Protein C Mutation (A267T) Results in ER Retention and Unfolded Protein Response Activation

Abstract

BackgroundProtein C (PC) deficiency is associated with a high risk of venous thrombosis. Recently, we identified the PC-A267T mutation in a patient with PC deficiency and revealed by in vitro studies decreased intracellular and secreted levels of the mutant. The aim of the present study was to characterize the underlying mechanism(s).Methodology/Principal FindingsCHO-K1 cells stably expressing the wild-type (PC-wt) or the PC mutant were generated. In order to examine whether the PC mutant was subjected to increased intracellular degradation, the cells were treated with several inhibitors of various degradation pathways and pulse-chase experiments were performed. Protein-chaperone complexes were analyzed by treating the cells with a cross-linker followed by Western blotting (WB). Expression levels of the immunoglobulin-binding protein (BiP) and the phosphorylated eukaryotic initiation factor 2α (P-eIF2α), both common ER stress markers, were determined by WB to examine if the mutation induced ER stress and unfolded protein response (UPR) activation. We found no major differences in the intracellular degradation between the PC variants. The PC mutant was retained in the endoplasmic reticulum (ER) and had increased association with the Grp-94 and calreticulin chaperones. Retention of the PC-A267T in ER resulted in UPR activation demonstrated by increased expression levels of the ER stress markers BiP and P-eIF2α and caused also increased apoptotic activity in CHO-K1 cells as evidenced by elevated levels of DNA fragmentation.Conclusions/SignificanceThe reduced intracellular level and impaired secretion of the PC mutant were due to retention in ER. In contrast to other PC mutations, retention of the PC-A267T in ER resulted in minor increased proteasomal degradation, rather it induced ER stress, UPR activation and apoptosis.

Molecular Characterization of Two Homozygous Factor VII Variants Associated with Intracranial Bleeding

“…These misfolded proteins can accumulate inside the cells and lead to endoplasmic reticulum (ER) stress. We have previously reported induction of ER stress in cells expressing missense, microdeletion, or elongated FVII variants [16], and similar results are reported for other coagulation factors and inhibitors [17,18].…”

The chemical chaperone sodium 4-phenylbutyrate improves the secretion of the protein CA267T mutant in CHO-K1 cells trough the GRASP55 pathway

“…In order to determine whether chemical chaperones were able to restore secretion of the PC A267T mutant, we treated Chinese hamster ovary cells (CHO-K1) stably expressing PC wild type (PC wt ) or PC A267T [ 1 ] with 3 different compounds: Sodium 4-phenylbutyrate (PBA), trimethylamine N -oxide (TMAO) and taurourosdeoxycholic acid (TUDCA). While no effect was seen with TMAO or TUDCA, treatment with PBA for 48 h enhanced the secretion of PC A267T .…”

“…We found that this mutant, PC A267T , was retained in the endoplasmic reticulum (ER), most likely due to misfolding of the protein. This caused ER stress, activation of the unfolded protein response and apoptosis [ 1 , 4 ]. Moreover, in hemophilia A, a domain-specific misfolding in the FVIII A3 domain caused ER retention of the mutant FVIII with poor secretion of the functional protein [ 5 ].…”

Trp250‐hK2 is defective in intracellular trafficking and activates the unfolded protein response

“…; Tjeldhorn et al . ). Unlike these mutant proteins, SNP rs198977 is not a disease‐causing genetic polymorphism but only a risk factor of prostate cancer, which leads to the same biological result, namely a decrease in serum level of the mutant protein compared to wild type.…”