The chemical chaperone sodium 4-phenylbutyrate improves the secretion of the protein CA267T mutant in CHO-K1 cells trough the GRASP55 pathway
© Chollet et al. 2015
Received: 22 June 2015
Accepted: 1 October 2015
Published: 9 October 2015
Some inherited coagulation factor deficiencies are caused by intracellular retention or degradation of misfolded proteins, and chemical chaperones have been shown to reverse protein misfolding. The purpose of the present study was to investigate whether chemical chaperones may improve secretion of the protein CA267T (PCA267T) mutant in a cellular model. Using stably transfected Chinese hamster ovary cells (CHO-K1) expressing PCA267T we demonstrate that sodium 4-phenylbutyrate (PBA) increased the secretion of PCA267T by approximately 4-fold in comparison with untreated cells, and that this secretion seemed to follow an unconventional pathway via the Golgi reassembly stacking protein (GRASP55).
In order to determine whether chemical chaperones were able to restore secretion of the PCA267T mutant, we treated Chinese hamster ovary cells (CHO-K1) stably expressing PC wild type (PCwt) or PCA267T  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 PCA267T. Our data indicate that PBA can rescue the trafficking of this mutant PC via an unconventional pathway involving the Golgi reassembly stacking protein GRASP55.
Several diseases are caused by defective protein folding leading to excessive protein degradation by proteasomes or to aggregation of misfolded proteins inside or outside the cell .
In some inherited coagulation factor deficiencies, failure of the mutant protein to adopt its properly folded state has been demonstrated. Our group has previously described the protein CA267T (PCA267T) mutant in a patient with congenital PC deficiency and recurrent deep vein thrombosis . We found that this mutant, PCA267T, 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 .
Recently, several therapeutic strategies using small molecules able to correct or prevent misfolding of a protein, have been explored. Amongst them are chemical chaperones, which are low molecular mass compounds that effectively are able to inhibit the formation of misfolded structures and reverse the intracellular retention of misfolded proteins . Therefore, chemical chaperones could be effective in reversing misfolding of blood coagulation proteins.
Since our previous studies have shown a defective trafficking of the PC A267T mutant  we tested a possible effect of chemical chaperones on the secretion of this mutant protein. Thus, CHO-K1 cells stably expressing PCwt and the mutant PCA267T were treated with PBA at 1, 2, and 5 mM for 48 h, trimethylamine N-oxide (TMAO) at 25 and 50 mM for 24 h and taurourosdeoxycholic acid (TUDCA) at 0.2 and 0.5 mM for 16 h. Untreated cells were used as control. The concentrations of PC antigen in the culture medium were measured using the Zymutest Protein C kit and the total protein concentration of the cell lysates was measured by the BCA Protein Assay kit. PC antigen levels in culture medium were normalized to the total concentration of protein of the respective cell lysates. Three independent experiments were performed in triplicates. Results were tested for statistical significance using one-way ANOVA or Student’s t test. P values <0.05 were considered statistically significant. GraphPad Prism version 5 was used for statistical analysis.
Studies have shown that PBA can reverse the cellular mislocalization or misfolding of proteins. For example, PBA treatment increased the secretion of ER retained α1-ATZ in a cell and mouse model of mutant alpha 1-antitrypsin-Z (α1-ATZ) . Thus, for the PCA267T mutant, the positive effect of PBA on secretion of the mutant protein can probably be related to a stabilization of the protein conformation by PBA, which can facilitate the trafficking of the protein. This might be a general effect of PBA since the secretion of the PCwt was also found to be slightly increased.
In conclusion, we report here for the first time that PBA improved the secretion of the PCA267T mutant through an unconventional GRASP55 dependent way. Most likely, this is achieved by a process involving stabilization of the protein conformation, which would facilitate the intracellular trafficking of the mutant. Functional studies are required to verify the activity of the secreted PCA267T mutant and also to determine a possible beneficial therapeutic role of PBA in protein C deficiency and other coagulation factor deficiencies in which the genetic defect causes a misfolding of the protein.
MEC and GS designed the research, analyzed and interpreted the data. MEC wrote the manuscript and performed the experiments. ES performed the confocal microscopy study and participated in the interpretation of the results. GS, NI and PMS interpreted the data and edited the manuscript. PMS obtained funding. All authors read and approved the final manuscript.
The authors thank Lena Tjeldhorn for establishing the cell lines overexpressing protein C wild type or the protein C A267V mutant.
The study was supported by Grants from the South-Eastern Norway Regional Health Authority.
The authors declare they have no competing interests.
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- Tjeldhorn L, Iversen N, Sandvig K, Bergan J, Sandset PM, Skretting G. Protein C mutation (A267T) results in ER retention and unfolded protein response activation. PLoS One. 2011;6:e24009.PubMed CentralView ArticlePubMedGoogle Scholar
- Balch WE, Morimoto RI, Dillin A, Kelly JW. Adapting proteostasis for disease intervention. Science. 2008;319:916–9.View ArticlePubMedGoogle Scholar
- Tjeldhorn L, Sandset PM, Haugbro K, Skretting G. Hereditary protein C deficiency caused by the Ala267Thr mutation in the protein C gene is associated with symptomatic and asymptomatic venous thrombosis. Thromb Res. 2010;125:230–4.View ArticlePubMedGoogle Scholar
- Tjeldhorn L, Iversen N, Sandvig K, Bergan J, Sandset PM, Skretting G. Functional characterization of the protein C A267T mutation: evidence for impaired secretion due to defective intracellular transport. BMC Cell Biol. 2010;11:67.PubMed CentralView ArticlePubMedGoogle Scholar
- Summers RJ, Meeks SL, Healey JF, Brown HC, Parker ET, Kempton CL, et al. Factor VIII A3 domain substitution N1922S results in hemophilia A due to domain-specific misfolding and hyposecretion of functional protein. Blood. 2011;117:3190–8.PubMed CentralView ArticlePubMedGoogle Scholar
- Chaudhuri TK, Paul S. Protein-misfolding diseases and chaperone-based therapeutic approaches. FEBS J. 2006;273:1331–49.View ArticlePubMedGoogle Scholar
- Burrows JA, Willis LK, Perlmutter DH. Chemical chaperones mediate increased secretion of mutant alpha 1-antitrypsin (alpha 1-AT) Z: a potential pharmacological strategy for prevention of liver injury and emphysema in alpha 1-AT deficiency. Proc Natl Acad Sci USA. 2000;97:1796–801.PubMed CentralView ArticlePubMedGoogle Scholar
- Zemskov EA, Mikhailenko I, Hsia RC, Zaritskaya L, Belkin AM. Unconventional secretion of tissue transglutaminase involves phospholipid-dependent delivery into recycling endosomes. PLoS One. 2011;6:e19414.PubMed CentralView ArticlePubMedGoogle Scholar
- Giuliani F, Grieve A, Rabouille C. Unconventional secretion: a stress on GRASP. Curr Opin Cell Biol. 2011;23:498–504.View ArticlePubMedGoogle Scholar
- Gee HY, Noh SH, Tang BL, Kim KH, Lee MG. Rescue of DeltaF508-CFTR trafficking via a GRASP-dependent unconventional secretion pathway. Cell. 2011;146:746–60.View ArticlePubMedGoogle Scholar
- Xiang Y, Zhang X, Nix DB, Katoh T, Aoki K, Tiemeyer M, et al. Regulation of protein glycosylation and sorting by the Golgi matrix proteins GRASP55/65. Nat Commun. 2013;4:1659–70.PubMed CentralView ArticlePubMedGoogle Scholar