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P Gros

Publications and source records attributed to P Gros.

At least 37 records · Page 2Linked to original sources
PubMed2001

The structure of leech anti-platelet protein, an inhibitor of haemostasis.

Leech anti-platelet protein (LAPP) from the leech Haementeria officinalis is a collagen-binding protein that inhibits the collagen-mediated adhesion of blood platelets. The crystal structure of recombinant LAPP has been determined using single isomorphous replacement with anomalous scattering combined with solvent flattening and threefold molecular averaging. The model of LAPP has been refined to 2.2 A resolution (R factor 21.5%; free R factor 24.0%). LAPP contains an 89-residue C-terminal domain consisting of a central six-stranded antiparallel beta-sheet flanked on one side by an alpha-helix and on the other side by two extended loops with little secondary structure. A 36-residue N-terminal region is not visible in the electron-density map. This region is rich in glycine and lacks hydrophobic residues. It probably does not have a compact globular fold, but instead has an extended conformation and is flexible. The crystal packing suggests that LAPP may form tightly interacting dimers. The fold of the C-terminal domain of LAPP closely resembles that of the N-domain of hepatocyte growth factor (HGF), which classifies LAPP as a PAN domain. However, no significant sequence homology exists between LAPP and other PAN domains. Common structural features between LAPP and the HGF N-domain include two disulfide bonds that link the alpha-helix to the central region of the protein and five residues with a conserved hydrophobic nature that are located in the core of the domain. These conserved structural features may be an important determinant of the PAN-domain type of fold.

Amino Acid Sequence↗
PubMed2001

Cysteines 431 and 1074 are responsible for inhibitory disulfide cross-linking between the two nucleotide-binding sites in human P-glycoprotein.

Human wild-type and Cys-less P-glycoproteins were expressed in Pichia pastoris and purified in high yield in detergent-soluble form. Both ran on SDS gels as a single 140-kDa band in the presence of reducing agent and showed strong verapamil-stimulated ATPase activity in the presence of added lipid. The wild type showed spontaneous formation of higher molecular mass species in the absence of reducing agent, and its ATPase was activated by dithiothreitol. Oxidation with Cu(2+) generated the same higher molecular mass species, primarily at 200 and approximately 300 kDa, in high yield. Cross-linking was reversed by dithiothreitol and prevented by pretreatment with N-ethylmaleimide. Using proteins containing different combinations of naturally occurring Cys residues, it was demonstrated that an inhibitory intramolecular disulfide bond forms between Cys-431 and Cys-1074 (located in the Walker A sequences of nucleotide-binding sites 1 and 2, respectively), giving rise to the 200-kDa species. In addition, dimeric P-glycoprotein species ( approximately 300 kDa) form by intermolecular disulfide bonding between Cys-431 and Cys-1074. The ready formation of the intramolecular disulfide between Cys-431 and Cys-1074 establishes that the two nucleotide-binding sites of P-glycoprotein are structurally very close and capable of intimate functional interaction, consistent with available information on the catalytic mechanism. Formation of such a disulfide in vivo could, in principle, underlie a regulatory mechanism and might provide a means of intervention to inhibit P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Recombinant congenic strains derived from A/J and C57BL/6J: a tool for genetic dissection of complex traits.

Complex genetic traits can be dissected in mice, using well-defined sets of recombinant inbred strains, congenic strains, and recombinant congenic strains (RCS). We report the creation of a series of 37 independent RCS derived from the commonly used inbred strains of laboratory mouse A/J (A) and C57BL/6J (B6). These RCS were derived by systematic inbreeding of independent pairs of animals from a (F1 x A) x A and a (F1 x B) x B double backcross (N3), to create AcB and BcA strains, respectively. Fifteen AcB strains and 22 BcA strains at between 18 and 30 generations of inbreeding have been generated, are healthy, and show stable breeding performance. These strains have been genotyped for a total of 625 informative microsatellite DNA markers covering the entire genome, with an average spacing of 2.6 cM. Haplotype analyses indicate that on average, AcB and BcA strains contain 13.25% of the donor genome, a value close to the 12.5% expected from the breeding scheme used in their creation. In the AcB set, approximately 79% of the B6 genome has been transferred in independent strains, while in the BcA set approximately 84% of the A genome is represented on the B6 background. This represents an excellent coverage of congenic segments from both parental genomes in the two sets of strains, which can now be used to map simple and complex traits in a genome-wide fashion. As an example of the power of AcB/BcA strains as a mapping tool, the 37 strains were typed for susceptibility to infection with Legionella pneumophila, a monogenic trait controlled by the Lgn1 locus on Chromosome 13. Analysis of the strain distribution pattern of L. pneumophila susceptibility allowed direct mapping of Lgn1 to a 3-cM interval. The AcB/BcA set should prove a useful tool with which to investigate the complex genetic basis of known interstrain differences between A and B6 for many important diseases.

Animals↗
PubMed2001

Crystallization and preliminary X-ray analysis of Drosophila glutathione S-transferase-2.

The sigma-class glutathione S-transferase-2 (GST-2) from Drosophila melanogaster is predominantly found within the indirect flight muscles (IFMs), where it is bound to the 'heavy' subunit of the IFM thin filament troponin complex (Tn-H). An N-terminal extension found in GST-2 is unique within the sigma GST class and may be involved in its interaction with Tn-H or modulate its enzymatic function. The recombinant protein has been crystallized at room temperature using ammonium sulfate as precipitant. Synchrotron radiation was used to measure a complete native data set to 1.75 A resolution from flash-cooled crystals. The crystals belong to one of the trigonal space groups P3(1)21 or P3(2)21, with unit-cell parameters a = b = 89.7, c = 131.8 A. The self-rotation function is consistent with a GST-2 dimer in the asymmetric unit.

Animals↗
PubMed2001

Identification of a new chemically induced allele (Lp(m1Jus)) at the loop-tail locus: morphology, histology, and genetic mapping.

Loop-tail (Lp) is a semidominant mutation that affects neurulation in mice. Heterozygous animals are characterized by a looped-tail appearance (pig tail) and wobbly head movements while homozygous embryos exhibit a neural tube closure defect that extends from the caudal midbrain to the tip of the tail. The Lp gene has been finely mapped to the distal part of chromosome 1, and a positional cloning strategy has been initiated to isolate the defective gene. This study represents the characterization of a new Lp allele (Lp(m1Jus)) induced by N-ethyl-N-nitrosurea mutagenesis. Lp(m1Jus)/+ mice have a looped-tail appearance, and both Lp(m1Jus)/Lp(m1Jus) homozygotes and Lp/Lp(m1Jus) compound heterozygotes fail to initiate neural tube closure along most of the embryonic axis. These data indicate that the Lp(m1Jus) allele causes a neural tube defect and overall phenotype similar to that of the original Lp allele. Segregation analysis of 90 (Lp(m1Jus)/+ x C57BL/6J)F(1) x C57BL/6J looped-tail mice with seven markers that define the Lp genetic map (D1Mit455/D1Mit146/D1Mit148/D1Mit270-1 cM-D1Mit113-0.4 cM-Lp-0.2 cM-D1Mit149-0.8 cM-D1Mit115) showed significant linkage between Lp(m1Jus) and all loci analyzed (P < 0.0001). Eight crossovers were detected with the proximal cluster of D1Mit455, D1Mit146, D1Mit148, and D1Mit270, indicating a recombination rate higher than expected in this region, and a single recombinant was encountered with the distal markers D1Mit149 and D1Mit115. Based on these phenotypic and genetic data, Lp(m1Jus) is most likely allelic to Lp, thereby representing a valuable additional tool for the positional cloning of the Lp gene and its subsequent molecular characterization.

Alkylating Agents↗
PubMed2001

Activation of phosphatidylinositol transfer protein alpha and beta isoforms from inclusion bodies.

Fully active phosphatidylinositol transfer protein (PI-TP) isoforms alpha and beta have been obtained from Escherichia coli inclusion bodies. Folding and activation of PI-TPalpha was achieved in the presence of DiC7:0-phosphatidylcholine-Triton X-114 (PtdCho-TX114) mixed micelles. Replacement of DiC7:0-PtdCho with the natural ligands of PI-TPalpha, i.e. long-chain PtdCho and phosphatidylinositol, did not stimulate activation. Efficient activation of PI-TPalpha required a low temperature (4 degrees C), the presence of dithiothreitol, and was achieved at a relatively high protein concentration (i.e. up to 500 microg ml(-1)). The inclusion bodies yielded 10 mg homogeneous PI-TPalpha per liter of E. coli culture. Conditions for full activation of PI-TPbeta were similar to those for PI-TPalpha except that long-chain PtdCho-TX114 mixed micelles and a very low protein concentration (i.e. 10 microg ml(-1)) were required. In contrast to PI-TPalpha, PI-TPbeta lost its lipid transfer activity within a few days. This inactivation could be prevented by addition of beta-alanine. In summary, despite 94% sequence similarity, PI-TPalpha and PI-TPbeta display a striking difference both in their preference for the PtdCho acyl chain length required for activation, and in their conformational stability after folding.

Amino Acid Sequence↗
PubMed2001

Expression of the DMT1 (NRAMP2/DCT1) iron transporter in mice with genetic iron overload disorders.

Iron overload is highly prevalent, but its molecular pathogenesis is poorly understood. Recently, DMT1 was shown to be a major apical iron transporter in absorptive cells of the duodenum. In vivo, it is the only transporter known to be important for the uptake of dietary non-heme iron from the gut lumen. The expression and subcellular localization of DMT1 protein in 3 mouse models of iron overload were examined: hypotransferrinemic (Trf(hpx)) mice, Hfe knockout mice, and B2m knockout mice. Interestingly, in Trf(hpx) homozygotes, DMT1 expression was strongly induced in the villus brush border when compared to control animals. This suggests that DMT1 expression is increased in response to iron deficiency in the erythron, even in the setting of systemic iron overload. In contrast, no increase was seen in DMT1 expression in animals with iron overload resembling human hemochromatosis. Therefore, it does not appear that changes in DMT1 levels are primarily responsible for iron loading in hemochromatosis.

Animals↗
PubMed2001

Major photoaffinity drug binding sites in multidrug resistance protein 1 (MRP1) are within transmembrane domains 10-11 and 16-17.

MRP1 is an ABC (or ATP binding cassette) membrane transport protein shown to confer resistance to structurally dissimilar drugs. Studies of MRP1 topology suggested the presence of a hydrophobic N-domain with five potential membrane-spanning domains linked to an MDR1-like core (MSD1-NBD1-L1-MSD2-NBD2) by an intracellular linker domain (L0). MRP1-mediated multidrug resistance is thought to be due to enhanced drug efflux. However, little is known about MRP1-drug interaction and its drug binding site(s). We previously developed several photoreactive probes to study MRP1-drug interactions. In this report, we have used eight MRP1-HA variants that were modified to have hemagglutinin A (HA) epitopes inserted at different sites in MRP1 sequence. Exhaustive in-gel digestion of all IAARh123 photoaffinity-labeled MRP1-HA variants revealed the same profile of photolabeled peptides as seen for wild type MRP1. Photolabeling of the different MRP1-HA variants followed by digestion with increasing concentrations of trypsin or Staphylococcus aureus V8 protease (1:800 to 1:5 w/w) and immunoprecipitation with anti-HA mAb identified two small photolabeled peptides ( approximately 6-7 kDa) from MRP1-HA(574) and MRP1-HA(1222). Based on the location of the HA epitopes in the latter variants together with molecular masses of the two peptides, the photolabeled amino acid residues were localized to MRP1 sequences encoding transmembranes 10 and 11 of MSD1 (Ser(542)-Arg(593)) and transmembranes 16 and 17 of MSD2 (Cys(1205)-Glu(1253)). Interestingly, the same sequences in MRP1 were also photolabeled with a structurally different photoreactive drug, IACI, confirming the significance of transmembranes 10, 11, 16 and 17 in MRP1 drug binding. Taken together, the results in this study provide the first delineation of the drug binding site(s) of MRP1. Furthermore, our findings suggest the presence of common drug binding site(s) for structurally dissimilar drugs.

ATP-Binding Cassette Transporters↗
PubMed2001

Biophysical characterization of the influence of salt on tetrameric SecB.

SecB is a tetrameric chaperone, with a monomeric molecular mass of 17 kDa, that is involved in protein translocation in Escherichia coli. It has been hypothesized that SecB undergoes a conformational change as a function of the salt concentration. To gain more insight into the salt-dependent behavior of SecB, we studied the protein in solution by dynamic light scattering, size exclusion chromatography, analytical ultracentrifugation, and small angle neutron scattering. The results clearly demonstrate the large influence of the salt concentration on the behavior of SecB. At high salt concentration, SecB is a non-spherical protein with a radius of gyration of 3.4 nm. At low salt concentration the hydrodynamic radius of the protein is apparently decreased, whereas the ratio of the frictional coefficients is increased. The protein solution behaves in a non-ideal way at low salt concentrations, as was shown by the analytical ultracentrifugation data and a pronounced interparticle effect observed by small angle neutron scattering. A possible explanation is a change in surface charge distribution dependent on the salt concentration in the solvent. We summarize our data in a model for the salt-dependent conformation of tetrameric SecB.

Bacterial Proteins↗
PubMed2001

Divalent-metal transport by NRAMP proteins at the interface of host-pathogen interactions.

The NRAMP family of divalent-metal transporters plays a key role in the homeostasis of iron and other metals. NRAMP2 (DMT1) acts as an iron-uptake protein in both the duodenum and in peripheral tissues. NRAMP1 functions as a divalent-metal efflux pump at the phagosomal membrane of macrophages and neutrophils, and mutations in NRAMP1 cause susceptibility to several intracellular pathogens. NRAMP homologues have been identified in bacteria and are involved in acquiring divalent metals from the extracellular environment. Interestingly, bacterial and mammalian NRAMP proteins would compete for the same essential substrates within the microenvironment of the phagosome, at the interface of host-pathogen interactions.

Animals↗
PubMed2001

Ltap, a mammalian homolog of Drosophila Strabismus/Van Gogh, is altered in the mouse neural tube mutant Loop-tail.

Neural tube defects (NTDs) such as spina bifida and anencephaly are common congenital malformations in humans (1/1,000 births) that result from failure of the neural tube to close during embryogenesis. The etiology of NTDs is complex, with both genetic and environmental contributions; the genetic component has been extensively studied with mouse models. Loop-tail (Lp) is a semidominant mutation on mouse chromosome 1 (ref. 4). In the two known Lp alleles (Lp, Lpm1Jus), heterozygous mice exhibit a characteristic looped tail, and homozygous embryos show a completely open neural tube in the hindbrain and spinal region, a condition similar to the severe craniorachischisis defect in humans. Morphological and neural patterning studies indicate a role for the Lp gene product in controlling early morphogenesis and patterning of both axial midline structures and the developing neural plate. The 0.6-cM/0.7-megabase (Mb) Lp interval is delineated proximally by D1Mit113/Apoa2/Fcer1g and distally by Fcer1a/D1Mit149/Spna1 and contains a minimum of 17 transcription units. One of these genes, Ltap, encodes a homolog of Drosophila Strabismus/Van Gogh (Stbm/Vang), a component of the frizzled/dishevelled tissue polarity pathway. Ltap is expressed broadly in the neuroectoderm throughout early neurogenesis and is altered in two independent Lp alleles, identifying this gene as a strong candidate for Lp.

Amino Acid Sequence↗
PubMed2001

Susceptibility to mouse cytomegalovirus is associated with deletion of an activating natural killer cell receptor of the C-type lectin superfamily.

Cytomegalovirus is the leading cause of congenital viral disease and the most important opportunistic infection in immunocompromised patients. We have used a mouse experimental infection model (MCMV) to study the genetic parameters of host/virus interaction. Susceptibility to infection with MCMV is controlled by Cmv1, a chromosome 6 locus that regulates natural killer (NK) cell activity against virally infected targets. Here, we use a positional cloning strategy to isolate the gene mutated at the Cmv1 locus. Cmv1 maps within a 0.35-cM interval defined by markers D6Ott8 and D6Ott115, which corresponds to a physical distance of 1.6 Mb (refs. 6-8). A transcript map of the region identified 19 genes, including members of the killer cell lectin-like receptor family a (Klra, formerly Ly49; refs. 9-12), which encode inhibitory or activating NK cell receptors that interact with MHC class I molecules. Klra genes have different copy numbers and genomic organization, and are highly polymorphic among inbred strains, making it difficult to distinguish between normal allelic variants and distinct Klra genes, or possible mutations associated with Cmv1. The recombinant inbred strain BXD-8/Ty (BXD-8; ref. 18), derived from Cmv1r C57BL/6 (B6, resistant) and Cmv1s DBA/2 (susceptible), is of particular interest because it is highly susceptible to MCMV infection despite having a B6 haplotype at Cmv1. We determined that MCMV susceptibility in BXD-8 is associated with the deletion of Klra8 (formerly Ly49h).

Animals↗
PubMed2001

Solvent behaviour in flash-cooled protein crystals at cryogenic temperatures.

The solvent behaviour of flash-cooled protein crystals was studied in the range 100--180 K by X-ray diffraction. If the solvent is within large channels it crystallizes at 155 K, as identified by a sharp change in the increase of unit-cell volume upon temperature increase. In contrast, if a similar amount of solvent is confined to narrow channels and/or individual cavities it does not crystallize in the studied temperature range. It is concluded that the solvent in large channels behaves similarly to bulk water, whereas when confined to narrow channels it is mainly protein-associated. The analogy with the behaviour of pure bulk water provides circumstantial evidence that only solvent in large channels undergoes a glass transition in the 100--180 K temperature range. These studies reveal that flash-cooled protein crystals are arrested in a metastable state up to at least 155 K, thus providing an upper temperature limit for their storage and handling. The results are pertinent to the development of rational crystal annealing procedures and to the study of temperature-dependent radiation damage to proteins. Furthermore, they suggest an experimental paradigm for studying the correlation between solvent behaviour, protein dynamics and protein function.

Acetylcholinesterase↗
PubMed2001

Specific protein dynamics near the solvent glass transition assayed by radiation-induced structural changes.

The nature of the dynamical coupling between a protein and its surrounding solvent is an important, yet open issue. Here we used temperature-dependent protein crystallography to study structural alterations that arise in the enzyme acetylcholinesterase upon X-ray irradiation at two temperatures: below and above the glass transition of the crystal solvent. A buried disulfide bond, a buried cysteine, and solvent exposed methionine residues show drastically increased radiation damage at 155 K, in comparison to 100 K. Additionally, the irradiation-induced unit cell volume increase is linear at 100 K, but not at 155 K, which is attributed to the increased solvent mobility at 155 K. Most importantly, we observed conformational changes in the catalytic triad at the active site at 155 K but not at 100 K. These changes lead to an inactive catalytic triad conformation and represent, therefore, the observation of radiation-inactivation of an enzyme at the atomic level. Our results show that at 155 K, the protein has acquired--at least locally--sufficient conformational flexibility to adapt to irradiation-induced alterations in the conformational energy landscape. The increased protein flexibility may be a direct consequence of the solvent glass transition, which expresses as dynamical changes in the enzyme's environment. Our results reveal the importance of protein and solvent dynamics in specific radiation damage to biological macromolecules, which in turn can serve as a tool to study protein flexibility and its relation to changes in a protein's environment.

Acetylcholinesterase↗
PubMed2001

[Isolated facial palsy and occult adenoid cystic carcinoma of the parotid].

We report a case of adenoid cystic carcinoma of the parotid disclosed by facial palsy alone. No tumefaction could be detected clinically or at imaging. The diagnosis was established at surgical exploration of the facial nerve. Total extended parotidectomy was completed by radiotherapy of the tumor site.

Carcinoma, Adenoid Cystic↗
PubMed2000

Rhodamine 123 binds to multiple sites in the multidrug resistance protein (MRP1).

The mechanisms of MRP1-drug binding and transport are not clear. In this study, we have characterized the interaction between MRP1 and rhodamine 123 (Rh123) using the photoreactive-iodinated analogue, [(125)I]iodoaryl azido-rhodamine 123 (or IAARh123). Photoaffinity labeling of plasma membranes from HeLa cells transfected with MRP1 cDNA (HeLa-MRP1) with IAARh123 shows the photolabeling of a 190 kDa polypeptide not labeled in HeLa cells transfected with the vector alone. Immunoprecipitation of a 190 kDa photolabeled protein with MRP1-sepcific monoclonal antibodies (QCRL-1, MRPr1, and MRPm6) confirmed the identity of this protein as MRP1. Analysis of MRP1-IAARh123 interactions showed that photolabeling of membranes from HeLa-MRP1 with increasing concentrations of IAARh123 was saturable, and was inhibited with excess of IAARh123. Furthermore, the photoaffinity labeling of MRP1 with IAARh123 was greatly reduced in the presence of excess Leukotreine C(4) or MK571, but to a lesser extent with excess doxorubicin, colchicine or chloroquine. Cell growth assays showed 5-fold and 14-fold increase in the IC(50) of HeLa-MRP1 to Rh123 and the Etoposide VP16 relative to HeLa cells, respectively. Analysis of Rh123 fluorescence in HeLa and HeLa-MRP1 cells with or without ATP suggests that cross-resistance to Rh123 is in part due to reduced drug accumulation in the cytosol of HeLa-MRP1 cells. Mild digestion of purified IAARh123-photolabeled MRP1 with trypsin showed two large polypeptides (approximately 111 and approximately 85 kDa) resulting from cleavage in the linker domain (L1) connecting the multiple-spanning domains MSD0 and MSD1 to MSD2. Exhaustive proteolysis of purified IAARh123-labeled 85 and 111 kDa polypeptides revealed one (6 kDa) and two (approximately 6 plus 4 kDa) photolabeled peptides, respectively. Resolution of total tryptic digest of IAARh123-labeled MRP1 by HPLC showed three radiolabeled peaks consistent with the three Staphylococcus aureus V8 cleaved peptides from the Cleveland maps. Together, the results of this study show direct binding of IAARh123 to three sites that localize to the N- and C-domains of MRP1. Moreover, IAARh123 provides a sensitive and specific probe to study MRP1-drug interactions.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

The Nramp2/DMT1 iron transporter is induced in the duodenum of microcytic anemia mk mice but is not properly targeted to the intestinal brush border.

Microcytic anemia (mk) mice and Belgrade (b) rats are severely iron deficient because of impaired intestinal iron absorption and defective iron metabolism in peripheral tissues. Both animals carry a glycine to arginine substitution at position 185 in the iron transporter known as Nramp2/DMT1 (divalent metal transporter 1). DMT1 messenger RNA (mRNA) and protein expression has been examined in the gastrointestinal tract of mk mice. Northern blot analysis indicates that, by comparison to mk/+ heterozygotes, mk/mk homozygotes show a dramatic increase in the level of DMT1 mRNA in the duodenum. This increase in RNA expression is paralleled by a concomitant increase of the 100-kd DMT1 isoform I protein expression in the duodenum. Immunohistochemical analyses show that, as for normal mice on a low-iron diet, DMT1 expression in enterocytes of mk/mk mice is restricted to the duodenum. However, and in contrast to normal enterocytes, little if any expression of DMT1 is seen at the apical membrane in mk/mk mice. These results suggest that the G185R mutation, which was shown to impair the transport properties of DMT1, also affects the membrane targeting of the protein in mk/mk enterocytes. This loss of function of DMT1 is paralleled by a dramatic increase in expression of the defective protein in mk/mk mice. This is consistent with a feedback regulation of DMT1 expression by iron stores. (Blood. 2000;96:3964-3970)

Alleles↗