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P Béguin

Publications and source records attributed to P Béguin.

At least 19 recordsLinked to original sources
PubMed2004

Chronic intermittent hypoxia increases infarction in the isolated rat heart.

Coronary heart disease is frequently associated with obstructive sleep apnea syndrome and treating obstructive sleep apnea appears to significantly improve the outcome in coronary heart disease. Thus we have developed a rat model of chronic intermittent hypoxia (IH) to study the influence of this condition on myocardial ischemia-reperfusion tolerance and on functional vascular reactivity. Wistar male rats were divided in three experimental groups (n = 12 each) subjected to chronic IH (IH group), normoxia (N group), or control conditions (control group). IH consisted of repetitive cycles of 1 min (40 s with inspired O(2) fraction 5% followed by 20 s normoxia) and was applied for 8 h during daytime, for 35 days. Normoxic cycles were applied in the same conditions, inspired O(2) fraction remaining constant at 21%. On day 36, mean arterial blood pressure (MABP) was measured before isolated hearts were submitted to an ischemia-reperfusion protocol. The thoracic aorta and left carotid artery were also excised for functional reactivity studies. MABP was not significantly different between the three experimental groups. Infarct sizes (in percent of ventricles) were significantly higher in IH group (46.9 +/- 3.6%) compared with N (26.1 +/- 2.8%) and control (21.7 +/- 2.1%) groups. Vascular smooth muscle function was similar in aorta and carotid arteries from all groups. The endothelium-dependent relaxation in response to acetylcholine was also similar in aorta and carotid arteries from all groups. Chronic IH increased heart sensitivity to infarction, independently of a significant increase in MABP, and did not affect vascular reactivity of aorta and carotid arteries.

Animals↗
PubMed2001

CHIF, a member of the FXYD protein family, is a regulator of Na,K-ATPase distinct from the gamma-subunit.

The biological role of small membrane proteins of the new FXYD family is largely unknown. The best characterized FXYD protein is the gamma-subunit of the Na,K-ATPase (NKA) that modulates the Na,K-pump function in the kidney. Here, we report that, similarly to gamma(a) and gamma(b) splice variants, the FXYD protein CHIF (corticosteroid-induced factor) is a type I membrane protein which is associated with NKA in renal tissue, and modulates the Na,K-pump transport when expressed in Xenopus oocytes. In contrast to gamma(a) and gamma(b), which both decrease the apparent Na+ affinity of the Na,K-pump, CHIF significantly increases the Na+ affinity and decreases the apparent K+ affinity due to an increased Na+ competition at external binding sites. The extracytoplasmic FXYD motif is required for stable gamma-subunit and CHIF interaction with NKA, while cytoplasmic, positively charged residues are necessary for the gamma-subunit's association efficiency and for CHIF's functional effects. These data document that CHIF is a new tissue-specific regulator of NKA which probably plays a crucial role in aldosterone-responsive tissues responsible for the maintenance of body Na+ and K+ homeostasis.

Alternative Splicing↗
PubMed2001

Regulation of Ca2+ channel expression at the cell surface by the small G-protein kir/Gem.

Voltage-dependent calcium (Ca2+) channels are involved in many specialized cellular functions, and are controlled by intracellular signals such as heterotrimeric G-proteins, protein kinases and calmodulin (CaM). However, the direct role of small G-proteins in the regulation of Ca2+ channels is unclear. We report here that the GTP-bound form of kir/Gem, identified originally as a Ras-related small G-protein that binds CaM, inhibits high-voltage-activated Ca2+ channel activities by interacting directly with the beta-subunit. The reduced channel activities are due to a decrease in alpha1-subunit expression at the plasma membrane. The binding of Ca2+/CaM to kir/Gem is required for this inhibitory effect by promoting the cytoplasmic localization of kir/Gem. Inhibition of L-type Ca2+ channels by kir/Gem prevents Ca2+-triggered exocytosis in hormone-secreting cells. We propose that the small G-protein kir/Gem, interacting with beta-subunits, regulates Ca2+ channel expression at the cell surface.

Animals↗
PubMed2000

Cellulose-binding domains promote hydrolysis of different sites on crystalline cellulose.

The cohesin-dockerin interaction in Clostridium thermocellum cellulosome mediates the tight binding of cellulolytic enzymes to the cellulosome-integrating protein CipA. Here, this interaction was used to study the effect of different cellulose-binding domains (CBDs) on the enzymatic activity of C. thermocellum endoglucanase CelD (1,4-beta-d endoglucanase, EC) toward various cellulosic substrates. The seventh cohesin domain of CipA was fused to CBDs originating from the Trichoderma reesei cellobiohydrolases I and II (CBD(CBH1) and CBD(CBH2)) (1,4-beta-d glucan-cellobiohydrolase, EC), from the Cellulomonas fimi xylanase/exoglucanase Cex (CBD(Cex)) (beta-1,4-d glucanase, EC), and from C. thermocellum CipA (CBD(CipA)). The CBD-cohesin hybrids interacted with the dockerin domain of CelD, leading to the formation of CelD-CBD complexes. Each of the CBDs increased the fraction of cellulose accessible to hydrolysis by CelD in the order CBD(CBH1) < CBD(CBH2) approximately CBD(Cex) < CBD(CipA). In all cases, the extent of hydrolysis was limited by the disappearance of sites accessible to CelD. Addition of a batch of fresh cellulose after completion of the reaction resulted in a new burst of activity, proving the reversible binding of the intact complexes despite the apparent binding irreversibility of some CBDs. Furthermore, burst of activity also was observed upon adding new batches of CelD-CBD complexes that contained a CBD differing from the first one. This complementation between different CBDs suggests that the sites made available for hydrolysis by each of the CBDs are at least partially nonoverlapping. The only exception was CBD(CipA), whose sites appeared to overlap all of the other sites.

Amino Acid Sequence↗
PubMed2000

Is phosphorylation of the alpha1 subunit at Ser-16 involved in the control of Na,K-ATPase activity by phorbol ester-activated protein kinase C?

The alpha1 subunit of Na,K-ATPase is phosphorylated at Ser-16 by phorbol ester-sensitive protein kinase(s) C (PKC). The role of Ser-16 phosphorylation was analyzed in COS-7 cells stably expressing wild-type or mutant (T15A/S16A and S16D-E) ouabain-resistant Bufo alpha1 subunits. In cells incubated at 37 degrees C, phorbol 12, 13-dibutyrate (PDBu) inhibited the transport activity and decreased the cell surface expression of wild-type and mutant Na,K-pumps equally ( approximately 20-30%). This effect of PDBu was mimicked by arachidonic acid and was dependent on PKC, phospholipase A(2), and cytochrome P450-dependent monooxygenase. In contrast, incubation of cells at 18 degrees C suppressed the down-regulation of Na,K-pumps and revealed a phosphorylation-dependent stimulation of the transport activity of Na,K-ATPase. Na,K-ATPase from cells expressing alpha1-mutants mimicking Ser-16 phosphorylation (S16D or S16E) exhibited an increase in the apparent Na affinity. This finding was confirmed by the PDBu-induced increase in Na sensitivity of the activity of Na,K-ATPase measured in permeabilized nontransfected COS-7 cells. These results illustrate the complexity of the regulation of Na,K-ATPase alpha1 isozymes by phorbol ester-sensitive PKCs and reveal 1) a phosphorylation-independent decrease in cell surface expression and 2) a phosphorylation-dependent stimulation of the transport activity attributable to an increase in the apparent Na affinity.

Animals↗
PubMed2000

Endoplasmic reticulum quality control of oligomeric membrane proteins: topogenic determinants involved in the degradation of the unassembled Na,K-ATPase alpha subunit and in its stabilization by beta subunit assembly.

The molecular nature of determinants that mediate degradation of unassembled, polytopic subunits of oligomeric membrane proteins and their stabilization after partner subunit assembly is largely unknown. Expressing truncated Na,K-ATPase alpha subunits alone or together with beta subunits, we find that in unassembled alpha subunits neither the four N-terminal transmembrane segments acting as efficient alternating signal anchor-stop transfer sequences nor the large, central cytoplasmic loop exposes any degradation signal, whereas poor membrane insertion efficiency of C-terminal membrane domains M5, M7, and M9 coincides with the transient exposure of degradation signals to the cytoplasmic side. beta assembly with an alpha domain comprising at least D902 up to Y910 in the extracytoplasmic M7/M8 loop is necessary to stabilize Na,K-ATPase alpha subunits by favoring M7/M8 membrane pair formation and by protecting a degradation signal recognized from the endoplasmic reticulum (ER) lumenal side. Thus our results suggest that ER degradation of Na,K-ATPase alpha subunits is 1) mainly mediated by folding defects caused by inefficient membrane insertion of certain membrane domains, 2) a multistep process, which involves proteolytic and/or chaperone components acting from the ER lumenal side in addition to cytosolic, proteasome-related factors, and 3) prevented by partner subunit assembly because of direct protection and retrieval of degradation signals from the cytoplasm to the ER lumenal side. These results likely represent a paradigm for the ER quality control of unassembled, polytopic subunits of oligomeric membrane proteins.

Animals↗
PubMed1999

PKA-mediated phosphorylation of the human K(ATP) channel: separate roles of Kir6.2 and SUR1 subunit phosphorylation.

ATP-sensitive potassium (K(ATP)) channels play important roles in many cellular functions such as hormone secretion and excitability of muscles and neurons. Classical ATP-sensitive potassium (K(ATP)) channels are heteromultimeric membrane proteins comprising the pore-forming Kir6.2 subunits and the sulfonylurea receptor subunits (SUR1 or SUR2). The molecular mechanism by which hormones and neurotransmitters modulate K(ATP) channels via protein kinase A (PKA) is poorly understood. We mutated the PKA consensus sequences of the human SUR1 and Kir6.2 subunits and tested their phosphorylation capacities in Xenopus oocyte homogenates and in intact cells. We identified the sites responsible for PKA phosphorylation in the C-terminus of Kir6.2 (S372) and SUR1 (S1571). Kir6.2 can be phosphorylated at its PKA phosphorylation site in intact cells after G-protein (Gs)-coupled receptor or direct PKA stimulation. While the phosphorylation of Kir6.2 increases channel activity, the phosphorylation of SUR1 contributes to the basal channel properties by decreasing burst duration, interburst interval and open probability, and also increasing the number of functional channels at the cell surface. Moreover, the effect of PKA could be mimicked by introducing negative charges in the PKA phosphorylation sites. These data demonstrate direct phosphorylation by PKA of the K(ATP) channel, and may explain the mechanism by which Gs-coupled receptors stimulate channel activity. Importantly, they also describe a model of heteromultimeric ion channels in which there are functionally distinct roles of the phosphorylation of the different subunits.

ATP-Binding Cassette Transporters↗
PubMed1999

beta-subunit assembly is essential for the correct packing and the stable membrane insertion of the H,K-ATPase alpha-subunit.

The alpha-subunits of H,K-ATPase (HKAalpha) and Na,K-ATPase require a beta-subunit for maturation. We investigated the role of the beta-subunit in the membrane insertion and stability of the HKAalpha expressed in Xenopus oocytes. Individual membrane segments M1, M2, M3, M4, and M9 linked to a glycosylation reporter act as signal anchor (SA) motifs, and M10 acts as a partial stop transfer motif. In combined HKAalpha constructs, M2 acts as an efficient stop transfer sequence, and M3 acts as a SA sequence. However, M5 and M9 have only partial SA function, and M7 has no SA function. Consistent with the membrane insertion properties of segments in combined alpha constructs, M1-3 alpha-proteins are resistant to cellular degradation, and M1-5 up to M1-10 alpha-proteins are not resistant to cellular degradation. However, co-expression with beta-subunits increases the membrane insertion of M9 in a M1-9 alpha-protein and completely protects M1-10 alpha-proteins against cellular degradation. Our results indicate that HKAalpha N-terminal (M1-M4) membrane insertion and stabilization are mediated by intrinsic molecular characteristics; however, the C-terminal (M5-M10) membrane insertion and thus the stabilization of the entire alpha-subunit depend on intramolecular and intermolecular beta-subunit interactions that are similar but not identical to data obtained for the Na,K-ATPase alpha-subunit.

Animals↗
PubMed1999

Hybrid enzymes.

Combining structural elements belonging to different proteins is a powerful method for generating proteins with new properties. Progress based on detailed structural and functional analysis enables a better integration of the elements to be fitted together while preserving or creating functional interactions between them.

Allosteric Regulation↗
PubMed1999

Insulin-induced stimulation of Na+,K(+)-ATPase activity in kidney proximal tubule cells depends on phosphorylation of the alpha-subunit at Tyr-10.

Phosphorylation of the alpha-subunit of Na+,K(+)-ATPase plays an important role in the regulation of this pump. Recent studies suggest that insulin, known to increase solute and fluid reabsorption in mammalian proximal convoluted tubule (PCT), is stimulating Na+,K(+)-ATPase activity through the tyrosine phosphorylation process. This study was therefore undertaken to evaluate the role of tyrosine phosphorylation of the Na+,K(+)-ATPase alpha-subunit in the action of insulin. In rat PCT, insulin and orthovanadate (a tyrosine phosphatase inhibitor) increased tyrosine phosphorylation level of the alpha-subunit more than twofold. Their effects were not additive, suggesting a common mechanism of action. Insulin-induced tyrosine phosphorylation was prevented by genistein, a tyrosine kinase inhibitor. The site of tyrosine phosphorylation was identified on Tyr-10 by controlled trypsinolysis in rat PCTs and by site-directed mutagenesis in opossum kidney cells transfected with rat alpha-subunit. The functional relevance of Tyr-10 phosphorylation was assessed by 1) the abolition of insulin-induced stimulation of the ouabain-sensitive (86)Rb uptake in opossum kidney cells expressing mutant rat alpha1-subunits wherein tyrosine was replaced by alanine or glutamine; and 2) the similarity of the time course and dose dependency of the insulin-induced increase in ouabain-sensitive (86)Rb uptake and tyrosine phosphorylation. These findings indicate that phosphorylation of the Na+,K(+)-ATPase alpha-subunit at Tyr-10 likely participates in the physiological control of sodium reabsorption in PCT.

Amino Acid Substitution↗
PubMed1998

Role of beta-subunit domains in the assembly, stable expression, intracellular routing, and functional properties of Na,K-ATPase.

The beta-subunit of Na,K-ATPase (betaNK) interacts with the catalytic alpha-subunit (alphaNK) in the ectodomain, the transmembrane, and the cytoplasmic domain. The functional significance of these different interactions was studied by expressing alphaNK in Xenopus oocytes along with N-terminally modified betaNK or with chimeric betaNK/betaH,K-ATPase (betaHK). Complete truncation of the betaNK N terminus allows for cell surface-expressed, functional Na,K-pumps that exhibit, however, reduced apparent K+ and Na+ affinities as assessed by electrophysiological measurements. A mutational analysis suggests that these functional effects are not related to a direct interaction of the beta N terminus with the alphaNK but rather that N-terminal truncation induces a conformational change in another functionally relevant beta domain. Comparison of the functional properties of alphaNK.betaNK, alphaNK.betaHK, or alphaNK. betaNK/betaHK complexes shows that the effect of the betaNK on K+ binding is mainly mediated by its ectodomain. Finally, betaHK/NK containing the transmembrane domain of betaHK produces stable but endoplasmic reticulum-retained alphaNK.beta complexes, while alphaNK/betaHK complexes can leave the ER but exhibit reduced ouabain binding capacity and transport function. Thus, interactions of both the transmembrane and the ectodomain of betaNK with alphaNK are necessary to form correctly folded Na,K-ATPase complexes that can be targeted to the plasma membrane and/or become functionally competent. Furthermore, the beta N terminus plays a role in the beta-subunit's folding necessary for correct interactions with the alpha-subunit.

Amino Acid Sequence↗
PubMed1998

Membrane integration of Na,K-ATPase alpha-subunits and beta-subunit assembly.

The control of membrane insertion of polytopic proteins is still poorly understood. We carried out in vivo translation/insertion experiments in Xenopus oocytes with combined wild type or mutant membrane segments of the alpha-subunit of the heterodimeric Na, K-ATPase linked to a glycosylation reporter sequence. We confirm that the four N-terminal hydrophobic segments of the alpha-subunit behave as alternating signal anchor/stop transfer motifs necessary for two lipid-inserted membrane pairs. For the six C-terminal membrane segments, however, proper packing depends on specific sequence information and association with the beta-subunit. M5 is a very inefficient signal anchor sequence due to the presence of prolines and polar amino acids. Its correct membrane insertion is probably mediated by posttranslational hairpin formation with M6, which is favored by a proline pair in the connecting loop. M7 has partial signal anchor function, which may be mediated by the presence of glycine and glutamine residues. The formation of a transmembrane M7/M8 pair requires the association of the beta-subunit, which induces a conformational change in the connecting extracytoplasmic loop that favors M7/M8 packing. The formation of the M9/M10 pair appears to be predominantly mediated by the efficient stop transfer function of M10. Mutations that provide signal anchor function to M5, M7, and M9 abolish or impede the transport activity of the enzyme. These data illustrate the importance of specific amino acids near or within hydrophobic regions as well as of subunit oligomerization for correct topographical alignment that is necessary for proper folding and/or activity of oligomeric membrane proteins.

Animals↗
PubMed1998

Cellulase and hemicellulase genes of Clostridium thermocellum from five independent collections contain few overlaps and are widely scattered across the chromosome.

Five independent collections, comprising a total of 34 clones encoding cellulases, hemicellulases and cell surface proteins of Clostridium thermocellum, were searched for overlapping or contiguous DNA fragments. The clones were hybridized to large genomic restriction fragments separated by pulse-field electrophoresis. Clones hybridizing to the same fragment were further compared by hybridization to smaller fragments, by cross-hybridization and by restriction mapping. The probes hybridized to loci which were usually not clustered and were scattered over at least one third of the chromosome. Besides previously identified clusters, only two clones were found to be adjacent. Two pairs of clones appeared to contain the same genes cloned in duplicate, and one of the genes was shown to be cloned in triplicate.

Cellulase↗
PubMed1998

Regulation of expression and function by subunits of oligomeric P-type ATPases.

Na,K-ATPase activity must be finely controlled to meet the constantly changing physiological demands and to avoid destabilization of body homeostasis. Recent experimental evidence suggests that certain regulatory mechanisms are closely linked to the multisubunit structure of the Na,K-pump molecule. Na,K-ATPase is composed of a catalytic alpha and a glycoprotein beta subunit and sometimes of a third component, the gamma subunit. The beta subunit is a fundamental element of Na,K-ATPase in that its assembly in the ER is required for the structural and functional maturation of the catalytic alpha subunit and in consequence the beta subunit controls the expression of functional pumps at the cell surface. Furthermore, beta subunits influence the transport properties of the mature catalytic alpha subunits. Distinct interaction sites mediate the two functions of the beta subunit. Recently, we have started to characterize the gamma subunit, the functional role of which is yet not known. Immuno-radiolabeling of epitope-tagged gamma subunits expressed in Xenopus oocytes shows that the gamma subunits is a type I membrane protein which specifically associates only with Na,K-ATPase but not with other oligomeric P-type ATPases. The gamma peptide does not influence the formation or the cell surface expression of functional alpha-beta complexes. On the other hand, the gamma peptide itself needs association with Na,K-ATPase to be stably expressed and to be efficiently transported to the plasma membrane. Finally, the gamma subunit can modulate the K activation of Na,K-pumps. In conclusion, processes such as subunit assembly or the subunit composition of the cell surface expressed Na,K-pumps appear to cooperate with hormones in the control of the expression and the activity of Na,K-ATPase.

Adenosine Triphosphatases↗
PubMed1997

The crystal structure of a type I cohesin domain at 1.7 A resolution.

The quaternary organization of the cellulosome, a multi-enzymatic extracellular complex produced by cellulolytic bacteria, depends on specific interactions between dockerin domains, double EF-hand subunits carried by the catalytic components, and cohesin domains, individual receptor subunits linearly arranged within a non-catalytic scaffolding polypeptide. Cohesin-dockerin complexes with distinct specificities are also thought to mediate the attachment of cellulosomes to the cell membrane. We report here the crystal structure of a single cohesin domain from the scaffolding protein of Clostridium thermocellum. The cohesin domain folds into a nine-stranded beta-sandwich with an overall "jelly roll" topology, similar to that observed in bacterial cellulose-binding domains. Surface-exposed patches of conserved residues promote extensive intermolecular contacts in the crystal, and suggest a possible binding target for the EF-hand pair of the cognate dockerin domain. Comparative studies of cohesin domains indicate that, in spite of low sequence similarities and different functional roles, all cohesin domains share a common nine-stranded beta-barrel fold stabilized by a conserved hydrophobic core. The formation of stable cohesin-dockerin complexes requires the presence of Ca2+. However, the structure of the cohesin domain reported here reveals no obvious Ca2+-binding site, and previous experiments have failed to detect high affinity binding of Ca2+ to the unliganded dockerin domain of endoglucanase CelD. Based on structural and biochemical evidence, we propose a model of the cohesin-dockerin complex in which the dockerin domain requires complexation with its cohesin partner for protein stability and high-affinity Ca2+ binding.

Amino Acid Sequence↗
PubMed1997

Synergism between the cellulosome-integrating protein CipA and endoglucanase CelD of Clostridium thermocellum.

The cellulosome-integrating protein CipA of Clostridium thermocellum was produced from a recombinant clone of Escherichia coli and purified by cellulose affinity and anion exchange chromatography. Two active forms of C. thermocellum endoglucanase CelD were tested for binding and hydrolytic activity on Avicel in the presence and in the absence of CipA. One was 68 kDa CelD, which contains an intact dockerin domain. The other was 65 kDa CelD, in which the dockerin domain is partially deleted. CipA promoted quantitative binding of 68 kDa CelD to Avicel and enhanced its Avicelase activity by at least ten-fold. By contrast, CipA had no effect on the activity nor on the cellulose-binding affinity of the truncated 65 kDa form. These results show that interaction between CipA and the catalytic component CelD is needed for the degradation of cellulose and confirm that the interaction is mediated by the dockerin domain of CelD.

Bacterial Proteins↗
PubMed1997

Interaction between Clostridium thermocellum endoglucanase CelD and polypeptides derived from the cellulosome-integrating protein CipA: stoichiometry and cellulolytic activity of the complexes.

Four mini-scaffoldins were constructed from modules derived from the Clostridium thermocellum cellulosome-integrating protein CipA. Cip7 and Cip6 contained one and two cohesin modules respectively. Cip14 and Cip16, also containing one and two cohesin modules respectively, were flanked by a cellulose-binding domain. Endoglucanase CelD formed stable complexes with all mini-scaffoldins. Analytical ultracentrifugation of the complexes showed that 1 mol of CelD bound per mol of Cip14, and 2 mol of CelD bound per mol of Cip16. Under the conditions used for assaying cellulase activity, 96% of CelD alone bound to Avicel. Association with Cip14 or Cip16 increased the cellulose binding of CelD to 99%, while association with Cip7 or Cip6 decreased binding to 79 and 75% respectively. The hydrolytic activity of CelD against Avicel was increased 3-fold in complexes with Cip14 and Cip16, but remained substantially the same in complexes with Cip6 and Cip7. Addition of whole CipA also enhanced the efficiency of Avicel hydrolysis by CelD. However, even at an optimal ratio of the components, CelD-CipA complexes were somewhat less active than complexes of CelD with Cip14 or Cip16. These results suggest that the synergism observed between CelD and Cip14 or Cip16 is mostly due to the presence of the cellulose-binding domain, which promotes productive binding of the enzyme.

Adsorption↗