Biomedical subjects

F Gauthier

Publications and source records attributed to F Gauthier.

At least 37 records · Page 2Linked to original sources

PubMed2000

Interaction of the third helix of Antennapedia homeodomain and a phospholipid monolayer, studied by ellipsometry and PM-IRRAS at the air-water interface.

The penetratin peptide, a 16 amino acid sequence extracted from Antennapedia homeodomain, is able to translocate across a neural cell membrane through an unknown mechanism, most likely a non-specific interaction with membrane lipids. Beyond its potential application as vector targeting small hydrophilic molecules and enabling them to reach a cell nucleus, this observation raises intriguing questions concerning the physico-chemistry of peptide-lipid interactions. Here we present a study of the role of lipid surface pressure and head charge on the mechanism of interaction. This was performed using optical techniques: surface infrared spectroscopy and ellipsometry, applied to a monolayer of phospholipids deposited at the air-water interface. Determination of the structure and orientation of peptides and lipids (separately or together) evidenced that electrostatic rather than amphiphilic interactions determine the peptide adsorption and its action on lipids.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2000

Modulation of Rho and cytoskeletal protein attachment to membranes by a prenylcysteine analog.

The GTPases Rho regulate the assembly of polymerized actin structures. Their C-terminal sequences end with the CAAX motif that undergo a lipidation of the cysteine residue. Analogs to the C-terminal ends of Rho proteins, N-acetyl-S-all-trans, trans-farnesyl-L-cysteine and N-acetyl-S-all-trans-geranylgeranyl-L-cysteine, wereused to analyze the role of prenylation in their membrane association. Silver-stained gels indicated that N-acetyl-S-all-trans-geranylgeranyl-L-cysteine treatment released only a few proteins of 20, 46, and 60 kDa. Western blot analysis showed that N-acetyl-S-all-trans-geranylgeranyl-L-cysteine released RhoB (10%), RhoA (28%), and Cdc42 (95%) from membranes, whereas N-acetyl-S-all-trans and trans-farnesyl-L-cysteine did not. Rab1, which possesses two geranylgeranyl groups, was also strongly extracted by N-acetyl-S-all-trans-geranylgeranyl-L-cysteine, whereas Ras, which is farnesylated, was not. Furthermore, N-acetyl-S-all-trans-geranylgeranyl-L-cysteine was very efficient (95%) in dissociating actin and tubulin from membranes but not integral membrane protein P-glycoprotein and sodium/phosphate cotransporter NaP(i)-2. The extraction of Rho and cytoskeletal proteins occurred below the critical micellar concentration of N-acetyl-S-all-trans-geranylgeranyl-L-cysteine. Membrane treatments with 0.7 m KI totally extracted actin, whereas 70% of Cdc42 was released. Actin was, however, insoluble in Triton X-100-treated membranes, whereas this detergent extracted (80%) Cdc42. These data show that Rho proteins and actin are not physically bound together and suggest that their extraction from membranes by N-acetyl-S-all-trans-geranylgeranyl-L-cysteine likely occurs via different mechanisms.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

The glycosylphosphatidyl inositol-anchored adhesion molecule F3/contactin is required for surface transport of paranodin/contactin-associated protein (caspr).

Paranodin/contactin-associated protein (caspr) is a transmembrane glycoprotein of the neurexin superfamily that is highly enriched in the paranodal regions of myelinated axons. We have investigated the role of its association with F3/contactin, a glycosylphosphatidyl inositol (GPI)-anchored neuronal adhesion molecule of the Ig superfamily. Paranodin was not expressed at the cell surface when transfected alone in CHO or neuroblastoma cells. Cotransfection with F3 resulted in plasma membrane delivery of paranodin, as analyzed by confocal microscopy and cell surface biotinylation. The region that mediates association with paranodin was mapped to the Ig domains of F3 by coimmunoprecipitation experiments. The association of paranodin with F3 allowed its recruitment to Triton X-100-insoluble microdomains. The GPI anchor of F3 was necessary, but not sufficient for surface expression of paranodin. F3-Ig, a form of F3 deleted of the fibronectin type III (FNIII) repeats, although GPI-linked and expressed at the cell surface, was not recovered in the microdomain fraction and was unable to promote cell surface targeting of paranodin. Thus, a cooperative effect between the GPI anchor, the FNIII repeats, and the Ig regions of F3 is required for recruitment of paranodin into lipid rafts and its sorting to the plasma membrane.

Animals↗

PubMed2000

Activation of beta-catenin in epithelial and mesenchymal hepatoblastomas.

Wnt/beta-catenin signaling is frequently activated in cancer cells by stabilizing mutations of beta-catenin or loss-of-function mutations of the APC tumor suppressor gene. We have analysed the role of beta-catenin in the pathogenesis of hepatoblastoma (HB), an embryonic liver tumor occurring mainly in children under 2 years of age. Sequence analysis of the beta-catenin NH2-terminal domain in 18 epithelial and mixed HBs revealed missense mutations in the GSK3beta phosphorylation motif or interstitial deletions in 12 tumors (67%). In the remaining cases, no truncating mutation of APC could be evidenced. Immunohistochemical analysis of beta-catenin in 11 HBs demonstrated nuclear/cytoplasmic accumulation of the protein in all tumors analysed, with predominant nuclear beta-catenin immunostaining in undifferentiated cells. Membranous beta-catenin localization was preserved only in fetal-type tumoral hepatocytes and was associated with E-cadherin expression. Moreover, we show that beta-catenin is aberrantly overexpressed in a large spectrum of tumor components, including hepatocyte-like cells at various differentiation stages and heterologous elements such as squamous, osteoid and chrondroid tissues, and in occasional other mesenchymally-derived cells. These data strongly suggest that activation of beta-catenin signaling is an obligatory step in HB pathogenesis, and raise the possibility that it interferes with developmental signals that specify different tissue types at early stages of hepatic differentiation.

Biological Transport↗

PubMed2000

Altered expression of cruzipain and a cathepsin B-like target in a Trypanosoma cruzi cell line displaying resistance to synthetic inhibitors of cysteine-proteinases.

The therapeutic potential of synthetic inhibitors to the major cysteine-proteinase from Trypanosoma cruzi (cruzain or cruzipain) was recently demonstrated in animal models of Chagas' disease. A possible limitation of this strategy would be the emergence of parasite populations developing resistance to cysteine-proteinase inhibitors. Here, we describe the properties of a phenotypically stable T. cruzi cell line (R-Dm28) that displays increased resistance to Z-(SBz)Cys-Phe-CHN2, an irreversible cysteine-proteinase inhibitor which preferentially inactivates cathepsin L-like enzymes. Isolated from axenic cultures of the parental cells (IC50 1.5 microM), R-Dm28 epimastigotes exhibited 13-fold (IC50) 20 microM) higher resistance to this inhibitor and did not display cross-resistance to unrelated trypanocidal drugs, such as benznidazol and nifurtimox. Western blotting (with mAb), affinity labeling (with biotin-LVG-CHN2) and FACS analysis of R-Dm28 log-phase epimastigotes revealed that the cruzipain target was expressed at lower levels, as compared with Dm28c. Interestingly, this deficit was paralleled by increased expression of an unrelated Mr 30 000 cysteine-proteinase whose activity was somewhat refractory to inhibition by Z-(SBz)Cys-Phe-CHN,. N-terminal sequencing of the affinity-purified biotin-LVG-proteinase complex allowed its identification as a cathepsin B-like enzyme. Increased antigenic deposits of this proteinase were found in the grossly enlarged and electron dense reservosomes from R-Dm28 epimastigotes. Our data suggest that R-Dm28 resistance to toxic effects induced by the synthetic inhibitor may result from decreased availability of the most sensitive cysteine-proteinase target, cruzipain. The deficit in metabolic functions otherwise mediated by this cathepsin L-like proteinase is likely compensated by increased expression/accumulation of a cathepsin B-like target.

Animals↗

PubMed2000

Coliform bacteria and nitrogen fixation in pulp and paper mill effluent treatment systems.

The majority of pulp and paper mills now biotreat their combined effluents using activated sludge. On the assumption that their wood-based effluents have negligible fixed N, and that activated-sludge microorganisms will not fix significant N, these mills routinely spend large amounts adding ammonia or urea to their aeration tanks (bioreactors) to permit normal biomass growth. N(2) fixation in seven Eastern Canadian pulp and paper mill effluent treatment systems was analyzed using acetylene reduction assays, quantitative nitrogenase (nifH) gene probing, and bacterial isolations. In situ N(2) fixation was undetectable in all seven bioreactors but was present in six associated primary clarifiers. One primary clarifier was studied in greater detail. Approximately 50% of all culturable cells in the clarifier contained nifH, of which >90% were Klebsiella strains. All primary-clarifier coliform bacteria growing on MacConkey agar were identified as klebsiellas, and all those probed contained nifH. In contrast, analysis of 48 random coliform isolates from other mill water system locations showed that only 24 (50%) possessed the nifH gene, and only 13 (27%) showed inducible N(2)-fixing activity. Thus, all the pulp and paper mill primary clarifiers tested appeared to be sites of active N(2) fixation (0.87 to 4.90 mg of N liter(-1) day(-1)) and a microbial community strongly biased toward this activity. This may also explain why coliform bacteria, especially klebsiellas, are indigenous in pulp and paper mill water systems.

Base Sequence↗

PubMed2000

Risk of gastrointestinal bleeding during adolescence and early adulthood in children with portal vein obstruction.

OBJECTIVE: To estimate the risk of bleeding during adolescence and early adulthood in a group of children with portal vein obstruction who had not undergone an effective treatment beforehand. STUDY DESIGN: Children (n = 44) were followed up from age 12 years to a mean age of 20 years (range, 15-34 years). Actuarial risk of bleeding, related to previous occurrence of gastrointestinal bleeding and to pattern of varices at age 12, was calculated yearly. RESULTS: Twenty-four children presented with gastrointestinal bleeding after age 12, and 20 did not bleed. The overall actuarial probability of bleeding was 49% at age 16 and 76% at age 24. Probability of bleeding at age 23 was higher in children who had bled before age 12 than in children who had not bled (93% vs 56%; P =.007). Probabilities of bleeding at age 18 and at age 23 were 60% and 85%, respectively, in patients who had grade II or III esophageal varices at age 12. The 9 children without varices or with grade I varices only on endoscopy did not bleed between the ages of 12 and 20 years. CONCLUSIONS: Children with portal vein obstruction have a >50% risk of bleeding during adolescence; the pattern of varices on endoscopy at age 12 may have a prognostic value.

Actuarial Analysis↗

PubMed1999

New, sensitive fluorogenic substrates for human cathepsin G based on the sequence of serpin-reactive site loops.

Cathepsin G has both trypsin- and chymotrypsin-like activity, but studies on its enzymatic properties have been limited by a lack of sensitive synthetic substrates. Cathepsin G activity is physiologically controlled by the fast acting serpin inhibitors alpha1-antichymotrypsin and alpha1-proteinase inhibitor, in which the reactive site loops are cleaved during interaction with their target enzymes. We therefore synthesized a series of intramolecularly quenched fluorogenic peptides based on the sequence of various serpin loops. Those peptides were assayed as substrates for cathepsin G and other chymotrypsin-like enzymes including chymotrypsin and chymase. Peptide substrates derived from the alpha1-antichymotrypsin loop were the most sensitive for cathepsin G with kcat/Km values of 5-20 mM-1 s-1. Substitutions were introduced at positions P1 and P2 in alpha1-antichymotrypsin-derived substrates to tentatively improve their sensitivity. Replacement of Leu-Leu in ortho-aminobenzoyl (Abz)-Thr-Leu-Leu-Ser-Ala-Leu-Gln-N-(2, 4-dinitrophenyl)ethylenediamine (EDDnp) by Pro-Phe in Abz-Thr-Pro-Phe-Ser-Ala-Leu-Gln-EDDnp produced the most sensitive substrate of cathepsin G ever reported. It was cleaved with a specificity constant kcat/Km of 150 mM-1 s-1. Analysis by molecular modeling of a peptide substrate bound into the cathepsin G active site revealed that, in addition to the protease S1 subsite, subsites S1' and S2' significantly contribute to the definition of the substrate specificity of cathepsin G.

Amino Acid Sequence↗

PubMed1999

Revisiting the S2 specificity of papain by structural analogs of Phe.

Papain characteristically has a strong preference for encoded L-aromatic amino acids (Phe > Tyr) at P2 position. We re-examined papain S2 specificity using structural analogs of Phe, in fluorogenic substrates of the series: dansyl-Xaa-Arg-Ala-Pro-Trp (Xaa = P2 residue). Kinetic analyses showed that the S2 pocket accommodates a broad spectrum of Phe derivatives. Papain is poorly stereoselective towards Dns-(D/L)-Phe-Arg-Ala-Pro-Trp and binding is not critically affected by replacement of the benzyl ring by the non-aromatic lateral chain of cyclohexylalanine. The Km was significantly improved by mono- and di-chlorination of Phe, or by its substitution by an electronegative group-like NO2, but the specificity constant was unchanged. Shortening or lengthening the side chain by adding or removing a methylene group impairs the P2/S2 interactions significantly, as do constrained structural analogs of Phe. Incorporation of benzyl-substituted phenylalanyl amino acid could help to design peptide-derived inhibitors with greater affinity and bioavailability.

Binding Sites↗

PubMed1999

Outcome of cystic fibrosis-associated liver cirrhosis: management of portal hypertension.

BACKGROUND/AIM: Variceal bleeding is the most severe complication in patients with cystic fibrosis-associated liver cirrhosis, who often do not have severe respiratory failure. The advent of liver transplantation has broadened the treatment options. The purpose of this study was to report our experience with the management of portal hypertension. METHODS: Clinical and biochemical features, outcome of liver disease and management of portal hypertension were analyzed retrospectively in 44 children with cystic fibrosis-associated liver cirrhosis. RESULTS: The mean age at diagnosis of liver cirrhosis was 9 years. Eighty-six per cent of the children developed esophageal varices, 50% of whom bled early in their second decade. Injection sclerotherapy of esophageal varices did not prevent recurrence of bleeding in five of seven children. Elective surgical portosystemic shunting was successfully performed in nine of 11 patients considered being at high risk of bleeding or with recurrent bleeding episodes but without severe pulmonary failure and liver dysfunction, allowing prolonged post-operative survival up to 15 years. Two of three children who underwent isolated liver transplantation for severe portal hypertension died post-operatively. CONCLUSIONS: Management emphasis in cystic fibrosis patients with liver cirrhosis should be on control of bleeding and variceal decompression. These results suggest that surgical portosystemic shunting may be considered to relieve portal hypertension in patients without progressive liver failure and severe lung disease as an alternative to liver transplantation. With this policy, patients may be stabilized for many years until progression of liver or lung diseases indicates liver or lung-liver transplantation.

Adolescent↗

PubMed1999

Discrimination of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, and mammalian cathepsins B and L, by a pH-inducible fluorogenic substrate of trypanosomal cysteine proteinases.

The substrate specificity of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, was investigated using a series of dansyl-peptides based on the putative autoproteolytic sequence of the proteinase (VVG-GP) located at the hinge region between the catalytic domain and the C-terminal extension. Replacing Val with Pro at P2 in this sequence greatly improved the rate of cleavage by cruzipain. Tyr and Val residues are preferred at P3 by all cysteine proteinases whatever their origin, whereas only cruzipain and cathepsin L cleaved substrate with a His at that position. The combination of a Pro at P2 and His at P3 abolished cleavage by cathepsin L, so that only cruzipain was able to cleave the HPGGP peptide at the GG bond. A substrate with intramolecularly quenched fluorescence was raised on this sequence (Abz-HPGGPQ-EDDnp) which was also specifically cleaved by cruzipain (kcat/Km of 157 000 m-1. s-1) and by a homologous proteinase from Trypanosoma congolense. The pH activity profile of cruzipain on Abz-HPGGPQ-EDDnp showed a narrow peak with a maximum at pH 5.5 and no cleavage above pH 6.8, although trypanosomal cysteine proteinases remain active at basic pH. The lack of activity at neutral and basic pH was due to a decrease in kcat, while the Km remained essentially unchanged, demonstrating that the substrate still binds to the enzyme and therefore behaves as an inhibitor. Changing the substrate into an inhibitor depended on the deprotonation of the His residue in the substrate, as deduced from a comparison of the pH activity profile with that of a related, but uncharged, substrate. Abz-HPGGPQ-EDDnp also inhibited mammalian cathepsins B and L but was not cleaved by these proteinases at any pH. The importance of the His residue at P3 for cleavage by cruzipain was confirmed by substituting Lys for His at that position. The resulting peptide was not cleaved by cruzipain in spite of the presence of a positively charged group at P3, but still interacted with the enzyme. It was concluded that the presence of an imidazolium group at P3 was essential to endow the HPGGPQ sequence with the properties of a cruzipain substrate.

Animals↗

PubMed1999

Heme and acute inflammation role in vivo of heme in the hepatic expression of positive acute-phase reactants in rats.

Acute-phase protein synthesis in the liver during inflammation is regulated via cytokines and glucocorticoids. Using quantitative reverse transcription (RT)-PCR analysis and immunoassay, we explored, in the rat, the response of the acute-phase protein, alpha-2 macroglobulin (A2M), after systemic inflammation induced by lipopolysaccharide (LPS) or localized inflammation induced by turpentine oil (TO). The results indicate that synthesis of A2M is higher following TO-induced inflammation than LPS-induced inflammation and is not correlated with interleukin (IL)-6 or glucocorticoid levels. We studied the putative role of heme in this differential A2M expression following localized vs. systemic inflammation; addition of heme during LPS-induced inflammation can boost the expression of A2M, whereas blocking heme synthesis (by succinyl acetone) or enhancing its consumption in parallel biosynthetic pathways (cytochrome P450 induction by phenobarbital) decreases A2M expression. This decrease was abolished by exogenous heme supplementation. Finally, we demonstrate that heme supplementation is also able to increase the A2M response in female rats to a level similar to that in male rats providing a new insight into the puzzling sexual dimorphism observed previously during localized inflammation. We propose that heme should be considered a new regulatory element in controlling liver A2M expression during inflammation.

Acute-Phase Proteins↗

PubMed1999

Chicken cystatin stimulates nitric oxide release from interferon-gamma-activated mouse peritoneal macrophages via cytokine synthesis.

Cystatins are natural tight-binding, reversible inhibitors of cysteine proteases. We have shown that cystatins also stimulate nitric oxide (NO) production by interferon-gamma-activated mouse peritoneal macrophages [Verdot, L., Lalmanach, G., Vercruysse, V., Hartman, S., Lucius, R., Hoebeke, J., Gauthier F. & Vray, B. (1996) J. Biol. Chem. 271, 28077-28081]. The present study was undertaken to further document this new function. Macrophages activated with interferon-gamma and then stimulated with interferon-gamma plus chicken cystatin generated increased amounts of NO in comparison with macrophages only activated with interferon-gamma. Interferon-gamma-activated macrophages must be incubated with chicken cystatin for at least 8 h to upregulate NO production. NO induction was due to increased inducible nitric oxide synthase protein synthesis. Macrophages incubated with chicken cystatin alone or with interferon-gamma plus chicken cystatin produced increased amounts of both tumor necrosis factor alpha and interleukin 10. The addition of recombinant murine tumor necrosis factor alpha alone or in combination with recombinant murine interleukin-10 mimicked the effect of chicken cystatin. The addition of neutralizing anti-(tumor necrosis factor alpha) antibodies reduced sharply NO production by chicken cystatin/interferon-gamma-activated mouse peritoneal macrophages. Taken together, these data suggest that chicken cystatin induces the synthesis of tumor necrosis factor alpha and interleukin 10. In turn, these two cytokines stimulate the production of NO by interferon-gamma-activated macrophages. The findings point to a new relationship between cystatins, cytokines, inflammation and the immune response.

Animals↗

PubMed1999

Paediatric liver transplantation with a split graft: experience at Bicêtre.

UNLABELLED: This study evaluates the results of paediatric liver transplantation (PLT) with split liver grafts at Bicêtre hospital. PATIENTS AND METHODS: Between January 1, 1988 and December 31, 1995, 205 PLT were performed in 180 children. One auxiliary PLT was excluded from the study. The graft was a whole (WLG), reduced (RLG) and split liver graft (SLG) in 76, 112 and 16 cases respectively. The SLG consisted of segments II + III in 14 cases, and II + III + IV in 2 cases. Results of PLT with SLT, RLT and WLG were retrospectively compared. Minimal follow-up was 12 months. RESULTS: In elective PLT, actual 1 year patient (graft) survival were 93.3% (84.4%) with WLG (n = 64), 84.1% (76.4%) with RLG (n = 72), 81.8% (81.8%) with SLG (n = 11). In urgent LT, actual patient (graft) survival were 100% (83.3%) with WLG (n = 6), 58.6% (52.5%) with RLG (n = 40), 25% (20%) with SLG (n = 5). Specific complications of the splitting technique were: 2 Budd-Chiari syndromes in 2 early patients, without any new case after modification of the technique of left hepatic vein to inferior vena cava anastomosis; 4 bile leaks (25%) from the left hepatic duct to Roux-en-Y loop anastomosis. DISCUSSION: In our experience, the results of PLT with SLG were satisfactory in the elective situation, but disappointing in urgent cases. In the current context of liver graft shortage, appropriate use of this sophisticated and demanding technique depends on the experience of the team, recipient's condition, and logistic considerations.

Adolescent↗

PubMed1999

[Smooth muscle mesentery associated with Epstein-Barr virus (EBV). Report of a case in a child with a liver transplant under FK506 regimen].

Smooth-muscle tumors, benign and malignant, are increasingly recognized in children who are immunocompromised because of HIV infection and organ transplantation. We report a case of an EBV-associated smooth-muscle tumor, of unusual location arising in a seven-year-old post-transplant patient who was previously treated for a lymphoproliferative disease. Five years after liver transplantation, a mesenteric tumor was diagnosed. The tumor was composed of spindle cells with smooth-muscle features. Immunohistochemical analysis was positive for muscle-specific actin and desmin, negative for EBV latent membrane protein (LMP-1). In situ hybridization revealed nuclear EBV sequences. This case underlines the role of EBV infection in the development of unusual smooth-muscle tumors after organ transplantation. The evolution of these rare tumors is uncertain.

Child↗

PubMed1999

[Unusual site of an embryonal rhabdomyosarcoma of the mesenchymal hepatic pedicle].

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of children. Tumors arising in the extrahepatic biliary tree are extremely rare (less than 1% of cases). In this location, most are RMS of the botryoid type. We report a case of a 10-year-old child with embryonal RMS arising in the mesenchyma of the hepatic pedicle. Most tumor cells were large, round with abundant eosinophilic cytoplasm. A few cells were small round or spindle-shaped. Tumor cells showed positive immunostaining for muscle markers: desmin and sarcomeric actin. Electron microscopy revealed 2 types of cells: some were undifferentiated and others showed striated muscle differentiation features.

Adult↗

PubMed1998

Inhibition of trypanosomal cysteine proteinases by their propeptides.

The ability of the prodomains of trypanosomal cysteine proteinases to inhibit their active form was studied using a set of 23 overlapping 15-mer peptides covering the whole prosequence of congopain, the major cysteine proteinase of Trypanosoma congolense. Three consecutive peptides with a common 5-mer sequence YHNGA were competitive inhibitors of congopain. A shorter synthetic peptide consisting of this 5-mer sequence flanked by two Ala residues (AYHNGAA) also inhibited purified congopain. No residue critical for inhibition was identified in this sequence, but a significant improvement in Ki value was obtained upon N-terminal elongation. Procongopain-derived peptides did not inhibit lysosomal cathepsins B and L but did inhibit native cruzipain (from Dm28c clone epimastigotes), the major cysteine proteinase of Trypanosoma cruzi, the proregion of which also contains the sequence YHNGA. The positioning of the YHNGA inhibitory sequence within the prosegment of trypanosomal proteinases is similar to that covering the active site in the prosegment of cysteine proteinases, the three-dimensional structure of which has been resolved. This strongly suggests that trypanosomal proteinases, despite their long C-terminal extension, have a prosegment that folds similarly to that in related mammal and plant cysteine proteinases, resulting in reverse binding within the active site. Such reverse binding could also occur for short procongopain-derived inhibitory peptides, based on their resistance to proteolysis and their ability to retain inhibitory activity after prolonged incubation. In contrast, homologous peptides in related cysteine proteinases did not inhibit trypanosomal proteinases and were rapidly cleaved by these enzymes.

Amino Acid Sequence↗

PubMed1998

Localization of RhoA GTPase to endothelial caveolae-enriched membrane domains.

Caveolae are small microdomains of the plasma membrane that are thought to play important roles in signal transduction processes. In this work, we have investigated the association of Rho proteins with caveolae-enriched membrane domains isolated from cultured endothelial cells. Fractionation of ECV304 cells by sucrose gradient density centrifugation in the absence of detergent resulted in the co-sedimentation of a significant proportion of RhoA and Cdc42 with known caveolae marker proteins, including caveolin, but not with other non-caveolae membrane proteins such as the angiotensin-converting enzyme. Immunoprecipitation experiments carried on crude endothelial cell lysates as well as with solubilized caveolae-enriched membrane domains showed the coimmunoprecipitation of caveolin with RhoA but not with Cdc42. Incubation of endothelial cell lysates with a glutathione-S-transferase (GST)-RhoA fusion protein resulted in the specific precipitation of caveolin, while addition of GST-caveolin-1 to the lysates promoted the precipitation of RhoA. Moreover, incubation of bacterially expressed RhoA with GST-caveolin-1 resulted in the precipitation of RhoA, indicating that RhoA directly interacts with caveolin-1. This interaction was found to be nucleotide-independent and was not affected by prior modification of RhoA with the C3 exoenzyme from C. botulinium or with the cytotoxic necrotinizing factor from E. coli. Taken together, these results suggest the association of RhoA with endothelial caveolae-enriched membrane domains, likely through physical interaction with caveolin-1. These findings may provide new insights into the functions played by Rho proteins and caveolae in signal transduction events.

Caveolin 1↗