The chaperone‐like sodium phenylbutyrate improves factor IX intracellular trafficking and activity impaired by the frequent p.R294Q mutation

Abstract

Background Missense mutations often impair protein folding and intracellular processing, which can be improved by small compounds with chaperone-like activity. However, little has been done in coagulopathies, where even modest increases of functional levels could have therapeutic implications. Objectives To rescue the expression of factor IX (FIX) variants affected by missense mutations associated with type I hemophilia B (HB) through chaperone-like compounds. Methods Expression studies of recombinant (r)FIX variants and evaluation of secreted levels (ELISA), intracellular trafficking (immunofluorescence) and activity (coagulant assays) before and after treatment of cells with chaperone-like compounds. Results As a model we chose the most frequent HB mutation (p.R294Q, ~100 patients), compared with other recurrent mutations associated with severe/moderate type I HB. Immunofluorescence studies revealed retention of rFIX variants in the endoplasmic reticulum and negligible localization in the Golgi, thus indicating impaired intracellular trafficking. Consistently, and in agreement with coagulation phenotypes in patients, all missense mutations resulted in impaired secretion (< 1% wild-type rFIX). Sodium phenylbutyrate (NaPBA) quantitatively improved trafficking to the Golgi and dose dependently promoted secretion (from 0.3 ± 0.1% to 1.5 ± 0.3%) only of the rFIX-294Q variant. Noticeably, this variant displayed a specific coagulant activity that was higher (~2.0 fold) than that of wild-type rFIX in all treatment conditions. Importantly, coagulant activity was concurrently increased to levels (3.0 ± 0.9%) that, if achieved in patients, would ameliorate the bleeding phenotype. Conclusions Altogether, our data detail molecular mechanisms underlying type I HB and candidate NaPBA as affordable 'personalized' therapeutics for patients affected by the highly frequent p.R294Q mutation, and with reduced access to substitutive therapy.

Production and control of coagulation proteins for factor X activation in human endothelial cells and fibroblasts

“…Plots of the green intensity values (PDI or Golgi) and the red intensity values (coagulation proteins) at the same locations allowed a visual demonstration of signal colocalization. Similar methodology has been previously demonstrated as a measure of intracellular protein co-localization 66 . This same analysis was used to show negative co-localized detection of intracellular FIX with VWF in both LSECs and HUVECs.…”

The effect of the chemical chaperone 4-phenylbutyrate on secretion and activity of the p.Q160R missense variant of coagulation factor FVII

“…It was also tested in a short-term clinical trial as a therapeutic approach for cystic fibrosis in patients harbouring the ΔF508 variant of the cystic fibrosis transmembrane conductance regulator [26]. Interestingly, 4-PBA has been reported to increase the secretion of other coagulation factor variants such as FVIII-F309S [27], FIX-R294Q [17], and protein C-A267T [16]. Previously, it was demonstrated that 4-PBA could rescue various mutant proteins arrested in the ER allowing them to be expressed on the cell surface and to be functionally correct [28, 29].…”

“…Chemical chaperones are small compounds that can stabilize misfolded proteins and improve the protein-folding capacity of cells and thereby increase protein secretion [13, 14]. Despite their therapeutic potential, only a few studies have been performed on their effect on mutated coagulation proteins [15–17], and no studies have so far been performed on FVII. In the present study, we therefore examined the potential effects of chemical chaperones on trafficking and secretion of the FVII variant p.Q160R.…”

Molecular Mechanisms and Determinants of Innovative Correction Approaches in Coagulation Factor Deficiencies

“…Nonsense and missense mutations, which overall represent the most frequent gene alterations arising from point mutations, may affect protein synthesis at translational or post-translational levels or both. In general, nonsense mutations may result in the production of potentially unstable truncated proteins with loss-of-function features, whereas missense mutations may exert pleiotropic effects on protein biosynthesis/processing and/or secretion/function as well as affect pre-mRNA splicing when falling into exonic sequences, as exemplified by the several variants characterized in the coagulation field [105,110,127,128,129,130].…”

“…In the first model, cells expressing the A267T PC variant with defective intracellular transport [206] were treated with several compounds, but only Na-PBA resulted in a dose-dependent (up to ~3 fold) and significant increase in PC antigen levels [207]. In the second model, Na-PBA appreciably improved, in a dose-dependent manner, the intracellular trafficking and secretion of the most frequent FIX missense variant p.R294Q associated with proportionally low FIX activity and antigen levels [130]. In particular, secreted protein levels of the R294Q variant significantly increased (up to ~6 fold) upon treatment of stably-expressing cells with Na-PBA, a finding that was further confirmed at the intracellular level by the observation of improved trafficking to the Golgi compartment.…”