radiation, or block MDM2 synthesis and thereby reduce cellular MDM2 levels, as in the case of UV radiation. It is therefore likely that MDM2, which is known to be modified by ubiquitination, SUMOylation and multi-site phosphorylation, may itself be a target for stress signalling (SUMO is small ubiquitin-related modifier-1). In the present study we show that, like p53, the MDM2 protein is a substrate for phosphorylation by the protein kinase CK2 (CK2) in vitro. CK2 phosphorylates a single major site, Ser267, which lies within the central acidic domain of MDM2. Fractionation of cellular extracts revealed the presence of a single Ser267 protein kinase which co-purified with CK2 on ion-exchange chromatography and, like CK2, was subject to inhibition by micromolar concentrations of the CK2-specific inhibitor 5,6-dichlororibofuranosylbenzimidazole. Radiolabelling of cells expressing tagged recombinant wild-type MDM2 or a S267A (Ser267 → Ala) mutant, followed by phosphopeptide analysis, confirmed that Ser267 is a cellular target for phosphorylation. Ser267 mutants are still able to direct the degradation of p53, but in a slightly reduced capacity. These data highlight a potential route by which one of several physiological modifications occurring within the central acidic domain of the MDM2 protein can occur.
Differences in tissue-specific and embryonic expression of mouse Ceacam1 and Ceacam2 genes
Authors: HAN Eric , PHAN Dillon , LO Piao , POY Matthew N. , BEHRINGER Richard , NAJJAR Sonia M. , LIN Sue-Hwa
Published: August 2015

The intercellular adhesion molecule CEACAM1, also known as C-CAM1 (where CAM is cell-adhesion molecule), can function as a tumour suppressor in several carcinomas, including those of the prostate, breast, bladder and colon. This suggests that CEACAM1 may play an important role in the regulation of normal cell growth and differentiation. However, there is no direct evidence to support this putative function of CEACAM1. To elucidate its physiological function by targeted gene deletion, we isolated the Ceacam genes from a mouse 129 Sv/Ev library. Although there is only one Ceacam1 gene in humans and one in rats, two homologous genes (Ceacam1 and Ceacam2) have been identified in the mouse. Our sequence analysis revealed that the genes encoded nine exons and spanned approx. 16-17 kb (Ceacam1) and 25kb (Ceacam2). The genes were highly similar (79.6%). The major differences in the protein-coding regions were located in exons 2, 5 and 6 (76.9%, 87.0% and 78.5% similarity respectively). In addition, introns 2, 5 and 7 were also significantly different, being 29.7%, 59.8% and 64.5% similar respectively. While most of these differences were due to nucleotide substitutions, two insertions of 418 and 5849bp occurred in intron 2 of Ceacam2, and another two insertions of 1384 and 197bp occurred in introns 5 and 7 respectively. To determine whether functional redundancy exists between Ceacam1 and Ceacam2, we examined their expression in 16 mouse tissues by using semi-quantitative reverse transcription-PCR. As in human and rat, in the mouse Ceacam1 mRNA was highly abundant in the liver, small intestine, prostate and spleen. In contrast, Ceacam2 mRNA was only detected in kidney, testis and, to a lesser extent, spleen. Reverse transcription-PCR using testis RNA indicated that Ceacam2 in the testis is an alternatively spliced form containing only exons 1, 2, 5, 6, 8 and 9. In the mouse embryo, Ceacam1 mRNA was detected at day 8.5, disappeared between days 9.5 and 12.5, and re-appeared at day 19. On the other hand, no Ceacam2 mRNA was detected throughout embryonic development. The different tissue expression patterns and regulation during embryonic development suggest that the CEACAM1 and CEACAM2 proteins, although highly similar, may have different functions both during mouse development and in adulthood.

The Ceacam1 and Ceacam2 sequences have been deposited in the GenBank®/EMBL/DDBJ/GSDB Nucleotide Sequence Databases with accession numbers AF287911 and AF287912 respectively.
Modulation of epidermal growth factor receptor phosphorylation by endogenously expressed gangliosides
Authors: ZURITA Adolfo R. , MACCIONI Hugo J. F. , DANIOTTI Jose L.
Published: August 2015

The effect of changing the ganglioside composition of Chinese hamster ovary K1 cells on the function of the epidermal growth factor receptor (EGFr) was examined by studying the signalling pathway generated after the binding of epidermal growth factor (EGF) both in cells depleted of glycosphingolipids by inhibiting glucosylceramide synthase activity and in cell lines expressing different gangliosides as the result of stable transfection of appropriate ganglioside glycosyltransferases. After stimulation with EGF, cells depleted of glycolipids showed EGFr phosphorylation and extracellular signal-regulated protein kinase 2 (ERK2) activity as parental cells expressing GM3 [ganglioside nomenclature follows Svennerholm (1963) J. Neurochem. 10, 613-623] or as transfected cells expressing mostly GM2 and GD1a as the result of stable transfection of UDP-GalNAc:LacCer/GM3/GD3 N-acetylgalactosaminyltransferase. However, cells stably transfected with CMP-NeuAc:GM3 sialyltransferase and expressing GD3 at the cell surface showed both decreased EGFr phosphorylation and ERK2 activation after stimulation with EGF. Results suggest that changes in the ganglioside composition of cell membranes might be important in the regulation of the EGF signal transduction.
The potential mechanism of the diabetogenic action of streptozotocin: inhibition of pancreatic β-cell O-GlcNAc-selective N-acetyl-β-d-glucosaminidase
Authors: KONRAD Robert J. , MIKOLAENKO Irina , TOLAR Joseph F. , LIU Kan , KUDLOW Jeffrey E.
Published: August 2015

Streptozotocin (STZ), an analogue of GlcNAc, inhibits purified rat spleen O-GlcNAc-selective N-acetyl-β-d-glucosaminidase (O-GlcNAcase), the enzyme that removes O-GlcNAc from protein. We have shown previously that STZ increases pancreatic islet O-linked protein glycosylation. In light of these data, we investigated the possibility further that STZ causes β-cell death by inhibiting O-GlcNAcase. In isolated islets, the time course and dose curve of STZ-induced O-glycosylation correlated with β-cell toxicity. STZ inhibition of rat islet O-GlcNAcase activity also paralleled that of its β-cell toxicity, with significant inhibition occurring at a concentration of 1mM. In contrast, STZ inhibition of rat brain O-GlcNAcase and β-TC3 insulinoma cell O-GlcNAcase was significantly right-shifted compared with islets, with STZ only significantly inhibiting activity at a concentration of 5mM, the same concentration required for β-TC3 cell toxicity. In comparison, N-methyl-N-nitrosourea, the nitric oxide-donating portion of STZ, did not cause increased islet O-glycosylation, β-cell toxicity or inhibition of β-cell O-GlcNAcase. Enhanced STZ sensitivity of islet O-GlcNAcase compared with O-GlcNAcase from other tissues or an insulinoma cell line suggests why actual islet β-cells are particularly sensitive to STZ. Confirming this idea, STZ-induced islet β-cell toxicity was completely blocked by GlcNAc, which also prevented STZ-induced O-GlcNAcase inhibition, but was not even partially blocked by glucose, glucosamine or GalNAc. Together, these data demonstrate that STZ's inhibition of β-cell O-GlcNAcase is the mechanism that accounts for its diabetogenic toxicity.
Pterin interactions with distinct reductase activities of NO synthase
Authors: PANTKE Morten M. , REIF Andreas , VALTSCHANOFF Juli G. , SHUTENKO Zhanna , FREY Armin , WEINBERG Richard J. , PFLEIDERER Wolfgang , SCHMIDT Harald H. H. W.
Published: August 2015

Besides oxidizing l-arginine, neuronal NO synthase (NOS) NADPH-dependently reduces various electron acceptors, including cytochrome c and tetrazolium salts. The latter NADPH diaphorase reaction is used as a NOS-specific histochemical stain. Both reductase activities have been utilized to analyse electron transfer mechanisms within NOS. Basal l-arginine turnover by homodimeric NOS is enhanced by exogenous tetrahydrobiopterin, and the intra-subunit electron flow may include intermediate trihydrobiopterin. In the present work we have investigated the possible role of the tetrahydrobiopterin binding site of NOS in its reductase activities by examining the effects of anti-pterin type (PHS) NOS inhibitors. Although the type I anti-pterin, PHS-32, which does not affect basal dimeric NOS activity, also had no effect on either reductase activity, the type II anti-pterin, PHS-72, which inhibits basal NOS activity, inhibited both reductase activities and the NADPH diaphorase histochemical stain. Pterin-free NOS monomers catalysed both cytochrome c and tetrazolium salt reduction. Our data suggest that both NOS reductase activities are independent of tetrahydrobiopterin. However, occupation of an exosite near the pterin site in NOS by type II anti-pterins may interfere with the electron flow within the active centre, suggesting that steric perturbation of the pterin binding pocket or reductase interaction contribute to the mechanism of inhibition by this class of NOS inhibitors.
Human tear lipocalin acts as an oxidative-stress-induced scavenger of potentially harmful lipid peroxidation products in a cell culture system
Authors: LECHNER Markus , WOJNAR Petra , REDL Bernhard
Published: August 2015

Human tear lipocalin [lipocalin 1 (lcn-1); von Ebner's gland protein] is a member of the lipocalin superfamily that is known to bind an unusual variety of lipophilic ligands. Because of its properties and its tissue-specific expression it has been suggested that lcn-1 might act as a physiological protection factor of epithelia. Overexpression of lcn-1 under certain disease conditions supported such a function. However, experimental investigations into its exact biological role and its mode of expression were impeded because lcn-1 was previously found to be produced only in serous glands. To overcome this problem we therefore sought a cell line that produced lcn-1 endogenously. Using reverse-transcriptase-mediated PCR analysis we found expression of lcn-1 in the human teratocarcinoma-derived NT2 precursor cells. Under normal conditions the production of lcn-1 is low. However, treatment of the cells with H2O2 or FeSO4, which typically induce lipid peroxidation, significantly enhanced the expression of lcn-1. Binding studies revealed that arachidonic acid and several lipid peroxidation products including 7β-hydroxycholesterol, 8-isoprostane and 13-hydroxy-9,11-octadecadienoic acid specifically bind to lcn-1. To investigate the physiological consequence of this observation we purified holo-(lcn-1) from culture medium and extracted the bound ligands. The presence of F2-isoprostanes in the extracts obtained from the fractions containing lcn-1 indicates that these typical lipid peroxidation products are indeed ligands of the protein in vivo. These results support the idea that lcn-1 acts as a physiological scavenger of potentially harmful lipophilic molecules; lcn-1 might therefore be a novel member of the cellular defence against the deleterious effects of oxidative stress.
Substitution of murine ferrochelatase glutamate-287 with glutamine or alanine leads to porphyrin substrate-bound variants
Authors: FRANCO Ricardo , PEREIRA Alice S. , TAVARES Pedro , MANGRAVITA Arianna , BARBER Michael J. , MOURA Isabel , FERREIRA Gloria C.
Published: August 2015

Ferrochelatase (EC 4.99.1.1) is the terminal enzyme of the haem biosynthetic pathway and catalyses iron chelation into the protoporphyrin IX ring. Glutamate-287 (E287) of murine mature ferrochelatase is a conserved residue in all known sequences of ferrochelatase, is present at the active site of the enzyme, as inferred from the Bacillus subtilis ferrochelatase three-dimensional structure, and is critical for enzyme activity. Substitution of E287 with either glutamine (Q) or alanine (A) yielded variants with lower enzymic activity than that of the wild-type ferrochelatase and with different absorption spectra from the wild-type enzyme. In contrast to the wild-type enzyme, the absorption spectra of the variants indicate that these enzymes, as purified, contain protoporphyrin IX. Identification and quantification of the porphyrin bound to the E287-directed variants indicate that approx. 80% of the total porphyrin corresponds to protoporphyrin IX. Significantly, rapid stopped-flow experiments of the E287A and E287Q variants demonstrate that reaction with Zn2+ results in the formation of bound Zn-protoporphyrin IX, indicating that the endogenously bound protoporphyrin IX can be used as a substrate. Taken together, these findings suggest that the structural strain imposed by ferrochelatase on the porphyrin substrate as a critical step in the enzyme catalytic mechanism is also accomplished by the E287A and E287Q variants, but without the release of the product. Thus E287 in murine ferrochelatase appears to be critical for the catalytic process by controlling the release of the product.
Chimaeras reveal the role of the catalytic core in the activation of the plasma membrane Ca2+ pump
Authors: BA-THEIN William , CARIDE Ariel J. , ENYEDI Ágnes , PÁSZTY Katalin , CROY Cynthia L. , FILOTEO Adelaida G. , PENNISTON John T.
Published: August 2015

Isoform 2b of the plasma membrane calcium pump differs from the ubiquitous isoform 4b in the following: (a) higher basal activity in the absence of calmodulin; (b) higher affinity for calmodulin; and (c) higher affinity for Ca2+ in the presence of calmodulin [Elwess, Filoteo, Enyedi and Penniston (1997) J. Biol. Chem. 272, 17981–17986]. To investigate which parts of the molecule determine these kinetic differences, we made four chimaeric constructs in which portions of isoform 2b were grafted into isoform 4b: chimaera I contains only the C-terminal regulatory region of isoform 2b; chimaera II contains the N-terminal moiety of isoform 2b, including both cytoplasmic loops; chimaera III contains the sequence of isoform 2b starting from the N-terminus to after the end of the first (small) cytoplasmic loop; and chimaera IV contains only the second (large) cytoplasmic loop. Surprisingly, chimaera I showed low basal activity in the absence of calmodulin and low affinity for calmodulin, unlike isoform 2b. In contrast, the chimaera containing both loops showed high basal activity, and Ca2+ activation curves (both in the absence and in the presence of calmodulin) similar to those of isoform 2b. The rates of activation by calmodulin and of inactivation by calmodulin removal were measured, and the apparent Kd for calmodulin was calculated from the ratio between these rate constants. The order of affinity was: 2b = II>4b = IV>III = I. From these results it is clear that the construct that most closely resembles isoform 2b is chimaera II. This shows that, in order to obtain an enzyme with properties similar to those of isoform 2b, both cytoplasmic loops are needed.
Effects of the human papilloma virus HPV-16 E7 oncoprotein on glycolysis and glutaminolysis: role of pyruvate kinase type M2 and the glycolytic-enzyme complex
Authors: MAZUREK Sybille , ZWERSCHKE Werner , JANSEN-DÜRR Pidder , EIGENBRODT Erich
Published: August 2015

Proliferating and tumour cells express the glycolytic isoenzyme, pyruvate kinase type M2 (M2-PK), which occurs in a highly active tetrameric form and in a dimeric form with low affinity for phosphoenolpyruvate. The switch between the two forms regulates glycolytic phosphometabolite pools and the interaction between glycolysis and glutaminolysis. In the present study, we show the effects of oncoprotein E7 of the human papilloma virus (HPV)-16 (E7)-transformation on two NIH 3T3 cell strains with different metabolic characteristics. E7-transformation of the high glycolytic NIH 3T3 cell strain led to a shift of M2-PK to the dimeric form and, in consequence, to a decrease in the cellular pyruvate kinase mass-action ratio, the glycolytic flux rate and the (ATP+GTP)/(UTP+CTP) ratio, as well as to an increase in fructose 1,6-bisphosphate (FBP) levels, glutamine consumption and cell proliferation. The low glycolytic NIH 3T3 cell strain is characterized by high pyruvate and glutamine consumption rates and by an intrinsically large amount of the dimeric form of M2-PK, which is correlated with high FBP levels, a low (ATP+GTP)/(CTP+UTP) ratio and a high proliferation rate. E7-transformation of this cell strain led to an alteration in the glycolytic-enzyme complex that correlates with an increase in pyruvate and glutamine consumption and a slight increase in the flow of glucose to lactate. The association of phosphoglyceromutase within the glycolytic-enzyme complex led to an increase of glucose and serine consumption and a disruption of the linkage between glucose consumption and glutaminolysis. In both NIH 3T3 cell lines, transformation increased glutaminolysis and the positive correlation between alanine and lactate production.
Functional architecture in the cell nucleus
Authors: DUNDR Miroslav , MISTELI Tom
Published: August 2015

The major functions of the cell nucleus, including transcription, pre-mRNA splicing and ribosome assembly, have been studied extensively by biochemical, genetic and molecular methods. An overwhelming amount of information about their molecular mechanisms is available. In stark contrast, very little is known about how these processes are integrated into the structural framework of the cell nucleus and how they are spatially and temporally co-ordinated within the three-dimensional confines of the nucleus. It is also largely unknown how nuclear architecture affects gene expression. In order to understand how genomes are organized, and how they function, the basic principles that govern nuclear architecture and function must be uncovered. Recent work combining molecular, biochemical and cell biological methods is beginning to shed light on how the nucleus functions and how genes are expressed in vivo. It has become clear that the nucleus contains distinct compartments and that many nuclear components are highly dynamic. Here we describe the major structural compartments of the cell nucleus and discuss their established and proposed functions. We summarize recent observations regarding the dynamic properties of chromatin, mRNA and nuclear proteins, and we consider the implications these findings have for the organization of nuclear processes and gene expression. Finally, we speculate that self-organization might play a substantial role in establishing and maintaining nuclear organization.
Use of fluorescent Ca2+ dyes with green fluorescent protein and its variants: problems and solutions
Authors: BOLSOVER Stephen , IBRAHIM Ozbek , O'LUANAIGH Niamh , WILLIAMS Helen , COCKCROFT Shamshad
Published: August 2015

We have studied the degree to which fluorescent Ca2+ indicator dyes, and green fluorescent protein and its variants, can be used together. We find that the most commonly used fluorescent protein, enhanced green fluorescent protein (EGFP), seriously contaminates fura 2 signals. We suggest two alternative combinations for which there is no detectable contamination of the Ca2+ indicator signal by the fluorescent protein. Blue fluorescent protein can be used with the Ca2+ indicator Fura Red; EGFP can be used with the Ca2+ indicator X-Rhod 1. The use of these combinations will permit the accurate measurement of Ca2+ signals in cells transfected with fluorescent proteins.
Effects of histatin 5 and derived peptides on Candida albicans
Authors: RUISSEN Anita L. A. , GROENINK Jasper , HELMERHORST Eva J. , WALGREEN-WETERINGS Els , van't HOF Wim , VEERMAN Enno C. I. , NIEUW AMERONGEN Arie V.
Published: August 2015

Three anti-microbial peptides were compared with respect to their killing activity against Candida albicans and their ability to disturb its cellular and internal membranes. Histatin 5 is an anti-fungal peptide occurring naturally in human saliva, while dhvar4 and dhvar5 are variants of its active domain, with increased anti-microbial activity. dhvar4has increased amphipathicity compared with histatin 5, whereas dhvar5has amphipathicity comparable with that of histatin 5. All three peptides caused depolarization of the cytoplasmic and/or mitochondrial membrane, indicating membranolytic activity. For the variant peptides both depolarization and killing occurred at a faster rate. With FITC-labelled peptides, no association with the cytoplasmic membrane was observed, contradicting the formation of permanent transmembrane multimeric peptide pores. Instead, the peptides were internalized and act on internal membranes, as demonstrated with mitochondrion- and vacuole-specific markers. In comparison with histatin 5, the variant peptides showed a more destructive effect on mitochondria. Entry of the peptides and subsequent killing were dependent on the metabolic state of the cells. Blocking of the mitochondrial activity led to complete protection against histatin 5 activity, whereas that of dhvar4 was hardly affected and that of dhvar5 was affected only intermediately.
Uterocalin, a lipocalin provisioning the preattachment equine conceptus: fatty acid and retinol binding properties, and structural characterization
Authors: SUIRE Sabine , STEWART Francesca , BEAUCHAMP Jeremy , KENNEDY Malcolm W.
Published: August 2015

The equine conceptus is surrounded by a fibrous capsule that persists until about day 20 of pregnancy, whereupon the capsule is lost, the conceptus attaches to the endometrium and placentation proceeds. Before attachment, the endometrium secretes in abundance a protein of the lipocalin family, uterocalin. The cessation of secretion coincides with the end of the period during which the conceptus is enclosed in its capsule, suggesting that uterocalin is essential for the support of the embryo before direct contact between maternal and foetal tissues is established. Using recombinant protein and fluorescence-based assays, we show that equine uterocalin binds the fluorescent fatty acids 11-(dansylamino)undecanoic acid, dansyl-d,l-α-amino-octanoic acid and cis-parinaric acid, and, by competition, oleic, palmitic, arachidonic, docosahexaenoic, γ-linolenic, cis-eicosapentaenoic and linoleic acids. Uterocalin also binds all-trans-retinol, the binding site for which is coincident or interactive with that for fatty acids. Molecular modelling and intrinsic fluorescence analysis of the wild-type protein and a Trp → Glu mutant protein indicated that uterocalin has an unusually solvent-exposed Trp side chain projecting from its large helix directly into solvent. This feature is unusual among lipocalins and might relate to binding to, and uptake by, the trophoblast. Uterocalin therefore has the localization and binding activities for the provisioning of the equine conceptus with lipids including those essential for morphogenesis and pattern formation. The possession of a fibrous capsule surrounding the conceptus might be an ancestral condition in mammals; homologues of uterocalin might be essential for early development in marsupials and in eutherians in which there is a prolonged preimplantation period.
The neurofibromatosis 2 protein product merlin selectively binds F-actin but not G-actin, and stabilizes the filaments through a lateral association
Authors: JAMES Marianne F. , MANCHANDA Nitasha , GONZALEZ-AGOSTI Charo , HARTWIG John H. , RAMESH Vijaya
Published: August 2015

The neurofibromatosis 2 protein product merlin, named for its relatedness to the ezrin, radixin and moesin (ERM) family of proteins, is a tumour suppressor whose absence results in the occurrence of multiple tumours of the nervous system, particularly schwannomas and meningiomas. Merlin's similarity to ERMs suggests that it might share functions, acting as a link between cytoskeletal components and the cell membrane. The N-terminus of merlin has strong sequence identity to the N-terminal actin-binding region of ezrin; here we describe in detail the merlin–actin interaction. Employing standard actin co-sedimentation assays, we have determined that merlin isoform 2 binds F-actin with an apparent binding constant of 3.6μM and a stoichiometry of 1mol of merlin per 11.5mol of actin in filaments at saturation. Further, solid-phase binding assays reveal that merlin isoforms 1 and 2 bind actin filaments differentially, suggesting that the intramolecular interactions in isoform 1 might hinder its ability to bind actin. However, merlin does not bind G-actin. Studies of actin filament dynamics show that merlin slows filament disassembly with no influence on the assembly rate, indicating that merlin binds along actin filament lengths. This conclusion is supported by electron microscopy, which demonstrates that merlin binds periodically along cytoskeletal actin filaments. Comparison of these findings with those reported for ERM proteins reveal a distinct role for merlin in actin filament dynamics.
Threonine synthesis from aspartate in Escherichia coli cell-free extracts: pathway dynamics
Authors: RAÏS Badr , CHASSAGNOLE Christophe , LETELLIER Thierry , FELL David A. , MAZAT Jean-Pierre
Published: August 2015

We have developed an experimental model of the whole threonine pathway that allows us to study the production of threonine from aspartate under different conditions. The model consisted of a desalted crude extract of Escherichia coli to which we added the substrates and necessary cofactors of the pathway: aspartate, ATP and NADPH. In this experimental model we measured not only the production of threonine, but also the time dependence of all the intermediate metabolites and of the initial substrates, aspartate, ATP and NADPH. A stoichiometric conversion of precursors into threonine was observed. We have derived conditions in which a quasi steady state can be transiently observed and used to simulate physiological conditions of functioning of the pathway in the cell. The dependence of threonine synthesis and of the aspartate and NADPH consumption on the initial aspartate and threonine concentrations exhibits greater sensitivity to the aspartate concentration than to the threonine concentration in these non-steady-state conditions. A response to threonine is only observed in a narrow concentration range from 0.23 to 2mM.
Regulation by retinoic acid of acylation-stimulating protein and complement C3 in human adipocytes
Authors: SCANTLEBURY Thea , SNIDERMAN Allan D. , CIANFLONE Katherine
Published: August 2015

Acylation-stimulating protein (ASP), a product of complement C3, stimulates triacylglycerol synthesis in adipocytes. Previous studies have identified transthyretin, associated with chylomicrons, as a stimulator of C3 and ASP production. Since both transthyretin and chylomicrons transport retinyl ester/retinol, our goal was to investigate whether retinoic acid (RA) could be a potential hormonal mediator of the effect. Inhibitors of protein synthesis and protein secretion eliminated the stimulatory effects of chylomicrons on both C3 and ASP production in human differentiated adipocytes, suggesting that de novo protein synthesis and secretion are both required. Incubation with chylomicrons increased C3 mRNA levels (37±1.5%). RA alone or with chylomicrons had a stimulatory effect on C3 production (29-fold at 16.6nM RA) and ASP production. An RA receptor antagonist blocked stimulation of C3 mRNA and C3 secretion by both RA and chylomicrons. Finally, RA and chylomicrons activated a 1.8kb C3-promoter–luciferase construct transfected into 3T3-F442 and 3T3-L1 cells (by 41±0.2% and 69±0.3% respectively), possibly via RA receptor half-sites identified by sequence analysis. This is the first evidence documenting stimulation by RA of the C3 gene. Thus we propose RA as a novel cellular trigger in chylomicrons that subsequently results in increased ASP production by adipocytes after a meal.
Triacylglycerol-rich lipoproteins alter the secretion, and the cholesterol-effluxing function, of apolipoprotein E-containing lipoprotein particles from human (THP-1) macrophages
Authors: LINDHOLM E. Marie , PALMER Anna M. , GRAHAM Annette
Published: August 2015

Elevated plasma levels of triacylglycerol-rich lipoproteins (TGRLP) are associated with increased risk of atherogenesis and abnormal reverse cholesterol transport, as illustrated in Type II diabetes. Here we examine the effect of plasma triacylglycerol-rich or cholesteryl ester-rich lipoproteins on the secretion of nascent apolipoprotein E (apoE)-containing lipoprotein E (LpE) particles by human (THP-1) macrophages. As expected, preincubation with low-density lipoprotein (LDL) yielded small but significant increases in total cellular cholesterol content and also the secretion of apoE by macrophages. By contrast, preincubation with TGRLP resulted in higher, dose-dependent, increases in apoE secretion that reflected, but were not dependent on, cellular triacylglycerol accumulation. Secreted apoE was incorporated into a pre-β migrating LpE fraction that differed in lipid composition and flotation density depending on preincubation conditions. Specifically, the LpE-containing lipoprotein fraction produced by macrophages preincubated with TGRLP was cholesterol-poor, markedly heterogeneous and of higher peak flotation density (d 1.14–1.18) when compared with particles produced after preincubation with LDL. Both the conditioned medium and the isolated (d < 1.21) LpE-containing fraction, yielded by macrophages preincubated with TGRLP, seemed poorer at inducing cholesterol efflux than the equivalent fractions from cells preincubated with LDL, as judged by [3H]cholesterol efflux from untreated ‘naïve’ macrophages. Thus, although the interaction of TGRLP with macrophages can enhance apoE output from these cells, the LpE particles produced seem to be relatively inefficient mediators of cholesterol efflux. These factors might contribute to the increased risk of atherosclerosis in individuals with Type II diabetes.
Human NADPH-P450 oxidoreductase modulates the level of cytochrome P450 CYP2D6 holoprotein via haem oxygenase-dependent and -independent pathways
Authors: DING Shaohong , YAO Denggao , DEENI Yusuf Y. , BURCHELL Brian , WOLF C. Roland , FRIEDBERG Thomas
Published: August 2015

NADPH-P450 oxidoreductase (CPR) is essential for the activity of cytochrome P450 (P450). Previous studies demonstrated that CPR regulates the levels of various P450 isoforms in vitro. We investigated the mechanistic basis for this regulation. By transfection of Chinese hamster ovary DUKXB11 cells we obtained the cell line DUKX/2D6, which expressed human CYP2D6, a P450 isoform. Subsequently, DUKX/2D6 cells were transfected with human CPR cDNA to generate the cell line DUKX/2D6/CPR-3. Expression of recombinant CPR decreased the level of spectrally detectable CYP2D6 holoprotein in DUKX/2D6/CPR-3 cells by 70%, whereas the level of immunodetectable apoprotein remained unchanged. Addition of the radical scavenger DMSO increased levels of CYP2D6 holoenzyme in DUKX/2D6/CPR-3 cells but not in DUKX/2D6 cells. A similar effect was noted when cells were grown in the presence of hemin. Importantly, combined treatment with DMSO and hemin increased levels of CYP2D6 holoenzyme in DUKX/2D6/CPR-3 but not in DUKX/2D6 cells even further than either treatment alone. None of these treatments affected the level of immunodetectable CYP2D6. This demonstrates that expression of CPR increases production of damaging radicals but also that CPR may alter haem homoeostasis. In agreement with this, the activity of haem oxygenase, a rate-limiting enzyme in haem metabolism, was compared with that in DUKX/DHFR control cells (expressing dihydrofolate reductase), and was 3-fold higher in DUKX/2D6/CPR-3 but similar in DUKX/2D6 cells. Furthermore, treatment of cells with sodium arsenite increased levels of haem oxygenase concomitant with a marked decrease of spectrally detectable CYP2D6 and a rise in levels of ferritin, which sequesters free iron released from the destruction of haem. These data demonstrate that CPR regulates P450 activity by supplying electrons and also by altering P450 levels via radical-and haem oxygenase-mediated pathways.
What the structure of a calcium pump tells us about its mechanism
Authors: LEE Anthony G. , EAST J. Malcolm
Published: August 2015

The report of the crystal structure of the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum in its Ca2+-bound form [Toyoshima, Nakasako and Ogawa (2000) Nature (London) 405, 647–655] provides an opportunity to interpret much kinetic and mutagenic data on the ATPase in structural terms. There are no large channels leading from the cytoplasmic surface to the pair of high-affinity Ca2+ binding sites within the transmembrane region. One possible access pathway involves the charged residues in transmembrane α-helix M1, with a Ca2+ ion passing through the first site to reach the second site. The Ca2+-ATPase also contains a pair of binding sites for Ca2+ that are exposed to the lumen. In the four-site model for transport, phosphorylation of the ATPase leads to transfer of the two bound Ca2+ ions from the cytoplasmic to the lumenal pair of sites. In the alternating four-site model for transport, phosphorylation leads to release of the bound Ca2+ ions directly from the cytoplasmic pair of sites, linked to closure of the pair of lumenal binding sites. The lumenal pair of sites could involve a cluster of conserved acidic residues in the loop between M1 and M2. Since there is no obvious pathway from the high-affinity sites to the lumenal surface of the membrane, transport of Ca2+ ions must involve a significant change in the packing of the transmembrane α-helices. The link between the phosphorylation domain and the pair of high-affinity Ca2+ binding sites is probably provided by two small helices, P1 and P2, in the phosphorylation domain, which contact the loop between transmembrane α-helices M6 and M7.
Characterization of matrix metalloproteinase-26, a novel metalloproteinase widely expressed in cancer cells of epithelial origin
Authors: MARCHENKO George N. , RATNIKOV Boris I. , ROZANOV Dmitry V. , GODZIK Adam , DERYUGINA Elena I. , STRONGIN Alex Y.
Published: August 2015

Identification of expanding roles for matrix metalloproteinases (MMPs) in complex regulatory processes of tissue remodelling has stimulated the search for genes encoding proteinases with unique functions, regulation and expression patterns. By using a novel cloning strategy, we identified three previously unknown human MMPs, i.e. MMP-21, MMP-26 and MMP-28, in comprehensive gene libraries. The present study is focused on the gene and the protein of a novel MMP, MMP-26. Our findings show that MMP-26 is specifically expressed in cancer cells of epithelial origin, including carcinomas of lung, prostate and breast. Several unique structural and regulatory features, including an unusual ‘cysteine-switch’ motif, discriminate broad-spectrum MMP-26 from most other MMPs. MMP-26 efficiently cleaves fibrinogen and extracellular matrix proteins, including fibronectin, vitronectin and denatured collagen. Protein sequence, minimal modular domain structure, exon–intron mapping and computer modelling demonstrate similarity between MMP-26 and MMP-7 (matrilysin). However, substrate specificity and transcriptional regulation, as well as the functional role of MMP-26 and MMP-7 in cancer, are likely to be distinct. Despite these differences, matrilysin-2 may be a suitable trivial name for MMP-26. Our observations suggest an important specific function for MMP-26 in tumour progression and angiogenesis, and confirm and extend the recent findings of other authors [Park, Ni, Gerkema, Liu, Belozerov and Sang (2000) J. Biol. Chem. 275, 20540–20544; Uría and López-Otín (2000) Cancer Res. 60, 4745–4751; de Coignac, Elson, Delneste, Magistrelli, Jeannin, Aubry, Berthier, Schmitt, Bonnefoy and Gauchat (2000) Eur. J. Biochem. 267, 3323–3329].