A Therapeutic Chemical Chaperone Inhibits Cholera Intoxication and Unfolding/Translocation of the Cholera Toxin A1 Subunit

Cholera toxin (CT) travels as an intact AB5 protein toxin from the cell surface to the endoplasmic reticulum (ER) of an intoxicated cell. In the ER, the catalytic A1 subunit dissociates from the rest of the toxin. Translocation of CTA1 from the ER to the cytosol is then facilitated by the quality control mechanism of ER-associated degradation (ERAD). Thermal instability in the isolated CTA1 subunit generates an unfolded toxin conformation that acts as the trigger for ERAD-mediated translocation to the cytosol. In this work, we show by circular dichroism and fluorescence spectroscopy that exposure to 4-phenylbutyric acid (PBA) inhibited the thermal unfolding of CTA1. This, in turn, blocked the ER-to-cytosol export of CTA1 and productive intoxication of either cultured cells or rat ileal loops. In cell culture studies PBA did not affect CT trafficking to the ER, CTA1 dissociation from the holotoxin, or functioning of the ERAD system. PBA is currently used as a therapeutic agent to treat urea cycle disorders. Our data suggest PBA could also be used in a new application to prevent or possibly treat cholera

Expression of hereditary hemochromatosis C282Y HFE protein in HEK293 cells activates specific endoplasmic reticulum stress responses

<p>Abstract</p> <p>Background</p> <p>Hereditary Hemochromatosis (HH) is a genetic disease associated with iron overload, in which individuals homozygous for the mutant C282Y <it>HFE </it>associated allele are at risk for the development of a range of disorders particularly liver disease. Conformational diseases are a class of disorders associated with the expression of misfolded protein. HFE C282Y is a mutant protein that does not fold correctly and consequently is retained in the Endoplasmic Reticulum (ER). In this context, we sought to identify ER stress signals associated with mutant C282Y HFE protein expression, which may have a role in the molecular pathogenesis of HH.</p> <p>Results</p> <p>Vector constructs of Wild type HFE and Mutant C282Y HFE were made and transfected into HEK293 cell lines. We have shown that expression of C282Y HFE protein triggers both an unfolded protein response (UPR), as revealed by the increased GRP78, ATF6 and CHOP expression, and an ER overload response (EOR), as indicated by NF-κB activation. Furthermore, C282Y HFE protein induced apoptotic responses associated with activation of ER stress. Inhibition studies demonstrated that tauroursodeoxycholic acid, an endogenous bile acid, downregulates these events. Finally, we found that the co-existence of both C282Y HFE and Z alpha 1-antitrypsin protein (the protein associated with the liver disease of Z alpha 1-antitrypsin deficiency) expression on ER stress responses acted as potential disease modifiers with respect to each other.</p> <p>Conclusion</p> <p>Our novel observations suggest that both the ER overload response (EOR) and the unfolded protein response (UPR) are activated by mutant C282Y HFE protein.</p

Alpha-1-Antitrypsin in Pathogenesis of Hepatocellular Carcinoma

Context: Alpha-1-antitrypsin (A1AT) is the most abundant liver-derived, highly polymorphic, glycoprotein in plasma. Hereditary deficiency of alpha-1-antitrypsin in plasma (A1ATD) is a consequence of accumulation of polymers of A1AT mutants in endoplasmic reticulum of hepatocytes and other A1AT-producing cells. One of the clinical manifestations of A1ATD is liver disease in childhood and cirrhosis and/or hepatocellular carcinoma (HCC) in adulthood. Epidemiology and pathophysiology of liver failure in early childhood caused by A1ATD are well known, but the association with hepatocellular carcinoma is not clarified. The aim of this article is to review different aspects of association between A1AT variants and hepatocellular carcinoma, with emphasis on the epidemiology and molecular pathogenesis. The significance of A1AT as a biomarker in the diagnosis of HCC is also discussed. Evidence Acquisitions: Search for relevant articles were performed through Pub Med, HighWire, and Science Direct using the keywords "alpha-1-antitrypsin", "liver diseases", "hepatocellular carcinoma", "SERPINA1". Articles published until 2011 were reviewed. Results: Epidemiology studies revealed that severe A1ATD is a significant risk factor for cirrhosis and HCC unrelated to the presence of HBV or HCV infections. However, predisposition to HCC in moderate A1ATD is rare, and probably happens in combination with HBV and/or HCV infections or other unknown risk factors. It is assumed that accumulation of polymers of A1ATD variants in endoplasmic reticulum of hepatocytes leads to damage of hepatocytes by gain-of-function mechanism. Also, increased level of A1AT was recognized as diagnostic and prognostic marker of HCC. Conclusions: Clarification of a carcinogenic role for A1ATD and identification of pro-inflammatory or some still unknown factors that lead to increased susceptibility to HCC associated with A1ATD may contribute to a better understanding of hepatic carcinogenesis and to the development of new drugs

Protein quality control: the who’s who, the where’s and therapeutic escapes

In cells the quality of newly synthesized proteins is monitored in regard to proper folding and correct assembly in the early secretory pathway, the cytosol and the nucleoplasm. Proteins recognized as non-native in the ER will be removed and degraded by a process termed ERAD. ERAD of aberrant proteins is accompanied by various changes of cellular organelles and results in protein folding diseases. This review focuses on how the immunocytochemical labeling and electron microscopic analyses have helped to disclose the in situ subcellular distribution pattern of some of the key machinery proteins of the cellular protein quality control, the organelle changes due to the presence of misfolded proteins, and the efficiency of synthetic chaperones to rescue disease-causing trafficking defects of aberrant proteins