bluuebunny/crossref_metadata_2025 huggingface dataset:
Oxidative dimerization in metallothionein is a result of intermolecular disulphide bonds between cysteines in the α-domain
Authors: ZANGGER Klaus , SHEN Gong , ÖZ Gülin , OTVOS James D. , ARMITAGE Ian M.
Published: August 2015

Upon storage under aerobic conditions metallothioneins (MTs) form a new species, which is characterized by a molecular mass approximately twice the size of monomeric MT and shifted 113/111Cd- and 1H-NMR resonances. The investigation of this oxidative dimerization process by NMR spectroscopy allowed us to structurally characterize this MT species that has been described to occur in vivo and might be synthesized under conditions of oxidative stress. The oxidative dimer was characterized by the formation of an intermolecular cysteine disulphide bond involving the α-domain, and a detailed analysis of chemical shift changes and intermolecular nuclear Overhauser effects points towards a disulphide bond involving Cys36. In contrast to the metal-bridged (non-oxidative) dimerization, the metal–cysteine cluster structures in both MT domains remain intact and no conformational exchange or metal–metal exchange was observed. Also in contrast to the many recently reported oxidative processes which involve the β-domain cysteine groups and result in the increased dynamics of the bound metal ions in this N-terminal domain, we found no evidence for any increased dynamics in the α-domain metals following this oxidation. Therefore these findings provide additional corroboration that metal binding in the C-terminal α-domain is rather tight, even under conditions of a changing cellular oxidation potential, compared with the more labile/dynamic nature of the metals in the N-terminal β-domain cluster under similar conditions.
Identification of a novel integral plasma membrane protein induced during adipocyte differentiation
Authors: ALBREKTSEN Tatjana , RICHTER Henrijette E. , CLAUSEN Jes T. , FLECKNER Jan
Published: August 2015

Adipocyte differentiation is co-ordinately regulated by several transcription factors and is accompanied by changes in the expression of a variety of genes. Using mRNA differential display analysis, we have isolated a novel mRNA, DD16, specifically induced during the course of adipocyte differentiation. DD16 mRNAs are present in several tissues, but among the tissues tested, a remarkably higher level of expression was found in white adipose tissue. The DD16 cDNA encoded a polypeptide of 415 amino acids containing a single N-glycosylation site and an N-terminal hydrophobic stretch of 19 amino acids forming a transmembrane segment, indicating that DD16 is a glycosylated membrane-bound protein. Polyclonal antibodies raised against the DD16 peptide detected immunoreactive DD16 in membrane fractions, notably the plasma membrane. Association of DD16 with the plasma membrane was further confirmed by biotinylation studies of cell surface proteins, suggesting that DD16 is an integral plasma membrane protein. Therefore we propose to give DD16 the name APMAP (Adipocyte Plasma Membrane-Associated Protein). Although the biological function of this polypeptide is presently unknown, our data suggest that APMAP may function as a novel protein involved in the cross-talk of mature adipocytes with the environment.
Functional analysis of the interleukin-1-receptor-associated kinase (IRAK-1) in interleukin-1β-stimulated nuclear factor κB (NF-κB) pathway activation: IRAK-1 associates with the NF-κB essential modulator (NEMO) upon receptor stimulation
Authors: COOKE Emma-Louise , UINGS Iain J. , XIA Chulin L. , WOO Patricia , RAY Keith P.
Published: August 2015

The interleukin-1 (IL-1)-receptor-associated kinase (IRAK-1) is essential for IL-1-stimulated nuclear factor κB (NF-κB) activation. To study the role of IRAK-1 in IL-1β signalling, we have generated a set of IRAK-1 variants that express distinct domains of IRAK-1 either alone or in combination and have examined their effects on an NF-κB-responsive reporter in HeLa cells. Unlike full-length IRAK-1, the deletion mutants were unable to activate NF-κB in the absence of cytokine stimulation. However, an IRAK-1 variant lacking only the N-terminal domain retained the ability of the full-length protein to potentiate both IL-1β and tumour necrosis factor α (TNFα)-induced NF-κB activation. In contrast, expression of the N-terminus or the C-terminus of IRAK-1, or a fusion protein incorporating both domains, inhibited both IL-1β- and TNFα-induced effects. Expression of an IRAK-1 variant lacking only the C-terminal domain preferentially inhibited IL-1β versus TNFα-induced NF-κB activation. These data suggest that the C-terminal domain may link IRAK-1 to downstream signalling components common to both the IL-1 and TNF pathways. Furthermore, we have demonstrated that endogenous IRAK-1 becomes phosphorylated upon IL-1β treatment and can be detected along with NF-κB essential modulator (NEMO) and IκB kinase β (IKKβ) in high-molecular-mass complexes of 600–800kDa. Moreover, IRAK-1 could be detected in NEMO immunoprecipitates from IL-1β-stimulated cells. We conclude that IRAK-1 mediates IL-1β signal transduction through a ligand-dependent association of IRAK-1 with the IKK complex.
The result of equilibrium-constant calculations strongly depends on the evaluation method used and on the type of experimental errors
Authors: FUCHS Hendrik , GESSNER Reinhard
Published: August 2015

The determination of equilibrium constants is a widespread tool both to understand and to characterize protein–protein interactions. A variety of different methods, among them Scatchard analysis, is used to calculate these constants. Although more than 1000 articles dealing with equilibrium constants are published every year, the effects of experimental errors on the results are often disregarded when interpreting the data. In the present study we theoretically analysed the effect of various types of experimental errors on equilibrium constants derived by three different methods. A computer simulation clearly showed that certain experimental errors, namely inaccurate background correction, inexact calibration, saturation effects, slow kinetics and simple scattering, can adversely affect the result. The analysis further revealed that, for a given type of error, the same data set can produce different results depending on the method used.
Different combinations of the heat-shock cognate protein 70 (hsc70) C-terminal functional groups are utilized to interact with distinct tetratricopeptide repeat-containing proteins
Authors: WU Shin-Jen , LIU Fu-Hwa , HU Su-Ming , WANG Chung
Published: August 2015

A group of tetratricopeptide repeat (TPR)-containing proteins has been shown to interact with the C-terminal domain of the 70kDa heat-shock cognate protein (hsc70). In the present study, the effect of the TPR-containing proteins, including the C-terminus of hsc70-interacting protein (CHIP), TPR1 and human glutamine-rich TPR-containing protein (hSGT), on refolding of luciferase by DnaJ and hsc70 was investigated. These proteins inhibited the restoration of luciferase activity by the chaperones. The inhibitory effect exerted by TPR1 and hSGT depended upon their binding to hsc70. However, the interaction with hsc70 did not appear to be required for the inhibition of luciferase refolding by CHIP. We also demonstrate that the peptide, GPTIEEVD, corresponding to the C-terminal end of hsc70, abolished the association of [3H]hsc70 with CHIP, TPR1 and hSGT. This implied that the GPTIEEVD motif of hsc70 was responsible for interacting with these TPR-containing proteins. However, the GGXP-repeats (where X is any aliphatic residue), another C-terminal conserved motif of vertebrate hsc70s, were not essential for interacting with the TPR-containing proteins. On the basis of mutagenesis studies, it was clear that a unique combination of the functional groups in the GPTIEEVD motif were utilized to interact with each TPR-containing protein, suggesting that inhibitors can be designed and used to elucidate the functional role of these interactions.
Functional properties of Drosophila inositol trisphosphate receptors
Authors: SWATTON Jane E. , MORRIS Stephen A. , WISSING Frank , TAYLOR Colin W.
Published: August 2015

The functional properties of the only inositol trisphosphate (IP3) receptor subtype expressed in Drosophila were examined in permeabilized S2 cells. The IP3 receptors of S2 cells bound (1,4,5)IP3 with high affinity (Kd = 8.5±1.1nM), mediated positively co-operative Ca2+ release from a thapsigargin-sensitive Ca2+ store (EC50 = 75±4nM, Hill coefficient = 2.1±0.2), and they were recognized by an antiserum to a peptide conserved in all IP3 receptor subtypes in the same way as mammalian IP3 receptors. As with mammalian IP3 receptors, (2,4,5)IP3 (EC50 = 2.3±0.3μM) and (4,5)IP2 (EC50 approx. 10μM) were approx. 20- and 100-fold less potent than (1,4,5)IP3. Adenophostin A, which is typically approx. 10-fold more potent than IP3 at mammalian IP3 receptors, was 46-fold more potent than IP3 in S2 cells (EC50 = 1.67±0.07nM). Responses to submaximal concentrations of IP3 were quantal and IP3-evoked Ca2+ release was biphasically regulated by cytosolic Ca2+. Using rapid superfusion to examine the kinetics of IP3-evoked Ca2+ release from S2 cells, we established that IP3 (10μM) maximally activated Drosophila IP3 receptors within 400ms. The activity of the receptors then slowly decayed (t1/2 = 2.03±0.07s) to a stable state which had 47±1% of the activity of the maximally active state. We conclude that the single subtype of IP3 receptor expressed in Drosophila has similar functional properties to mammalian IP3 receptors and that analyses of IP3 receptor function in this genetically tractable organism are therefore likely to contribute to understanding the roles of mammalian IP3 receptors.
Identification of the autophosphorylation sites and characterization of their effects in the protein kinase DYRK1A
Authors: HIMPEL Sunke , PANZER Pascal , EIRMBTER Klaus , CZAJKOWSKA Hanna , SAYED Muhammed , PACKMAN Len C. , BLUNDELL Tom , KENTRUP Heiner , GRÖTZINGER Joachim , JOOST Hans-G. , BECKER Walter
Published: August 2015

Protein kinases of the DYRK (‘dual-specificity tyrosine-regulated kinase’) family are characterized by a conserved Tyr-Xaa-Tyr motif (Tyr-319–Tyr-321) in a position exactly corresponding to the activation motif of the mitogen-activated protein kinase (MAP kinase) family (Thr-Xaa-Tyr). In a molecular model of the catalytic domain of DYRK1A, the orientation of phosphorylated Tyr-321 is strikingly similar to that of Tyr-185 in the known structure of the activated MAP kinase, extracellular-signal-regulated kinase 2. Consistent with our model, substitution of Tyr-321 but not of Tyr-319 by phenylalanine markedly reduced the enzymic activity of recombinant DYRK1A expressed in either Escherichia coli or mammalian cells. Direct identification of phosphorylated residues by tandem MS confirmed that Tyr-321, but not Tyr-319, was phosphorylated. When expressed in COS-7 cells, DYRK1A was found to be fully phosphorylated on Tyr-321. A catalytically inactive mutant of DYRK1A contained no detectable phosphotyrosine, indicating that Tyr-321 is autophosphorylated by DYRK1A. MS identified Tyr-111 and Ser-97 as additional autophosphorylation sites in the non-catalytic N-terminal domain of bacterially expressed DYRK1A. Enzymic activity was not affected in the DYRK1A-Y111F mutant. The present experimental data and the molecular model indicate that the activity of DYRK1A is dependent on the autophosphorylation of a conserved tyrosine residue in the activation loop.
Cladribine induces apoptosis in human leukaemia cells by caspase-dependent and -independent pathways acting on mitochondria
Authors: MARZO Isabel , PÉREZ-GALÁN Patricia , GIRALDO Pilar , RUBIO-FÉLIX Daniel , ANEL Alberto , NAVAL Javier
Published: August 2015

We have studied the role of caspases and mitochondria in apoptosis induced by 2-chloro-2′-deoxyadenosine (cladribine) in several human leukaemic cell lines. Cladribine treatment induced mitochondrial transmembrane potential (ΔΨm) loss, phosphatidylserine exposure, caspase activation and development of typical apoptotic morphology in JM1 (pre-B), Jurkat (T) and U937 (promonocytic) cells. Western-blot analysis of cell extracts revealed the activation of at least caspases 3, 6, 8 and 9. Co-treatment with Z-VAD-fmk (benzyloxy-carbonyl-Val-Ala-Asp-fluoromethylketone), a general caspase inhibitor, significantly prevented cladribine-induced death in JM1 and Jurkat cells for the first ≈ 40h, but not for longer times. Z-VAD-fmk also partly prevented some morphological and biochemical features of apoptosis in U937 cells, but not cell death. Co-incubation with selective caspase inhibitors Ac-DEVD-CHO (N-acetyl-Asp-Glu-Val-Asp-aldehyde),Ac-LEHD-CHO(N-acetyl-Leu-Glu-His-Asp-aldehyde) or Z-IETD-fmk (benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone), inhibition of protein synthesis with cycloheximide or cell-cycle arrest with aphidicolin did not prevent cell death. Overexpression of Bcl-2, but not CrmA, efficiently prevented death in Jurkat cells. In all cell lines, death was always preceded by ΔΨm loss and accompanied by the translocation of the protein apoptosis-inducing factor (AIF) from mitochondria to the nucleus. These results suggest that caspases are differentially involved in induction and execution of apoptosis depending on the leukaemic cell lineage. In any case, ΔΨm loss marked the point of no return in apoptosis and may be caused by two different pathways, one caspase-dependent and the other caspase-independent. Execution of apoptosis was always performed after ΔΨm loss by a caspase-9-triggered caspase cascade and the action of AIF.
Unfolding and refolding of a quinone oxidoreductase: α-crystallin, a molecular chaperone, assists its reactivation
Authors: GOENKA Shradha , RAMAN Bakthisaran , RAMAKRISHNA Tangirala , RAO Ch. Mohan
Published: August 2015

α-Crystallin, a member of the small heat-shock protein family and present in vertebrate eye lens, is known to prevent the aggregation of other proteins under conditions of stress. However, its role in the reactivation of enzymes from their non-native inactive states has not been clearly demonstrated. We have studied the effect of α-crystallin on the refolding of ∊-crystallin, a quinone oxidoreductase, from its different urea-denatured states. Co-refolding ∊-crystallin from its denatured state in 2.5M urea with either calf eye lens α-crystallin or recombinant human αB-crystallin could significantly enhance its reactivation yield. αB-crystallin was found to be more efficient than αA-crystallin in chaperoning the refolding of ∊-crystallin. In order to understand the nature of the denatured state(s) of ∊-crystallin that can interact with α-crystallin, we have investigated the unfolding pathway of ∊-crystallin. We find that it unfolds through three distinct intermediates: an altered tetramer, a partially unfolded dimer, which is competent to fold back to its active state, and a partially unfolded monomer. The partially unfolded monomer is inactive, exhibits highly exposed hydrophobic surfaces and has significant secondary structural elements with little or no tertiary structure. This intermediate does not refold into the active state without assistance. α-Crystallin provides the required assistance and improves the reactivation yield several-fold.
A distal region, hypersensitive to DNase I, plays a key role in regulating rabbit whey acidic protein gene expression
Authors: MILLOT Benjamin , FONTAINE Marie-Louise , THEPOT Dominique , DEVINOY Eve
Published: August 2015

The aim of the present study was to identify the functional domains of the upstream region of the rabbit whey acidic protein (WAP) gene, which has been used with considerable efficacy to target the expression of several foreign genes to the mammary gland. We have shown that this region exhibits three sites hypersensitive to DNase I digestion in the lactating mammary gland, and that all three sites harbour elements which can bind to Stat5 in vitro in bandshift assays. However, not all hypersensitive regions are detected at all stages from pregnancy to weaning, and the level of activated Stat5 detected in the rabbit mammary gland is low except during lactation. We have studied the role of the distal site, which is only detected during lactation, in further detail. It is located within a 849bp region that is required to induce a strong expression of the chloramphenicol acetyltransferase reporter gene in transfected mammary cells. Taken together, these results suggest that this region, centred around a Stat5-binding site and surrounded by a variable chromatin structure during the pregnancy–lactation cycle, may play a key role in regulating the expression of this gene in vivo. Furthermore, this distal region exhibits sequence similarity with a region located around 3kb upstream of the mouse WAP gene. The existence of such a distal region in the mouse WAP gene may explain the differences in expression between 4.1 and 2.1kb mouse WAP constructs.
GLUT4 translocation precedes the stimulation of glucose uptake by insulin in muscle cells: potential activation of GLUT4 via p38 mitogen-activated protein kinase
Authors: SOMWAR Romel , KIM David Y. , SWEENEY Gary , HUANG Carol , NIU Wenyan , LADOR Crina , RAMLAL Toolsie , KLIP Amira
Published: August 2015

We previously reported that SB203580, an inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), attenuates insulin-stimulated glucose uptake without altering GLUT4 translocation. These results suggested that insulin might activate GLUT4 via a p38 MAPK-dependent pathway. Here we explore this hypothesis by temporal and kinetic analyses of the stimulation of GLUT4 translocation, glucose uptake and activation of p38 MAPK isoforms by insulin. In L6 myotubes stably expressing GLUT4 with an exofacial Myc epitope, we found that GLUT4 translocation (t1/2 = 2.5min) preceded the stimulation of 2-deoxyglucose uptake (t1/2 = 6min). This segregation of glucose uptake from GLUT4 translocation became more apparent when the two parameters were measured at 22°C. Preincubation with the p38 MAPK inhibitors SB202190 and SB203580 reduced insulin-stimulated transport of either 2-deoxyglucose or 3-O-methylglucose by 40–60%. Pretreatment with SB203580 lowered the apparent transport Vmax of insulin-mediated 2-deoxyglucose and 3-O-methylglucose without any significant change in the apparent Km for either hexose. The IC50 values for the partial inhibition of 2-deoxyglucose uptake by SB202190 and SB203580 were 1 and 2μM respectively, and correlated with the IC50 for full inhibition of p38 MAPK by the two inhibitors in myotubes (2 and 1.4μM, respectively). Insulin caused a dose- (EC50 = 15nM) and time- (t1/2 = 3min) dependent increase in p38 MAPK phosphorylation, which peaked at 10min (2.3±0.3-fold). In vitro kinase assay of immunoprecipitates from insulin-stimulated myotubes showed activation of p38α (2.6±0.3-fold) and p38β (2.3±0.2-fold) MAPK. These results suggest that activation of GLUT4 follows GLUT4 translocation and that both mechanisms contribute to the full stimulation of glucose uptake by insulin. Furthermore, activation of GLUT4 may occur via an SB203580-sensitive pathway, possibly involving p38 MAPK.
Superficial disposition of the N-terminal region of the surfactant protein SP-C and the absence of specific SP-B–SP-C interactions in phospholipid bilayers
Authors: PLASENCIA Inés , CRUZ Antonio , CASALS Cristina , PÉREZ-GIL Jesús
Published: August 2015

A dansylated form of porcine surfactant-associated protein C (Dns-SP-C), bearing a single dansyl group at its N-terminal end, has been used to characterize the lipid–protein and protein–protein interactions of SP-C reconstituted in phospholipid bilayers, using fluorescence spectroscopy. The fluorescence emission spectrum of Dns-SP-C in phospholipid bilayers is similar to the spectrum of dansyl-phosphatidylethanolamine, and indicates that the N-terminal end of the protein is located at the surface of the membranes and is exposed to the aqueous environment. In membranes containing phosphatidylglycerol (PG), the fluorescence of Dns-SP-C shows a 3-fold increase with respect to the fluorescence of phosphatidylcholine (PC), suggesting that electrostatic lipid–protein interactions induce important effects on the structure and disposition of the N-terminal segment of the protein in these membranes. This effect saturates above 20% PG molar content in the bilayers. The parameters for the interaction of Dns-SP-C with PC or PG have been estimated from the changes induced in the fluorescence emission spectrum of the protein. The protein had similar Kd values for its interaction with the different phospholipids tested, of the order of a few micromolar. Cooling of Dns-SP-C-containing dipalmitoyl PC bilayers to temperatures below the phase transition of the phospholipid produced a progressive blue-shift of the fluorescence emission of the protein. This effect is interpreted as a consequence of the transfer of the N-terminal segment of the protein into less polar environments that originate during protein lateral segregation. This suggests that conformation and interactions of the N-terminal segment of SP-C could be important in regulating the lateral distribution of the protein in surfactant bilayers and monolayers. Potential SP-B–SP-C interactions have been explored by analysing fluorescence resonance energy transfer (RET) from the single tryptophan in porcine SP-B to dansyl in Dns-SP-C. RET has been detected in samples where native SP-B and Dns-SP-C were concurrently reconstituted in PC or PG bilayers. However, the analysis of the dependence of RET on the protein density excluded specific SP-B–Dns-SP-C associations.
Human acyl-CoA:diacylglycerol acyltransferase is a tetrameric protein
Authors: CHENG Dong , MEEGALLA Rupalie L. , HE Bokang , CROMLEY Debra A. , BILLHEIMER Jeffery T. , YOUNG Peter R.
Published: August 2015

Acyl-CoA:diacylglycerol acyltransferase (DGAT) is an integral membrane enzyme that catalyses the last step of triacylglycerol synthesis from diacylglycerol and acyl-CoA. Here we provide experimental evidence that DGAT is a homotetramer. Although the predicted molecular mass of human DGAT protein is 55kDa, CHAPS-solubilized recombinant human DGAT was eluted in fractions over 150kDa on gel-filtration chromatography. Cross-linking of recombinant DGAT in membranes with disuccinimidyl suberate yielded bands corresponding to the dimer (108kDa) and the tetramer (214kDa) in SDS/PAGE. Finally, when two differently epitope-tagged forms of DGAT were co-transfected into mammalian cells, they could be co-immunoprecipitated. From a human adipose tissue cDNA library we cloned a cDNA encoding a novel splice variant of DGAT (designated DGATsv) that contained a 77nt insert of unspliced intron with an in-frame stop codon. This resulted in a truncated form of DGAT that terminated at Arg-387, deleting 101 residues from the C-terminus containing the putative active site. DGATsv was enzymically inactive when transfected in HEK-293E cells but was still able to form dimer and tetramer on cross-linking, indicating that the ability to form tetramers resides in the N-terminal region. When co-expressed in HEK-293E cells, DGATsv did not inhibit the activity of full-length DGAT, suggesting that the subunits of DGAT catalyse triacylglycerol synthesis independently.
Cannabinoid-receptor-independent cell signalling by N-acylethanolamines
Authors: BERDYSHEV Evgueni V. , SCHMID Patricia C. , KREBSBACH Randy J. , HILLARD Cecilia J. , HUANG Chuanshu , CHEN Nanyue , DONG Zigang , SCHMID Harald H.O.
Published: August 2015

Anandamide and other polyunsaturated N-acylethanolamines (NAEs) exert biological activity by binding to cannabinoid receptors. These receptors are linked to Gi/o proteins and their activation leads to extracellular-signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAP kinase) activation, inhibition of cAMP-dependent signalling and complex changes in the expression of various genes. Saturated and monounsaturated NAEs cannot bind to cannabinoid receptors and may thus mediate cell signalling through other targets. Here we report that both saturated/monounsaturated NAEs and anandamide (20:4n-6 NAE) stimulate cannabinoid-receptor-independent ERK phosphorylation and activator protein-1 (AP-1)-dependent transcriptional activity in mouse epidermal JB6 cells. Using a clone of JB6 P+ cells with an AP-1 collagen–luciferase reporter construct, we found that 16:0, 18:1n-9, 18:1n-7, 18:2n-6 and 20:4n-6 NAEs stimulated AP-1-dependent transcriptional activity up to 2-fold, with maximal stimulation at approx. 10–15μM. Higher NAE concentrations had toxic effects mediated by alterations in mitochondrial energy metabolism. The AP-1 stimulation appeared to be mediated by ERK but not JNK or p38 signalling pathways, because all NAEs stimulated ERK1/ERK2 phosphorylation without having any effect on JNK or p38 kinases. Also, overexpression of dominant negative ERK1/ERK2 kinases completely abolished NAE-induced AP-1 activation. In contrast with 18:1n-9 NAE and anandamide, the cannabinoid receptor agonist WIN 55,212-2 did not stimulate AP-1 activity and inhibited ERK phosphorylation. The NAE-mediated effects were not attenuated by pertussis toxin and appeared to be NAE-specific, as a close structural analogue, oleyl alcohol, failed to induce ERK phosphorylation. The data support our hypothesis that the major saturated and monounsaturated NAEs are signalling molecules acting through intracellular targets without participation of cannabinoid receptors.
Angiotensin II inhibits insulin-stimulated phosphorylation of eukaryotic initiation factor 4E-binding protein-1 in proximal tubular epithelial cells
Authors: SENTHIL Duraisamy , FAULKNER Jennifer L. , GHOSH CHOUDHURY Goutam , ABBOUD Hanna E. , KASINATH Balakuntalam S.
Published: August 2015

Interaction between angiotensin II, which binds a G-protein-coupled receptor, and insulin, a ligand for receptor tyrosine kinase, was examined in renal proximal tubular epithelial cells. Augmented protein translation by insulin involves activation of eukaryotic initiation factor 4E (eIF4E) which follows the release of the factor from a heterodimeric complex by phosphorylation of its binding protein, 4E-BP1. Angiotensin II (1nM) or insulin (1nM) individually stimulated 4E-BP1 phosphorylation. However, pre-incubation with angiotensin II abrogated insulin-induced phosphorylation of 4E-BP1, resulting in persistent binding to eIF4E. Although angiotensin II and insulin individually activated phosphoinositide 3-kinase and extracellular signal-regulated kinase (ERK)-1/−2-type mitogen-activated protein (MAP) kinase, pre-incubation with angiotensin II abolished insulin-induced stimulation of these kinases, suggesting more proximal events in insulin signalling may be intercepted. Pretreatment with angiotensin II markedly inhibited insulin-stimulated tyrosine phosphorylation of insulin-receptor β-chain and insulin-receptor substrate 1. Losartan prevented angiotensin II inhibition of insulin-induced ERK-1/−2-type MAP kinase activation and 4E-BP1 phosphorylation, suggesting mediation of the effect of angiotensin II by its type 1 receptor. Insulin-stimulated de novo protein synthesis was also abolished by pre-incubation with angiotensin II. These data show that angiotensin II inhibits 4E-BP1 phosphorylation and stimulation of protein synthesis induced by insulin by interfering with proximal events in insulin signalling. Our data provide a mechanistic basis for insulin insensitivity induced by angiotensin II.
BFIT, a unique acyl-CoA thioesterase induced in thermogenic brown adipose tissue: cloning, organization of the human gene and assessment of a potential link to obesity
Authors: ADAMS Sean H. , CHUI Clarissa , SCHILBACH Sarah L. , YU Xing Xian , GODDARD Audrey D. , GRIMALDI J. Christopher , LEE James , DOWD Patrick , COLMAN Steven , LEWIN David A.
Published: August 2015

We hypothesized that certain proteins encoded by temperature-responsive genes in brown adipose tissue (BAT) contribute to the remarkable metabolic shifts observed in this tissue, thus prompting a differential mRNA expression analysis to identify candidates involved in this process in mouse BAT. An mRNA species corresponding to a novel partial-length gene was found to be induced 2–3-fold above the control following cold exposure (4°C), and repressed ≈ 70% by warm acclimation (33°C, 3 weeks) compared with controls (22°C). The gene displayed robust BAT expression (i.e. ≈ 7–100-fold higher than other tissues in controls). The full-length murine gene encodes a 594 amino acid (≈ 67kDa) open reading frame with significant homology to the human hypothetical acyl-CoA thioesterase KIAA0707. Based on cold-inducibility of the gene and the presence of two acyl-CoA thioesterase domains, we termed the protein brown-fat-inducible thioesterase (BFIT). Subsequent analyses and cloning efforts revealed the presence of a novel splice variant in humans (termed hBFIT2), encoding the orthologue to the murine BAT gene. BFIT was mapped to syntenic regions of chromosomes 1 (human) and 4 (mouse) associated with body fatness and diet-induced obesity, potentially linking a deficit of BFIT activity with exacerbation of these traits. Consistent with this notion, BFIT mRNA was significantly higher (≈ 1.6–2-fold) in the BAT of obesity-resistant compared with obesity-prone mice fed a high-fat diet, and was 2.5-fold higher in controls compared with ob/ob mice. Its strong, cold-inducible BAT expression in mice suggests that BFIT supports the transition of this tissue towards increased metabolic activity, probably through alteration of intracellular fatty acyl-CoA concentration.
Identification of cathepsin L as a differentially expressed message associated with skeletal muscle wasting
Authors: DEVAL Christiane , MORDIER Sylvie , OBLED Christiane , BECHET Daniel , COMBARET Lydie , ATTAIX Didier , FERRARA Marc
Published: August 2015

Alteration of skeletal muscle protein breakdown is a hallmark of a set of pathologies, including sepsis, with negative consequences for recovery. The aim of the present study was to search for muscle markers associated with protein loss, which could help in predicting and understanding pathological wasting. With the use of differential display reverse transcription-PCR, we screened differentially expressed genes in muscle from septic rats in a long-lasting catabolic state. One clone was isolated, confirmed as being overexpressed in septic skeletal muscle and identified as encoding the lysosomal cysteine endopeptidase cathepsin L. Northern- and Western-blot analysis of cathepsin L in gastrocnemius or tibialis anterior muscles of septic rats confirmed an elevation (up to 3-fold) of both mRNA and protein levels as early as 2 days post-infection, and a further increase 6 days post-infection (up to 13-fold). At the same time, the increase in mRNAs encoding other lysosomal endopeptidases or components of the ubiquitin–proteasome pathway did not exceed 4-fold. Cathepsin L mRNA was also increased in tibialis anterior muscle of rats treated with the glucocorticoid analogue, dexamethasone, or rats bearing the Yoshida Sarcoma. The increase in cathepsin L mRNA was reduced by 40% when the tumour-bearing animals were treated with pentoxifylline, an inhibitor of tumour necrosis factor-α production. In conclusion, these results demonstrate a positive and direct correlation between cathepsin L mRNA and protein level and the intensity of proteolysis, and identify cathepsin L as an appropriate early marker of muscle wasting. Cathepsin L presumably participates in the pathological response leading to muscle loss, with glucocorticoids and tumour necrosis factor-α potentially being involved in the up-regulation of cathepsin L.
An active immunization approach to generate protective catalytic antibodies
Authors: WANG Jun , HAN Yunqing , WILKINSON Miles F.
Published: August 2015

We report that mice immunized with a phosphate immunogen produced polyclonal catalytic antibodies (PCAbs) that catalysed the hydrolysis of carbaryl, a widely used broad-spectrum carbamate insecticide that exerts toxic effects in animals and humans. The reaction catalysed by the PCAbs (IgGs) obeyed Michaelis–Menten kinetics in vitro with the following values at pH8.0 and 25°C: Km≈ 8.0μM, kcat = 4.8×10−3–5.8×10−1, kcat/knon-cat = 5.6×101–6.8×103 (where knon-cat is the rate constant of the reaction in the absence of added catalyst). The PCAbs were also active in whole sera under physiological conditions in vitro. The PCAbs induced in vivo were also active in vivo, as immunization with the phosphate immunogen decreased the mouse blood concentration of carbaryl. To our knowledge, this is the first report demonstrating that active immunization generates antibodies possessing therapeutic catalytic function in vivo. We propose that active immunization schemes that induce enzymically active antibodies may provide a highly specific therapeutic approach for degrading toxic substances.
Fatty-acid-binding proteins do not protect against induced cytotoxicity in a kidney cell model
Authors: ZIMMERMAN Aukje W. , VEERKAMP Jacques H.
Published: August 2015

Intracellular accumulation of fatty acids (FAs) is a well-described consequence of renal ischaemia and may lead to lethal cell injury. Fatty-acid-binding proteins (FABPs) are small cytosolic proteins with high affinity for FAs. They may protect vital cellular functions by binding to and promoting the metabolism of FAs, thereby reducing their intracellular concentration. In this study we investigated the putative cytoprotective role of FABPs in a Madin–Darby canine kidney (MDCK) cell model for renal damage. We studied the effects of transfection with cDNA encoding heart FABP, adipocyte FABP or liver FABP on cytotoxicity induced by chemical anoxia or FAs. Transfection of MDCK type II cells with these cDNA types caused a 5–20-fold increase in FABP content, but did not change the rate or extent of palmitate uptake. After 1h of incubation with KCN, all cell types showed reduced viability and cellular ATP content and an intracellular accumulation of non-esterified FAs. High extracellular concentrations of oleate, but not palmitate, caused a markedly decreased cell viability and cellular ATP content. Oleate accumulated in non-esterified form in these cells. Simultaneous addition of glucose ameliorated the damaging effects of KCN or oleate, indicating that glycolytic ATP could substitute for uncoupled oxidative phosphorylation. No significant differences in the effects of chemical anoxia or oleate were observed between non-transfected, mock-transfected and FABP-cDNA-transfected cells. Non-esterified FA accumulation was not reduced in any of the FABP-cDNA-transfected cell lines. In conclusion, our data do not provide evidence for a cytoprotective role of FABP in this kidney cell model.
Hormone-triggered conformational changes within the insulin-receptor ectodomain: requirement for transmembrane anchors
Authors: FLÖRKE Ralf-Rudiger , SCHNAITH Kerstin , PASSLACK Waltraud , WICHERT Marc , KUEHN Lothar , FABRY Marlies , FEDERWISCH Matthias , REINAUER Hans
Published: August 2015

Interaction between two αβ half-receptors within the (αβ)2 holoreceptor complex is required for insulin binding with high affinity and for insulin-triggered changes of size and shape. To understand the underlying structure–function relationship, two truncated receptor constructs have been characterized. Reduction in the Stokes radius and increase in the sedimentation coefficient, which are characteristic for wild-type receptors, were entirely lacking for the recombinant human insulin receptor (HIR) ectodomain (HIR-ED). Stokes radii of about 5.8nm and sedimentation coefficients of 10.2S were found for both insulin-bound and free HIR-EDs. However, attaching the membrane anchors to the ectodomain, as with the recombinant membrane-anchored ectodomain (HIR-MAED) construct, was sufficient to restore not only high-affinity hormone binding but also the marked insulin-inducible alterations in hydrodynamic properties. The Stokes radii of HIR-MAED complexes, as assessed by non-denaturing PAGE, decreased upon insulin binding from 9.5nm to 7.9nm. In parallel, the sedimentation coefficient was increased from 9.0S to 9.8S. CD and fluorescence spectroscopy of HIR-MAED revealed only minor insulin-induced changes in the secondary structure. Similarity with wild-type receptors has also been demonstrated by the differential insertion of insulin-bound and free HIR-MAED complexes into artificial bilayer membranes of Triton X-114. The results are consistent with a model of receptor function that ensures a global insulin-triggered reorientation of subdomains within the ectodomain moieties while the secondary structure is essentially retained. For the rearrangement of such subdomains, the transmembrane anchors confer essential structural constraints on the receptor ectodomain.
Canatoxin, a toxic protein from jack beans (Canavalia ensiformis), is a variant form of urease (EC 3.5.1.5): biological effects of urease independent of its ureolytic activity
Authors: FOLLMER Cristian , BARCELLOS Grace B. S. , ZINGALI Russolina B. , MACHADO Olga L. T. , ALVES Elias W. , BARJA-FIDALGO Christina , GUIMARÃES Jorge A. , CARLINI Célia R.
Published: August 2015

Canatoxin is a toxic protein from Canavalia ensiformis seeds, lethal to mice (LD50 = 2mg/kg) and insects. Further characterization of canatoxin showed that its main native form (184kDa) is a non-covalently linked dimer of a 95kDa polypeptide containing zinc and nickel. Partial sequencing of internal peptides indicated homology with urease (EC 3.5.1.5) from the same seed. Canatoxin has approx. 30% of urease's activity for urea, and Km of 2–7mM. The proteins differ in their affinities for metal ions and were separated by affinity chromatography on a Zn2+ matrix. Similar to canatoxin, urease activates blood platelets and interacts with glycoconjugates. In contrast with canatoxin, no lethality was seen in mice injected with urease (10mg/kg). Pretreatment with p-hydroxymercuribenzoate irreversibly abolished the ureolytic activity of both proteins. On the other hand, p-hydroxymercuribenzoate-treated canatoxin was still lethal to mice, and both treated proteins were fully active in promoting platelet aggregation and binding to glycoconjugates. Taken together, our data indicate that canatoxin is a variant form of urease. Moreover, we show for the first time that these proteins display several biological effects that are unrelated to their enzymic activity for urea.
Implications of adenylate kinase-governed equilibrium of adenylates on contents of free magnesium in plant cells and compartments
Authors: IGAMBERDIEV Abir U. , KLECZKOWSKI Leszek A.
Published: August 2015

On the basis of the equilibrium of adenylate kinase (AK; EC 2.7.4.3), which interconverts MgATP and free AMP with MgADP and free ADP, an approach has been worked out to calculate concentrations of free magnesium (Mg2+), based on concentrations of total ATP, ADP and AMP in plant tissues and in individual subcellular compartments. Based on reported total adenylate contents, [Mg2+] in plant tissues and organelles varies significantly depending on light and dark regimes, plant age and developmental stage. In steady-state conditions, [Mg2+] in chloroplasts is similar in light and darkness (in the millimolar range), whereas in the cytosol it is very low in the light and increases to about 0.4mM in darkness. During the dark-to-light transition (photosynthetic induction), the [Mg2+] in chloroplasts falls to low values (0.2mM or less), corresponding to a delay in photosynthetic oxygen evolution. This delay is considered to result from lower activities of Mg-dependent enzymes in the Calvin cycle. In mitochondria, the changes in [Mg2+] are similar but smoother. On the other hand, when the transition from light to darkness is considered, an initial increase in [Mg2+] occurs in both chloroplasts and mitochondria, which may be of importance for the control of key regulatory enzymes (e.g. mitochondrial malic enzyme and pyruvate dehydrogenase complex) and for processes connected with light-enhanced dark respiration. A rationale is presented for a possible role of [MgATP]/[MgADP] ratio (rather than [ATPtotal]/[ADPtotal]) as an important component of metabolic cellular control. It is postulated that assays of total adenylates may provide an accurate measure of [Mg2+] in plant tissues/cells and subcellular compartments, given that the adenylates are equilibrated by AK.
Neuronal nitric oxide synthase generates superoxide from the oxygenase domain
Authors: YONEYAMA Hirohito , YAMAMOTO Akira , KOSAKA Hiroaki
Published: August 2015

When l-arginine is depleted, neuronal nitric oxide synthase (nNOS) has been reported to generate superoxide. A flavoprotein module construct of nNOS has been demonstrated to be sufficient for superoxide production. In contrast, nNOS was reported not to be involved in superoxide formation, because such formation occurred with a mixture of the boiled enzyme and redox-active cofactors. We aimed to resolve these controversial issues by examining superoxide generation, without the addition of redox-active cofactors, by recombinant wild-type nNOS and by C415A-nNOS, which has a mutation in the haem proximal site. In a superoxide-sensitive adrenochrome assay, the initial lag period of C415A-nNOS was increased 2-fold compared with that of native nNOS. With ESR using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide, prominent signals of the superoxide adduct were obtained with wild-type nNOS, whereas an enzyme preparation boiled for 5min did not produce superoxide. Higher concentrations of NaCN (10mM) decreased superoxide formation by 63%. Although the activity of the reductase domain was intact, superoxide generation from C415A-nNOS was decreased markedly, to only 10% of that of the wild-type enzyme. These results demonstrate that nNOS truly catalyses superoxide formation, that this involves the oxygenase domain, and that full-length nNOS hinders the reductase domain from producing superoxide.
TPIP: a novel phosphoinositide 3-phosphatase
Authors: WALKER Steven M. , DOWNES C. Peter , LESLIE Nick R.
Published: August 2015

The PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumour suppressor is a phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] 3-phosphatase that plays a critical role in regulating many cellular processes by antagonizing the phosphoinositide 3-kinase signalling pathway. We have identified and characterized two human homologues of PTEN, which differ with respect to their subcellular localization and lipid phosphatase activities. The previously cloned, but uncharacterized, TPTE (transmembrane phosphatase with tensin homology) is localized to the plasma membrane, but lacks detectable phosphoinositide 3-phosphatase activity. TPIP (TPTE and PTEN homologous inositol lipid phosphatase) is a novel phosphatase that occurs in several differentially spliced forms of which two, TPIPα and TPIPβ, appear to be functionally distinct. TPIPα displays similar phosphoinositide 3-phosphatase activity compared with PTEN against PtdIns(3,4,5)P3, PtdIns(3,5)P2, PtdIns(3,4)P2 and PtdIns(3)P, has N-terminal transmembrane domains and appears to be localized on the endoplasmic reticulum. This is unusual as most signalling-lipid-metabolizing enzymes are not integral membrane proteins. TPIPβ, however, lacks detectable phosphatase activity and is cytosolic. TPIP has a wider tissue distribution than the testis-specific TPTE, with specific splice variants being expressed in testis, brain and stomach. TPTE and TPIP do not appear to be functional orthologues of the Golgi-localized and more distantly related murine PTEN2. We suggest that TPIPα plays a role in regulating phosphoinositide signalling on the endoplasmic reticulum, and might also represent a tumour suppressor and functional homologue of PTEN in some tissues.
Structural and functional characterization of the mouse fatty acid translocase promoter: activation during adipose differentiation
Authors: TEBOUL Lydia , FEBBRAIO Maria , GAILLARD Danielle , AMRI Ez-Zoubir , SILVERSTEIN Roy , GRIMALDI Paul A.
Published: August 2015

Fatty acid translocase (FAT/CD36) is a cell-surface glycoprotein that functions as a receptor/transporter for long-chain fatty acids (LCFAs), and interacts with other protein and lipid ligands. FAT/CD36 is expressed by various cell types, including platelets, monocytes/macrophages and endothelial cells, and tissues with an active LCFA metabolism, such as adipose, small intestine and heart. FAT/CD36 expression is induced during adipose cell differentiation and is transcriptionally up-regulated by LCFAs and thiazolidinediones in pre-adipocytes via a peroxisome-proliferator-activated receptor (PPAR)-mediated process. We isolated and analysed the murine FAT/CD36 promoter employing C2C12N cells directed to differentiate to either adipose or muscle. Transient transfection studies revealed that the 309bp upstream from the start of exon 1 confer adipose specific activity. Sequence analysis of this DNA fragment revealed the presence of two imperfect direct repeat-1 elements. Electrophoretic mobility-shift assay demonstrated that these elements were peroxisome-proliferator-responsive elements (PPREs). Mutagenesis and transfection experiments indicated that both PPREs co-operate to drive strong promoter activity in adipose cells. We conclude that murine FAT/CD36 expression in adipose tissue is dependent upon transcriptional activation via PPARs through binding to two PPREs located at −245 to −233bp and −120 to −108bp from the transcription start site.
myo-Inositol oxygenase: molecular cloning and expression of a unique enzyme that oxidizes myo-inositol and d-chiro-inositol
Authors: ARNER Ryan J. , PRABHU K. Sandeep , THOMPSON Jerry T. , HILDENBRANDT George R. , LIKEN Andrew D. , REDDY C. Channa
Published: August 2015

myo-Inositol oxygenase (MIOX) catalyses the first committed step in the only pathway of myo-inositol catabolism, which occurs predominantly in the kidney. The enzyme is a non-haem-iron enzyme that catalyses the ring cleavage of myo-inositol with the incorporation of a single atom of oxygen. A full-length cDNA was isolated from a pig kidney library with an open reading frame of 849bp and a corresponding protein subunit molecular mass of 32.7kDa. The cDNA was expressed in a bacterial pET expression system and an active recombinant MIOX was purified from bacterial lysates to electrophoretic homogeneity. The purified enzyme displayed the same catalytic properties as the native enzyme with Km and kcat values of 5.9mM and 11min−1 respectively. The pI was estimated to be 4.5. Preincubation with 1mM Fe2+ and 2mM cysteine was essential for the enzyme's activity. d-chiro-Inositol, a myo-inositol isomer, is a substrate for the recombinant MIOX with an estimated Km of 33.5mM. Both myo-inositol and d-chiro-inositol have been implicated in the pathogenesis of diabetes. Thus an understanding of the regulation of MIOX expression clearly represents a potential window on the aetiology of diabetes as well as on the control of various intracellular phosphoinositides and key signalling pathways.