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

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.

Up‐regulation of golgi α‐mannosidase IA and down‐regulation of golgi α‐mannosidase IC activates unfolded protein response during hepatocarcinogenesis

“…Thus, we treated MAN1A1‐ overexpressing cells with ER stress inhibitors, including the chemical chaperone sodium 4‐phenylbutyrate (4‐PBA) and tauroursodeoxycholic acid (TUDCA), both of which ameliorate ER stress,35 to examine whether they can block or revert the expression of cell cycle‐ and migration‐related genes as well as the UPR regulators in the MAN1A1 ‐overexpressing cells. On treatment with the ER stress inhibitors 4‐PBA and TUDCA, the up‐regulation of these genes was reversed in Hep3B cells, PLC5 cells, HepG2 cells, and 293T cells (Supporting Fig.…”

Unfolding the therapeutic potential of chemical chaperones for age-related macular degeneration

“…Logically, then, interventions that shift the ER stress-UPR balance towards decreased ER stress might prove useful in the treatment of AMD. A variety of data have demonstrated that chemical and pharmacological chaperones can decrease signs of ER stress [31][32][33]. Taken together, this suggests a potential for such compounds in the treatment of AMD.…”

“…Both in vivo and in vitro studies of these diseases have elucidated the role of particular chaperone compounds in decreasing misfolded protein aggregation, enhancing proper trafficking of the protein(s) in question to target sites, and ameliorating the overall disease phenotype. The success of chaperones in other disorders, such as nephrogenic diabetes insipidus and Parkinson's disease, is encouraging [21,31,32,38,50]. However, to date, no published studies have examined the use of chemical chaperones as a treatment for AMD in humans or in AMD models.…”

“…De Almeida et al found that PBA given to transfected HFE C282Y cells decreased aggregates of the HFE C282Y mutant protein involved in hereditary hemochromatosis. It was hypothesized that PBA contributed to a more efficient disposal of the mutant by chemically enhancing the ER capacity to cope with the expression of misfolded HFE [32]. Recently, Yam et al tested the effect of PBA on myocilin aggregates.…”

Next-generation sequencing of hereditary hemochromatosis-related genes: Novel likely pathogenic variants found in the Portuguese population

“…In fact, at least seven missense mutations causing disease were reported at the HFE exon 4 (which codes for the α3 domain of the protein) including the major p.Cys282Tyr mutation, making this exon a hot spot for mutations. In the case of this mutation, the tyrosine for cysteine substitution disrupts the formation of the disulfide bond that physiologically occurs between Cys225 and Cys282 residues and that is essential for HFE association with β2-microglobulin [1,16,17]. Therefore, the p.Cys282Tyr-mutated HFE protein is unable to bind to the chaperone β2-microglobulin and to be transported to cell surface where it would interact with TfR1 and TfR2 in order to regulate hepcidin expression.…”