bluuebunny/crossref_metadata_2025 huggingface dataset:
Age-related accumulation of Maillard reaction products in human articular cartilage collagen
Authors: VERZIJL Nicole , DEGROOT Jeroen , OLDEHINKEL Esther , BANK Ruud A. , THORPE Suzanne R. , BAYNES John W. , BAYLISS Michael T. , BIJLSMA Johannes W. J. , LAFEBER Floris P. J. G. , TEKOPPELE Johan M.
Published: August 2015

Non-enzymic modification of tissue proteins by reducing sugars, the so-called Maillard reaction, is a prominent feature of aging. In articular cartilage, relatively high levels of the advanced glycation end product (AGE) pentosidine accumulate with age. Higher pentosidine levels have been associated with a stiffer collagen network in cartilage. However, even in cartilage, pentosidine levels themselves represent < 1 cross-link per 20 collagen molecules, and as such cannot be expected to contribute substantially to the increase in collagen network stiffness. In the present study, we investigated a broad range of Maillard reaction products in cartilage collagen in order to determine whether pentosidine serves as an adequate marker for AGE levels. Not only did the well-characterized AGEs pentosidine, Nε-(carboxymethyl)lysine, and Nε-(carboxyethyl)lysine increase with age in cartilage collagen (all P < 0.0001), but also general measures of AGE cross-linking, such as browning and fluorescence (both P < 0.0001), increased. The levels of these AGEs are all higher in cartilage collagen than in skin collagen. As a functional measure of glycation the digestibility of articular collagen by bacterial collagenase was investigated; digestibility decreased linearly with age, proportional to the extent of glycation. Furthermore, the arginine content and the sum of the hydroxylysine and lysine content of cartilage collagen decrease significantly with age (P < 0.0001 and P < 0.01 respectively), possibly due to modification by the Maillard reaction. The observed relationship between glycation and amino acid modification has not been reported previously in vivo. Our present results indicate that extensive accumulation of a variety of Maillard reaction products occurs in cartilage collagen with age. Altogether our results support the hypothesis that glycation contributes to stiffer and more brittle cartilage with advancing age.
A novel transcriptional factor with Ser/Thr kinase activity involved in the transforming growth factor (TGF)-β signalling pathway
Authors: OHTA Shigeki , TAKEUCHI Masakazu , DEGUCHI Masashi , TSUJI Toshinaga , GAHARA Yoshinari , NAGATA Kiyoshi
Published: August 2015

Transforming growth factor-β (TGF-β) shows a variety of biological activities in various organs or cells. Recently some factors such as Smads (Sma and Mad proteins) and TGF-β activating kinase 1 (‘TAK1’) have been characterized as signalling molecules downstream of TGF-β. Several TGF-β response elements have been identified such as cAMP response element, Smad binding element, and recognition sites for activating protein-1 and stimulating protein-1 in various gene promoters. We also reported a TGF-β response element in the human C-type natriuretic peptide (CNP) gene promoter. In this paper, we report on a novel factor which regulates the TGF-β response promoter. This factor, named TSF1 (TGF-β stimulated factor 1), possessed DNA-binding ability and activated the TGF-β responsive CNP promoter or vascular endothelial growth factor gene promoter which possesses a sequence element analogous to the TGF-β responsive GC-rich element of the CNP promoter. TSF1 did not directly activate a Smads-dependent promoter from plasminogen activator inhibitor 1 gene, but it showed enhancement in co-operation with Smad3 and Smad4. Interestingly, this factor had the structural features of a Ser/Thr kinase and actually exhibited protein kinase activity. TSF1 mRNA as well as its protein level were stimulated by TGF-β treatment. Thus, TSF1 is an unique factor with two biological functions, transcriptional regulation and protein phosphorylation, that may be involved in TGF-β signals.
Human sphingosine kinase: purification, molecular cloning and characterization of the native and recombinant enzymes
Authors: PITSON Stuart M. , J. D'ANDREA Richard , VANDELEUR Lucianne , MORETTI Paul A. B. , XIA Pu , GAMBLE Jennifer R. , VADAS Mathew A. , WATTENBERG Binks W.
Published: August 2015

Sphingosine 1-phosphate (S1P) is a novel lipid messenger that has important roles in a wide variety of mammalian cellular processes including growth, differentiation and death. Basal levels of S1P in mammalian cells are generally low, but can increase rapidly and transiently when cells are exposed to mitogenic agents and other stimuli. This increase is largely due to increased activity of sphingosine kinase (SK), the enzyme that catalyses its formation. In the current study we have purified, cloned and characterized the first human SK to obtain a better understanding of its biochemical activity and possible activation mechanisms. The enzyme was purified to homogeneity from human placenta using ammonium sulphate precipitation, anion-exchange chromatography, calmodulin-affinity chromatography and gel-filtration chromatography. This resulted in a purification of over 106-fold from the original placenta extract. The enzyme was cloned and expressed in active form in both HEK-293T cells and Escherichia coli, and the recombinant E. coli-derived SK purified to homogeneity. To establish whether post-translational modifications lead to activation of human SK activity we characterized both the purified placental enzyme and the purified recombinant SK produced in E. coli, where such modifications would not occur. The premise for this study was that post-translational modifications are likely to cause conformational changes in the structure of SK, which may result in detectable changes in the physico-chemical or catalytic properties of the enzyme. Thus the enzymes were characterized with respect to substrate specificity and kinetics, inhibition kinetics and various other physico-chemical properties. In all cases, both the native and recombinant SKs displayed remarkably similar properties, indicating that post-translational modifications are not required for basal activity of human SK.
Platelet-derived-growth-factor-induced signalling in human platelets: phosphoinositide-3-kinase-dependent inhibition of platelet activation
Authors: SELHEIM Frode , FUKAMI Miriam H. , HOLMSEN Holm , VASSBOTN Flemming S.
Published: August 2015

Human platelets release platelet-derived growth factor (PDGF) from α-granules during platelet activation. We have previously shown that platelets have PDGF α-receptors, a transmembrane tyrosine kinase that takes part in negative feedback regulation during platelet activation. Here we have described a study of PDGF-induced tyrosine phosphorylation of platelet substrates and phosphoinositide 3-kinase (PI-3K) activity in collagen-stimulated platelets. By immunoblotting with phosphotyrosine antibodies of collagen-activated platelets we found that PDGF increased the phosphorylation of several platelet substrates, e.g. pp140, pp120 and pp85. PDGF inhibited collagen-induced platelet activation in the presence of inhibitors of autocrine stimulation, thus blocking the pure collagen-induced signal transduction. PDGF enhanced the collagen-induced formation of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 as measured by HPLC. Wortmannin and LY294002, two unrelated inhibitors of PI-3K, were used to investigate the role of PI-3K in PDGF-induced platelet signalling. Incubation of platelets with wortmannin and LY294002 blocked the formation of three phosphorylated inositides as well as the inhibitory effect of PDGF on collagen-induced platelet activation. We conclude that the inhibitory effect of PDGF on platelet activation is PI-3K dependent. This is the first demonstration of a negative regulatory function of 3-phosphorylated inositides in platelets.
New type of starch-binding domain: the direct repeat motif in the C-terminal region of Bacillus sp. no. 195 α-amylase contributes to starch binding and raw starch degrading
Authors: SUMITANI Jun-ichi , TOTTORI Tadashi , KAWAGUCHI Takashi , ARAI Motoo
Published: August 2015

The α-amylase from Bacillus sp. no. 195 (BAA) consists of two domains: one is the catalytic domain similar to α-amylases from animals and Streptomyces in the N-terminal region; the other is the functionally unknown domain composed of an approx. 90-residue direct repeat in the C-terminal region. The gene coding for BAA was expressed in Streptomyces lividans TK24. Three active forms of the gene products were found. The pHand thermal profiles of BAAs, and their catalytic activities for p-nitrophenyl maltopentaoside and soluble starch, showed almost the same behaviours. The largest, 69kDa, form (BAA-α) was of the same molecular mass as that of the mature protein estimated from the nucleotide sequence, and had raw-starch-binding and -degrading abilities. The second largest, 60kDa, form (BAA-β), whose molecular mass was the same as that of the natural enzyme from Bacillus sp. no. 195, was generated by proteolytic processing between the two repeat sequences in the C-terminal region, and had lower activities for raw starch binding and degrading than those of BAA-α. The smallest, 50kDa, form (BAA-γ) contained only the N-terminal catalytic domain as a result of removal of the C-terminal repeat sequence, which led to loss of binding and degradation of insoluble starches. Thus the starch adsorption capacity and raw-starch-degrading activity of BAAs depends on the existence of the repeat sequence in the C-terminal region. BAA-α was specifically adsorbed on starch or dextran (α-1,4 or α-1,6glucan), and specifically desorbed with maltose or β-cyclodextrin. These observations indicated that the repeat sequence of the enzyme was functional in the starch-binding domain (SBD). We propose the designation of the homologues to the SBD of glucoamylase from Aspergillus niger as family I SBDs, the homologues to that of glucoamylase from Rhizopus oryzae as family II, and the homologues of this repeat sequence of BAA as family III.
Transcription of genes encoding pregnancy-specific glycoproteins is regulated by negative promoter-selective elements
Authors: PANZETTA-DUTARI Graciela M. , KORITSCHONER Nicolás P. , BOCCO José L. , NORES Rodrigo , DUMUR Catherine I. , PATRITO Luis C.
Published: August 2015

The human pregnancy-specific glycoprotein (PSG) genes comprise a family of 11 highly conserved members whose expression is maximal in placental cells and marginal in other cell types. We have investigated here the molecular basis of PSG regulation by analysing a large regulatory region of the PSG-5 gene in cells that do and do not express these genes. The promoter region (-254 to -43), which does not contain a TATA-box, large GC-rich sequences or a classical initiator, was active in all cell types analysed. Additional upstream sequences up to position -3204 repressed promoter activity. Two independent repressor regions were identified and found to operate effectively in HeLa, COS-7 and HTR8/SVneo placental cells. More significantly, these negatively acting modules failed to repress a heterologous TATA-containing thymidine kinase promoter. Detailed transcriptional and DNA–protein analyses of the proximal repressor region (-605 to -254) revealed the presence of both negative and positive cis-acting elements. Disruption of the repressive functions resulted in an enhanced transcription of the reporter constructs. In conclusion, these results demonstrate that PSG-5 gene transcription is highly repressed by promoter-selective negative regulatory regions and the relief of repression allows enhanced PSG-5 gene transcription irrespective of the cell type. Furthermore, our findings suggest that PSG genes are expressed mainly through a derepression mechanism.
Multidrug resistance protein 1 regulates lipid asymmetry in erythrocyte membranes
Authors: DEKKERS David W. C. , COMFURIUS Paul , VAN GOOL Rein G. J. , BEVERS Edouard M. , ZWAAL Robert F. A.
Published: August 2015

The role of multidrug resistance protein 1 (MRP1) in the maintenance of transbilayer lipid asymmetry in the erythrocyte membrane was investigated. The transbilayer distribution of endogenous phospholipids and [(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl (NBD)-labelled lipid analogues was compared in the absence and the presence of inhibitors of MRP1. At equilibrium the transbilayer distribution of the NBD analogues (in the absence of MRP1 inhibitors) was very similar to that of the endogenous lipids. Inhibition of MRP1 by verapamil or indomethacin resulted in a shift in the amount of probe that was internalized: approx. 50% of NBD-labelled phosphatidylcholine (PtdCho) and 9% of NBD-sphingomyelin (NBD-Spm) were no longer extractable by BSA in cells treated with inhibitor, in comparison with 25% and 3% for control cells respectively. To verify whether inhibition of MRP1 also affected the distribution of the endogenous phospholipids, phospholipase A2 and sphingomyelinase were used to assess the amount of each of the various lipid classes present in the membrane outer leaflet. No shift in phospholipid distribution was observed after 5h of incubation with verapamil or indomethacin. However, after 48h of incubation with these inhibitors, significantly smaller amounts of PtdCho and Spm were present in the outer membrane leaflet. No appreciable change was observed in the distribution of phosphatidylethanolamine or phosphatidylserine. Decreased hydrolysis of PtdCho and Spm was not due to endovesicle formation, as revealed by electron microscopy. This is the first report to show that MRP1 has a role in the maintenance of the outwards orientation of endogenous choline-containing phospholipids in the erythrocyte membrane.
NMR structural determination of viscotoxin A3 from Viscum album L.
Authors: ROMAGNOLI Silvia , UGOLINI Raffaella , FOGOLARI Federico , SCHALLER Gerhard , URECH Konrad , GIANNATTASIO Matteo , RAGONA Laura , MOLINARI Henriette
Published: August 2015

The high-resolution three-dimensional structure of the plant toxin viscotoxin A3, from Viscum album L., has been determined in solution by 1H NMR spectroscopy at pH 3.6 and 12°C (the structure has been deposited in the Protein Data Bank under the id. code 1ED0). Experimentally derived restraints including 734 interproton distances from nuclear Overhauser effect measurements, 22 hydrogen bonds, 32ϕ angle restraints from J coupling measurements, together with three disulphide bridge constraints were used as input in restrained molecular dynamics, followed by minimization, using DYANA and Discover. Backbone and heavy atom root-mean-square deviations were 0.47±0.11Å (1Å = 10-10 m) and 0.85±0.13Å respectively. Viscotoxin A3 consists of two α-helices connected by a turn and a short stretch of antiparallel β-sheet. This fold is similar to that found in other thionins, such as crambin, hordothionin-α and -β, phoratoxin A and purothionin-α and -β. The difference in the observed biological activity for thionins of known structure is discussed in terms of the differences in the calculated surface potential distribution, playing an important role in their function through disruption of cell membranes. In addition, the possible role in DNA binding of the helix–turn–helix motif of viscotoxin A3 is discussed.
Overexpression of antizyme in the hearts of transgenic mice prevents the isoprenaline-induced increase in cardiac ornithine decarboxylase activity and polyamines, but does not prevent cardiac hypertrophy
Authors: MACKINTOSH Caroline A. , FEITH David J. , SHANTZ Lisa M. , PEGG Anthony E.
Published: August 2015

Two lines of transgenic mice were produced with constitutive expression of antizyme-1 in the heart, driven from the cardiac α-myosin heavy chain promoter. The use of engineered antizyme cDNA in which nucleotide 205 had been deleted eliminated the need for polyamine-mediated frameshifting, normally necessary for translation of antizyme mRNA, and thus ensured the constitutive expression of antizyme. Antizyme-1 is thought to be a major factor in regulating cellular polyamine content, acting both to inhibit ornithine decarboxylase (ODC) activity and to target it for degradation, as well as preventing polyamine uptake. The two transgenic lines had substantial, but different, levels of antizyme in the heart, as detected by Western blotting and by the ability of heart extracts to inhibit exogenous purified ODC. Despite the high levels of antizyme, endogenous ODC activity was not completely abolished, with 10– 39% remaining, depending on the transgenic line. Additionally, a relatively small decrease (30–32%) in cardiac spermidine content was observed, with levels of putrescine and spermine unaffected. Interestingly, although the two lines of transgenic mice had different antizyme expression levels, they had almost identical cardiac polyamine content. When treated with a single acute dose of isoprenaline (isoproterenol), cardiac ODC activity and putrescine content were substantially increased (by 14-fold and 4.7-fold respectively) in non-transgenic littermate mice, but these increases were completely prevented in the transgenic mice from both founder lines. Prolonged exposure to isoprenaline also caused increases in cardiac ODC activity and polyamine content, as well as an increase in cardiac growth, in non-transgenic mice. Although the increases in cardiac ODC activity and polyamine content were prevented in the transgenic mice from both founder lines, the increase in cardiac growth was unaffected. These transgenic mice thus provide a valuable model system in which to study the importance of polyamine levels in cardiac growth and electrophysiology in response to stress.
Oxygen tension limits nitric oxide synthesis by activated macrophages
Authors: MCCORMICK Charles C. , LI Wai Ping , CALERO Monica
Published: August 2015

Previous studies have established that constitutive calcium-dependent (‘low-output’) nitric oxide synthase (NOS) is regulated by oxygen tension. We have investigated the role of oxygen tension in the synthesis of NO by the ‘high-output’ calcium-independent NOS in activated macrophages. Hypoxia increased macrophage NOS gene expression in the presence of one additional activator, such as lipopolysaccharide or interferon-γ, but not in the presence of both. Hypoxia markedly reduced the synthesis of NO by activated macrophages (as measured by accumulation of nitrite and citrulline), such that, at 1% oxygen tension, NO accumulation was reduced by 80–90%. The apparent Km for oxygen calculated from cells exposed to a range of oxygen tensions was found to be 10.8%, or 137µM, O2 This value is considerably higher than the oxygen tension in tissues, and is virtually identical to that reported recently for purified recombinant macrophage NOS. The decrease in NO synthesis did not appear to be due to diminished arginine or cofactor availability, since arginine transport and NO synthesis during recovery in normoxia were normal. Analysis of NO synthesis during hypoxia as a function of extracellular arginine indicated that an altered Vmax, but not KmArg, accounted for the observed decrease in NO synthesis. We conclude that oxygen tension regulates the synthesis of NO in macrophages by a mechanism similar to that described previously for the calcium-dependent low-output NOS. Our data suggest that oxygen tension may be an important physiological regulator of macrophage NO synthesis in vivo.
Calcium-dependent conformational stability of modules 1 and 2 of human gelsolin
Authors: ZAPUN André , GRAMMATYKA Stéphane , DÉRAL Gaël , VERNET Thierry
Published: August 2015

Gelsolin modulates the actin cytoskeleton in the cytoplasm and clears the circulation of stray filaments. In vitro, gelsolin cleaves, nucleates and caps actin filaments, activities that are calcium-dependent. Both cellular and secreted forms share a sequence of 730 residues comprising six homologous modules termed G1–G6. A disulphide bond is formed in secreted G2, whereas in the cytoplasm it remains reduced. A point mutation in G2 causes an amyloidosis with neurological, ophthalmological and dermatological symptoms. This mutation does not affect the cytoplasmic form, while the secreted form is proteolysed. As a first step towards understanding how gelsolin folds and functions in different cellular compartments, we have characterized at equilibrium the urea-induced unfolding of G1 and G2, with or without calcium and/or disulphide bond. G1 and G2 both exhibit two-state unfolding behaviour and are stabilized by calcium. The disulphide bond also contributes to the stability of G2. In the absence of Ca2+ and disulphide bond, G2 adopts a non-native conformation, suggesting that folding of G2 in the cytoplasm relies on the presence of surrounding modules or other molecular partners.
Human 3α-hydroxysteroid dehydrogenase isoforms (AKR1C1–AKR1C4) of the aldo-keto reductase superfamily: functional plasticity and tissue distribution reveals roles in the inactivation and formation of male and female sex hormones
Authors: PENNING Trevor M. , BURCZYNSKI Michael E. , JEZ Joseph M. , HUNG Chien-Fu , LIN Hseuh-Kung , MA Haiching , MOORE Margaret , PALACKAL Nisha , RATNAM Kapila
Published: August 2015

The kinetic parameters, steroid substrate specificity and identities of reaction products were determined for four homogeneous recombinant human 3α-hydroxysteroid dehydrogenase (3α-HSD) isoforms of the aldo-keto reductase (AKR) superfamily. The enzymes correspond to type 1 3α-HSD (AKR1C4), type 2 3α(17β)-HSD (AKR1C3), type 3 3α-HSD (AKR1C2) and 20α(3α)-HSD (AKR1C1), and share at least 84% amino acid sequence identity. All enzymes acted as NAD(P)(H)-dependent 3-, 17- and 20-ketosteroid reductases and as 3α-, 17β- and 20α-hydroxysteroid oxidases. The functional plasticity of these isoforms highlights their ability to modulate the levels of active androgens, oestrogens and progestins. Salient features were that AKR1C4 was the most catalytically efficient, with kcat/Km values for substrates that exceeded those obtained with other isoforms by 10–30-fold. In the reduction direction, all isoforms inactivated 5α-dihydrotestosterone (17β-hydroxy-5α-androstan-3-one; 5α-DHT) to yield 5α-androstane-3α,17β-diol (3α-androstanediol). However, only AKR1C3 reduced ∆4-androstene-3,17-dione to produce significant amounts of testosterone. All isoforms reduced oestrone to 17β-oestradiol, and progesterone to 20α-hydroxy-pregn-4-ene-3,20-dione (20α-hydroxyprogesterone). In the oxidation direction, only AKR1C2 converted 3α-androstanediol to the active hormone 5α-DHT. AKR1C3 and AKR1C4 oxidized testosterone to ∆4-androstene-3,17-dione. All isoforms oxidized 17β-oestradiol to oestrone, and 20α-hydroxyprogesterone to progesterone. Discrete tissue distribution of these AKR1C enzymes was observed using isoform-specific reverse transcriptase-PCR. AKR1C4 was virtually liver-specific and its high kcat/Km allows this enzyme to form 5α/5β-tetrahydrosteroids robustly. AKR1C3 was most prominent in the prostate and mammary glands. The ability of AKR1C3 to interconvert testosterone with ∆4-androstene-3,17-dione, but to inactivate 5α-DHT, is consistent with this enzyme eliminating active androgens from the prostate. In the mammary gland, AKR1C3 will convert ∆4-androstene-3,17-dione to testosterone (a substrate aromatizable to 17β-oestradiol), oestrone to 17β-oestradiol, and progesterone to 20α-hydroxyprogesterone, and this concerted reductive activity may yield a pro-oesterogenic state. AKR1C3 is also the dominant form in the uterus and is responsible for the synthesis of 3α-androstanediol which has been implicated as a parturition hormone. The major isoforms in the brain, capable of synthesizing anxiolytic steroids, are AKR1C1 and AKR1C2. These studies are in stark contrast with those in rat where only a single AKR with positional- and stereo-specificity for 3α-hydroxysteroids exists.
Specificity and mechanism of action of some commonly used protein kinase inhibitors
Authors: DAVIES Stephen P. , REDDY Helen , CAIVANO Matilde , COHEN Philip
Published: August 2015

The specificities of 28 commercially available compounds reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases have been examined against a large panel of protein kinases. The compounds KT 5720, Rottlerin and quercetin were found to inhibit many protein kinases, sometimes much more potently than their presumed targets, and conclusions drawn from their use in cell-based experiments are likely to be erroneous. Ro 318220 and related bisindoylmaleimides, as well as H89, HA1077 and Y 27632, were more selective inhibitors, but still inhibited two or more protein kinases with similar potency. LY 294002 was found to inhibit casein kinase-2 with similar potency to phosphoinositide (phosphatidylinositol) 3-kinase. The compounds with the most impressive selectivity profiles were KN62, PD 98059, U0126, PD 184352, rapamycin, wortmannin, SB 203580 and SB 202190. U0126 and PD 184352, like PD 98059, were found to block the mitogen-activated protein kinase (MAPK) cascade in cell-based assays by preventing the activation of MAPK kinase (MKK1), and not by inhibiting MKK1 activity directly. Apart from rapamycin and PD 184352, even the most selective inhibitors affected at least one additional protein kinase. Our results demonstrate that the specificities of protein kinase inhibitors cannot be assessed simply by studying their effect on kinases that are closely related in primary structure. We propose guidelines for the use of protein kinase inhibitors in cell-based assays.
Cloning, expression and subcellular localization of two novel splice variants of mouse transient receptor potential channel 2
Authors: HOFMANN Thomas , SCHAEFER Michael , SCHULTZ Günter , GUDERMANN Thomas
Published: August 2015

Transient receptor potential channels (TRPCs) are known as candidate molecular correlates of receptor-activated or store-operated calcium entry. While functional roles for most TRPCs have been suggested, the physiological relevance of TRPC2 remains obscure. Whereas human and bovine TRPC2 are candidate pseudogenes, full-length rodent TRPC2 transcripts have been reported. There is, however, considerable controversy concerning mRNA splicing, tissue distribution and the function of these proteins. We report the molecular cloning of two novel murine TRPC2 splice variants, mTRPC2α and mTRPC2β. mTRPC2α RNA is expressed at low levels in many tissues and cell systems, while mTRPC2β is exclusively and abundantly expressed in the vomeronasal organ (VNO). When expressed in human embryonic kidney (HEK)-293 cells, mTRPC2 did not enhance receptor- or store-activated calcium entry. In order to investigate the basis of such a functional defect, mTRPC2–green fluorescent protein fusion proteins were examined by confocal microscopy. Fusion proteins were retained in endomembranes when expressed in HEK-293 or other cells of epithelial or neuronal origin. Co-expression of TRPC2 with other TRPCs did not restore plasma-membrane trafficking. We conclude that TRPC2 may form functional channels in the cellular context of the VNO, but is unlikely to have a physiological function in other tissues.

The sequences of mTRPC2α and mTRPC2β have been submitted to GenBank under the accession numbers AF230802 and AF230803 respectively.
Novel ribofuranosylnucleoside lead compounds for potent and selective inhibitors of mitochondrial thymidine kinase-2
Authors: BALZARINI Jan , ZHU Chaoyong , DE CLERCQ Erik , PÉREZ-PÉREZ Maria-Jesus , CHAMORRO Christina , CAMARASA Maria-José , KARLSSON Anna
Published: August 2015

The ribonucleoside analogues (E)-5-(2-bromovinyl)uridine (5-BV-Urd) and 3´-spiro-(4´-amino-1´,2´-oxathiole-2´,2´-dioxide)-5-methyluridine (3´-AOD-5-MeUrd) emerged as potent and selective competitive inhibitors of mitochondrial thymidine kinase (TK)-2 with respect to thymidine (Ki/Km values of 9.0 and 1.2 respectively). Cytosolic TK-1 did not show measurable affinity for these compounds. [32P]Phosphate transfer studies from [γ-32P]ATP to 5-BV-Urd and 3´-AOD-5-MeUrd revealed extremely poor substrate activity but potent inhibitory potential of the compounds. It was concluded that the ribonucleosides 5-BV-Urd and 3´-AOD-5-MeUrd represent two new lead compounds for potent and selective inhibitors of mitochondrial TK-2.
Regulation of the Rac1-specific exchange factor Tiam1 involves both phosphoinositide 3-kinase-dependent and -independent components
Authors: FLEMING Ian N. , GRAY Alexander , DOWNES C. Peter
Published: August 2015

The small GTPase Rac1 is involved in regulating membrane ruffling, gene transcription, cell-cycle progression and cell transformation, and some of these events are blocked by inhibitors of phosphoinositide 3-kinase (PI 3-kinase). Moreover, Rac1 can be activated by several guanine nucleotide exchange factors, which facilitate the release of GDP. We therefore investigated the ability of PI 3-kinase lipid products to regulate Tiam1, a Rac1-specific exchange factor. Tiam1 bound to polyphosphorylated inositol lipids in the rank order PtdIns(3,4,5)P3 > PtdIns(3,4)P2 ≫ PtdIns(4,5)P2, and this binding could be attributed to the N-terminal pleckstrin-homology (N-PH) domain. Both PtdIns(3,4,5)P3 and PtdIns(3,4)P2 enhanced Tiam1 guanine nucleotide exchange activity in vitro, but PtdIns(4,5)P2 had no effect. Co-expression of a constitutively active PI 3-kinase with Tiam1 increased the amount of GTP-bound Rac1 in vivo, a response which required the N-PH domain of Tiam1. Ectopic expression of Tiam1 caused membrane ruffling in Swiss 3T3 cells that was characterized by wortmannin-sensitive and -insensitive components, which required the N-PH domain and the C-terminal PH domain of Tiam1 respectively. These results reveal novel facets of Tiam1-dependent regulation of Rac1 function.
Mitochondrial phospholipid hydroperoxide glutathione peroxidase inhibits the release of cytochrome c from mitochondria by suppressing the peroxidation of cardiolipin in hypoglycaemia-induced apoptosis
Authors: NOMURA Kazuhiro , IMAI Hirotaka , KOUMURA Tomoko , KOBAYASHI Toshihide , NAKAGAWA Yasuhito
Published: August 2015

Cytochrome c (cyt. c) is a proapoptotic factor that binds preferentially to cardiolipin (CL), a mitochondrial lipid, but not to cardiolipin hydroperoxide (CL-OOH). Cyt. c that had bound to CL liposomes was liberated on peroxidation of the liposomes by a radical. The generation of CL-OOH in mitochondria occurred before the release of cyt. c in rat basophile leukaemia (RBL)2H3 cells that had been induced to undergo apoptosis by exposure to hypoglycaemia with 2-deoxyglucose (2DG). The amount of cyt. c bound to CL prepared from the mitochondria of 2DG-treated cells was lower than that of untreated cells. The release of cyt. c was completely suppressed when the production of CL-OOH in mitochondria was inhibited by the overexpression of mitochondrial phospholipid hydroperoxide glutathione peroxidase (PHGPx). The fluorescence from CL-labelling dye (10-N-nonyl Acridine Orange) decreased on the induction of apoptosis by 2DG. However, no decrease in fluorescence was observed in PHGPx-overexpressing cells. Cyt. c was released from mitochondria that had been isolated from control cells on peroxidation by t-butylhydroperoxide, but no similar liberation of cyt. c from mitochondria isolated from mitochondrial PHGPx-overexpressing cells was observed. These findings suggest that the generation of CL-OOH in mitochondria might be a primary event that triggers the release of cyt. c from mitochondria in the apoptotic process in which mitochondrial PHGPx participates as an anti-apoptotic factor by preventing the formation of CL-OOH.
Phospholamban remains associated with the Ca2+- and Mg2+-dependent ATPase following phosphorylation by cAMP-dependent protein kinase
Authors: NEGASH Sewite , YAO Qing , SUN Hongye , LI Jinhui , BIGELOW Diana J. , SQUIER Thomas C.
Published: August 2015

We have used fluorescence and spin-label EPR spectroscopy to investigate how the phosphorylation of phospholamban (PLB) by cAMP-dependent protein kinase (PKA) modifies structural interactions between PLB and the Ca2+- and Mg2+-dependent ATPase (Ca-ATPase) that result in enzyme activation. Following covalent modification of N-terminal residues of PLB with dansyl chloride or the spin label 4-isothiocyanato-2,2,6,6-tetramethylpiperidine-N-oxyl (‘ITC-TEMPO’), we have co-reconstituted PLB with affinity-purified Ca-ATPase isolated from skeletal sarcoplasmic reticulum (SR) with full retention of catalytic function. The Ca2+-dependence of the ATPase activity of this reconstituted preparation is virtually identical with that observed using native cardiac SR before and after PLB phosphorylation, indicating that co-reconstituted sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase 1 (SERCA1) and PLB provide an equivalent experimental model for SERCA2a–PLB interactions. Phosphorylation of PLB in the absence of the Ca-ATPase results in a greater amplitude of rotational mobility, suggesting that the structural linkage between the transmembrane region and the N-terminus is destabilized. However, whereas co-reconstitution with the Ca-ATPase restricts the amplitude of rotational motion of PLB, subsequent phosphorylation of PLB does not significantly alter its rotational dynamics. Thus structural interactions between PLB and the Ca-ATPase that restrict the rotational mobility of the N-terminus of PLB are retained following the phosphorylation of PLB by PKA. On the other hand, the fluorescence intensity decay of bound dansyl is sensitive to the phosphorylation state of PLB, indicating that there are changes in the tertiary structure of PLB coincident with enzyme activation. These results suggest that PLB phosphorylation alters its structural interactions with the Ca-ATPase by inducing structural rearrangements between PLB and the Ca-ATPase within a defined complex that modulates Ca2+-transport function.
The catabolic function of the α-aminoadipic acid pathway in plants is associated with unidirectional activity of lysine–oxoglutarate reductase, but not saccharopine dehydrogenase
Authors: ZHU Xiaohong , TANG Guiliang , GALILI Gad
Published: August 2015

Whereas plants and animals use the α-aminoadipic acid pathway to catabolize lysine, yeast and fungi use the very same pathway to synthesize lysine. These two groups of organisms also possess structurally distinct forms of two enzymes in this pathway, namely lysine–oxoglutarate reductase (lysine–ketoglutarate reductase; LKR) and saccharopine dehydrogenase (SDH): in plants and animals these enzymes are linked on to a single bifunctional polypeptide, while in yeast and fungi they exist as separate entities. In addition, yeast LKR and SDH possess bi-directional activities, and their anabolic function is regulated by complex transcriptional and post-transcriptional controls, which apparently ascertain differential accumulation of intermediate metabolites; in plants, the regulation of the catabolic function of these two enzymes is not known. To elucidate the regulation of the catabolic function of plant bifunctional LKR/SDH enzymes, we have used yeast as an expression system to test whether a plant LKR/SDH also possesses bi-directional LKR and SDH activities, similar to the yeast enzymes. The Arabidopsis enzyme complemented a yeast SDH, but not LKR, null mutant. Identical results were obtained when deletion mutants encoding only the LKR or SDH domains of this bifunctional polypeptide were expressed individually in the yeast cells. Moreover, activity assays showed that the Arabidopsis LKR possessed catabolic, but not anabolic, activity, and its uni-directional activity stems from its structure rather than its linkage to SDH. Our results suggest that the uni-directional activity of LKR plays an important role in regulating the catabolic function of the α-amino adipic acid pathway in plants.
Effect of human neuronal tau on denaturation and reactivation of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase
Authors: CHEN Yong-Hui , HE Rong-Qiao , LIU Ying , LIU Yang , XUE Zhi-Gang
Published: August 2015

Human neuronal tau-40 (htau-40) has been used to study denaturation and renaturation of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12). Inactivation of GAPDH incubated with tau was more distinguishably detected than that of control GAPDH during thermal and guanidine hydrochloride (GdnHCl) denaturation. However, tau did not influence the activity of GAPDH at room temperature or in solution without GdnHCl. A marked change in both the emission intensity and emission maximum of the intrinsic fluorescence at 335nm of GAPDH with tau was observed when GdnHCl concentration was 0.8M, but that of the control without tau occurred in 1.2M GdnHCl. The first-order rate of the decrease in the fluorescence intensity of the enzyme with tau was approximately twice as great as that of GAPDH without tau. Kinetics of inactivation of GAPDH with tau in 0.2M GdnHCl was a monophasic procedure, instead of the biphasic procedure followed by the control, as described before [He, Zhao, Yan and Li (1993) Biochim. Biophys. Acta 1163, 315–320]. Similar results were obtained when the enzyme was thermally denatured at 45°C. It revealed that tau bound to the denatured GAPDH but not the native molecule. On the other hand, tau suppressed refolding and reactivation of GAPDH when this enzyme was reactivated by dilution of GdnHCl solution. Furthermore, tau improved the aggregation of the non-native GAPDH in solutions. It suggested that tau acted in an anti-chaperone-like manner towards GAPDH in vitro. However, tau lost that function when it was aggregated or phosphorylated by neuronal cdc2-like protein kinase. It showed that tau's anti-chaperone-like function depended on its native conformation.
‘pH-jump’ crystallographic analyses of γ-lactam–porcine pancreatic elastase complexes
Authors: WRIGHT Penny A. , WILMOUTH Rupert C. , CLIFTON Ian J. , SCHOFIELD Christopher J.
Published: August 2015

β-Lactams inhibit a range of enzymes via acylation of nucleophilic serine residues. Certain γ-lactam analogues of monocyclic β-lactams have also been shown to be reversible inhibitors of porcine pancreatic elastase (PPE), forming acyl-enzyme complexes that are stable with respect to hydrolysis. Crystallographic analysis at pH 5 of an acyl-enzyme complex formed with PPE and one of these inhibitors revealed the ester carbonyl located in the oxyanion hole in a similar conformation to that observed in the structure of a complex formed between a heptapeptide (β-casomorphin-7) and PPE. Only weak electron density was observed for the His-57 side chain in its ‘native’conformation. Instead, the His-57 side chain predominantly adopted a conformation rotated approx. 90° from its normal position. PPE–γ-lactam crystals were subjected to ‘pH-jumps’by placing the crystals in a buffer of increased pH prior to freezing for data collection. The results indicate that the conformation of the γ-lactam-derived acyl-enzyme species in the PPE active site is dependent on pH, a result having implications for the analysis of other serine protease–inhibitor structures at non-catalytic pH values. The results help to define the stereoelectronic relationship between the ester of the acyl-enzyme complex, the side chain of His-57 and the incoming nucleophile during the reversible (de)acylation steps, implying it is closely analogous to the hydrolytic deacylation step during catalytic peptide hydrolysis.
Receptor-activity-modifying protein 1 forms heterodimers with two G-protein-coupled receptors to define ligand recognition
Authors: LEUTHÄUSER Kerstin , GUJER Remo , ALDECOA Amaya , McKINNEY R. ANNE , MUFF Roman , FISCHER Jan A. , BORN Walter
Published: August 2015

Receptor-activity-modifying proteins (RAMPs) with single transmembrane domains define the function of two G-protein-coupled receptors of the B family. Cell-surface complexes of human RAMP1 (hRAMP1) and human calcitonin (CT) receptor isotype 2 (hCTR2) or rat CT-receptor-like receptor (rCRLR) have now been identified through protein cross-linking, co-immunoprecipitation and confocal microscopy. They are two distinct CT-gene-related peptide (CGRP) receptors coupled to cAMP production and pharmacologically distinguished by the CT and CGRP antagonists salmon CT(8-32) and human or rat CGRP(8-37). Thus direct molecular interactions of hRAMP1 with hCTR2 or rCRLR are required for CGRP recognition. hCTR2, moreover, adopts non-traditional functions through its association with hRAMP1.
Identification and characterization of a silencer regulatory element in the 3′-flanking region of the murine CD46 gene
Authors: NOMURA Midori , TSUJIMURA Akira , BEGUM Nasim A. , MATSUMOTO Misako , WABIKO Hiroetsu , TOYOSHIMA Kumao , SEYA Tsukasa
Published: August 2015

The murine membrane cofactor protein (CD46) gene is expressed exclusively in testis, in contrast to human CD46, which is expressed ubiquitously. To elucidate the mechanism of differential CD46 gene expression among species, we cloned entire murine CD46 genomic DNA and possible regulatory regions were placed in the flanking region of the luciferase reporter gene. The reporter gene assay revealed a silencing activity not in the promoter, but in the 3´-flanking region of the gene and the silencer-like element was identified within a 0.2-kb region between 0.6 and 0.8kb downstream of the stop codon. This silencer-like element was highly similar to that of the pig MHC class-I gene. The introduction of a mutation into this putative silencer element of murine CD46 resulted in an abrogation of the silencing effect. Electrophoretic mobility-shift assay indicated the presence of the binding molecule(s) for this silencer sequence in murine cell lines and tissues. A size difference of the protein–silencer-element complex was observed depending upon the solubilizers used for preparation of the nuclear extracts. A mutated silencer sequence failed to interact with the binding molecules. The level of the binding factor was lower in the testicular germ cells compared with other organs. Thus the silencer element and its binding factor may play a role in transcriptional regulation of murine CD46 gene expression. These results imply that the effects of the CD46 silencer element encompass the innate immune and reproductive systems, and in mice may determine the testicular germ-cell-dominant expression of CD46.
A mammalian cytochrome fused to a chloroplast transit peptide is a functional haemoprotein and is imported into isolated chloroplasts
Authors: LIU Yan-Yun , KADERBHAI Naheed , KADERBHAI Mustak A.
Published: August 2015

The small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a major chloroplast stromal protein that is cytosolically synthesized as a precursor with an N-terminal extension, known as the transit sequence or transit peptide (Tp). The Tp is essential for the post-translational uptake of the precursor by the chloroplast. The Tp is thought to influence the conformation of the precursor protein and to facilitate polypeptide translocation across the chloroplast envelope barrier via a Tp-selective translocon. To address these issues we have devised a novel strategy to generate substrate amounts of a chloroplast targeting sequence as a fusion with the chromogenic globular domain of cytochrome b5 (Cyt). The chimaeric protein is an ideal probe for investigating the conformation of a preprotein and events surrounding protein import into isolated chloroplasts. The Cyt of liver endoplasmic reticulum was fused at its N-terminus with the Tp of the small subunit of Rubisco of Pisum sativum (pea). To enhance its production by clearance from the cytoplasm of Escherichia coli, the chimaera was engineered by further N-terminal linkage of a prokaryotic secretory signal. Expression of this tripartite fusion resulted in mg quantities of the signal sequence–processed Tp–Cyt protein, which was eventually targeted to the membranes. The chromogenic nature of the chimaera and its localization to the bacterial membrane facilitated the biochemical isolation of the precursor in a soluble and functional form. The purified preprotein displayed spectral and enzymic properties that were indistinguishable from the native parental Cyt, implying an absence of observable influence of the Tp on the conformation of the haemoprotein. The chimaeric precursor was imported into the stroma of the isolated chloroplasts in a dose-dependent manner. Import was also strongly dependent upon exogenously supplied ATP. The stromally imported chimaeric precursor protein was processed to a size characteristic of Cyt.
Molecular characterization of human tensin
Authors: CHEN Huaiyang , ISHII Akiko , WONG Wai-Keung , CHEN Lan Bo , LO Su Hao
Published: August 2015

Tensin is a focal-adhesion molecule that binds to actin filaments and interacts with phosphotyrosine-containing proteins. To analyse tensin's function in mammals, we have cloned tensin cDNAs from human and cow. The isolated approx. 7.7-kb human cDNA contains an open reading frame encoding 1735 amino acid residues. The amino acid sequence of human tensin shares 60% identity with chicken tensin, and contains all the structural features described previously in chicken tensin. This includes the actin-binding domains, the Src homology domain 2, and the region similar to a tumour suppressor, PTEN. Two major differences between human and chicken tensin are (i) the lack of the first 54 residues present in chicken tensin, and (ii) the addition of 34- and 38-residue inserts in human and bovine tensin. In addition, our interspecies sequencing data have uncovered the presence of a glutamine/CAG repeat that appears to have expanded in the course of evolution. Northern-blot analysis reveals a 10-kb message in most of the human tissues examined. An additional 9-kb message is detected in heart and skeletal muscles. The molecular mass predicted from the human cDNA is 185kDa, although both endogenous and recombinant human tensin migrate as 220-kDa proteins on SDS/PAGE. The discrepancy is due to the unusually low electrophoretic mobility of the central region of the tensin polypeptide (residues 306–981). A survey of human prostate and breast cancer cell lines by Western-blot analysis shows a lack of tensin expression in most cancer cell lines, whereas these lines express considerable amounts of focal-adhesion molecules such as talin and focal-adhesion kinase. Finally, tensin is rapidly cleaved by a focal-adhesion protease, calpain II. Incubation of cells with a calpain inhibitor, MDL, prevented tensin cleavage and induced morphological change in these cells, suggesting that cleavage of tensin and other focal-adhesion constituents by calpain disrupts maintenance of normal cell shape.
Cloning and characterization of full-length mouse thymidine kinase 2: the N-terminal sequence directs import of the precursor protein into mitochondria
Authors: WANG Liya , ERIKSSON Staffan
Published: August 2015

The subcellular localization of mitochondrial thymidine kinase (TK2) has been questioned, since no mitochondrial targeting sequences have been found in cloned human TK2 cDNAs. Here we report the cloning of mouse TK2 cDNA from a mouse full-length enriched cDNA library. The mouse TK2 cDNA codes for a protein of 270 amino acids, with a 40-amino-acid presumed N-terminal mitochondrial targeting signal. In vitro translation and translocation experiments with purified rat mitochondria confirmed that the N-terminal sequence directed import of the precursor TK2 into the mitochondrial matrix. A single 2.4kb mRNA transcript was detected in most tissues examined, except in liver, where an additional shorter (1.0kb) transcript was also observed. There was no correlation between the tissue distribution of TK2 activity and the expression of TK2 mRNA. Full-length mouse TK2 protein and two N-terminally truncated forms, one of which corresponds to the mitochondrial form of TK2 and a shorter form corresponding to the previously characterized recombinant human TK2, were expressed in Escherichia coli and affinity purified. All three forms of TK2 phosphorylated thymidine, deoxycytidine and 2′-deoxyuridine, but with different kinetic efficiencies. A number of cytostatic pyrimidine nucleoside analogues were also tested and shown to be good substrates for the various forms of TK2. The active form of full-length mouse TK2 was a dimer, as judged by Superdex 200 chromatography. These results enhance our understanding of the structure and function of TK2, and may help to explain the mitochondrial disorder, mitochondrial neurogastrointestinal encephalomyopathy.