References of "Journal of Biological Chemistry"
     in
Bookmark and Share    
Full Text
Peer Reviewed
See detailCloning and characterization of ADAMTS-14, a novel ADAMTS displaying high homology with ADAMTS-2 and ADAMTS-3.
Colige, Alain ULg; Vandenberghe, Isabel; Thiry, Marc ULg et al

in Journal of Biological Chemistry (2002), 277(8), 5756-66

The processing of amino- and carboxyl-propeptides of fibrillar collagens is required to generate collagen monomers that correctly assemble into fibrils. Mutations in the ADAMTS2 gene, the ... [more ▼]

The processing of amino- and carboxyl-propeptides of fibrillar collagens is required to generate collagen monomers that correctly assemble into fibrils. Mutations in the ADAMTS2 gene, the aminopropeptidase of procollagen I and II, result in the accumulation of non-fully processed type I procollagen, causing human Ehlers-Danlos syndrome type VIIC and animal dermatosparaxis. In this study, we show that the aminopropeptide of type I procollagen can be cleaved in vivo in absence of ADAMTS-2 activity and that this processing is performed at the cleavage site for ADAMTS-2. In an attempt to identify the enzyme responsible for this alternative aminoprocollagen peptidase activity, we have cloned the cDNA and determined the primary structure of human and mouse ADAMTS-14, a novel ADAMTS displaying striking homologies with ADAMTS-2 and -3. The structure of the human gene, which maps to 10q21.3, and the mechanisms of generation of the various transcripts are described. The existence of two sites of initiation of transcription, in two different promoter contexts, suggests that transcripts resulting from these two sites can be differently regulated. The tissue distribution of ADAMTS-14, the regulation of the gene expression by various cytokines and the activity of the recombinant enzyme are evaluated. The potential function of ADAMTS-14 as a physiological aminoprocollagen peptidase in vivo is discussed. [less ▲]

Detailed reference viewed: 19 (6 ULg)
Full Text
Peer Reviewed
See detailIdentification of a karyopherin alpha 2 recognition site in PLAG1, which functions as a nuclear localization signal.
Braem, Caroline V; Kas, Koen; Meyen, Eva et al

in Journal of Biological Chemistry (2002), 277(22), 19673-8

The activation of the pleomorphic adenoma gene 1 (PLAG1) is the most frequent gain-of-function mutation found in pleomorphic adenomas of the salivary glands. To gain more insight into the regulation of ... [more ▼]

The activation of the pleomorphic adenoma gene 1 (PLAG1) is the most frequent gain-of-function mutation found in pleomorphic adenomas of the salivary glands. To gain more insight into the regulation of PLAG1 function, we searched for PLAG1-interacting proteins. Using the yeast two-hybrid system, we identified karyopherin alpha2 as a PLAG1-interacting protein. Physical interaction between PLAG1 and karyopherin alpha2 was confirmed by an in vitro glutathione S-transferase pull-down assay. Karyopherin alpha2 escorts proteins into the nucleus via interaction with a nuclear localization sequence (NLS) composed of short stretches of basic amino acids. Two putative NLSs were identified in PLAG1. The predicted NLS1 (KRKR) was essential for physical interaction with karyopherin alpha2 in glutathione S-transferase pull-down assay, and its mutation resulted in decreased nuclear import of PLAG1. Moreover, NLS1 was able to drive the nuclear import of the cytoplasmic protein beta-galactosidase. In contrast, predicted NLS2 of PLAG1 (KPRK) was not involved in karyopherin alpha2 binding nor in its nuclear import. The residual nuclear import of PLAG1 after mutation of the NLS1 was assigned to the zinc finger domain of PLAG1. These observations indicate that the nuclear import of PLAG1 is governed by its zinc finger domain and by NLS1, a karyopherin alpha2 recognition site. [less ▲]

Detailed reference viewed: 37 (10 ULg)
Full Text
Peer Reviewed
See detailMolecular characterization of a specific thiamine triphosphatase widely expressed in mammalian tissues
Lakaye, Bernard ULg; Makarchikov, Alexander F; Antunes, Adelio F et al

in Journal of Biological Chemistry (2002), 277(16), 13771-13777

Thiamine triphosphate (ThTP) is found at low concentrations in most animal tissues, and recent data suggest that it may act as a phosphate donor for the phosphorylation of some proteins. In the mammalian ... [more ▼]

Thiamine triphosphate (ThTP) is found at low concentrations in most animal tissues, and recent data suggest that it may act as a phosphate donor for the phosphorylation of some proteins. In the mammalian brain, ThTP synthesis is rapid, but its steady-state concentration remains low, presumably because of rapid hydrolysis. In this report we purified a soluble thiamine triphosphatase (ThTPase; EC 3.6.1.28) from calf brain. The bovine ThTPase is a 24-kDa monomer, hydrolyzing ThTP with virtually absolute specificity. Partial sequence data obtained from the purified bovine enzyme by tandem mass spectrometry were used to search the GenBank(TM) data base. A significant identity was found with only one human sequence, the hypothetical 230-amino acid protein MGC2652. The coding regions from human and bovine brain mRNA were amplified by reverse transcription-PCR, cloned in Escherichia coli, and sequenced. The human open reading frame was expressed in E. coli as a GST fusion protein. Transformed bacteria had a high isopropyl-beta-D-thiogalactopyranoside-inducible ThTPase activity. The recombinant ThTPase had properties similar to those of human brain ThTPase, and it was specific for ThTP. The mRNA was expressed in most human tissues but at relatively low levels. This is the first report of a molecular characterization of a specific ThTPase. [less ▲]

Detailed reference viewed: 49 (15 ULg)
Full Text
Peer Reviewed
See detailMetal Ion Binding and Coordination Geometry for Wild Type and Mutants of Metallo-Beta -Lactamase from Bacillus Cereus 569/H/9 (Bcii): A Combined Thermodynamic, Kinetic, and Spectroscopic Approach
De Seny, Dominique ULg; Heinz, U.; Wommer, S. et al

in Journal of Biological Chemistry (2001), 276(48), 45065-78

One high affinity (nm) and one low affinity (microM) macroscopic dissociation constant for the binding of metal ions were found for the wild-type metallo-beta-lactamase from Bacillus cereus as well as six ... [more ▼]

One high affinity (nm) and one low affinity (microM) macroscopic dissociation constant for the binding of metal ions were found for the wild-type metallo-beta-lactamase from Bacillus cereus as well as six single-site mutants in which all ligands in the two metal binding sites were altered. Surprisingly, the mutations did not cause a specific alteration of the affinity of metal ions for the sole modified binding site as determined by extended x-ray absorption fine structure (EXAFS) and perturbed angular correlation of gamma-rays spectroscopy, respectively. Also UV-visible absorption spectra for the mono-cobalt enzymes clearly contain contributions from both metal sites. The observations of the very similar microscopic dissociation constants of both binding sites in contrast to the significantly differing macroscopic dissociation constants inevitably led to the conclusion that binding to the two metal sites exhibits negative cooperativity. The slow association rates for forming the binuclear enzyme determined by stopped-flow fluorescence measurements suggested that fast metal exchange between the two sites for the mononuclear enzyme hinders the binding of a second metal ion. EXAFS spectroscopy of the mono- and di-zinc wild type enzymes and two di-zinc mutants provide a definition of the metal ion environments, which is compared with the available x-ray crystallographic data. [less ▲]

Detailed reference viewed: 11 (1 ULg)
Full Text
Peer Reviewed
See detailStructural Determinants of Cold Adaptation and Stability in a Large Protein
D'Amico, Salvino ULg; Gerday, Charles ULg; Feller, Georges ULg

in Journal of Biological Chemistry (2001), 276(28), 25791-6

The heat-labile alpha-amylase from an antarctic bacterium is the largest known protein that unfolds reversibly according to a two-state transition as shown by differential scanning calorimetry. Mutants of ... [more ▼]

The heat-labile alpha-amylase from an antarctic bacterium is the largest known protein that unfolds reversibly according to a two-state transition as shown by differential scanning calorimetry. Mutants of this enzyme were produced, carrying additional weak interactions found in thermostable alpha-amylases. It is shown that single amino acid side chain substitutions can significantly modify the melting point T(m), the calorimetric enthalpy Delta H(cal), the cooperativity and reversibility of unfolding, the thermal inactivation rate constant, and the kinetic parameters k(cat) and K(m). The correlation between thermal inactivation and unfolding reversibility displayed by the mutants also shows that stabilizing interactions increase the frequency of side reactions during refolding, leading to intramolecular mismatches or aggregations typical of large proteins. Although all mutations were located far from the active site, their overall trend is to decrease both k(cat) and K(m) by rigidifying the molecule and to protect mutants against thermal inactivation. The effects of these mutations indicate that the cold-adapted alpha-amylase has lost a large number of weak interactions during evolution to reach the required conformational plasticity for catalysis at low temperatures, thereby producing an enzyme close to the lowest stability allowing maintenance of the native conformation. [less ▲]

Detailed reference viewed: 12 (1 ULg)
Full Text
Peer Reviewed
See detailFli-1 inhibits collagen type I production in dermal fibroblasts via an Sp1-dependent pathway.
Czuwara-Ladykowska, Joanna; Shirasaki, Fumiaki; Jackers, Pascale ULg et al

in Journal of Biological Chemistry (2001), 276(24), 20839-20848

Fibrosis is characterized by the excessive deposition of extracellular matrix (ECM), especially collagen. Because Ets factors are implicated in physiological and pathological ECM remodeling, the aim of ... [more ▼]

Fibrosis is characterized by the excessive deposition of extracellular matrix (ECM), especially collagen. Because Ets factors are implicated in physiological and pathological ECM remodeling, the aim of this study was to investigate the role of Ets factors in collagen production. We demonstrate that the expression of collagenous proteins and collagen alpha2(I) (COL1A2) mRNA was inhibited following stable transfection of Fli-1 in dermal fibroblasts. Subsequent analysis of the COL1A2 promoter identified a critical Ets binding site that mediates Fli-1 inhibition. In contrast, Ets-1 stimulates COL1A2 promoter activity. In vitro binding assays demonstrate that both Fli-1 and Ets-1 form DNA-protein complexes with sequences present in COL1A2 promoter. Furthermore, Fli-1 binding to the COL1A2 is enhanced via Sp1-dependent interaction. Studies using Fli-1 dominant interference and DNA binding mutants indicate that Fli-1 inhibition is mediated by both direct (DNA binding) and indirect (via protein-protein interaction) mechanisms and that Sp1 is an important mediator of the Fli-1 function. Furthermore, experiments using the Gal4 system in the context of different cell types as well as experiments with the COL1A2 promoter in different cell lines demonstrate that the direction and magnitude of the effect of Fli-1 is promoter- and cell context-specific. We propose that Fli-1 inhibits COL1A2 promoter activity by competition with Ets-1. In addition, we postulate that another factor (co-repressor) may be required for maximal inhibition after recruitment to the Fli-1-Sp1 complex. We conclude that the ratio of Fli-1 to Ets-1 and the presence of co-regulatory proteins ultimately control ECM production in fibroblasts. [less ▲]

Detailed reference viewed: 56 (30 ULg)
Peer Reviewed
See detailThiomandelic acid, a broad spectrum inhibitor of zinc beta-lactamases: kinetic and spectroscopic studies.
Mollard, C.; Moali, C.; Papamicael, C. et al

in Journal of Biological Chemistry (2001), 276(48), 45015-23

Resistance to beta-lactam antibiotics mediated by metallo-beta-lactamases is an increasingly worrying clinical problem. Candidate inhibitors include mercaptocarboxylic acids, and we report studies of a ... [more ▼]

Resistance to beta-lactam antibiotics mediated by metallo-beta-lactamases is an increasingly worrying clinical problem. Candidate inhibitors include mercaptocarboxylic acids, and we report studies of a simple such compound, thiomandelic acid. A series of 35 analogues were synthesized and examined as metallo-beta-lactamase inhibitors. The K(i) values (Bacillus cereus enzyme) are 0.09 microm for R-thiomandelic acid and 1.28 microm for the S-isomer. Structure-activity relationships show that the thiol is essential for activity and the carboxylate increases potency; the affinity is greatest when these groups are close together. Thioesters of thiomandelic acid are substrates for the enzyme, liberating thiomandelic acid, suggesting a starting point for the design of "pro-drugs." Importantly, thiomandelic acid is a broad spectrum inhibitor of metallo-beta-lactamases, with a submicromolar K(i) value for all nine enzymes tested, except the Aeromonas hydrophila enzyme; such a wide spectrum of activity is unprecedented. The binding of thiomandelic acid to the B. cereus enzyme was studied by NMR; the results are consistent with the idea that the inhibitor thiol binds to both zinc ions, while its carboxylate binds to Arg(91). Amide chemical shift perturbations for residues 30-40 (the beta(3)-beta(4) loop) suggest that this small inhibitor induces a movement of this loop of the kind seen for other larger inhibitors. [less ▲]

Detailed reference viewed: 15 (0 ULg)
Full Text
Peer Reviewed
See detailInteraction Between The N-Terminal Domain Of Gastric H,K-Atpase And The Spectrin Binding Domain Of Ankyrin Iii
Festy, F.; Robert, Jocelyne ULg; Brasseur, Robert ULg et al

in Journal of Biological Chemistry (2001), 276(11), 7721-6

We screened a cDNA bank of rabbit gastric fundic mucosa by two-hybrid assays looking for binding partners of the N-terminal domain of the rabbit gastric H,K-ATPase. We extracted five clones sharing more ... [more ▼]

We screened a cDNA bank of rabbit gastric fundic mucosa by two-hybrid assays looking for binding partners of the N-terminal domain of the rabbit gastric H,K-ATPase. We extracted five clones sharing more than 90% sequence identity. The longest clone codes for a protein sharing a high identity (96 and 96.8%, respectively) with a fragment of the membrane domain, from Arg-835 to Ser-873, plus the major part of the "spectrin binding domain" going from Glu-874 to Leu-1455 of human and mouse ankyrin III. We conclude that the membrane and spectrin binding domains of the rabbit ankyrin III are candidates for the binding partner of the N-terminal domain of the rabbit gastric H,K-ATPase. To validate the ankyrin-ATPase interaction and to test its specificity, we produced both domains in yeast and bacteria, coimmunoprecipitated them with an anti-ATPase antibody, and copurified them by affinity chromatography. The sequence of rabbit ankyrin III was not known, and this is the first report demonstrating that the ankyrin III and the H,K-ATPase interact with no intermediate. The interaction involves the N-terminal domain of the ATPase on one hand and the spectrin binding domain of the ankyrin on the other. [less ▲]

Detailed reference viewed: 14 (0 ULg)
Full Text
Peer Reviewed
See detailThe Human Vpac(1) Receptor - Three-Dimensional Model And Mutagenesis Of The N-Terminal Domain
Lins, Laurence ULg; Couvineau, A.; Rouyer-Fessard, C. et al

in Journal of Biological Chemistry (2001), 276(13),

The human VPAC(1) receptor for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide belongs to the class II family of G-protein-coupled receptors with seven transmembrane ... [more ▼]

The human VPAC(1) receptor for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide belongs to the class II family of G-protein-coupled receptors with seven transmembrane segments. Like for all class II receptors, the extracellular N-terminal domain of the human VPAC(1) receptor plays a predominant role in peptide ligand recognition. To determine the three-dimensional structure of this N-terminal domain (residues 1-144), the Protein Data Bank (PDB) was screened for a homologous protein. A subdomain of yeast lipase B was found to have 27% sequence identity and 50% sequence homology with the N-terminal domain (8) of the VPAC(1) receptor together with a good alignment of the hydrophobic clusters. A model of the N-terminal domain of VPAC(1) receptor was thus constructed by homology. It indicated the presence of a putative signal sequence in the N-terminal extremity. Moreover, residues (Glu(36), Trp(67), Asp(68), Trp(73), and Gly(109)) which were shown to be crucial for VIP binding are gathered around a groove that is essentially negatively charged. New putatively important residues for VIP binding were suggested from the model analysis. Site-directed mutagenesis and stable transfection of mutants in CHO cells indicated that Pro(74), Pro(87), Phe(90), and Trp(110) are indeed important for VIP binding and activation of adenylyl cyclase activation. Combination of molecular modeling and directed mutagenesis provided the first partial three-dimensional structure of a VIP-binding domain, constituted of an electronegative groove with an outspanning tryptophan shell at one end, in the N-terminal extracellular region of the human VPAC(1) receptor. [less ▲]

Detailed reference viewed: 8 (0 ULg)
Full Text
Peer Reviewed
See detailExpression and function of the collagen receptor GPVI during megakaryocyte maturation.
Lagrue-Lak-Hal, A. H.; Debili, N.; Kingbury, G. et al

in Journal of Biological Chemistry (2001), 276(18), 15316-25

In this report, the expression and function of the platelet collagen receptor glycoprotein VI (GPVI) were studied in human megakaryocytes during differentiation and maturation of mobilized blood and cord ... [more ▼]

In this report, the expression and function of the platelet collagen receptor glycoprotein VI (GPVI) were studied in human megakaryocytes during differentiation and maturation of mobilized blood and cord blood derived CD34(+) cells. By flow cytometry, using an anti-GPVI monoclonal antibody or convulxin, a GPVI-specific ligand, GPVI was detected only on CD41(+) cells including some CD41(+)/CD34(+) cells, suggesting expression at a stage of differentiation similar to CD41. These results were confirmed at the mRNA level using reverse transcription-polymerase chain reaction. GPVI expression was low during megakaryocytic differentiation but increased in the more mature megakaryocytes (CD41(high)). As in platelets, megakaryocyte GPVI associates with the Fc receptor gamma chain (FcRgamma). The FcR gamma chain was detected at the RNA and protein level at all stages of megakaryocyte maturation preceding the expression of GPVI. The other collagen receptor, alpha(2)beta(1) integrin (CD49b/CD29), had a pattern of expression similar to GPVI. Megakaryocytic GPVI was recognized as a 55-kDa protein by immunoblotting and ligand blotting, and thus it presented a slightly lower apparent molecular mass than platelet GPVI (58 kDa). Megakaryocytes began to adhere to immobilized convulxin via GPVI after only 8-10 days of culture, at a time when megakaryocytes were maturing. At this stage of maturation, they also adhered to immobilized collagen by alpha(2)beta(1) integrin-dependent and -independent mechanisms. Convulxin induced a very similar pattern of protein tyrosine phosphorylation in megakaryocytes and platelets including Syk, FcRgamma, and PLC(gamma)2. Our results showed that GPVI is expressed early during megakaryocytic differentiation but functionally allows megakaryocyte adherence to collagen only at late stages of differentiation when its expression increases. [less ▲]

Detailed reference viewed: 63 (2 ULg)
Full Text
Peer Reviewed
See detailDistinct carbohydrate recognition domains of an invertebrate defense molecule recognize Gram-negative and Gram-positive bacteria.
Bilej, M.; De Baetselier, P.; Van Dijck, E. et al

in Journal of Biological Chemistry (2001), 276(49), 45840-7

Coelomic fluid of Eisenia foetida earthworms (Oligochaeta, Annelida) contains a 42-kDa defense molecule named CCF for coelomic cytolytic factor. By binding microbial antigens, namely the O-antigen of ... [more ▼]

Coelomic fluid of Eisenia foetida earthworms (Oligochaeta, Annelida) contains a 42-kDa defense molecule named CCF for coelomic cytolytic factor. By binding microbial antigens, namely the O-antigen of lipopolysaccharide (LPS), beta-1,3-glucans, or N,N'-diacetylchitobiose present, respectively, on Gram-negative bacteria or yeast cell walls, CCF triggers the prophenoloxidase activating pathway. We report that CCF recognizes lysozyme-predigested Gram-positive bacteria or the peptidoglycan constituent muramyl dipeptide as well as muramic acid. To identify the pattern recognition domains of CCF, deletion mutants were tested for their ability to reconstitute the prophenoloxidase cascade in E. foetida coelomic fluid depleted of endogenous CCF in the presence of LPS, beta-1,3-glucans, N,N'-diacetylchitobiose, and muramic acid. In addition, affinity chromatography of CCF peptides was performed on immobilized beta-1,3-glucans or N,N'-diacetylchitobiose. We found that the broad specificity of CCF for pathogen-associated molecular patterns results from the presence of two distinct pattern recognition domains. One domain, which shows homology with the polysaccharide and glucanase motifs of beta-1,3-glucanases and invertebrate defense molecules located in the central part of the CCF polypeptide chain, interacts with LPS and beta-1,3-glucans. The C-terminal tryptophan-rich domain mediates interactions of CCF with N,N'-diacetylchitobiose and muramic acid. These data provide evidence for the presence of spatially distinct carbohydrate recognition domains within this invertebrate defense molecule. [less ▲]

Detailed reference viewed: 12 (0 ULg)
Full Text
Peer Reviewed
See detailProcollagen II amino propeptide processing by ADAMTS-3. Insights on dermatosparaxis.
Fernandes, R. J.; Hirohata, S.; Engle, J. M. et al

in Journal of Biological Chemistry (2001), 276(34), 31502-9

The amino and carboxyl propeptides of procollagens I and II are removed by specific enzymes as a prerequisite for fibril assembly. Null mutations in procollagen I N-propeptidase (ADAMTS-2) cause ... [more ▼]

The amino and carboxyl propeptides of procollagens I and II are removed by specific enzymes as a prerequisite for fibril assembly. Null mutations in procollagen I N-propeptidase (ADAMTS-2) cause dermatosparaxis in cattle and the Ehlers-Danlos syndrome (dermatosparactic type) in humans by preventing proteolytic excision of the N-propeptide of procollagen I. We have found that procollagen II is processed normally in dermatosparactic nasal cartilage, suggesting the existence of another N-propeptidase(s). We investigated such a role for ADAMTS-3 in Swarm rat chondrosarcoma RCS-LTC cells, which fail to process the procollagen II N-propeptide. Stable transfection of RCS-LTC cells with bovine ADAMTS-2 or human ADAMTS-3 partially rescued the processing defect, suggesting that ADAMTS-3 has procollagen II N-propeptidase activity. Human skin and skin fibroblasts showed 30-fold higher mRNA levels of ADAMTS-2 than ADAMTS-3, whereas ADAMTS-3 mRNA was 5-fold higher than ADAMTS-2 mRNA in human cartilage. We propose that both ADAMTS-2 and ADAMTS-3 process procollagen II, but ADAMTS-3 is physiologically more relevant, given its preferred expression in cartilage. The findings provide an explanation for the sparing of cartilage in dermatosparaxis and, perhaps, for the relative sparing of some procollagen I-containing tissues. [less ▲]

Detailed reference viewed: 14 (0 ULg)
Full Text
Peer Reviewed
See detailInfluence of moderate temperatures on myristoyl-CoA metabolism and acyl-CoA thioesterase activity in the psychrophilic antarctic yeast Rhodotorula aurantiaca.
Sabri, Ahmed ULg; Bare, G.; Jacques, Philippe ULg et al

in Journal of Biological Chemistry (2001), 276(16), 12691-6

The inability of psychrophilic microorganisms to grow at moderate temperatures (>20 degrees C) presently represents an unresolved thermodynamic paradox. Here we report for the psychrophilic yeast ... [more ▼]

The inability of psychrophilic microorganisms to grow at moderate temperatures (>20 degrees C) presently represents an unresolved thermodynamic paradox. Here we report for the psychrophilic yeast Rhodotorula aurantiaca A19, isolated from Antarctic ice, that the inability to grow at temperatures close to 20 degrees C is associated with profound alterations in cell morphology and integrity. High performance liquid chromatography analysis of the intracellular acyl-CoA esters revealed an abnormal accumulation of myristoyl-CoA (C14-CoA) in cells cultivated close to the nonpermissive temperature. Its concentration (500 microm) was found to be 28-fold higher than in cells cultivated at 0 degrees C. If one considers its ability to disrupt membrane bilayers and to inhibit many cellular enzymes and functions, intracellular myristoyl-CoA accumulation in the psychrophile R. aurantiaca represents one of the principal causes of growth arrest at moderate temperatures. Intracellular acyl-CoA concentrations are believed to be regulated by thioesterase activity. Thus in an attempt to explore the mechanism by which temperature disrupts myristoyl-CoA metabolism, we isolated and characterized a long chain acyl-CoA thioesterase. The monomeric 80-kDa thioesterase from the psychrophilic yeast shows a very strong specificity for myristoyl-CoA. The affinity for substrate and the catalytic efficiency of the thioesterase are optimal below 5 degrees C (temperatures habitually experienced by the strain) and dramatically decrease with increasing temperature. The loss of affinity for substrate is related to the intracellular increase of myristoyl-CoA concentration. Our observations reveal one of the probable mechanisms by which temperature fixes the limit of growth for this psychrophilic yeast. [less ▲]

Detailed reference viewed: 83 (18 ULg)
Full Text
Peer Reviewed
See detailGene activation by varicella-zoster virus IE4 protein requires its dimerization and involves both the arginine-rich sequence, the central part, and the carboxyl-terminal cysteine-rich region
Baudoux, Laurence; Defechereux, Patricia; Rentier, Bernard ULg et al

in Journal of Biological Chemistry (2000), 275(42), 32822-32831

Varicella-zoster virus (VZV) open reading frame 4-encoded protein (IE4) possesses transactivating properties for VZV genes as well as for those of heterologous viruses. Since most transcription factors ... [more ▼]

Varicella-zoster virus (VZV) open reading frame 4-encoded protein (IE4) possesses transactivating properties for VZV genes as well as for those of heterologous viruses. Since most transcription factors act as dimers, IE4 dimerization was studied using the mammalian two-hybrid system. Introduction of mutations in the IE4 open reading frame demonstrated that both the central region and the carboxyl-terminal cysteine-rich domain were important for efficient dimerization. Within the carboxyl-terminal domain, substitution of amino acids encompassing residues 443-447 totally abolished dimerization. Gene activation by IE4 was studied by transient transfection with an IE4 expression plasmid and a reporter gene under the control of either the human immunodeficiency virus, type 1, long terminal repeat or the VZV thymidine kinase promoter. Regions of IE4 important for dimerization were also shown to be crucial for transactivation. In addition, the arginine-rich domains Rb and Re of the amino-terminal region were also demonstrated to be important for transactivation, whereas the Ra domain as well as an acidic and bZIP-containing regions were shown to be dispensable for gene transactivation. A nucleocytoplasmic shuttling of IE4 has also been characterized, involving a nuclear localization signal identified within the Rb domain and a nuclear export mechanism partially depending on Crm-1. [less ▲]

Detailed reference viewed: 20 (3 ULg)
Full Text
Peer Reviewed
See detailMembrane Type 1 Matrix Metalloproteinase-Associated Degradation of Tissue Inhibitor of Metalloproteinase 2 in Human Tumor Cell Lines
Maquoi, Erik ULg; Frankenne, Francis ULg; Baramova, Eugénia et al

in Journal of Biological Chemistry (2000), 275(15), 11368-78

Tissue inhibitor of metalloproteinase 2 (TIMP-2) is required for the membrane type 1 matrix metalloproteinase (MT1-MMP)-dependent activation of pro-MMP-2 on the cell surface. MT1-MMP-bound TIMP-2 has been ... [more ▼]

Tissue inhibitor of metalloproteinase 2 (TIMP-2) is required for the membrane type 1 matrix metalloproteinase (MT1-MMP)-dependent activation of pro-MMP-2 on the cell surface. MT1-MMP-bound TIMP-2 has been shown to function as a receptor for secreted pro-MMP-2, resulting in the formation of a trimolecular complex. In the presence of uncomplexed active MT1-MMP, the prodomain of cell surface-associated MMP-2 is cleaved, and activated MMP-2 is released. However, the behavior of MT1-MMP-bound TIMP-2 during MMP-2 activation is currently unknown. In this study, (125)I-labeled recombinant TIMP-2 ((125)I-rTIMP-2) was used to investigate the fate of TIMP-2 during pro-MMP-2 activation by HT1080 and transfected A2058 cells. HT1080 and A2058 cells transfected with MT1-MMP cDNA (but not vector-transfected A2058 cells) were able to bind (125)I-rTIMP-2, to activate pro-MMP-2, and to process MT1-MMP into an inactive 43-kDa form. Under these conditions, (125)I-rTIMP-2 bound to the cell surface was rapidly internalized and degraded in intracellular organelles through a bafilomycin A(1)-sensitive mechanism, and (125)I-bearing low molecular mass fragment(s) were released in the culture medium. These different processes were inhibited by hydroxamic acid-based synthetic MMP inhibitors and rTIMP-2, but not by rTIMP-1 or cysteine, serine, or aspartic proteinase inhibitors. These results support the concept that the MT1-MMP-dependent internalization and degradation of TIMP-2 by some tumor cells might be involved in the regulation of pericellular proteolysis. [less ▲]

Detailed reference viewed: 18 (3 ULg)
Full Text
Peer Reviewed
See detailStructural, Kinetic, and Calorimetric Characterization of the Cold-Active Phosphoglycerate Kinase from the Antarctic Pseudomonas Sp. Tacii18
Bentahir, Mostafa; Feller, Georges ULg; Aittaleb, Mohamed et al

in Journal of Biological Chemistry (2000), 275(15), 11147-53

The gene encoding the phosphoglycerate kinase (PGK) from the Antarctic Pseudomonas sp. TACII18 has been cloned and found to be inserted between the genes encoding for glyceraldhyde-3-phosphate ... [more ▼]

The gene encoding the phosphoglycerate kinase (PGK) from the Antarctic Pseudomonas sp. TACII18 has been cloned and found to be inserted between the genes encoding for glyceraldhyde-3-phosphate dehydrogenase and fructose aldolase. The His-tagged and the native recombinant PGK from the psychrophilic Pseudomonas were expressed in Escherichia coli. The wild-type and the native recombinant enzymes displayed identical properties, such as a decreased thermostability and a 2-fold higher catalytic efficiency at 25 degrees C when compared with the mesophilic PGK from yeast. These properties, which reflect typical features of cold-adapted enzymes, were strongly altered in the His-tagged recombinant PGK. The structural model of the psychrophilic PGK indicated that a key determinant of its low stability is the reduced number of salt bridges, surface charges, and aromatic interactions when compared with mesophilic and thermophilic PGK. Differential scanning calorimetry of the psychrophilic PGK revealed unusual variations in its conformational stability for the free and substrate-bound forms. In the free form, a heat-labile and a thermostable domain unfold independently. It is proposed that the heat-labile domain acts as a destabilizing domain, providing the required flexibility around the active site for catalysis at low temperatures. [less ▲]

Detailed reference viewed: 26 (0 ULg)
Full Text
Peer Reviewed
See detailA natural dominant negative P2X1 receptor due to deletion of a single amino acid residue.
Oury, Cécile ULg; Toth-Zsamboki, Emese; Van Geet, Chris et al

in Journal of Biological Chemistry (2000)

We describe a naturally occurring dominant negative P2X1 mutant. This mutant lacks one leucine within a stretch of four leucine residues in its second transmembrane domain (TM2) (amino acids 351-354 ... [more ▼]

We describe a naturally occurring dominant negative P2X1 mutant. This mutant lacks one leucine within a stretch of four leucine residues in its second transmembrane domain (TM2) (amino acids 351-354). Confocal microscopy revealed proper plasma membrane localization of the mutant in stably transfected HEK293 cells. Nevertheless, voltage-clamped HEK293 cells expressing mutated P2X1 channels failed to develop an ATP or ADP-induced current. Furthermore, when co-expressed with the wild type receptor in Xenopus oocytes, the mutated protein exhibited a dose-dependent dominant negative effect on the normal ATP or ADP-induced P2X1 channel activity. These data indicate that deletion of a single apolar amino acid residue at the inner border of the P2X1 TM2 generates a nonfunctional channel. [less ▲]

Detailed reference viewed: 24 (0 ULg)
Full Text
Peer Reviewed
See detailProteolytic cleavage confers nitric oxide synthase inducing activity upon prolactin
Corbacho, A. M.; Nava, G.; Eiserich, J. P. et al

in Journal of Biological Chemistry (2000), 275(18), 13183-6

Prolactin (PRL), originally associated with milk secretion, is now known to possess a wide variety of biological actions and diverse sites of production beyond the pituitary. Proteolytic cleavage is a ... [more ▼]

Prolactin (PRL), originally associated with milk secretion, is now known to possess a wide variety of biological actions and diverse sites of production beyond the pituitary. Proteolytic cleavage is a common post-translational modification that can either activate precursor proteins or confer upon the peptide fragment unique biological actions not exerted by the parent molecule. Recent studies have demonstrated that the 16-kDa N-terminal proteolytic cleavage product of PRL (16K-PRL) acts as a potent inhibitor of angiogenesis. Despite previous demonstrations of 16K-PRL production in vivo, biological functions beyond its antiangiogenic actions remain unknown. Here we show that 16K-PRL, but not full-length PRL, acts to promote the expression of the inducible isoform of nitric oxide synthase (iNOS) and nitric oxide (*NO) production by pulmonary fibroblasts and alveolar type II cells with potency comparable with the proinflammatory cytokines interleukin-1beta, interferon gamma, and tumor necrosis factor alpha. The differential effect of 16K-PRL versus PRL occurs through a receptor distinct from known PRL receptors. Additionally, pulmonary fibroblasts express the PRL gene and endogenously produce 16K-PRL, suggesting that this pathway may serve both autocrine and paracrine roles in the regulation of *NO production. These results reveal that proteolytic cleavage of PRL confers upon this classical hormone potent iNOS inducing activity, suggesting its role in inflammatory/immune processes. [less ▲]

Detailed reference viewed: 19 (1 ULg)
Full Text
Peer Reviewed
See detailProton re-uptake partitioning between uncoupling protein and ATP synthase during benzohydroxamic acid-resistant state 3 respiration in tomato fruit mitochondria.
Jarmuszkiewicz, W.; Almeida, A.; Vercesi, A. et al

in Journal of Biological Chemistry (2000), 275(18), 13315-13320

The yield of oxidative phosphorylation in isolated tomato fruit mitochondria depleted of free fatty acids remains constant when respiratory rates are decreased by a factor of 3 by the addition of n-butyl ... [more ▼]

The yield of oxidative phosphorylation in isolated tomato fruit mitochondria depleted of free fatty acids remains constant when respiratory rates are decreased by a factor of 3 by the addition of n-butyl malonate. This constancy makes the determination of the contribution of the linoleic acid-induced energy-dissipating pathway by the ADP/O method possible. No decrease in membrane potential is observed in state 3 respiration with increasing concentration of n-butyl malonate, indicating that the rate of ATP synthesis is steeply dependent on membrane potential. Linoleic acid decreases the yield of oxidative phosphorylation in a concentration-dependent manner by a pure protonophoric process like that in the presence of FCCP. ADP/O measurements allow calculation of the part of respiration leading to ATP synthesis and the part of respiration sustained by the dissipative H(+) re-uptake induced by linoleic acid. Respiration sustained by this energy-dissipating process remains constant at a given LA concentration until more than 50% inhibition of state 3 respiration by n-butyl malonate is achieved. The energy dissipative contribution to oxygen consumption is proposed to be equal to the protonophoric activity of plant uncoupling protein divided by the intrinsic H(+)/O of the cytochrome pathway. It increases with linoleic acid concentration, taking place at the expense of ADP phosphorylation without an increase in the respiration. [less ▲]

Detailed reference viewed: 8 (1 ULg)
Peer Reviewed
See detailThe Arabidopsis Thaliana Pin1at Gene Encodes A Single-Domain Phosphorylation-Dependent Peptidyl Prolyl Cis/Trans Isomerase
Landrieu, I.; De Veylder, L.; Fruchart, Js. et al

in Journal of Biological Chemistry (2000), 275(14),

Detailed reference viewed: 8 (2 ULg)