Biomedical subjects

Jérôme Garin

Publications and source records attributed to Jérôme Garin.

At least 19 recordsLinked to original sources

PubMed2007

Biochemical characterization and lysosomal localization of the mannose-6-phosphate protein p76 (hypothetical protein LOC196463).

Most soluble lysosomal proteins carry Man6P (mannose 6-phosphate), a specific carbohydrate marker that enables their binding to cellular MPRs (Man6P receptors) and their subsequent targeting towards the lysosome. This characteristic was exploited to identify novel soluble lysosomal proteins by proteomic analysis of Man6P proteins purified from a human cell line. Among the proteins identified during the course of the latter study [Journet, Chapel, Kieffer, Roux and Garin (2002) Proteomics, 2, 1026-1040], some had not been previously described as lysosomal proteins. We focused on a protein detected at 76 kDa by SDS/PAGE. We named this protein 'p76' and it appeared later in the NCBI protein database as the 'hypothetical protein LOC196463'. In the present paper, we describe the identification of p76 by MS and we analyse several of its biochemical characteristics. The presence of Man6P sugars was confirmed by an MPR overlay experiment, which showed the direct and Man6P-dependent interaction between p76 and the MPR. The presence of six N-glycosylation sites was validated by progressive peptide-N-glycosidase F deglycosylation. Experiments using N- and C-termini directed anti-p76 antibodies provided insights into p76 maturation. Most importantly, we were able to demonstrate the lysosomal localization of this protein, which was initially suggested by its Man6P tags, by both immunofluorescence and sub-cellular fractionation of mouse liver homogenates.

Animals↗

PubMed2007

Regulation of phagocyte NADPH oxidase activity: identification of two cytochrome b558 activation states.

Activation of the phagocyte NADPH oxidase (phox) requires the association of cytosolic proteins (p67-phox, p47-phox, p40-phox, and Rac1/2) with the membrane cytochrome b558, leading to a hemoprotein conformation change. To clarify this mechanism, the phagocyte NADPH oxidase complex was isolated through cytochrome b558 purification after three chromatographic steps. The purified neutrophil complex was constitutively active in the absence of an amphiphile agent with a maximum turnover (125 mol O2(-) x s(-1) x mol heme b(-1)), indicating that cytochrome b558 has been activated by cytosolic proteins and is in an "open conformation," able to transfer a maximum rate of electrons. In contrast, the phox complex prepared with B lymphocyte cytosol shows a lower constitutive turnover (approximately 50 mol O2(-) x s(-1) x mol heme b(-1)). Analysis of phox complex components by Western blot and mass spectrometry showed the presence of cytosolic factors (especially p67-phox) and structural proteins (moesin, ezrin). To investigate the difference in activity of phox complexes, we evaluated the effect of MRP8 and MRP14, specifically expressed in neutrophils, on the activity of the B lymphocyte complex. MRPs induce the switch between the partially and the fully "open" cytochrome b558 conformation. Moreover, their effect was independent of p67-phox. Data point out two potential cytochrome b558 activation states.

ATP-Binding Cassette Transporters↗

PubMed2007

The Crl-RpoS regulon of Escherichia coli.

The RpoS subunit of RNA polymerase controls the expression of numerous genes involved in stationary phase and in response to different stress conditions. The regulatory protein Crl increases the activity of RpoS by direct interaction with the RpoS holoenzyme. To define the extent of the Crl regulon, we used two-dimensional SDS-PAGE to measure the role of Crl in regulating the expression of the Escherichia coliproteome in stationary phase at 30 degrees C. By comparing the proteome of four strains (wild type, crl(-), rpoS(-), and crl(-)rpoS(-)), we observed that the intensity of 74 spots was modified in at least one mutant context. 62 spots were identified by mass spectrometry and correspond to 40 distinct proteins. They were classified in four main categories: DNA metabolism, central metabolism, response to environmental modifications, and miscellaneous. Three proteins were specifically involved in quorum sensing: TnaA (the tryptophanase that converts tryptophan to indole), WrbA (Trp repressor-binding protein), and YgaG (homologous to LuxS, autoinducer-2 synthase). Because little is known about the regulation of Crl expression, we investigated the influence of diffusible molecules on the expression of Crl. Using Western blotting experiments, we showed that, at 30 degrees C, a diffusible molecule(s) produced during the transition phase between the exponential and stationary phases induces a premature expression of Crl. Indole was tested as one of the potential candidates: at 37 degrees C, it is present in the extracellular medium at a constant concentration, but at 30 degrees C, its concentration peaks during the transition phase. When indole was added to the culture medium, it also induced prematurely the expression of Crl at both the transcriptional and translational levels in a Crl-dependent manner. Crl may thus be considered a new environmental sensor via the indole concentration.

Bacterial Proteins↗

PubMed2007

Mutual regulation of Crl and Fur in Escherichia coli W3110.

The small regulatory protein Crl controls the expression of curli. Recently we have shown that Crl interacts directly with one of the most global regulators of Escherichia coli, the stress-related sigma factor RpoS, suggesting a more global role for Crl. We show here by a proteomics analysis that the expression of at least nine cellular proteins was considerably modified when Crl was overexpressed. We assessed the part of transcriptional and post-transcriptional regulation for five of these genes. The results showed that Crl regulates the expression of another global regulator, the central regulator of iron homeostasis, Fur. A molecular analysis revealed that Crl and Fur affect their own and each other's expression. We provide physical evidence for the binding of Fur to the crl and fur promoter regions. Crl modulated the affinity of Fur at the fur promoter but not at the crl promoter. The triad RpoS-Crl-Fur may thus represent the centerpiece of a global regulatory system of response to different stresses.

Bacterial Proteins↗

PubMed2006

A proteomics dissection of Arabidopsis thaliana vacuoles isolated from cell culture.

To better understand the mechanisms governing cellular traffic, storage of various metabolites, and their ultimate degradation, Arabidopsis thaliana vacuole proteomes were established. To this aim, a procedure was developed to prepare highly purified vacuoles from protoplasts isolated from Arabidopsis cell cultures using Ficoll density gradients. Based on the specific activity of the vacuolar marker alpha-mannosidase, the enrichment factor of the vacuoles was estimated at approximately 42-fold with an average yield of 2.1%. Absence of significant contamination by other cellular compartments was validated by Western blot using antibodies raised against specific markers of chloroplasts, mitochondria, plasma membrane, and endoplasmic reticulum. Based on these results, vacuole preparations showed the necessary degree of purity for proteomics study. Therefore, a proteomics approach was developed to identify the protein components present in both the membrane and soluble fractions of the Arabidopsis cell vacuoles. This approach includes the following: (i) a mild oxidation step leading to the transformation of cysteine residues into cysteic acid and methionine to methionine sulfoxide, (ii) an in-solution proteolytic digestion of very hydrophobic proteins, and (iii) a prefractionation of proteins by short migration by SDS-PAGE followed by analysis by liquid chromatography coupled to tandem mass spectrometry. This procedure allowed the identification of more than 650 proteins, two-thirds of which copurify with the membrane hydrophobic fraction and one-third of which copurifies with the soluble fraction. Among the 416 proteins identified from the membrane fraction, 195 were considered integral membrane proteins based on the presence of one or more predicted transmembrane domains, and 110 transporters and related proteins were identified (91 putative transporters and 19 proteins related to the V-ATPase pump). With regard to function, about 20% of the proteins identified were known previously to be associated with vacuolar activities. The proteins identified are involved in ion and metabolite transport (26%), stress response (9%), signal transduction (7%), and metabolism (6%) or have been described to be involved in typical vacuolar activities, such as protein and sugar hydrolysis. The subcellular localization of several putative vacuolar proteins was confirmed by transient expression of green fluorescent protein fusion constructs.

Arabidopsis↗

PubMed2006

Post-meiotic shifts in HSPA2/HSP70.2 chaperone activity during mouse spermatogenesis.

HSPA2 (formerly HSP70.2) is a testis-specific member of the HSP70 family known to play a critical role in the completion of meiosis during male germ cell differentiation. Although abundantly present in post-meiotic cells, its function during spermiogenesis remained obscure. Here, using a global proteomic approach to identify genome-organizing proteins in condensing spermatids, we discovered an unexpected role for HSPA2, which acquires new functions and becomes tightly associated with major spermatid DNA-packaging proteins, transition proteins 1 and 2. Hence, HSPA2 is identified here as the first transition protein chaperone, and these data shed a new light on the yet totally unknown process of genome-condensing structure assembly in spermatids.

Animals↗

PubMed2006

Plant seed peroxygenase is an original heme-oxygenase with an EF-hand calcium binding motif.

A growing body of evidence indicates that phytooxylipins play important roles in plant defense responses. However, many enzymes involved in the biosynthesis of these metabolites are still elusive. We have purified one of these enzymes, the peroxygenase (PXG), from oat microsomes and lipid droplets. It is an integral membrane protein requiring detergent for its solubilization. Proteinase K digestion showed that PXG is probably deeply buried in lipid droplets or microsomes with only about 2 kDa at the C-terminal region accessible to proteolytic digestion. Sequencing of the N terminus of the purified protein showed that PXG had no sequence similarity with either a peroxidase or a cytochrome P450 but, rather, with caleosins, i.e. calcium-binding proteins. In agreement with this finding, we demonstrated that recombinant thale cress and rice caleosins, expressed in yeast, catalyze hydroperoxide-dependent mono-oxygenation reactions that are characteristic of PXG. Calcium was also found to be crucial for peroxygenase activity, whereas phosphorylation of the protein had no impact on catalysis. Site-directed mutagenesis studies revealed that PXG catalytic activity is dependent on two highly conserved histidines, the 9 GHz EPR spectrum being consistent with a high spin pentacoordinated ferric heme.

Amino Acid Sequence↗

PubMed2006

Dendritic cell derived-exosomes: biology and clinical implementations.

Exosomes are nanometer-sized membrane vesicles invaginating from multivesicular bodies and secreted from different cell types. They represent an "in vitro" discovery, but vesicles with the hallmarks of exosomes are present in vivo in germinal centers and biological fluids. Their protein and lipid composition is unique and could account for their expanding functions such as eradication of obsolete proteins, antigen presentation, or "Trojan horses" for viruses or prions. The potential of dendritic cell-derived exosomes (Dex) as cell-free cancer vaccines is addressed in this review. Lessons learned from the pioneering clinical trials allowed reassessment of the priming capacities of Dex in preclinical models, optimizing clinical protocols, and delineating novel, biological features of Dex in cancer patients.

Animals↗

PubMed2006

Parallel changes in global protein profiles during long-term experimental evolution in Escherichia coli.

Twelve populations of Escherichia coli evolved in and adapted to a glucose-limited environment from a common ancestor. We used two-dimensional protein electrophoresis to compare two evolved clones, isolated from independently derived populations after 20,000 generations. Exceptional parallelism was detected. We compared the observed changes in protein expression profiles with previously characterized global transcription profiles of the same clones; this is the first time such a comparison has been made in an evolutionary context where these changes are often quite subtle. The two methodologies exhibited some remarkable similarities that highlighted two different levels of parallel regulatory changes that were beneficial during the evolution experiment. First, at the higher level, both methods revealed extensive parallel changes in the same global regulatory network, reflecting the involvement of beneficial mutations in genes that control the ppGpp regulon. Second, both methods detected expression changes of identical gene sets that reflected parallel changes at a lower level of gene regulation. The protein profiles led to the discovery of beneficial mutations affecting the malT gene, with strong genetic parallelism across independently evolved populations. Functional and evolutionary analyses of these mutations revealed parallel phenotypic decreases in the maltose regulon expression and a high level of polymorphism at this locus in the evolved populations.

Adaptation, Physiological↗

PubMed2006

Intracellular localization of p40, a protein identified in a preparation of lysosomal membranes.

Unlike lysosomal soluble proteins, few lysosomal membrane proteins have been identified. Rat liver lysosomes were purified by centrifugation on a Nycodenz density gradient. The most hydrophobic proteins were extracted from the lysosome membrane preparation and were identified by MS. We focused our attention on a protein of approx. 40 kDa, p40, which contains seven to ten putative transmembrane domains and four lysosomal consensus sorting motifs in its sequence. Knowing that preparations of lysosomes obtained by centrifugation always contain contaminant membranes, we combined biochemical and morphological methods to analyse the subcellular localization of p40. The results of subcellular fractionation of mouse liver homogenates validate the lysosomal residence of p40. In particular, a density shift of lysosomes induced by Triton WR-1339 similarly affected the distributions of p40 and beta-galactosidase, a lysosomal marker protein. We confirmed by fluorescence microscopy on eukaryotic cells transfected with p40 or p40-GFP (green fluorescent protein) constructs that p40 is localized in lysosomes. A first molecular characterization of p40 in transfected Cos-7 cells revealed that it is an unglycosylated protein tightly associated with membranes. Taken together, our results strongly support the hypothesis that p40 is an authentic lysosomal membrane protein.

Amino Acid Sequence↗

PubMed2006

Pyruvate formate-lyase and a novel route of eukaryotic ATP synthesis in Chlamydomonas mitochondria.

Pyruvate formate-lyase (PFL) catalyzes the non-oxidative conversion of pyruvate to formate and acetyl-CoA. PFL and its activating enzyme (PFL-AE) are common among strict anaerobic and microaerophilic prokaryotes but are very rare among eukaryotes. In a proteome survey of isolated Chlamydomonas reinhardtii mitochondria, we found several PFL-specific peptides leading to the identification of cDNAs for PFL and PFL-AE, establishing the existence of a PFL system in this photosynthetic algae. Anaerobiosis and darkness led to increased PFL transcripts but had little effect on protein levels, as determined with antiserum raised against C. reinhardtii PFL. Protein blots revealed the occurrence of PFL in both chloroplast and mitochondria purified from aerobically grown cells. Mass spectrometry sequencing of C. reinhardtii mitochondrial proteins, furthermore, identified peptides for phosphotransacetylase and acetate kinase. The phosphotransacetylase-acetate kinase pathway is a common route of ATP synthesis or acetate assimilation among prokaryotes but is novel among eukaryotes. In addition to PFL and pyruvate dehydrogenase, the algae also expresses pyruvate:ferredoxin oxidoreductase and bifunctional aldehyde/alcohol dehydrogenase. Among eukaryotes, the oxygen producer C. reinhardtii has the broadest repertoire of pyruvate-, ethanol-, and acetate-metabolizing enzymes described to date, many of which were previously viewed as specific to anaerobic eukaryotic lineages.

Acetylesterase↗

PubMed2006

Dynamics of Arabidopsis thaliana soluble proteome in response to different nutrient culture conditions.

In an effort to determine the best extraction procedure compatible with the high-reproducible 2-DE, different methods of soluble protein extraction from Arabidopsis cell culture suspensions grown in Gamborg B5 medium were tested. A reference 2-DE map was established for this soluble extract revealing 1184 spots. The most abundant protein spots were excised, trypsin-digested, and mass spectra obtained via MALDI-TOF and/or LC coupled to ESI-MS. Three hundred and thirty one proteins were identified and their functions were defined based on sequence comparisons and classified in different protein families. In order to analyze the impact of culture medium on the Arabidopsis proteome, we performed the 2-DE map from Arabidopsis cell suspensions cultured in another growth medium Murashige and Skoog (M-S) and 327 major spots were identified. Using PDQuest imaging analysis, significant increases in the amount of several housekeeping enzymes, stress/defense proteins, and heat shock proteins were found in M-S medium. Modified expression of certain proteins and detection of new isoforms involved in nitrate assimilation, nitrogen, and sulfur metabolism were also observed in the M-S medium. This study provides the first 2-DE maps of the soluble proteome of Arabidopsis cell suspensions. The comparative analysis of the Arabidopsis proteome in respect to different nutrient supplies shows that the culture medium may significantly influence the expression pattern of major soluble proteins in Arabidopsis cells. This work also constitutes an important step for further proteomic analysis concerning cell responses to abiotic or biotic stresses.

Arabidopsis↗

PubMed2006

Analysis of the dynamic Bacillus subtilis Ser/Thr/Tyr phosphoproteome implicated in a wide variety of cellular processes.

The physiological role of proteins phosphorylated on serine/threonine/tyrosine (Ser/Thr/Tyr) residues or the identity of the corresponding kinases and phosphatases is generally poorly understood in bacteria. As a first step in analysing the importance of such phosphorylation, we sought to establish the nature of the Ser/Thr/Tyr phosphoproteome in Bacillus subtilis, using in vivo labelling with [(32)P]-orthophosphate, one-unit pH 2-DE, combined with MS. Highly reproducible 2-D profiles of phosphoproteins were obtained with early stationary-phase cells. The 2-D profiles contained at least 80 clearly labelled spots in the pH range 4-7. Forty-six spots were analysed by MS (confirmed in most cases by LC-MS/MS), identifying a total of 29 different proteins, with 19 identified for the first time as bacterial phosphoproteins. These phosphoproteins are implicated in a wide variety of cellular processes, including carbon and energy metabolism, transport, stress and development. Significant changes to the profiles were obtained as a result of cold, heat or osmotic shock, demonstrating that, in stationary-phase cells, the phosphoproteome is dynamic. An initial comparative study indicated that at least 25 [(32)P]-labelled spots were also stained by Pro-Q Diamond, with apparently six additional phosphoproteins uniquely detected by Pro-Q.

Bacillus subtilis↗

PubMed2006

The early responses of Arabidopsis thaliana cells to cadmium exposure explored by protein and metabolite profiling analyses.

To get more insight into plant cell response to cadmium (Cd) stress, both proteomic and metabolomic "differential display" analyses were performed on Arabidopsis thaliana cells exposed to different concentrations of the toxic chemical. After a 24 h treatment, soluble proteins extracted from untreated and treated cells were separated by 2-D-PAGE and image analyses were performed to quantify and compare protein levels. Proteins up- and down-regulated in response to Cd were identified by MS and mapped into specific metabolic pathways and cellular processes, highlighting probable activation of the carbon, nitrogen, and sulfur metabolic pathways. For some of these proteins, Northern blot and RT-PCR analyses were performed to test transcript accumulation in response to Cd. In parallel, metabolite profiling analyses by LC coupled to ESI MS were initiated to better characterize the metabolic adaptation to the chemical stress. This study revealed that the main variation at the metabolite level came from the presence of six different families of phytochelatins, in A. thaliana cells treated with Cd, whose accumulation increases with Cd concentrations. Taken together these data provide an overview of the molecular and cellular changes elicited by Cd exposure.

Amino Acids↗

PubMed2006

Proteomic analysis of the eukaryotic parasite Encephalitozoon cuniculi (microsporidia): a reference map for proteins expressed in late sporogonial stages.

The microsporidian Encephalitozoon cuniculi is a unicellular obligate intracellular parasite considered as an emerging opportunistic human pathogen. The differentiation phase of its life cycle leads to the formation of stress-resistant spores. The E. cuniculi genome (2.9 Mbp) having been sequenced, we undertook a descriptive proteomic study of a spore-rich cell population isolated from culture supernatants. A combination of 2-DE and 2-DE-free techniques was applied to whole-cell protein extracts. Protein identification was performed using an automated MALDI-TOF-MS platform and a nanoLC-MS/MS instrument. A reference 2-DE map of about 350 major spots with multiple isoforms was obtained, and for the first time in microsporidia, a large set of unique proteins (177) including proteins with unknown function in a proportion of 25.6% was identified. The data are mainly discussed with reference to secretion and spore structural features, energy and carbohydrate metabolism, cell cycle control and parasite survival in the environment.

Amino Acids↗

PubMed2005

An optimized strategy for ICAT quantification of membrane proteins.

The work presented here focuses on the development of a method adapting isotope labeling of proteins with ICAT to the study of highly hydrophobic proteins. Conditions for the labeling of proteins were first established using two standard soluble proteins and iodoacetamidyl-3,6-dioxaoctanediamine biotin (PEO-iodoacetyl biotin). Results demonstrated the efficiency of the labeling in the presence of high concentrations of both SDS and urea. These conditions were then used to label a highly hydrophobic mitochondrial membrane protein, the adenine nucleotide translocator ANT-1, with PEO-iodoacetyl biotin and then with the cleavable ICAT reagent. The results presented here show that labeling of proteins with cleavable ICAT is possible and may even be improved in strong denaturing buffers containing both SDS at a concentration higher than 0.5% (w/v) and urea. These results open the possibility of applying the ICAT strategy to complex samples containing very hydrophobic proteins solubilized in urea-SDS buffers. The adaptability of the developed method is demonstrated here with preliminary results obtained during the study of membrane-enriched fractions prepared from murine embryonic stem cells.

Affinity Labels↗

PubMed2005

Identification of proteins binding the native tubulin dimer.

Microtubules play an essential role in eukaryotic cells, where they perform a wide variety of functions. In this paper, we describe the characterization of proteins associated to tubulin dimer in its native form, using affinity chromatography and mass spectrometry. We used an immunoaffinity column with coupled-monoclonal antibody directed against the alpha-tubulin C-terminus. Tubulin was first loaded onto the column, then interphase and mitotic cell lysates were chromatographed. Tubulin-binding proteins were eluted using a peptide mimicking the alpha-tubulin C-terminus. Elution fractions were analyzed by SDS-PAGE, and a total of 14 proteins were identified with high confidence by mass spectrometry. These proteins could be grouped in four classes: known tubulin-binding proteins, one microtubule-associated protein, heat shock proteins, and proteins that were not shown previously to bind tubulin dimer or microtubules.

Animals↗

PubMed2005

Histone-modifying complexes regulate gene expression pertinent to the differentiation of the protozoan parasite Toxoplasma gondii.

Pathogenic apicomplexan parasites like Toxoplasma and Plasmodium (malaria) have complex life cycles consisting of multiple stages. The ability to differentiate from one stage to another requires dramatic transcriptional changes, yet there is a paucity of transcription factors in these protozoa. In contrast, we show here that Toxoplasma possesses extensive chromatin remodeling machinery that modulates gene expression relevant to differentiation. We find that, as in other eukaryotes, histone acetylation and arginine methylation are marks of gene activation in Toxoplasma. We have identified mediators of these histone modifications, as well as a histone deacetylase (HDAC), and correlate their presence at target promoters in a stage-specific manner. We purified the first HDAC complex from apicomplexans, which contains novel components in addition to others previously reported in eukaryotes. A Toxoplasma orthologue of the arginine methyltransferase CARM1 appears to work in concert with the acetylase TgGCN5, which exhibits an unusual bias for H3 [K18] in vitro. Inhibition of TgCARM1 induces differentiation, showing that the parasite life cycle can be manipulated by interfering with epigenetic machinery. This may lead to new approaches for therapy against protozoal diseases and highlights Toxoplasma as an informative model to study the evolution of epigenetics in eukaryotic cells.

Acetylation↗