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Biomedical subjects

Philippe Gros

Publications and source records attributed to Philippe Gros.

At least 19 recordsLinked to original sources
PubMed2007

Mutational analysis of conserved aromatic residues in the A-loop of the ABC transporter ABCB1A (mouse Mdr3).

The A-loop is a recently described conserved region in the NBDs of ABC transporters [Ambudkar, S.V., Kim, I.-W., Xia, D. and Sauna, Z.E. (2006) The A-loop, a novel conserved aromatic acid subdomain upstream of the Walker A motif in ABC transporters, is critical for ATP binding. FEBS Lett. 580, 1049-1055; Kim, I.W., Peng, X.H., Sauna, Z.E., FitzGerald, P.C., Xia, D., Muller, M., Nandigama, K. and Ambudkar, S.V. (2006) The conserved tyrosine residues 401 and 1044 in ATP sites of human P-glycoprotein are critical for ATP binding and hydrolysis: evidence for a conserved subdomain, the A-loop in the ATP-binding cassette. Biochemistry 45, 7605-7616]. In mouse P-glycoprotein (Abcb1a), the aromatic residue of the A-loop in both NBDs is a tyrosine: Y397 in NBD1 and Y1040 in NBD2. Another tyrosine residue (618 in NBD1 and 1263 in NBD2) also appears to lie in proximity to the ATP molecule. We have mutated residues Y397, Y618, Y1040, and Y1263 to tryptophan and analyzed the effect of these substitutions on transport properties, ATP binding, and ATP hydrolysis by Abcb1a (mouse Mdr3). Y618W and Y1263W enzymes had catalytic characteristics similar to WT Abcb1a. On the other hand, Y397W and Y1040W showed impaired transport and greatly reduced ATPase activity, including a approximately 10-fold increase in Km for MgATP. Thus, Y397 and Y1040 play an important role in Abcb1a catalysis.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2006

Birc1e/Naip5 rapidly antagonizes modulation of phagosome maturation by Legionella pneumophila.

Legionella survives intracellularly by preventing fusion with lysosomes, due to phagosome escape from the endocytic pathway at an early stage of phagosome maturation, and by creating a replicative organelle that acquires endoplasmic reticulum (ER) characteristics through sustained interactions and fusion with the ER. Intracellular replication of Legionella pneumophila in mouse macrophages is controlled by the Lgn1 locus. Functional complementation in vivo has identified the Birc1e/Naip5 gene as being responsible for the Lgn1 effect. To understand the function and temporal site of action of Birc1e/Naip5 in susceptibility to L. pneumophila, we examined the biogenesis of Legionella-containing vacuoles (LCVs) formed in permissive A/J macrophages and in their Birc1e/Naip5 transgenic non-permissive counterpart. Birc1e/Naip5 effects on acquisition of lysosomal and ER markers were evident within 1-2 h following infection. A significantly higher proportion of LCVs formed in Birc1e/Naip5 transgenic macrophages had acquired the lysosomal markers cathepsin D and Lamp1 by 2 h post infection, whereas a significantly higher proportion of LCVs formed in permissive macrophages were positively stained for the ER markers BAP31 and calnexin, 6 h post infection. Likewise, studies by electron microscopy showed acquisition of lysosomal contents (horseradish peroxidase), within the first hour following phagocytic uptake, by LCVs formed in Birc1e/Naip5 transgenic macrophages and delivery of the ER marker glucose 6-phosphatase (G6Pase) only to the lumen of LCVs formed in A/J macrophages. Finally, a larger proportion of LCVs formed in A/J macrophages were studded with ribosomes 24 h post infection, compared with LCVs formed in Birc1e/Naip5 transgenic macrophages. These results suggest that sensing of L. pneumophila products by Birc1e/Naip5 in macrophages occurs rapidly following phagocytosis, a process that antagonizes the ability of L. pneumophila to remodel its phagosome into a specialized vacuole with ER characteristics.

Animals↗
PubMed2006

Competition of bacteriophage polypeptides with native replicase proteins for binding to the DNA sliding clamp reveals a novel mechanism for DNA replication arrest in Staphylococcus aureus.

Bacteriophages have evolved specific mechanisms that redirect bacterial metabolic pathways to the bacteriophage reproduction cycle. In this study, we characterized the bactericidal mechanism of two polypeptides from bacteriophages Twort and G1 that target the DNA sliding clamp of Staphylococcus aureus. The DNA sliding clamp, which tethers DNA polymerase to its template and thereby confers processivity upon the enzyme, was found to be essential for the viability of S. aureus. Expression of polypeptides TwortORF168 and G1ORF240 in S. aureus selectively inhibited DNA replication which in turn resulted in cell death. Both polypeptides specifically inhibited the S. aureus DNA replicase that was reconstituted in vitro but not the corresponding replicase of Streptococcus pyogenes. We demonstrated that inhibition of DNA synthesis is multifaceted and occurs via binding the DNA sliding clamp: TwortORF168 and G1ORF240 bound tightly to the DNA sliding clamp and prevented both its loading onto DNA and its interaction with DNA polymerase C. These results elucidate the impact of bacteriophage polypeptide expression upon DNA replication in the growing cell.

Amino Acid Sequence↗
PubMed2006

Identification of a tyrosine-based motif (YGSI) in the amino terminus of Nramp1 (Slc11a1) that is important for lysosomal targeting.

In macrophages, Nramp1 (Slc11a1) is expressed in lysosomes and restricts replication of intracellular pathogens by removing divalent metals (Mn2+ and Fe2+) from the phagolysosome. Nramp2 (DMT1, Slc11a2) is expressed both at the duodenal brush border where it mediates uptake of dietary iron and ubiquitously at the plasma membrane/recycling endosomes of many cell types where it transports transferrin-associated iron across the endosomal membrane. In Nramp2, a carboxyl-terminal cytoplasmic motif ((555)YLLNT(559)) is critical for internalization and recycling of the transporter from the plasma membrane. Here we studied the subcellular trafficking properties of Nramp1 and investigated the cis-acting sequences responsible for targeting to lysosomes. For this, we constructed and studied Nramp1/Nramp2 chimeric proteins where homologous domains of each protein were exchanged. Chimeras exchanging the amino-(upstream TM1) and carboxyl-terminal (downstream TM12) cytoplasmic segments of both transporters were stably expressed in porcine LLC-PK1 kidney cells and were studied with respect to expression, maturation, stability, cell surface targeting, transport activity, and subcellular localization. An Nramp2 isoform II chimera bearing the amino terminus of Nramp1 was not expressed at the cell surface but was targeted to lysosomes. This lysosomal targeting was abolished by single alanine substitutions at Tyr15 and Ile18 of a (15)YGSI(18) motif present in the amino terminus of Nramp1. These results identify YGSI as a tyrosine-based sorting signal responsible for lysosomal targeting of Nramp1.

Amino Acid Motifs↗
PubMed2006

Proteomic analysis of Salmonella enterica serovar typhimurium isolated from RAW 264.7 macrophages: identification of a novel protein that contributes to the replication of serovar typhimurium inside macrophages.

To evade host resistance mechanisms, Salmonella enterica serovar Typhimurium (STM), a facultative intracellular pathogen, must alter its proteome following macrophage infection. To identify new colonization and virulence factors that mediate STM pathogenesis, we have isolated STM cells from RAW 264.7 macrophages at various time points following infection and used a liquid chromatography-mass spectrometry-based proteomic approach to detect the changes in STM protein abundance. Because host resistance to STM infection is strongly modulated by the expression of a functional host-resistant regulator, i.e. natural resistance-associated macrophage protein 1 (Nramp1, also called Slc11a1), we have also examined the effects of Nramp1 activity on the changes of STM protein abundances. A total of 315 STM proteins have been identified from isolated STM cells, which are largely housekeeping proteins whose abundances remain relatively constant during the time course of infection. However, 39 STM proteins are strongly induced after infection, suggesting their involvement in modulating colonization and infection. Of the 39 induced proteins, 6 proteins are specifically modulated by Nramp1 activity, including STM3117, as well as STM3118-3119 whose time-dependent abundance changes were confirmed using Western blot analysis. Deletion of the gene encoding STM3117 resulted in a dramatic reduction in the ability of STM to colonize wild-type RAW 264.7 macrophages, demonstrating a critical involvement of STM3117 in promoting the replication of STM inside macrophages. The predicted function common for STM3117-3119 is biosynthesis and modification of the peptidoglycan layer of the STM cell wall.

Animals↗
PubMed2006

The paired domain of Pax3 contains a putative homeodomain interaction pocket defined by cysteine scanning mutagenesis.

Pax3 is a transcription factor that plays an important regulatory role during neurogenesis, myogenesis, and formation of neural crest cell derived structures. Pax3 has two DNA binding domains, a paired domain (PD) and paired-type homeodomain (HD) that show complete interdependence for DNA binding, with mutations in one domain impairing DNA binding by the other domain. Cooperative interactions between the PD and HD of Pax3 suggest that the two domains may physically interact for DNA binding. Site-specific modification with thiol reagents in single cysteine Pax3 mutants was used to determine which segment of the PD may interact with the HD. Twenty-four single cysteine mutants were independently introduced in the second alpha-helix (alpha2, positions 59-80) and in the beta-hairpin structure (positions 40-41) at the amino terminal portion of the PD. These mutants were tested for their ability to bind to PD (P6CON, P3OPT) and HD-specific DNA targets (P2), and the effect of treatment with N-ethylmaleimide on these binding properties was established. In the PD, single cysteine mutants CL/Q40C, CL/I59C, CL/V60C, CL/P69C, CL/S70C, CL/I72C, CL/S73C, CL/L76C, CL/V78C, and CL/S79C displayed NEM sensitive DNA binding toward both PD and HD targets. Three PD mutants (CL/L41C, CL/A63C, and CL/H64C) showed unusual behavior, with DNA binding to PD targets being NEM insensitive while DNA binding by the HD was abrogated by NEM treatment. Three-dimensional modeling of the NEM sensitive PD cysteine mutants reveal that they are not randomly distributed, but rather that they cluster in a hydrophobic pocket. We propose that this hydrophobic pocket may serve as a docking site for the HD during DNA binding by the intact protein.

Amino Acid Sequence↗
PubMed2006

A new class of small molecule RNA polymerase inhibitors with activity against rifampicin-resistant Staphylococcus aureus.

The RNA polymerase holoenzyme is a proven target for antibacterial agents. A high-throughput screening program based on this enzyme from Staphylococcus aureus had previously identified a 2-ureidothiophene-3-carboxylate as a low micromolar inhibitor. An investigation of the relationships between the structures of this class of compounds and their inhibitory- and antibacterial activities is described here, leading to a set of potent RNA polymerase inhibitors with antibacterial activity. Characterization of this bioactivity, including studies of the mechanism of action, is provided, highlighting the power of the reverse chemical genetics approach in providing tools to inhibit the bacterial RNA polymerase.

Anti-Bacterial Agents↗
PubMed2006

A novel R416C mutation in human DMT1 (SLC11A2) displays pleiotropic effects on function and causes microcytic anemia and hepatic iron overload.

A patient suffering from microcytic anemia and hepatic iron overload was found to be compound heterozygote for polymorphisms in the iron transporter DMT1 (Nramp2, SLC11A2), including a 3-bp deletion (DMT1(delCTT)) in intron 4 that partially impairs splicing and an amino acid substitution (DMT1(C1246T), R416C) at a conserved residue in transmembrane domain 9 of the protein. The functional properties and possible contribution to disease of the DMT1 R416C mutation were studied in independent mutants at that position (R416C, R416A, R416K, R416E) expressed in LLC-PK(1) kidney cells. Non-conservative substitutions at R416 (C, A, E) cause multiple functional deficiencies including defective protein processing, loss of transport activity, impaired cell surface targeting, and recycling through endosomes, concomitant with retention of the transporter in the endoplasmic reticulum. Conversely, a conservative isoelectric substitution (R416K) was less vulnerable, resulting in a functional transporter that was properly processed and targeted to the cell surface and to recycling endosomes. We propose that DMT1(C1246T) (R416C) represents a complete loss-of-function, and that a quantitative reduction in DMT1 expression is the cause of the microcytic anemia and iron overload in the patient.

Amino Acid Substitution↗
PubMed2006

Distinct targeting and recycling properties of two isoforms of the iron transporter DMT1 (NRAMP2, Slc11A2).

The metal transporter DMT1 (Slc11a2) plays a vital role in iron metabolism. Alternative splicing of the 3' exon generates two DMT1 isoforms with different C-terminal protein sequences and a 3' untranslated region harboring (isoform I, +IRE) or not (isoform II, -IRE), an iron-responsive element. Isoform I is expressed at the plasma membrane of certain epithelial cells including the duodenum brush border, where it is essential for the absorption of nutritional iron. Isoform II is expressed in many cells and is essential for the acquisiton of transferrin iron from acidified endosomes. The targeting and trafficking properties of DMT1 isoforms I and II were studied in transfected LLC-PK(1) kidney cells, with respect to isoform-specific differences in function, subcellular localization, endocytosis kinetics, and fate upon internalization. Isoform I showed higher surface expression and was internalized from the plasma membrane with slower kinetics than that of isoform II. As opposed to isoform II, which is efficiently sorted to recycling endosomes upon internalization, isoform I was not efficiently recycled and was targeted to lysosomes. Thus, alternative splicing of DMT1 critically regulates the subcellular localization and site of Fe(2+) transport.

Alternative Splicing↗
PubMed2006

The impact of genomics on the analysis of host resistance to infectious disease.

The advent of new technologies and resources, including the complete sequence of mammalian genomes, has had a dramatic impact on the genetic analysis of susceptibility to infections in humans and in animal models of infection. Genes responsible for simple or complex control of susceptibility to infection with different pathogens have been recently identified and characterized, and are reviewed herein.

Animals↗
PubMed2006

Cardiac failure in C5-deficient A/J mice after Candida albicans infection.

The effect of a deficiency in the C5 component of complement on the pathophyisology of infection with the fungal pathogen Candida albicans was studied by using the A/J inbred mouse strain and the BcA17 congenic mouse strain. Acute infection caused by intravenous injection of C. albicans blastospores is associated with rapid fungal replication in the heart, brain, and, in particular, kidneys of C5-deficient mice. Histological studies and analysis of markers for tissue damage indicated that the heart is the organ that is most affected and that it ultimately fails in C5-deficient mice. In A/J and BcA17 mice, tissue damage is associated with (i) cellular infiltration in the heart, which is not seen in the kidney despite the higher fungal load in the latter organ, and (ii) a very strong inflammatory response, including elevated levels of many cytokines and chemokines. This results in cardiomyopathy, which is associated with elevated levels of creatine kinase and cardiac troponin I in the circulation. Damage to the cardiac muscle is associated with metabolic changes, including hypoglycemia, decreased lipid utilization resulting in elevated levels of cardiac triglycerides, and unproductive glucose utilization linked to a dramatic increase in the level of pyruvate dehydrogenase kinase 4 (Pdk4), a negative regulator of the pyruvate dehydrogenase complex.

Animals↗
PubMed2006

Influence of Slc11a1 on the outcome of Salmonella enterica serovar Enteritidis infection in mice is associated with Th polarization.

Genetic analyses identified Ses1 as a significant quantitative trait locus influencing the carrier state of 129S6 mice following a sublethal challenge with Salmonella enterica serovar Enteritidis. Previous studies have determined that Slc11a1 was an excellent candidate gene for Ses1. Kinetics of infection in 129S6 mice and Slc11a1-deficient (129S6-Slc11a1(tm1Mcg)) mice demonstrated that the wild-type allele of Slc11a1 contributed to the S. enterica serovar Enteritidis carrier state as early as 7 days postinfection. Gene expression profiling demonstrated that 129S6 mice had a significant up-regulation of proinflammatory genes associated with macrophage activation at day 10 postinfection, followed by a gradual increase in immunoglobulin transcripts, whereas 129S6-Slc11a1(tm1Mcg) mice had higher levels of immunoglobulins earlier in the infection. Quantitative reverse transcription-PCR revealed an increase in Th1 cytokine (Ifng and Il12) and Th1-specific transcription factor Tbx21 expression during infection in both the 129S6 and 129S6-Slc11a1(tm1Mcg) strains. However, the expression of Gata3, a transcription factor involved in Th2 polarization, Cd28, and Il4 was markedly increased in Slc11a1-deficient mice during infection, suggesting a predominant Th2 phenotype in 129S6-Slc11a1(tm1Mcg) animals following S. enterica serovar Enteritidis infection. A strong immunoglobulin G2a response, reflecting Th1 activity, was observed only in 129S6 mice. All together, these results are consistent with an impact of Slc11a1 on Th cell differentiation during chronic S. enterica serovar Enteritidis infection. The presence of a Th2 bias in Slc11a1-deficient mice is associated with improved bacterial clearance.

Animals↗
PubMed2006

Comparative genomic analysis of 18 Pseudomonas aeruginosa bacteriophages.

A genomic analysis of 18 P. aeruginosa phages, including nine newly sequenced DNA genomes, indicates a tremendous reservoir of proteome diversity, with 55% of open reading frames (ORFs) being novel. Comparative sequence analysis and ORF map organization revealed that most of the phages analyzed displayed little relationship to each other.

DNA, Viral↗
PubMed2005

Triaminotriazine DNA helicase inhibitors with antibacterial activity.

Screening of a chemical library in a DNA helicase assay involving the Pseudomonas aeruginosa DnaB helicase provided a triaminotriazine inhibitor with good antibacterial activity but associated cytotoxicity toward mammalian cells. Synthesis of analogs provided a few inhibitors that retained antibacterial activity and demonstrated a significant reduction in cytotoxicity. The impact of serum and initial investigations toward a mode of action highlight several features of this class of compounds as antibacterials.

Amination↗
PubMed2005

Visualization of vacuolar acidification-induced transcription of genes of pathogens inside macrophages.

The objective of these studies was to analyze the role of the ionic environment of phagosomal vacuoles in the control of pathogens by macrophages. Digital imaging and flow cytometry were used to follow the induction of the phoP promoter of Salmonella enterica Typhimurium within live macrophages. Manipulating the Mg2+ concentration within the Salmonella-containing vacuole (SCV) was without effect on the early induction of PhoPQ. Moreover, direct measurement of [Mg2+] within the SCV using nanosensor particles showed that, during this initial period of phoP activation, the concentration of the divalent cation is rapidly regulated and stabilizes around 1 mm. Extrusion of other divalent cations via the Nramp1 efflux pump was similarly ruled out as an important contributor to the activation of the regulon. By contrast, induction of PhoP was greatly attenuated when the pH gradient across the SCV membrane was dissipated. A second, more modest pH-independent component of PhoP induction was unmasked by inhibition of the vacuolar proton pump. This second component was eliminated by pretreatment of cells with IFNgamma, even though the cytokine augmented the overall PhoP response. These findings demonstrate the existence of at least three separate activators of phoP transcription: resting and IFNgamma-stimulated pH-sensitive components, plus a pH-independent component.

Acids↗
PubMed2005

Carboxyl-terminus determinants of the iron transporter DMT1/SLC11A2 isoform II (-IRE/1B) mediate internalization from the plasma membrane into recycling endosomes.

Mutations in DMT1 (Nramp2 and Slc11a2) impair iron metabolism and cause microcytic anemia. DMT1 is expressed at the duodenal brush border where it controls uptake of dietary iron and is present at the plasma membrane and in recycling endosomes of most cells, where it is necessary for acquisition of transferrin-associated iron. The goal of this study was to identify signal(s) in the cytoplasmic segments of DMT1 responsible for its subcellular targeting and internalization from the plasma membrane into recycling endosomes. We introduced mutations in the amino terminus (DeltaNT), carboxyl terminus (DeltaCT), as well as in NPAY28-31, YSCF62-65, and YLLNT555-559 motifs of a DMT1 construct bearing an exofacial epitope tag, which allowed labeling of the transporter at the cell surface for kinetic studies. Mutants were stably expressed in LLC-PK1 kidney cells and were studied for transport activity, subcellular localization, cell-surface and recycling pool distribution, and internalization from the plasma membrane. Kinetic studies showed that carboxyl-terminus mutants (DeltaCT and DeltaYLLNT) had an increased fraction of the "recycling pool" that was expressed at the cell surface because of impaired internalization from the plasma membrane. Further cell-surface-labeling and immunofluorescence studies in intact cells showed that the DeltaYLLNT and DeltaCT mutants were targeted to the lysosomal compartment upon internalization. These results suggest that the major signal for internalization and recycling of DMT1 isoform II (-IRE/1B) resides in its carboxyl terminus and that removal of this signal leads to a default lysosomal targeting.

Amino Acid Sequence↗
PubMed2005

Comparative studies of duodenal and macrophage ferroportin proteins.

Intestinal epithelial cells and reticuloendothelial macrophages are, respectively, involved in diet iron absorption and heme iron recycling from senescent erythrocytes, two critical processes of iron homeostasis. These cells appear to use the same transporter, ferroportin (Slc40a1), to export iron. The aim of this study was to compare the localization, expression, and regulation of ferroportin in both duodenal and macrophage cells. Using a high-affinity purified polyclonal antibody, we analyzed the localization and expression of ferroportin protein in the spleen, liver, and duodenum isolated from normal mice as well as from well-characterized mouse models of altered iron homeostasis. Ferroportin was found to be predominantly expressed in enterocytes of the duodenum, in splenic macrophages, and in liver Kupffer cells. Interestingly, the protein species detected in these cells migrated differently on SDS-PAGE. These differences in apparent molecular masses were partly explained by posttranslational complex N-linked glycosylations. In addition, in enterocytes, the transporter was mostly expressed at the basolateral membrane, whereas in bone marrow-derived macrophages, ferroportin was found predominantly localized in the intracellular vesicular compartment. However, some microdomains positive for ferroportin were also detected at the plasma membrane of macrophages. Despite these differences, we observed a parallel upregulation of ferroportin expression in tissue macrophages and enterocytes in response to iron-restricted erythropoiesis, suggesting that iron homeostasis is likely maintained through coordinate expression of the iron exporter in both intestinal and phagocytic cells. Our data also confirm a predominant regulation of ferroportin through systemic regulator(s) likely including hepcidin.

Animals↗