Characterization of the Effect of the Mitochondrial Protein Hint2 on Intracellular Ca2+ dynamics

Hint2, one of the five members of the superfamily of the histidine triad AMP-lysine hydrolase proteins, is expressed in mitochondria of various cell types. In human adrenocarcinoma cells, Hint2 modulates Ca2+ handling by mitochondria. As Hint2 is highly expressed in hepatocytes, we investigated if this protein affects Ca2+ dynamics in this cell type. We found that in hepatocytes isolated from Hint2−/− mice, the frequency of Ca2+ oscillations induced by 1 μM noradrenaline was 150% higher than in the wild-type. Using spectrophotometry, we analyzed the rates of Ca2+ pumping in suspensions of mitochondria prepared from hepatocytes of either wild-type or Hint2−/− mice; we found that Hint2 accelerates Ca2+ pumping into mitochondria. We then resorted to computational modeling to elucidate the possible molecular target of Hint2 that could explain both observations. On the basis of a detailed model for mitochondrial metabolism proposed in another study, we identified the respiratory chain as the most probable target of Hint2. We then used the model to predict that the absence of Hint2 leads to a premature opening of the mitochondrial permeability transition pore in response to repetitive additions of Ca2+ in suspensions of mitochondria. This prediction was then confirmed experimentally

Investigation of the role of sigma1-receptors in inositol 1,4,5-trisphosphate dependent calcium signaling in hepatocytes.

In hepatocytes, as in other cell types, Ca(2+) signaling is subject to complex regulations, which result largely from the intrinsic characteristics of the different inositol 1,4,5-trisphosphate receptor (InsP(3)R) isoforms and from their interactions with other proteins. Although sigma1 receptors (Sig-1Rs) are widely expressed in the liver, their involvement in hepatic Ca(2+) signaling remains unknown. We here report that in this cell type Sig-1R interact with type 1 isoforms of the InsP(3) receptors (InsP(3)R-1). These results obtained by immunoprecipitation experiments are confirmed by the observation that Sig-1R proteins and InsP(3)R-1 colocalize in hepatocytes. However, Sig-1R ligands have no effect on InsP(3)-induced Ca(2+) release in hepatocytes. This can be explained by the rather low expression level expression of InsP(3)R-1. In contrast, we find that Sig-1R ligands can inhibit agonist-induced Ca(2+) signaling via an inhibitory effect on InsP(3) synthesis. We show that this inhibition is due to the stimulation of PKC activity by Sig-1R, resulting in the well-known down-regulation of the signaling pathway responsible for the transduction of the extracellular stimulus into InsP(3) synthesis. The PKC sensitive to Sig-1R activity belongs to the family of conventional PKC, but the precise molecular mechanism of this regulation remains to be elucidated.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Successful Treatment with Rituximab and Immunoadsorption for an Auto-Antibody Induced Bile Salt Export Pump Deficiency in a Liver Transplanted Patient

We present an 8 years old girl who was diagnosed at 6 months of age of Progressive Familial Intrahepatic Cholestasis type 2. Although liver transplantation (LT) was classically considered curative for these patients, cholestasis recurrence with normal gamma-glutamyl transpeptidase (GGT), mediated by anti-bile salt export pump (BSEP) antibodies after LT (auto-antibody Induced BSEP Deficiency, AIBD) has been recently reported. Our patient underwent LT at 14 months. During her evolution, patient presented three episodes of acute rejection. Seven years after the LT, the patient presented pruritus with cholestasis and elevation of liver enzymes with persistent normal GGT. Liver biopsy showed intrahepatic cholestasis and giant-cell transformation with very low BSEP activity. Auto-antibodies against BSEP were detected therefore an AIBD was diagnosed. She was treated with Rituximab and immunoadsorption with resolution of the AIBD. As a complication of the treatment she developed a pneumocystis infection successfully treated with corticoids, cotrimoxazol and anidulafungin

Pharmacological premature termination codon readthrough of ABCB11 in bile salt export pump deficiency: an in vitro study

International audienceBACKGROUND & AIMS: Progressive familial intrahepatic cholestasis type 2 (PFIC2) is a severe hepatocellular cholestasis due to biallelic mutations in ABCB11 encoding the canalicular bile salt export pump (BSEP). Nonsense mutations are responsible for the most severe phenotypes. The aim was to assess the ability of drugs to induce readthrough of six nonsense mutations (p.Y354X, p.R415X, p.R470X, p.R1057X, p.R1090X and p.E1302X) identified in PFIC2 patients. APPROACH & RESULTS: The ability of G418, gentamicin and PTC124 to induce readthrough was studied using a dual gene reporter system in NIH3T3 cells. The ability of gentamicin to induce readthrough and to lead to the expression of a full-length protein was studied in HEK293, HepG2 and Can 10 cells, using immunodetection assays. The function of the gentamicin-induced full-length protein was studied by measuring the [3 H[3H] ]-taurocholate transcellular transport in stable MDCK clones co-expressing Ntcp. Combinations of gentamicin and chaperone drugs (UDCA, 4-PB) were investigated. In NIH3T3, aminoglycosides significantly increased readthrough level of all mutations studied, while PTC124 only slightly increased readthrough of p.E1302X. Gentamicin, induced a readthrough of p.R415X, p.R470X, p.R1057X, p.R1090X in HEK293 cells. The resulting full-length proteins localized within the cytoplasm, except for BsepR1090X that was also detected at the plasma membrane of HEK293 and at the canalicular membrane of Can 10 and HepG2 cells. Additional treatment with 4-PB and UDCA significantly increased the canalicular proportion of full-length BsepR1090X protein in Can 10 cells. In MDCK clones, gentamicin induced a 40% increase of the BsepR1090X [3 H[3H] ] [3H] -taurocholate transport, which was further increased with additional 4-PB treatment. CONCLUSION: This study constitutes a proof of concept for readthrough therapy in selected PFIC2 patients with nonsense mutations