Chaperone therapy for homocystinuria: the rescue of CBS mutations by heme arginate

Abstract

Classical homocystinuria is caused by mutations in the cystathionine β-synthase (CBS) gene. Previous experiments in bacterial and yeast cells showed that many mutant CBS enzymes misfold and that chemical chaperones enable proper folding of a number of mutations. In the present study, we tested the extent of misfolding of 27 CBS mutations previously tested in E. coli under the more folding-permissive conditions of mammalian CHO-K1 cells and the ability of chaperones to rescue the conformation of these mutations. Expression of mutations in mammalian cells increased the median activity 16-fold and the amount of tetramers 3.2-fold compared with expression in bacteria. Subsequently, we tested the responses of seven selected mutations to three compounds with chaperone-like activity. Aminooxyacetic acid and 4-phenylbutyric acid exhibited only a weak effect. In contrast, heme arginate substantially increased the formation of mutant CBS protein tetramers (up to sixfold) and rescued catalytic activity (up to ninefold) of five out of seven mutations (p.A114V, p.K102N, p.R125Q, p.R266K, and p.R369C). The greatest effect of heme arginate was observed for the mutation p.R125Q, which is non-responsive to in vivo treatment with vitamin B(6). Moreover, the heme responsiveness of the p.R125Q mutation was confirmed in fibroblasts derived from a patient homozygous for this genetic variant. Based on these data, we propose that a distinct group of heme-responsive CBS mutations may exist and that the heme pocket of CBS may become an important target for designing novel therapies for homocystinuria.

Mouse modeling and structural analysis of the p.G307S mutation in human cystathionine β-synthase (CBS) reveal effects on CBS activity but not stability

“…Previous work from our lab and others suggests that most missense CBS mutations affect protein stability and that treatment of these proteins with agents that promote proper folding (i.e. chaperones) can in many cases increase residual enzyme activity (21)(22)(23)(24). The process of protein folding may be thought of as an equilibrium between properly folded (functional) protein and unfolded or misfolded protein, and that many disease-associated missense mutations in CBS drive the equilibrium in favor of the misfolded protein.…”

“…In these systems, many mutant CBS proteins have been shown to be responsive to various treatments that alter the protein-folding environment, including the addition of chemical chaperones and the manipulation of cellular chaperone proteins (21)(22)(23)(24). However, cell based systems do not always reflect the true in vivo situation.…”

“…For example, the p.R266K and p.S466L mutations, show high levels of enzymatic activity in cell based heterologous expression systems, but cause massive protein instability when expressed in vivo in mouse liver (25,26). Recombinant human p.G307S CBS has been produced in several different heterologous expression systems, including E. coli, S. cerevisiae, and Chinese hamster ovary CHO-K1 cells (21,24,27). In almost all cases, the heterologous protein lacked enzyme activity.…”

Protein misfolding diseases: Prospects of pharmacological treatment

“…Classical homocystinuria is caused by mutations affecting the cystathionine beta‐synthase (CBS) protein. As 85% of these are of the missense type, it has been postulated that protein misfolding plays an important role in the pathogenesis of CBS deficiency . An important fraction of patients responds to pharmacological doses of pyridoxine, increasing hepatic CBS activity .…”

“…An important fraction of patients responds to pharmacological doses of pyridoxine, increasing hepatic CBS activity . Heme arginate also increases CBS residual activity and promotes proper enzyme assembly in vitro; although the clinical use of heme arginate may be limited by its cost and potential side effects . Recently, the first evidence has been reported for the use of S‐adenosylmethionine as a kinetic stabilizer for CBS, along with different molecules acting as chemical chaperones, such as betaine and taurine, Dimethyl sulfoxide (DMSO), glycerol, proline and Trimethylamine N‐oxide (TMAO) .…”