References of "Journal of Biological Chemistry"
     in
Bookmark and Share    
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: 15 (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: 17 (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: 19 (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: 12 (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: 16 (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: 6 (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: 4 (2 ULg)
Full Text
Peer Reviewed
See detailIdentification Of Key Residues For Interaction Of Vasoactive Intestinal Peptide With Human Vpac(1) And Vpac(2) Receptors And Development Of A Highly Selective Vpac(1) Receptor Agonist - Alanine Scanning And Molecular Modeling Of The Peptide
Nicole, P.; Lins, Laurence ULg; Rouyer-Fessard, C. et al

in Journal of Biological Chemistry (2000), 275(31), 24003-12

The widespread neuropeptide vasoactive intestinal peptide (VIP) has two receptors VPAC(1) and VPAC(2). Solid-phase syntheses of VIP analogs in which each amino acid has been changed to alanine (Ala scan ... [more ▼]

The widespread neuropeptide vasoactive intestinal peptide (VIP) has two receptors VPAC(1) and VPAC(2). Solid-phase syntheses of VIP analogs in which each amino acid has been changed to alanine (Ala scan) or glycine was achieved and each analog was tested for: (i) three-dimensional structure by ab initio molecular modeling; (ii) ability to inhibit (125)I-VIP binding (K(i)) and to stimulate adenylyl cyclase activity (EC(50)) in membranes from cell clones stably expressing human recombinant VPAC(1) or VPAC(2) receptor. The data show that substituting residues at 14 positions out of 28 in VIP resulted in a >10-fold increase of K(i) or EC(50) at the VPAC(1) receptor. Modeling of the three-dimensional structure of native VIP (central alpha-helice from Val(5) to Asn(24) with random coiled N and C terminus) and analogs shows that substitutions of His(1), Val(5), Arg(14), Lys(15), Lys(21), Leu(23), and Ile(26) decreased biological activity without altering the predicted structure, supporting that those residues directly interact with VPAC(1) receptor. The interaction of the analogs with human VPAC(2) receptor is similar to that observed with VPAC(1) receptor, with three remarkable exceptions: substitution of Thr(11) and Asn(28) by alanine increased K(i) for binding to VPAC(2) receptor; substitution of Tyr(22) by alanine increased EC(50) for stimulating adenylyl cyclase activity through interaction with the VPAC(2) receptor. By combining 3 mutations at positions 11, 22, and 28, we developed the [Ala(11,22,28)]VIP analog which constitutes the first highly selective (>1,000-fold) human VPAC(1) receptor agonist derived from VIP ever described. [less ▲]

Detailed reference viewed: 9 (0 ULg)
Full Text
Peer Reviewed
See detailBiological properties of human prolactin analogs depend not only on global hormone affinity, but also on the relative affinities of both receptor binding sites
Kinet, Sandrina; Bernichtein, Sophie; Kelly, Paul A. et al

in Journal of Biological Chemistry (1999), 274(37), 26033-43

Zinc increases the affinity of human growth hormone (hGH) for the human prolactin receptor (hPRLR) due to the coordination of one zinc ion involving Glu-174(hGH) and His-18(hGH). In contrast, binding of ... [more ▼]

Zinc increases the affinity of human growth hormone (hGH) for the human prolactin receptor (hPRLR) due to the coordination of one zinc ion involving Glu-174(hGH) and His-18(hGH). In contrast, binding of hPRL to the hPRLR is zinc-independent. We engineered in binding site 1 of hPRL a hGH-like zinc coordination site, by mutating Asp-183(hPRL) (homologous to Glu-174(hGH)) into Glu (D183E mutation). This mutation was also introduced into G129R hPRL, a binding site 2 mutant (Goffin, V., Kinet, S., Ferrag, F., Binart, N., Martial, J. A. , and Kelly, P. A. (1996) J. Biol. Chem. 271, 16573-16579). These analogs were characterized using a stable clone expressing both the hPRLR and a PRLR-responsive reporter gene. The D183E mutation per se decreases the binding affinity and transcriptional activity of hPRL. However, this loss is partially rescued by the addition of zinc and the effect is much more marked on bioactivity than on binding affinity. These data indicate that the D183E mutation confers zinc sensitivity to hPRL biological properties. Due to an impaired site 2, the agonistic activity of G129R analog is almost nil. Although the double mutant D183E/G129R displays lower affinity ( approximately 1 log) compared with G129R hPRL, it unexpectedly recovers partial agonistic activity in the absence of zinc. Moreover, whereas zinc increases the affinity of D183E/G129R, it paradoxically abolishes its agonistic activity. Our results demonstrate that the biological properties of hPRL analogs do not necessarily parallel their overall affinity. Rather, the relative affinities of the individual binding sites 1 and 2 may play an even more important role. [less ▲]

Detailed reference viewed: 7 (0 ULg)
Full Text
Peer Reviewed
See detailMurine Matrix Metalloproteinase 9 Gene. 5'-Upstream Region Contains Cis-Acting Elements for Expression in Osteoclasts and Migrating Keratinocytes in Transgenic Mice
Munaut, Carine ULg; Salonurmi, T.; Kontusaari, S. et al

in Journal of Biological Chemistry (1999), 274(9), 5588-96

Knowledge about the regulation of cell lineage-specific expression of extracellular matrix metalloproteinases is limited. In the present work, the murine matrix metalloproteinase 9 (MMP-9) gene was shown ... [more ▼]

Knowledge about the regulation of cell lineage-specific expression of extracellular matrix metalloproteinases is limited. In the present work, the murine matrix metalloproteinase 9 (MMP-9) gene was shown to contain 13 exons, and the 2.8-kilobase pair upstream region was found to contain several common promoter elements including a TATA box-like motif, three GC boxes, four AP-1-like binding sites, an AP-2 site, and three PEA3 consensus sequences that may be important for basic activity of the gene. In order to identify cell-specific regulatory elements, constructs containing varying lengths of the upstream region in front of a LacZ reporter gene were made and studied for expression in transgenic mice generated by microinjection into fertilized oocytes. Analyses of the mice revealed that the presence of sequences between -2722 and -7745 allowed for expression in osteoclasts and migrating keratinocytes, i. e. cells that have been shown to normally express the enzyme in vivo. The results represent the first in vivo demonstration of the location of cell-specific control elements in a matrix metalloproteinase gene and show that element(s) regulating most cell-specific activities of 92-kDa type collagenase are located in the -2722 to -7745 base pair region. [less ▲]

Detailed reference viewed: 7 (1 ULg)
Full Text
Peer Reviewed
See detailIkappab-Alpha Enhances Transactivation by the Hoxb7 Homeodomain-Containing Protein
Chariot, Alain ULg; Princen, Frédéric; Gielen, Jacques et al

in Journal of Biological Chemistry (1999), 274(9), 5318-25

Combinatorial interactions between distinct transcription factors generate specificity in the controlled expression of target genes. In this report, we demonstrated that the HOXB7 homeodomain-containing ... [more ▼]

Combinatorial interactions between distinct transcription factors generate specificity in the controlled expression of target genes. In this report, we demonstrated that the HOXB7 homeodomain-containing protein, which plays a key role in development and differentiation, physically interacted in vitro with IkappaB-alpha, an inhibitor of NF-kappaB activity. This interaction was mediated by the IkappaB-alpha ankyrin repeats and C-terminal domain as well as by the HOXB7 N-terminal domain. In transient transfection experiments, IkappaB-alpha markedly increased HOXB7-dependent transcription from a reporter plasmid containing a homeodomain consensus-binding sequence. This report therefore showed a novel function for IkappaB-alpha, namely a positive regulation of transcriptional activation by homeodomain-containing proteins. [less ▲]

Detailed reference viewed: 12 (3 ULg)
Full Text
Peer Reviewed
See detailThe Crystal Structure Of A Penicilloyl-Serine Transferase Of Intermediate Penicillin Sensitivity - The Dd-Transpeptidase Of Streptomyces K15
Fonze, E.; Vermeire, M.; Nguyen-Disteche, M. et al

in Journal of Biological Chemistry (1999), 274(31), 21853-60

The serine DD-transpeptidase/penicillin-binding protein of Streptomyces K15 catalyzes peptide bond formation in a way that mimics the penicillin-sensitive peptide cross-linking reaction involved in ... [more ▼]

The serine DD-transpeptidase/penicillin-binding protein of Streptomyces K15 catalyzes peptide bond formation in a way that mimics the penicillin-sensitive peptide cross-linking reaction involved in bacterial cell wall peptidoglycan assembly. The Streptomyces K15 enzyme is peculiar in that it can be considered as an intermediate between classical penicillin-binding proteins, for which benzylpenicillin is a very efficient inactivator, and the resistant penicillin-binding proteins that have a low penicillin affinity. With its moderate penicillin sensitivity, the Streptomyces K15 DD-transpeptidase would be helpful in the understanding of the structure-activity relationship of this penicillin-recognizing protein superfamily. The structure of the Streptomyces K15 enzyme has been determined by x-ray crystallography at 2.0-A resolution and refined to an R-factor of 18.6%. The fold adopted by this 262-amino acid polypeptide generates a two-domain structure that is close to those of class A beta-lactamases. However, the Streptomyces K15 enzyme has two particular structural features. It lacks the amino-terminal alpha-helix found in the other penicilloyl-serine transferases, and it exhibits, at its surface, an additional four-stranded beta-sheet. These two characteristics might serve to anchor the enzyme in the plasma membrane. The overall topology of the catalytic pocket of the Streptomyces K15 enzyme is also comparable to that of the class A beta-lactamases, except that the Omega-loop, which bears the essential catalytic Glu(166) residue in the class A beta-lactamases, is entirely modified. This loop adopts a conformation similar to those found in the Streptomyces R61 DD-carboxypeptidase and class C beta-lactamases, with no equivalent acidic residue. [less ▲]

Detailed reference viewed: 6 (1 ULg)
Peer Reviewed
See detailTranscriptional Regulation Of The Mn/Ca 9 Gene Coding For The Tumor-Associated Carbonic Anhydrase Ix - Identification And Characterization Of A Proximal Silencer Element
Kaluz, S.; Kaluzova, M.; Opavsky, R. et al

in Journal of Biological Chemistry (1999), 274(46),

The MN/CA 9 (MN) gene encodes a tumor-associated isoenzyme of the carbonic anhydrase family. Functional characterization of the 3. 5-kilobase pair MN 5' upstream region by deletion analysis led to the ... [more ▼]

The MN/CA 9 (MN) gene encodes a tumor-associated isoenzyme of the carbonic anhydrase family. Functional characterization of the 3. 5-kilobase pair MN 5' upstream region by deletion analysis led to the identification of the -173 to +31 fragment as the MN promoter. In vitro DNase I footprinting revealed the presence of five protected regions (PRs) within the MN promoter. Detailed deletion analysis of the promoter identified PR1 and PR2 (numbered from the transcription start) as the most critical for transcriptional activity. PR4 negatively affected transcription, since its deletion led to increased promoter activity and was confirmed to function as a promoter-, position-, and orientation-independent silencer element. Mutational analysis indicated that the direct repeat AGGGCacAGGGC is required for efficient repressor binding. Two components of the repressor complex (35 and 42 kDa) were found to be in direct contact with PR4 by UV cross-linking. Increased cell density, known to induce MN expression, did not affect levels of PR4 binding in HeLa cells. Significantly reduced repressor level seems to be responsible for MN up-regulation in the case of tumorigenic CGL3 as compared with nontumorigenic CGL1 HeLa x normal fibroblast hybrid cells. [less ▲]

Detailed reference viewed: 5 (1 ULg)
Full Text
Peer Reviewed
See detailFunctional and cooperative interactions between the homeodomain PDX1, Pbx, and Prep1 factors on the somatostatin promoter
Goudet, Ghylène; Delhalle, Sylvie; Biemar, Frédéric et al

in Journal of Biological Chemistry (1999), 274(7), 4067-73

Expression of the somatostatin gene in endocrine pancreatic cells is controlled by several regulatory cis-elements located in the promoter region. Among these, the adjacent UE-A and TSEI elements, located ... [more ▼]

Expression of the somatostatin gene in endocrine pancreatic cells is controlled by several regulatory cis-elements located in the promoter region. Among these, the adjacent UE-A and TSEI elements, located from -113 to -85 relative to the transcription initiation site, function in combination and act as a pancreas-specific mini-enhancer. The TSEI element is recognized by the pancreatic homeodomain factor PDX1. In the present study, we show that the UE-A element binds a heterodimeric complex composed of a Pbx factor and the Prep1 protein, both belonging to the atypical three-amino acid loop extension homeodomain family. Recombinant Pbx1 and Prep1 proteins bind cooperatively to the UE-A site, whereas neither protein can bind this site alone. Transient transfection experiments reveal that both Pbx1 and Prep1 are required to generate a strong transcriptional activation from the UE-A element when this element is inserted close to the TATA box. In contrast, in the context of the intact somatostatin promoter or mini-enhancer, Pbx1 and Prep1 alone have no effect, but they produce a drastic activation when the pancreatic homeodomain factor PDX1 is also coexpressed. Thus, the activity of the somatostatin mini-enhancer is mediated by a cooperative interaction between the Pbx-Prep1 heterodimeric complex and the pancreatic factor PDX1. [less ▲]

Detailed reference viewed: 18 (2 ULg)
Full Text
Peer Reviewed
See detailLys13 plays a crucial role in the functional adaptation of the thermophilic triose-phosphate isomerase from Bacillus stearothermophilus to high temperatures
Alvarez, Marco; Wouters, Johan; Maes, Dominique et al

in Journal of Biological Chemistry (1999), 274(27), 19181-7

The thermophilic triose-phosphate isomerases (TIMs) of Bacillus stearothermophilus (bTIM) and Thermotoga maritima (tTIM) have been found to possess a His12-Lys13 pair instead of the Asn12-Gly13 pair ... [more ▼]

The thermophilic triose-phosphate isomerases (TIMs) of Bacillus stearothermophilus (bTIM) and Thermotoga maritima (tTIM) have been found to possess a His12-Lys13 pair instead of the Asn12-Gly13 pair normally present in mesophilic TIMs. His12 in bTIM was proposed to prevent deamidation at high temperature, while the precise role of Lys13 is unknown. To investigate the role of the His12 and Lys13 pair in the enzyme's thermoadaptation, we reintroduced the "mesophilic residues" Asn and Gly into both thermophilic TIMs. Neither double mutant displayed diminished structural stability, but the bTIM double mutant showed drastically reduced catalytic activity. No similar behavior was observed with the tTIM double mutant, suggesting that the presence of the His12 and Lys13 cannot be systematically correlated to thermoadaptation in TIMs. We determined the crystal structure of the bTIM double mutant complexed with 2-phosphoglycolate to 2.4-A resolution. A molecular dynamics simulation showed that upon substitution of Lys13 to Gly an increase of the flexibility of loop 1 is observed, causing an incorrect orientation of the catalytic Lys10. This suggests that Lys13 in bTIM plays a crucial role in the functional adaptation of this enzyme to high temperature. Analysis of bTIM single mutants supports this assumption. [less ▲]

Detailed reference viewed: 8 (3 ULg)
Full Text
Peer Reviewed
See detailTEL is a sequence-specific transcriptional repressor.
Lopez, Rodolphe; Carron, Clémence; Oury, Cécile ULg et al

in Journal of Biological Chemistry (1999)

TEL is a gene frequently involved in specific chromosomal translocations in human leukemia and sarcoma that encodes a member of the ETS family of transcriptional regulators. TEL is unusual among other ETS ... [more ▼]

TEL is a gene frequently involved in specific chromosomal translocations in human leukemia and sarcoma that encodes a member of the ETS family of transcriptional regulators. TEL is unusual among other ETS proteins by its ability to self-associate in vivo, a property that is essential to the oncogenic activation of TEL-derived fusion proteins. We show here that TEL is a sequence-specific transcriptional repressor of ETS-binding site-driven transcription of model and natural promoters. [less ▲]

Detailed reference viewed: 4 (0 ULg)
Full Text
Peer Reviewed
See detailSubcellular compartmentalization of activation and desensitization of responses mediated by NK2 neurokinin receptors
Vollmer, J-Y; Alix, Philippe ULg; Chollet, A et al

in Journal of Biological Chemistry (1999), 274

A functional fluorescent neurokinin NK2 receptor was constructed by joining enhanced green fluorescent protein to the amino-terminal end of the rat NK2 receptor and was expressed in human embryonic kidney ... [more ▼]

A functional fluorescent neurokinin NK2 receptor was constructed by joining enhanced green fluorescent protein to the amino-terminal end of the rat NK2 receptor and was expressed in human embryonic kidney cells. On cell suspensions, the binding of fluorescent Bodipy-labeled neurokinin A results in a saturatable and reversible decrease of NK2 receptor fluorescence via fluorescence resonance energy transfer. This can be quantified for nM to mM agonist concentrations and monitored in parallel with intracellular calcium responses. On single cells, receptor site occupancy and local agonist concentration can be determined in real time from the decrease in receptor fluorescence. Simultaneous measurement of intracellular calcium responses and agonist binding reveals that partial receptor site occupancy is sufficient to desensitize cellular response to a second agonist application to the same membrane area. Subsequent stimulation of a distal membrane area leads to a second response to agonist, provided that it had not been exposed to agonist during the first application. Together with persistent translocation of fluorescent protein kinase C to the membrane area exposed to agonist, the present data support that not only homologous desensitization but also heterologous desensitization of NK2 receptors is compartmentalized to discrete membrane domains. [less ▲]

Detailed reference viewed: 9 (2 ULg)
Full Text
Peer Reviewed
See detailIdentification and characterization of a protozoan uncoupling protein in Acanthamoeba castellanii.
Jarmuszkiewicz, W.; Sluse-goffart, C.; Hryniewiecka, L. et al

in Journal of Biological Chemistry (1999), 274(33), 23198-23202

An uncoupling protein (UCP) has been identified in mitochondria from Acanthamoeba castellanii, a nonphotosynthetic soil amoeboid protozoon that, in molecular phylogenesis, appears on a branch basal to the ... [more ▼]

An uncoupling protein (UCP) has been identified in mitochondria from Acanthamoeba castellanii, a nonphotosynthetic soil amoeboid protozoon that, in molecular phylogenesis, appears on a branch basal to the divergence points of plants, animals, and fungi. The existence of UCP in A. castellanii (AcUCP) has been revealed using antibodies raised against plant UCP. Its molecular mass (32,000 Da) was similar to those of plant and mammalian UCPs. The activity of AcUCP has been investigated in mitochondria depleted of free fatty acids. Additions of linoleic acid stimulated state 4 respiration and decreased transmembrane electrical potential (DeltaPsi) in a manner expected from fatty acid cycling-linked H(+) reuptake. The half-maximal stimulation by linoleic acid was reached at 8.1 +/- 0.4 microM. Bovine serum albumin (fatty acid-free), which adsorbs linoleic acid, reversed the respiratory stimulation and correspondingly restored DeltaPsi. AcUCP was only weakly inhibited by purine nucleotides like UCP in plants. A single force-flow relationship has been observed for state 4 respiration with increasing concentration of linoleic acid or of an uncoupler and for state 3 respiration with increasing concentration of oligomycin, indicating that linoleic acid has a pure protonophoric effect. The activity of AcUCP in state 3 has been evidenced by ADP/oxygen atom determination. The discovery of AcUCP indicates that UCPs emerged, as specialized proteins for H(+) cycling, early during phylogenesis before the major radiation of phenotypic diversity in eukaryotes and could occur in the whole eukaryotic world. [less ▲]

Detailed reference viewed: 8 (1 ULg)
Full Text
Peer Reviewed
See detailX-ray analysis of the NMC-A beta-lactamase at 1.64-A resolution, a class A carbapenemase with broad substrate specificity
Swaren, Peter; Maveyraud, Laurent; Raquet, Xavier et al

in Journal of Biological Chemistry (1998), 273(41), 26714-26721

Detailed reference viewed: 10 (0 ULg)
Full Text
Peer Reviewed
See detailCharacterization of the C-Terminal Propeptide Involved in Bacterial Wall Spanning of Alpha-Amylase from the Psychrophile Alteromonas Haloplanctis
Feller, Georges ULg; D'Amico, Salvino ULg; Benotmane, A. M. et al

in Journal of Biological Chemistry (1998), 273(20), 12109-15

The antarctic psychrophile Alteromonas haloplanctis secretes a Ca2+- and Cl--dependent alpha-amylase. The nucleotide sequence of the amy gene and the amino acid sequences of the gene products indicate ... [more ▼]

The antarctic psychrophile Alteromonas haloplanctis secretes a Ca2+- and Cl--dependent alpha-amylase. The nucleotide sequence of the amy gene and the amino acid sequences of the gene products indicate that the alpha-amylase precursor is a preproenzyme composed by the signal peptide (24 residues), the mature alpha-amylase (453 residues, 49 kDa), and a long C-terminal propeptide or secretion helper (192 residues, 21 kDa). In cultures of the wild-type strain, the 70-kDa precursor is secreted at the mid-exponential phase and is cleaved by a nonspecific protease into the mature enzyme and the propeptide. The purified C-terminal propeptide displays several features common to beta-pleated transmembrane proteins. It has no intramolecular chaperone function because active alpha-amylase is expressed by Escherichia coli in the absence of the propeptide coding region. In E. coli, the 70-kDa precursor is directed toward the supernatant. When the alpha-amylase coding region is excised from the gene, the secretion helper can still promote its own membrane spanning. It can also accept a foreign passenger, as shown by the extracellular routing of a beta-lactamase-propeptide fusion protein. [less ▲]

Detailed reference viewed: 6 (0 ULg)