Search

Background The growing interest of the scientific community in trail running has highlighted the acute effects of practice at the time of these races on isolated aspects of physiological and structural systems; biological, physiological, cognitive, and muscular functions; and the psychological state of athletes. However, no integrative study has been conducted under these conditions with so many participants and monitoring of pre-, per-, and postrace variables for up to 10 days over a distance close to 100 miles. Objective The aim of this study was to evaluate the kinetics of the performance parameters during a 156 km trail run and 6000 m of elevation gain in pre-, per-, and postrace conditions. The general hypothesis is based on significant alterations in the psychological, physiological, mechanical, biological, and cognitive parameters. Methods The Trail Scientifique de Clécy took place on November 11, 2021. This prospective experimental study provides a comprehensive exploration of the constraints and adaptations of psychophysiological and sociological variables assessed in real race conditions during a trail running of 156 km on hilly ground and 6000 m of elevation gain (D+). The study protocol allowed for repeatability of study measurements under the same experimental conditions during the race, with the race being divided into 6 identical loops of 26 km and 1000 m D+. Measurements were conducted the day before and the morning of the race, at the end of each lap, after a pit stop, and up to 10 days after the race. A total of 55 participants were included, 43 (78%) men and 12 (22%) women, who were experienced in ultra–trail-running events and with no contraindications to the practice of this sport. Results The launch of the study was authorized on October 26, 2021, under the trial number 21-0166 after a favorable opinion from the Comité de Protection des Personnes Ouest III (21.09.61/SIRIPH 2G 21.01586.000009). Of the 55 runners enrolled, 41 (75%) completed the race and 14 (25%) dropped out for various reasons, including gastric problems, hypothermia, fatigue, and musculoskeletal injuries. All the measurements for each team were completed in full. The race times (ie, excluding the measurements) ranged from 17.8206 hours for the first runner to 35.9225 hours for the last runner. The average time to complete all measurements for each lap was 64 (SD 3) minutes. Conclusions The Trail Scientifique de Clécy, by its protocol, allowed for a multidisciplinary approach to the discipline. This approach will allow for the explanation of the studied parameters in relation to each other and observation of the systems of dependence and independence. The initial results are expected in June 2022. International Registered Report Identifier (IRRID) RR1-10.2196/38027

BACKGROUND The growing interest of the scientific community in trail running has highlighted the acute effects of practice at the time of these races on isolated aspects of physiological and structural systems; biological, physiological, cognitive, and muscular functions; and the psychological state of athletes. However, no integrative study has been conducted under these conditions with so many participants and monitoring of pre-, per-, and postrace variables for up to 10 days over a distance close to 100 miles. OBJECTIVE The aim of this study was to evaluate the kinetics of the performance parameters during a 156 km trail run and 6000 m of elevation gain in pre-, per-, and postrace conditions. The general hypothesis is based on significant alterations in the psychological, physiological, mechanical, biological, and cognitive parameters. METHODS The Trail Scientifique de Clécy took place on November 11, 2021. This prospective experimental study provides a comprehensive exploration of the constraints and adaptations of psychophysiological and sociological variables assessed in real race conditions during a trail running of 156 km on hilly ground and 6000 m of elevation gain (D+). The study protocol allowed for repeatability of study measurements under the same experimental conditions during the race, with the race being divided into 6 identical loops of 26 km and 1000 m D+. Measurements were conducted the day before and the morning of the race, at the end of each lap, after a pit stop, and up to 10 days after the race. A total of 55 participants were included, 43 (78%) men and 12 (22%) women, who were experienced in ultra–trail-running events and with no contraindications to the practice of this sport. RESULTS The launch of the study was authorized on October 26, 2021, under the trial number 21-0166 after a favorable opinion from the Comité de Protection des Personnes Ouest III (21.09.61/SIRIPH 2G 21.01586.000009). Of the 55 runners enrolled, 41 (75%) completed the race and 14 (25%) dropped out for various reasons, including gastric problems, hypothermia, fatigue, and musculoskeletal injuries. All the measurements for each team were completed in full. The race times (ie, excluding the measurements) ranged from 17.8206 hours for the first runner to 35.9225 hours for the last runner. The average time to complete all measurements for each lap was 64 (SD 3) minutes. CONCLUSIONS The Trail Scientifique de Clécy, by its protocol, allowed for a multidisciplinary approach to the discipline. This approach will allow for the explanation of the studied parameters in relation to each other and observation of the systems of dependence and independence. The initial results are expected in June 2022. INTERNATIONAL REGISTERED REPORT RR1-10.2196/38027

ABCB4 (ATP-binding cassette subfamily B member 4) is a hepatocanalicular floppase involved in biliary phosphatidylcholine (PC) secretion. Variations in the ABCB4 gene give rise to several biliary diseases, including progressive familial intrahepatic cholestasis type 3 (PFIC3), an autosomal recessive disease that can be lethal in the absence of liver transplantation. In this study, we investigated the effect and potential rescue of ten ABCB4 missense variations in NBD1:NBD2 homologous positions (Y403H/Y1043H, K435M/K1075M, E558K/E1200A, D564G/D1206G and H589Y/H1231Y) all localized at the conserved and functionally critical motifs of ABC transporters, six of which are mutated in patients. By combining structure analysis and in vitro studies, we found that all ten mutants were normally processed and localized at the canalicular membrane of HepG2 cells, but showed dramatically impaired PC transport activity that was significantly rescued by treatment with the clinically approved CFTR potentiator ivacaftor. Our results provide evidence that functional ABCB4 mutations are rescued by ivacaftor, paving the way for the repositioning of this potentiator for the treatment of selected patients with PFIC3 caused by mutations in the ATP-binding sites of ABCB4.

Annexin A2 (AnxA2) is a calcium- and phospholipid-binding protein that plays roles in cellular processes involving membrane and cytoskeleton dynamics and is able to associate to several partner proteins. However, the principal molecular partners of AnxA2 are negatively charged phospholipids such as phosphatidylserine and phosphatidyl-inositol-(4,5)-phosphate. Herein we have studied different aspects of membrane lipid rearrangements induced by AnxA2 membrane binding. X-ray diffraction data revealed that AnxA2 has the property to stabilize lamellar structures and to block the formation of highly curved lipid phases (inverted hexagonal phase, H

International audience; Background & aim: ABCB4 is expressed at the canalicular membrane of hepatocytes. This ATP-binding cassette (ABC) transporter is responsible for the secretion of phosphatidylcholine into bile canaliculi. Missense genetic variations of ABCB4 are correlated with several rare cholestatic liver diseases, the most severe being progressive familial intrahepatic cholestasis type 3 (PFIC3). In a repurposing strategy to correct intracellularly retained ABCB4 variants, we tested 16 compounds previously validated as cystic fibrosis transmembrane conductance regulator (CFTR) correctors.Methods: The maturation, intracellular localization and activity of intracellularly retained ABCB4 variants were analyzed in cell models after treatment with CFTR correctors. In addition, in silico molecular docking calculations were performed to test the potential interaction of CFTR correctors with ABCB4.Results: We observed that the correctors C10, C13, and C17, as well as the combinations of C3 + C18 and C4 + C18, allowed the rescue of maturation and canalicular localization of four distinct traffic-defective ABCB4 variants. However, such treatments did not permit a rescue of the phosphatidylcholine secretion activity of these defective variants and were also inhibitory of the activity of wild type ABCB4. In silico molecular docking analyses suggest that these CFTR correctors might directly interact with transmembrane domains and/or ATP-binding sites of the transporter.Conclusion: Our results illustrate the uncoupling between the traffic and the activity of ABCB4 because the same molecules can rescue the traffic of defective variants while they inhibit the secretion activity of the transporter. We expect that this study will help to design new pharmacological tools with potential clinical interest.

International audience; The ATP-binding cassette (ABC) transporters expressed at the canalicular membrane of hepatocytes mediate the secretion of several compounds into the bile canaliculi and therefore play a key role in bile secretion. Among these transporters, ABCB11 secretes bile acids, ABCB4 translocates phosphatidylcholine and ABCG5/G8 is responsible for cholesterol secretion, while ABCB1 and ABCC2 transport a variety of drugs and other compounds. The dysfunction of these transporters leads to severe, rare, evolutionary biliary diseases. The development of new therapies for patients with these diseases requires a deep understanding of the biology of these transporters. In this review, we report the current knowledge regarding the regulation of canalicular ABC transporters' folding, trafficking, membrane stability and function, and we highlight the role of molecular partners in these regulating mechanisms.

This study reports the prevalence of sporeforming bacteria and contamination levels of a variety of powdered dairy products from cows', ewes', goats' and mares’ milk produced in France. The concentration of mesophilic spores, thermophilic spores and highly heat-resistant thermophilic spores was assessed in 61 dairy powders. Thermophilic spore concentration was highly variable between powders, likely due to the different manufacturing processes used for transforming milk into dairy powders. The different stages of processing, particularly heat treatment close to 55 °C had a strong impact on selection of thermophilic bacteria contaminating dairy powders. For some products, thermophilic spore counts were as high as 5.89 log TSC g−1. Of the 313 thermophilic isolates selected, 93.3% belonged to the species Geobacillus stearothermophilus (43.5%), Bacillus licheniformis (30.0%) and Anoxybacillus flavithermus (19.8%). These results confirm the presence of the three majority species reported worldwide.

Introduction: Thermophilic sporeformers are present in raw milk at very low concentration and resist to pasteurisation applicated to destruct vegetative and pathogenic cells. Those spores can adhere to stainless steel due to their hydrophobicity and can form biofilms. Early stage biofilms are important because it can increase the matrix and the adhesion of other cells. Because of those biofilms, the three main species: Geobacillus stearothermophilus, Anoxybacillus flavithermus and Bacillus licheniformis can resists to Cleaning In Place (CIP) procedure, and contaminate a new process. Material and Methods: Early stage adhesion was conducted on stainless steel submerged by milk inoculated with a fresh culture of bacteria (G. stearothermophilus (N=15), A. flavithermus (N=32) and B. licheniformis (N=15)) for 6h of growth at 55°C under agitation. The ability of sporeformers to form biofilms under those conditions were measured by image analysis after a fluorescent coloration (acridine orange) and random photography. A coverage percentage was calculated by ImageJ ; and a positive threshold was set up at 5% of covering. The efficiency of CIP procedures were obtained after a caustic soda and nitric acid treatment during different duration and temperature of treatment. Tested biofilms were formed in milk during 12h at 55°C, in stainless steel microplates (96 wells) on the same species (3 strains for each) under agitation. Surviving spores were enumerated by the microcolony method. Results: Early stage adhesion shows that 62.5 % (N=20) of A. flavithermus strains can form biofilm within 6h, whereas only 6.7% (N=1) of G. stearothermophilus and 0% (N=0) of B. licheniformis biofilm in 6h at 55°C on submerged stainless steel. However, the maximum covering % on A. flavithermus was 35%; while on the only biofilm forming strain of G. stearothermophilus, this percentage reach 75%. Image analysis also shows biofilm structure from 2D to 3D. The presence and the resistance of spores to chemical cleaning was highly variable within strains. Nitric acid appears to be more effective than caustic soda against biofilms formed by vegetative cells and spores from these strains. Significance: Those results shows that strong biofilms are mainly composed of spore and are very resistant to CIP used in dairy industries. That is why a better understanding of control methods can lead to a finer and suitableness use of cleaning products.

International audience; Annexin A2 (AnxA2) is a calcium-and phospholipid-binding protein that plays roles in cellular processes involving membrane and cytoskeleton dynamics and is able to associate to several partner proteins. However, the principal molecular partners of AnxA2 are negatively charged phospholipids such as phosphatidylserine and phosphatidyl-inositol-(4,5)-phosphate. Herein we have studied different aspects of membrane lipid rearrangements induced by AnxA2 membrane binding. X-ray diffraction data revealed that AnxA2 has the property to stabilize lamellar structures and to block the formation of highly curved lipid phases (inverted hexagonal phase, HII). By using pyrene-labelled cholesterol and the environmental probe di-4-ANEPPDHQ, we observed that in model membranes, AnxA2 is able to modify both, cholesterol distribution and lipid compaction. In epithelial cells, we observed that AnxA2 localizes to membranes of different lipid order. The protein binding to membranes resulted in both, increases and/or decreases in membrane order depending on the cellular membrane regions. Overall, AnxA2 showed the capacity to modulate plasma membrane properties by inducing lipid redistribution that may lead to an increase in order or disorder of the membranes.

BACKGROUND & AIM The canalicular bile salt export pump (BSEP/ABCB11) of hepatocytes is the main adenosine triphosphate (ATP)-binding cassette (ABC) transporter responsible for bile acid secretion. Mutations in ABCB11 cause several cholestatic diseases, including progressive familial intrahepatic cholestasis type 2 (PFIC2) often lethal in absence of liver transplantation. We investigated in vitro the effect and potential rescue of a BSEP mutation by ivacaftor, a clinically approved cystic fibrosis transmembrane conductance regulator (CFTR/ABCC7) potentiator. METHODS The p.T463I mutation, identified in a PFIC2 patient and located in a highly conserved ABC transporter motif, was studied by 3D structure modelling. The mutation was reproduced in a plasmid encoding a rat Bsep-green fluorescent protein. After transfection, mutant expression was studied in Can 10 cells. Taurocholate transport activity and ivacaftor effect were studied in Madin-Darby canine kidney (MDCK) clones co-expressing the rat sodium-taurocholate co-transporting polypeptide (Ntcp/Slc10A1). RESULTS As the wild-type protein, BsepT463I was normally targeted to the canalicular membrane of Can 10 cells. As predicted by 3D structure modelling, taurocholate transport activity was dramatically low in MDCK clones expressing BsepT463I . Ivacaftor treatment increased by 1.7-fold taurocholate transport activity of BsepT463I (P

Adenosine triphosphate binding cassette transporter, subfamily B member 4 (ABCB4) is the transporter of phosphatidylcholine at the canalicular membrane of hepatocytes. ABCB4 deficiency, due to genetic variations, is responsible for progressive familial intrahepatic cholestasis type 3 (PFIC3) and other rare biliary diseases. Roscovitine is a molecule in clinical trial that was shown to correct the F508del variant of cystic fibrosis transmembrane conductance regulator (CFTR), another ABC transporter. In the present study, we hypothesized that roscovitine could act as a corrector of ABCB4 traffic-defective variants. Using HEK and HepG2 cells, we showed that roscovitine corrected the traffic and localisation at the plasma membrane of ABCB4-I541F, a prototypical intracellularly retained variant. However, roscovitine caused cytotoxicity, which urged us to synthesize non-toxic structural analogues. Roscovitine analogues were able to correct the intracellular traffic of ABCB4-I541F in HepG2 cells. Importantly, the phospholipid secretion activity of this variant was substantially rescued by three analogues (MRT2-235, MRT2-237 and MRT2-243) in HEK cells. We showed that these analogues also triggered the rescue of intracellular traffic and function of two other intracellularly retained ABCB4 variants, i.e. I490T and L556R. Our results indicate that structural analogues of roscovitine can rescue genetic variations altering the intracellular traffic of ABCB4 and should be considered as therapeutic means for severe biliary diseases caused by this class of variations.

ABCB4 (MDR3) is an ATP-binding cassette (ABC) transporter expressed at the canalicular membrane of hepatocytes where it mediates phosphatidylcholine (PC) secretion. Variations in the ABCB4 gene are responsible for several biliary diseases, including progressive familial intrahepatic cholestasis type 3 (PFIC3), a rare disease that can be lethal in the absence of liver transplantation. In this study, we investigated the effect and potential rescue of ABCB4 missense variations that reside in the highly conserved motifs of ABC transporters, involved in ATP binding. Five disease-causing variations in these motifs have been identified in ABCB4 (G535D, G536R, S1076C, S1176L and G1178S), three of which are homologous to the gating mutations of cystic fibrosis transmembrane conductance regulator (CFTR or ABCC7), (i.e. G551D, S1251N and G1349D), that were previously shown to be function-defective and corrected by ivacaftor (VX-770, Kalydeco®), a clinically approved CFTR potentiator. Three-dimension structural modeling predicted that all five ABCB4 variants would disrupt critical interactions in the binding of ATP and thereby impair ATP-induced nucleotide-binding domains (NBDs) dimerization and ABCB4 function. This prediction was confirmed by expression in cell models, which showed that the ABCB4 mutants were normally processed and targeted to the plasma membrane, whereas their PC secretion activity was dramatically decreased. As also hypothesized on the basis of molecular modeling, PC secretion activity of the mutants was rescued by the CFTR potentiator ivacaftor (VX-770). Conclusion: Disease-causing variations in the ATP-binding sites of ABCB4 cause defects in PC secretion, which can be rescued by ivacaftor. These results provide the first experimental evidence that ivacaftor is a potential therapy for selected patients who harbor mutations in the ATP-binding sites of ABCB4. This article is protected by copyright. All rights reserved.

Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare liver disease characterized by early onset of cholestasis that leads to cirrhosis and liver failure before adulthood. PFIC3 may be improved by chronic administration of ursodeoxycholic acid, although in many cases liver transplantation is the only therapy. The disease is caused by mutations of the adenosine triphosphate (ATP)–binding cassette, sub-family B, member 4 (ABCB4) [multidrug resistance 3 (MDR3)] gene encoding a specific hepatocellular canalicular transporter involved in biliary phosphatidylcholine secretion. Several mutations have been reported; however, the effect of individual mutations has not been investigated. ABCB4 is highly homologous to ATP-binding cassette, sub-family B, member 1 (ABCB1) (MDR1), the multidrug transporter responsible for drug resistance of cancer cells. We have studied the effect of mutation I541F localized to the first nucleotide-binding domain, which is highly conserved between ABCB4 and ABCB1. Plasmids encoding the wild-type human ABCB4 or rat ABCB1–green fluorescing protein (GFP) construct, and corresponding I541F-mutants, were expressed in hepatocellular carcinoma, human (HepG2) and Madin-Darby canine kidney (MDCK) cells. Expression studies showed that ABCB4 was localized at the bile canalicular membrane in HepG2 cells and at the apical surface in MDCK cells, whereas the I541F mutant was intracellular. In MDCK cells, ABCB1-I541F also accumulated intracellularly in compartments, which were identified as the endoplasmic reticulum and cis-Golgi, and remained partially endoH-sensitive. After shifting cells to 27°C, ABCB1-I541F was expressed at the apical cell surface in a mature and active form. Similarly, ABCB4 was significantly trafficked to the membrane of bile canaliculi in HepG2 cells. Conclusion: Mutation I541F causes mislocalization of both ABCB4 and ABCB1. Intracellular retention of ABCB4-I541F can explain the disease in PFIC3 patients bearing this mutation. The observation that plasma membrane expression and activity can be rescued by low temperature opens perspectives to develop novel therapies for the treatment of PFIC3. (HEPATOLOGY 2009.)

International audience; Progressive familial intrahepatic cholestasis type 3 is caused by biallelic variations of ABCB4, most often (≥70%) missense. In this study, we examined the effects of 12 missense variations identified in progressive familial intrahepatic cholestasis type 3 patients. We classified these variations on the basis of the defects thus identified and explored potential rescue of trafficking-defective mutants by pharmacological means. Variations were reproduced in the ABCB4 complementary DNA and the mutants, thus obtained, expressed in HepG2 and HEK293 cells. Three mutants were either fully (I541F and L556R) or largely (Q855L) retained in the endoplasmic reticulum, in an immature form. Rescue of the defect, i.e., increase in the mature form at the bile canaliculi, was obtained by cell treatments with cyclosporin A or C and, to a lesser extent, B, D, or H. Five mutations with little or no effect on ABCB4 expression at the bile canaliculi caused a decrease (F357L, T775M, and G954S) or almost absence (S346I and P726L) of phosphatidylcholine secretion. Two mutants (T424A and N510S) were normally processed and expressed at the bile canaliculi, but their stability was reduced. We found no defect of the T175A mutant or of R652G, previously described as a polymorphism. In patients, the most severe phenotypes appreciated by the duration of transplant-free survival were caused by ABCB4 variants that were markedly retained in the endoplasmic reticulum and expressed in a homozygous status. Conclusion: ABCB4 variations can be classified as follows: nonsense variations (I) and, on the basis of current findings, missense variations that primarily affect the maturation (II), activity (III), or stability (IV) of the protein or have no detectable effect (V); this classification provides a strong basis for the development of genotype-based therapies. (Hepatology 2015)

During prion infections, the cellular glycosylphosphatidylinositol-anchored glycoprotein PrP is converted into a conformational isoform. This abnormal conformer is thought to recruit and convert the normal cellular PrP into a likeness of itself and is proposed to be the infectious agent. We investigated the distribution of the PrP protein on the surface of Rov cells, an epithelial cell line highly permissive to prion multiplication, and we found that PrP is primarily expressed on the apical side. We further show that prion transmission to Rov cells is much more efficient if infectivity contacts the apical side, indicating that the apical and basolateral sides of Rov cells are not equally competent for prion infection and adding prions to the list of the conventional infectious agents (viruses and bacteria) that infect epithelial cells in a polarized manner. These data raise the possibility that apically expressed PrP may be involved in this polarized process of infection. This would add further support for a crucial role of PrP at the cell surface in prion infection of target cells.

International audience; : The ABCB4 transporter mediates phosphatidylcholine secretion at the canalicular membrane of hepatocytes and its genetic defects cause biliary diseases. While ABCB4 shares high sequence identity with the multidrug transporter ABCB1, its N-terminal domain is poorly conserved, leading us to hypothesize a functional specificity of this domain. A database of ABCB4 genotyping in a large series of patients was screened for variations altering residues of the N-terminal domain. Identified variants were then expressed in cell models to investigate their biological consequences. Two missense variations, T34M and R47G, were identified in patients with low-phospholipid associated cholelithiasis or intrahepatic cholestasis of pregnancy. The T34M and R47G mutated proteins showed no or minor defect, respectively, in maturation and targeting to the apical membrane, in polarized Madin-Darby Canine Kidney and HepG2 cells, while their stability was similar to that of wild type ABCB4. By contrast, the phosphatidylcholine secretion activity of both mutants was markedly decreased. In silico analysis indicated that the identified variants were likely to affect ABCB4 phosphorylation. Mass spectrometry analyses confirmed that the N-terminal domain of wild type ABCB4 could undergo phosphorylation in vitro and revealed that the T34M and R47G mutations impaired such phosphorylation. ABCB4-mediated phosphatidylcholine secretion was also increased by pharmacological activation of protein kinases A or C and decreased by the inhibition of these kinases. Furthermore, the secretion activity of the T34M and R47G mutants was less responsive than that of wild type ABCB4 to protein kinase modulators. Conclusion: We identified disease-associated variants of ABCB4 involved in the phosphorylation of its N-terminal domain and leading to decreased phosphatidylcholine secretion. Our results also indicate that ABCB4 activity is regulated by phosphorylation, in particular of N-terminal residues. (Hepatology 2014;).

We investigated the signals involved in the apical targeting of dipeptidyl peptidase IV (DPP IV/CD26), an archetypal type II transmembrane glycoprotein. A secretory construct, corresponding to the DPP IV ectodomain, was first stably expressed in both the enterocytic-like cell line Caco-2 and the epithelial kidney MDCK cells. Most of the secretory form of the protein was delivered apically in MDCK cells, whereas secretion was 60% basolateral in Caco-2 cells, indicating that DPP IV ectodomain targeting is cell-type-dependent. A chimera (CTM–GFP) containing only the cytoplasmic and transmembrane domains of mouse DPP IV plus the green fluorescent protein was then studied. In both cell lines, this chimera was preferentially expressed at the apical membrane. By contrast, a secretory form of GFP was randomly secreted, indicating that GFP by itself does not contain cryptic targeting information. Comparison of the sequence of the transmembrane domain of DPP IV and several other apically targeted proteins does not show any consensus, suggesting that the apical targeting signal may be conformational. Neither the DPP IV nor the CTM–GFP chimera was enriched in lipid rafts. Together these results indicate that, besides the well-known raft-dependent apical targeting pathway, the fate of the CTM domain of DPP IV may reveal a new raft-independent apical pathway.

Western blotting with U937 cell extracts as the substrate, and enzyme-linked immunosorbent assays (ELISA) with U937-, Jurkat- and Daudi cell-purified CD45 molecules were used to detect anti-CD45 reactivity in patients with systemic lupus erythematosus (SLE). By immunoblotting, 16 of 64 SLE sera were shown to be positive (25.0%). In the ELISAs, 13 out of 18 SLE sera reacted with the target CD45. Of these, three were not detectable on the blot. Importantly, 12 of these ELISA-positive sera contained IgM and IgG auto-antibodies. Neuraminidase-treatment of U937-precipitated CD45 molecules enhanced the reactivity to most of the isoforms, indicating that the antibodies may bind to asialylated polysaccharides.

Summary Targeting of glycosyl-phosphatidylinositol (GPI)-anchored proteins (GPI-APs) in polarized epithelial cells depends on their association with detergent-resistant membrane microdomains called rafts. In MDCK cells, GPI-APs associate with rafts in the trans-Golgi network and are directly delivered to the apical membrane. It has been shown that oligomerization is required for their stabilization in rafts and their apical targeting. In hepatocytes, GPI-APs are first delivered to the basolateral membrane and secondarily reach the apical membrane by transcytosis. We investigated whether oligomerization is required for raft association and apical sorting of GPI-APs in polarized HepG2 cells, and at which step of the pathway oligomerization occurs. Model proteins were wild-type GFP-GPI and a double cysteine GFP-GPI mutant, in which GFP dimerization was impaired. Unlike wild-type GFP-GPI, which was efficiently endocytosed and transcytosed to the apical surface, the double cysteine mutant was basolaterally internalized, but massively accumulated in early endosomes, and reached the bile canaliculi with delayed kinetics. The double cysteine mutant was less resistant to Triton X-100 extraction, and formed fewer high molecular weight complexes. We conclude from these results that, in hepatocytes, oligomerization plays a key role in targeting GPI-APs to the apical membrane, by increasing their affinity for rafts and allowing their transcytosis.

The ATP-binding cassette transporter ABCB4 is a phosphatidylcholine translocator specifically expressed at the bile canalicular membrane in hepatocytes, highly homologous to the multidrug transporter ABCB1. Variations in the ABCB4 gene sequence cause progressive familial intrahepatic cholestasis type 3. We have shown previously that the I541F mutation, when reproduced either in ABCB1 or in ABCB4, led to retention in the endoplasmic reticulum (ER)/Golgi. Here, Madin-Darby canine kidney cells expressing ABCB1-GFP were used as a model to investigate this mutant. We show that ABCB1-I541F is not properly folded and is more susceptible to in situ protease degradation. It colocalizes and coprecipitates with the ER chaperone calnexin and coprecipitates with the cytosolic chaperone Hsc/Hsp70. Silencing of calnexin or overexpression of Hsp70 have no effect on maturation of the mutant. We also tested potential rescue by chemical and pharmacological chaperones. Thapsigargin and sodium 4-phenyl butyrate were inefficient. Glycerol improved maturation and exit of the mutant from the ER. Cyclosporin A, a competitive substrate for ABCB1, restored maturation, plasma membrane expression, and activity of ABCB1-I541F. Cyclosporin A also improved maturation of ABCB4-I541F in Madin-Darby canine kidney cells. In HepG(2) cells transfected with ABCB4-I541F cDNA, cyclosporin A allowed a significant amount of the mutant protein to reach the membrane of bile canaliculi. These results show that the best strategy to rescue conformation-defective ABCB4 mutants is provided by pharmacological chaperones that specifically target the protein. They identify cyclosporin A as a potential novel therapeutic tool for progressive familial intrahepatic cholestasis type 3 patients.

A commonly-used method for analysing raft membrane domains is based on their resistance to extraction by non-ionic detergents at 4 °C. However, the selectivity of different detergents in defining raft membrane domains has been questioned. We have compared the lipid composition of detergent-resistant membranes (DRMs) obtained after Triton X-100 or Lubrol WX extraction in MDCK cells in order to understand the differential effect of these detergents on membranes and their selectivity in solubilizing or not proteins. Both Lubrol and Triton DRMs were enriched with cholesterol over the lysate, thus exhibiting characteristics consistent with the properties of membrane rafts. However, the two DRM fractions differed considerably in the ratio between lipids of the inner and outer membrane leaflets. Lubrol DRMs were especially enriched with phosphatidylethanolamine, including polyunsaturated species with long fatty acyl chains. Lubrol and Triton DRMs also differed in the amount of raft transmembrane proteins and raft proteins anchored to the cytoplasmic leaflet. Our results suggest that the inner side of rafts is enriched with phosphatidylethanolamine and cholesterol, and is more solubilized by Triton X-100 than by Lubrol WX.

Background & Aims In non-alcoholic fatty liver disease (NAFLD), hepatocytes can undergo necroptosis: a regulated form of necrotic cell death mediated by the receptor-interacting protein kinase (RIPK) 1. Herein, we assessed the potential for RIPK1 and its downstream effector mixed lineage kinase domain-like protein (MLKL) to act as therapeutic targets and markers of activity in NAFLD. Methods C57/BL6J-mice were fed a normal chow diet or a high-fat diet (HFD). The effect of RIPA-56, a highly specific inhibitor of RIPK1, was evaluated in HFD-fed mice and in primary human steatotic hepatocytes. RIPK1 and MLKL concentrations were measured in the serum of patients with NAFLD. Results When used as either a prophylactic or curative treatment for HFD-fed mice, RIPA-56 caused a downregulation of MLKL and a reduction of liver injury, inflammation and fibrosis, characteristic of non-alcoholic steatohepatitis (NASH), as well as of steatosis. This latter effect was reproduced by treating primary human steatotic hepatocytes with RIPA-56 or necrosulfonamide, a specific inhibitor of human MLKL, and by knockout (KO) of Mlkl in fat-loaded AML-12 mouse hepatocytes. Mlkl-KO led to activation of mitochondrial respiration and an increase in β-oxidation in steatotic hepatocytes. Along with decreased MLKL activation, Ripk3-KO mice exhibited increased activities of the liver mitochondrial respiratory chain complexes in experimental NASH. In patients with NAFLD, serum concentrations of RIPK1 and MLKL increased in correlation with activity. Conclusion The inhibition of RIPK1 improves NASH features in HFD-fed mice and reverses steatosis via an MLKL-dependent mechanism that, at least partly, involves an increase in mitochondrial respiration. RIPK1 and MLKL are potential serum markers of activity and promising therapeutic targets in NAFLD. Lay summary There are currently no pharmacological treatment options for non-alcoholic fatty liver disease (NAFLD), which is now the most frequent liver disease. Necroptosis is a regulated process of cell death that can occur in hepatocytes during NAFLD. Herein, we show that RIPK1, a gatekeeper of the necroptosis pathway that is activated in NAFLD, can be inhibited by RIPA-56 to reduce not only liver injury, inflammation and fibrosis, but also steatosis in experimental models. These results highlight the potential of RIPK1 as a therapeutic target in NAFLD.

Glycosylation was considered the major signal candidate for apical targeting of transmembrane proteins in polarized epithelial cells. However, direct demonstration of the role of glycosylation has proved difficult because non-glycosylated apical transmembrane proteins usually do not reach the cell surface. Here we were able to follow the targeting of the apical transmembrane glycoprotein NPP3 both when glycosylated and non-glycosylated. Transfected in polarized MDCK and Caco-2 cells, NPP3 was exclusively expressed at the apical membrane. The transport kinetics of the protein to the cell surface were studied after metabolic (35)S-labeling and surface immunoprecipitation. The newly synthesized protein was mainly targeted directly to the apical surface in MDCK cells, whereas 50% transited through the basolateral surface in Caco-2 cells. In both cell types, the basolaterally targeted pool was effectively transcytosed to the apical surface. In the presence of tunicamycin, NPP3 was not N-glycosylated. The non-glycosylated protein was partially retained intracellularly but the fraction that reached the cell surface was nevertheless predominantly targeted apically. However, transcytosis of the non-glycosylated protein was partially impaired in MDCK cells. These results provide direct evidence that glycosylation cannot be considered an apical targeting signal for NPP3, although glycosylation is necessary for correct trafficking of the protein to the cell surface.