Chemical Chaperones Reduce Endoplasmic Reticulum Stress and Prevent Mutant HFE Aggregate Formation

Sérgio F De Almeida,Gonçalo Picarote,John V Fleming,Maria Carmo-Fonseca,Jorge E Azevedo,Maria De Sousa

doi:10.1074/jbc.m702672200

Sérgio F De Almeida, Gonçalo Picarote + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m702672200

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Abstract

HFE C282Y, the mutant protein associated with hereditary hemochromatosis (HH), fails to acquire the correct conformation in the endoplasmic reticulum (ER) and is targeted for degradation. We have recently shown that an active unfolded protein response (UPR) is present in the cells of patients with HH. Now, by using HEK 293T cells, we demonstrate that the stability of HFE C282Y is influenced by the UPR signaling pathway that promotes its degradation. Treatment of HFE C282Y-expressing cells with tauroursodeoxycholic acid (TUDCA), a bile acid derivative with chaperone properties, or with the chemical chaperone sodium 4-phenylbutyrate (4PBA) impeded the UPR activation. However, although TUDCA led to an increased stability of the mutant protein, 4PBA contributed to a more efficient disposal of HFE C282Y to the degradation route. Fluorescence microscopy and biochemical analysis of the subcellular localization of HFE revealed that a major portion of the C282Y mutant protein forms intracellular aggregates. Although neither TUDCA nor 4PBA restored the correct folding and intracellular trafficking of HFE C282Y, 4PBA prevented its aggregation. These data suggest that TUDCA hampers the UPR activation by acting directly on its signal transduction pathway, whereas 4PBA suppresses ER stress by chemically enhancing the ER capacity to cope with the expression of misfolded HFE, facilitating its degradation. Together, these data shed light on the molecular mechanisms involved in HFE C282Y-related HH and open new perspectives on the use of orally active chemical chaperones as a therapeutic approach for HH.

Highlights

It has been known for some time that some compounds collectively called chemical chaperones have the ability to stabilize proteins in their native conformation contributing in some cases to rescue of the folding defect of mutant proteins (5)
Our goal in this study is to investigate the effect of the chemical chaperones tauroursodeoxycholic acid (TUDCA) and sodeoxycholic acid; 4PBA, 4-phenylbutyrate; ATF6, activating transcription factor-6; HA, hemagglutinin; GFP, green fluorescent protein; DMEM, Dulbecco’s modified Eagle’s medium; PBS, phosphate-buffered saline; Ab, antibody; mAb, monoclonal Ab; Endo H, endoglycosidase H; Heat shock proteins (HSPs), heat shock protein
We have recently shown that a unfolded protein response (UPR) is triggered following HFE C282Y expression (8)

Summary

Introduction

It has been known for some time that some compounds collectively called chemical chaperones have the ability to stabilize proteins in their native conformation contributing in some cases to rescue of the folding defect of mutant proteins (5). One well studied example of this mechanism is the restoration of the cell surface expression and function of the mutant cystic fibrosis transmembrane conductance regulator protein by chemical chaperones (6) It is thought, that these compounds may be effective in a number of other protein folding defects, providing an interesting therapeutic approach for a large number of different human diseases (7). We have recently reported that cells expressing HFE C282Y have an active unfolded protein response (UPR) (8) This specific ER stress response enhances the levels of molecular chaperones involved in protein folding and degradation and reduces the rate of protein synthesis (9). Investigation of the subcellular localization of HFE C282Y revealed that this misfolded protein forms aggregates and that 4PBA is effective in preventing their formation. These findings offer a potential new strategy for therapy designed to prevent the potential toxicity of the intracellular aggregates

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