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Sophie Roy

Publications and source records attributed to Sophie Roy.

At least 19 recordsLinked to original sources
PubMed2007

FXYD6 is a novel regulator of Na,K-ATPase expressed in the inner ear.

The exquisite sensitivity of the cochlea, which mediates the transduction of sound waves into nerve impulses, depends on the endolymph ionic composition and the endocochlear potential. A key protein in the maintenance of the electrochemical composition of the endolymph is the Na,K-ATPase. In this study, we have looked for the presence in the rat inner ear of members of the FXYD protein family, recently identified as tissue-specific modulators of Na,K-ATPase. Only FXYD6 is detected at the protein level. FXYD6 is expressed in various epithelial cells bordering the endolymph space and in the auditory neurons. FXYD6 co-localizes with Na,K-ATPase in the stria vascularis and can be co-immunoprecipitated with Na,K-ATPase. After expression in Xenopus oocytes, FXYD6 associates with Na,K-ATPase alpha1-beta1 and alpha1-beta2 isozymes, which are preferentially expressed in different regions of the inner ear and also with gastric and non-gastric H,K-ATPases. The apparent K(+) and Na(+) affinities of alpha1-beta1 and alpha1-beta2 isozymes are different. Association of FXYD6 with Na,K-ATPase alpha1-beta1 isozymes slightly decreases their apparent K(+) affinity and significantly decreases their apparent Na(+) affinity. On the other hand, association with alpha1-beta2 isozymes increases their apparent K(+) and Na(+) affinity. The effects of FXYD6 on the apparent Na(+) affinity of Na,K-ATPase and the voltage dependence of its K(+) effect are distinct from other FXYD proteins. In conclusion, this study defines the last FXYD protein of unknown function as a modulator of Na,K-ATPase. Among FXYD protein, FXYD6 is unique in its expression in the inner ear, suggesting a role in endolymph composition.

Amino Acid Sequence↗
PubMed2006

Structural and functional properties of two human FXYD3 (Mat-8) isoforms.

Six of 7 FXYD proteins have been shown to be tissue-specific modulators of Na,K-ATPase. In this study, we have identified two splice variants of human FXYD3, or Mat-8, in CaCo-2 cells. Short human FXYD3 has 72% sequence identity with mouse FXYD3, whereas long human FXYD3 is identical to short human FXYD3 but has a 26-amino acid insertion after the transmembrane domain. Short and long human FXYD3 RNAs and proteins are differentially expressed during differentiation of CaCo-2 cells. Long human FXYD3 is mainly expressed in nondifferentiated cells and short human FXYD3 in differentiated cells and both FXYD3 variants can be co-immunoprecipitated with a Na,K-ATPase antibody. In contrast to mouse FXYD3, which has two transmembrane domains for lack of cleavage of the signal peptide, human FXYD3 has a cleavable signal peptide and adopts a type I topology. After co-expression in Xenopus oocytes, both human FXYD3 variants associate stably only with Na,K-ATPase isozymes but not with H,K-ATPase or Ca-ATPase. Similar to mouse FXYD3, short human FXYD3 decreases the apparent K(+) and Na(+) affinity of Na,K-ATPase over a large range of membrane potentials. On the other hand, long human FXYD3 decreases the apparent K(+) affinity only at slightly negative and positive membrane potentials and increases the apparent Na(+) affinity of Na,K-ATPase. Finally, both short and long human FXYD3 induce a hyperpolarization activated current, similar to that induced by mouse FXYD3. Thus, we have characterized two human FXYD3 isoforms that are differentially expressed in differentiated and non-differentiated cells and show different functional properties.

Animals↗
PubMed2006

Hippi is essential for node cilia assembly and Sonic hedgehog signaling.

Hippi functions as an adapter protein that mediates pro-apoptotic signaling from poly-glutamine-expanded huntingtin, an established cause of Huntington disease, to the extrinsic cell death pathway. To explore other functions of Hippi we generated Hippi knock-out mice. This deletion causes randomization of the embryo turning process and heart looping, which are hallmarks of defective left-right (LR) axis patterning. We report that motile monocilia normally present at the surface of the embryonic node, and proposed to initiate the break in LR symmetry, are absent on Hippi-/- embryos. Furthermore, defects in central nervous system development are observed. The Sonic hedgehog (Shh) pathway is downregulated in the neural tube in the absence of Hippi, which results in failure to establish ventral neural cell fate. Together, these findings demonstrate a dual role for Hippi in cilia assembly and Shh signaling during development, in addition to its proposed role in apoptosis signal transduction in the adult brain under pathogenically stressful conditions.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Requirement for Map2k1 (Mek1) in extra-embryonic ectoderm during placentogenesis.

Map2k1(-/-) embryos die at mid-gestation from abnormal development and hypovascularization of the placenta. We now show that this phenotype is associated with a decreased labyrinth cell proliferation and an augmented cell apoptosis. Although the activation of MAP2K1 and MAP2K2 is widespread in the labyrinthine region, MAPK1 and MAPK3 activation is restricted to the cells lining the maternal sinuses, suggesting an important role for the ERK/MAPK cascade in these cells. In Map2k1(-/-) placenta, ERK/MAPK cascade activation is perturbed. Abnormal localization of the syncytiotrophoblasts is also observed in Map2k1(-/-) placenta, even though this cell lineage is specified at the correct time during placentogenesis. The placental phenotype can be rescued in tetraploid experiments. In addition, Map2k1-specific deletion in the embryo leads to normal embryo development and to the birth of viable Map2k1(-/-) mice. Altogether, these data enlighten the essential role of Map2k1 in extra-embryonic ectoderm during placentogenesis. In the embryo, the Map2k1 gene function appears dispensable.

Alleles↗
PubMed2006

Cleavage at the caspase-6 site is required for neuronal dysfunction and degeneration due to mutant huntingtin.

Cleavage of huntingtin (htt) has been characterized in vitro, and accumulation of caspase cleavage fragments represents an early pathological change in brains of Huntington's disease (HD) patients. However, the relationship between htt proteolysis and the pathogenesis of HD is unknown. To determine whether caspase cleavage of htt is a key event in the neuronal dysfunction and selective neurodegeneration in HD, we generated YAC mice expressing caspase-3- and caspase-6-resistant mutant htt. Mice expressing mutant htt, resistant to cleavage by caspase-6 but not caspase-3, maintain normal neuronal function and do not develop striatal neurodegeneration. Furthermore, caspase-6-resistant mutant htt mice are protected against neurotoxicity induced by multiple stressors including NMDA, quinolinic acid (QA), and staurosporine. These results are consistent with proteolysis of htt at the caspase-6 cleavage site being an important event in mediating neuronal dysfunction and neurodegeneration and highlight the significant role of htt proteolysis and excitotoxicity in HD.

Active Transport, Cell Nucleus↗
PubMed2006

Caspase-3 cleaves the formin-homology-domain-containing protein FHOD1 during apoptosis to generate a C-terminal fragment that is targeted to the nucleolus.

The formin homology (FH) proteins play a crucial role in cytoskeleton remodelling during many essential processes. In this study, we demonstrate for the first time that the formin-homology-domain-containing protein FHOD1 is cleaved by caspase-3 at the SVPD(616) site during apoptosis. Using confocal microscopy, we further demonstrate that while full length FHOD1 is mostly cytoplasmic, the FHOD1 N-terminal cleavage product is diffusely localized throughout the cytoplasm and the nucleoplasm, whereas the C-terminal cleavage product is almost exclusively nuclear with some nucleolar localization. Finally, using a run-on transcription assay we show that the C-terminal FHOD1 cleavage product has the ability to inhibit RNA polymerase I transcription when overexpressed in HeLa cells as shown by blockage of BrUTP incorporation.

Amino Acid Motifs↗
PubMed2005

Role of the transmembrane domain of FXYD7 in structural and functional interactions with Na,K-ATPase.

Members of the FXYD family are tissue-specific regulators of the Na,K-ATPase. Here, we have investigated the contribution of amino acids in the transmembrane (TM) domain of FXYD7 to the interaction with Na,K-ATPase. Twenty amino acids of the TM domain were replaced individually by tryptophan, and combined mutations and alanine insertion mutants were constructed. Wild type and mutant FXYD7 were expressed in Xenopus oocytes with Na,K-ATPase. Mutational effects on the stable association with Na,K-ATPase and on the functional regulation of Na,K-ATPase were determined by co-immunoprecipitation and two-electrode voltage clamp techniques, respectively. Most residues important for the structural and functional interaction of FXYD7 are clustered in a face of the TM helix containing the two conserved glycine residues, but others are scattered over two-thirds of the FXYD TM helix. Ile-35, Ile-43, and Ile-44 are only involved in the stable association with Na,K-ATPase. Glu-26, Met-30, and Ile-44 are important for the functional effect and/or the efficient association of FXYD7 with Na,K-ATPase, consistent with the prediction that these amino acids contact TM domain 9 of the alpha subunit (Li, C., Grosdidier, A., Crambert, G., Horisberger, J.-D., Michielin, O., and Geering, K. (2004) J. Biol. Chem. 279, 38895-38902). Several amino acids that are not implicated in the efficient association of FXYD7 with the Na,K-ATPase are specifically involved in the functional effect of FXYD7. Leu-32 and Phe-37 influence the apparent affinity for external K+, whereas Val-28 and Ile-42 are implicated in the apparent affinity for both external K+ and external Na+. These amino acids act in a synergistic way. These results highlight the important structural and functional role of the TM domain of FXYD7 and delineate the determinants that mediate the complex interactions of FXYD7 with Na,K-ATPase.

Adenosine Triphosphatases↗
PubMed2005

Lipophilic versus hydrogen-bonding effect in P3 on potency and selectivity of valine aspartyl ketones as caspase 3 inhibitors.

Caspase 3 is a cysteinyl protease that mediates apoptotic cell death. Its inhibition may have an important impact in the treatment of several degenerative diseases. The P1 aspartic acid residue is a required element of recognition for this enzyme that was maintained constant along with the adjacent natural valine as the P2 group. The thiobenzylmethylketone warhead on the aspartate was conveniently handled through solid-phase synthesis allowing modification in the P3 region that eventually led to simpler derivatives with increased potency against caspase 3. The key to such an effect is the introduction of hydroxyl group alpha to the P3 carbonyl.

Aspartic Acid↗
PubMed2005

Novel pyrazinone mono-amides as potent and reversible caspase-3 inhibitors.

The iterative process for the discovery of a series of pyrazinone mono-amides as potent, selective and reversible non-peptide caspase-3 inhibitors (e.g., M826 and M867) is reported. These compounds display potent anti apoptotic activities in a number of cell based systems in vitro as well as in several animal models in vivo.

Amides↗
PubMed2005

Huntingtin is cleaved by caspases in the cytoplasm and translocated to the nucleus via perinuclear sites in Huntington's disease patient lymphoblasts.

Accumulation of mutant Huntingtin (Htt), especially the N-terminal-cleaved Htt, participates in the pathophysiology of Huntington's disease (HD). It is difficult to elucidate temporal properties of the translocation of "endogenous" Htt using autopsy HD patient brains. Thus, we examined the cell biology of "endogenous" Htt cleavage and nuclear translocation in cultured lymphoblasts of HD patients and controls. Apoptotic stimulation of lymphoblasts elicits caspase-dependent cleavage and selective nuclear translocation of N-terminal portions of Htt. Discrete clusters of the N-terminal Htt accumulate at unique perinuclear sites prior to nuclear translocation. Our findings suggest that caspase cleavage of Htt is cytoplasmic and precedes sorting to specific perinuclear sites followed by nuclear translocation in HD patient tissue.

Active Transport, Cell Nucleus↗
PubMed2004

Caspase-7 expanded function and intrinsic expression level underlies strain-specific brain phenotype of caspase-3-null mice.

Caspase-3-deficient mice of the 129S1/SvImJ (129) strain show severe brain development defects resulting in brain overgrowth and perinatal lethality, whereas on the C57BL/6J (B6) background, these mice develop normally. We therefore sought to identify the strain-dependent ameliorating gene. We biochemically isolated caspase-7 from B6-caspase-3-null (Casp3-/-) tissues as being the enzyme with caspase-3-like properties and capability of performing a caspase-3 surrogate function, apoptotic DNA fragmentation. Moreover, we show that, in contrast to the human enzymes, mouse caspase-7 is as efficient as caspase-3 at cleaving and thus inactivating ICAD (inhibitor of caspase-activated DNase), the inhibitor of apoptotic DNA fragmentation. Low levels of caspase-7 expression and activation correlate with lack of DNA fragmentation in 129-Casp3-/- apoptotic precursor neurons, whereas B6-Casp3-/- cells, which can fragment their DNA, show higher levels of caspase-7 expression and activation. The amount of caspase-7 activation in apoptotic precursor neurons is independent of the presence of caspase-3. Together, our findings demonstrate for the first time a strong correlation between caspase-7 activity, normal brain development, and apoptotic DNA fragmentation in Casp3-/- mice.

Animals↗
PubMed2004

Reducing the peptidyl features of caspase-3 inhibitors: a structural analysis.

Caspases are cysteine proteases that specifically cleave Asp-Xxx bonds. They are key agents in inflammation and apoptosis and are attractive targets for therapy against inflammation, neurodegeneration, ischemia, and cancer. Many caspase structures are known, but most involve either peptide or protein inhibitors, unattractive candidates for drug development. We present seven crystal structures of inhibited caspase-3 that illustrate several approaches to reducing the peptidyl characteristics of the inhibitors while maintaining their potency and selectivity. The inhibitors reduce the peptidyl nature of inhibitors while preserving binding potency by (1). exploiting a hydrophobic binding site C-terminal to the cleavage site, (2). replacing the negatively charged aspartyl residue at P4 with neutral groups, and (3). using a peptidomimetic 5,6,7-tricyclic system or a pyrazinone at P2-P3. In addition, we have found that two nicotinic acid aldehydes induce a significant conformational change in the S2 and S3 subsites of caspase-3, revealing an unexpected binding mode. These results advance the search for caspase-directed drugs by revealing how unacceptable molecular features can be removed without loss of potency.

Caspase 3↗
PubMed2004

Differential modulation of endotoxin responsiveness by human caspase-12 polymorphisms.

Caspases mediate essential key proteolytic events in inflammatory cascades and the apoptotic cell death pathway. Human caspases functionally segregate into two distinct subfamilies: those involved in cytokine maturation (caspase-1, -4 and -5) and those involved in cellular apoptosis (caspase-2, -3, -6, -7, -8, -9 and -10). Although caspase-12 is phylogenetically related to the cytokine maturation caspases, in mice it has been proposed as a mediator of apoptosis induced by endoplasmic reticulum stress including amyloid-beta cytotoxicity, suggesting that it might contribute to the pathogenesis of Alzheimer's disease. Here we show that a single nucleotide polymorphism in caspase-12 in humans results in the synthesis of either a truncated protein (Csp12-S) or a full-length caspase proenzyme (Csp12-L). The read-through single nucleotide polymorphism encoding Csp12-L is confined to populations of African descent and confers hypo-responsiveness to lipopolysaccharide-stimulated cytokine production in ex vivo whole blood, but has no significant effect on apoptotic sensitivity. In a preliminary study, we find that the frequency of the Csp12-L allele is increased in African American individuals with severe sepsis. Thus, Csp12-L attenuates the inflammatory and innate immune response to endotoxins and in doing so may constitute a risk factor for developing sepsis.

Africa↗
PubMed2004

A caspase active site probe reveals high fractional inhibition needed to block DNA fragmentation.

Apoptotic markers consist of either caspase substrate cleavage products or phenotypic changes that manifest themselves as a consequence of caspase-mediated substrate cleavage. We have shown recently that pharmacological inhibitors of caspase activity prevent the appearance of two such apoptotic manifestations, alphaII-spectrin cleavage and DNA fragmentation, but that blockade of the latter required a significantly higher concentration of inhibitor. We investigated this phenomenon through the use of a novel radiolabeled caspase inhibitor, [(125)I]M808, which acts as a caspase active site probe. [(125)I]M808 bound to active caspases irreversibly and with high sensitivity in apoptotic cell extracts, in tissue extracts from several commonly used animal models of cellular injury, and in living cells. Moreover, [(125)I]M808 detected active caspases in septic mice when injected intravenously. Using this caspase probe, an active site occupancy assay was developed and used to measure the fractional inhibition required to block apoptosis-induced DNA fragmentation. In thymocytes, occupancy of up to 40% of caspase active sites had no effect on DNA fragmentation, whereas inhibition of half of the DNA cleaving activity required between 65 and 75% of active site occupancy. These results suggest that a high and persistent fractional inhibition will be required for successful caspase inhibition-based therapies.

Amino Acid Chloromethyl Ketones↗
PubMed2004

Neuroprotective effects of M826, a reversible caspase-3 inhibitor, in the rat malonate model of Huntington's disease.

1. Caspases, key enzymes in the apoptosis pathway, have been detected in the brain of HD patients and in animal models of the disease. In the present study, we investigated the neuroprotective properties of a new, reversible, caspase-3-specific inhibitor, M826 (3-([(2S)-2-[5-tert-butyl-3-[[(4-methyl-1,2,5-oxadiazol-3-yl)methyl]amino]-2-oxopyrazin-1(2H)-yl]butanoyl]amino)-5-[hexyl(methyl)amino]-4-oxopentanoic acid), in a rat malonate model of HD. 2. Pharmacokinetic and autoradiography studies after intrastriatal (i.str.) injection of 1.5 nmol of M826 or its tritiated analogue [(3)H]M826 indicated that the compound diffused within the entire striatum. The elimination half-life (T(1/2)) of M826 in the rat striatum was 3 h. 3. I.str. injection of 1.5 nmol of M826 10 min after malonate infusion induced a significant reduction (66%) in the number of neurones expressing active caspase-3 in the ipsilateral striatum. 4. Inhibition of active caspase-3 translated into a significant but moderate reduction (39%) of the lesion volume, and of cell death (24%), 24 h after injury. The efficacy of M826 at inhibiting cell death was comparable to that of the noncompetitive NMDA receptor antagonist MK801. 5. These data provide in vivo proof-of-concept of the neuroprotective effects of reversible caspase-3 inhibitors in a model of malonate-induced striatal injury in the adult rat.

Animals↗
PubMed2004

Differential efficacy of caspase inhibitors on apoptosis markers during sepsis in rats and implication for fractional inhibition requirements for therapeutics.

A rodent model of sepsis was used to establish the relationship between caspase inhibition and inhibition of apoptotic cell death in vivo. In this model, thymocyte cell death was blocked by Bcl-2 transgene, indicating that apoptosis was predominantly dependent on the mitochondrial pathway that culminates in caspase-3 activation. Caspase inhibitors, including the selective caspase-3 inhibitor M867, were able to block apoptotic manifestations both in vitro and in vivo but with strikingly different efficacy for different cell death markers. Inhibition of DNA fragmentation required substantially higher levels of caspase-3 attenuation than that required for blockade of other apoptotic events such as spectrin proteolysis and phosphatidylserine externalization. These data indicate a direct relationship between caspase inhibition and some apoptotic manifestations but that small quantities of uninhibited caspase-3 suffice to initiate genomic DNA breakdown, presumably through the escape of catalytic quantities of caspase-activated DNase. These findings suggest that putative caspase-independent apoptosis may be overestimated in some systems since blockade of spectrin proteolysis and other cell death markers does not accurately reflect the high degrees of caspase-3 inhibition needed to prevent DNA fragmentation. Furthermore, this requirement presents substantial therapeutic challenges owing to the need for persistent and complete caspase blockade.

Animals↗