Restoring assembly and activity of cystathionine β‐synthase mutants by ligands and chemical chaperones

Abstract

Misfolding and aggregation of mutant enzymes have been proposed to play role in the pathogenesis of homocystinuria due to cystathionine β-synthase (CBS) deficiency. Chemical chaperones have been recently shown to facilitate proper assembly of several CBS mutants. To asses the number of patients that may respond to chaperone therapy, we examined the effect of selected CBS ligands and osmolytes on assembly and activity of 27 CBS mutants that represent 70% of known CBS alleles. The mutant enzymes were expressed in a bacterial system, and their properties were assessed by native Western blotting and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) assay, respectively. We studied the chaperoning activity of δ-aminolevulinic acid (δ-ALA)—a heme precursor—and of three osmolytes betaine, 2-aminoethanesulfonic acid (taurine), and glycerol. Fourteen mutants responded by at least 30% increase in the amount of correctly assembled tetramers and enzymatic activity to the coexpressional presence of either 0.5 mM δ-ALA, 100 mM betaine, and/or 750 mM glycerol. Eight of these mutants (p.R266K, p.P49L, p.R125Q, p.K102N, p.R369C, p.V180A, p.P78R, p.S466L) were rescuable by all of these three substances. Four mutants showed increased formation of tetramers that was not accompanied by changes in activity. Topology of mutations appeared to determine the chaperone responsiveness, as 11 of 14 solvent-exposed mutations were substantially more responsive than three of 13 buried mutations. This study identified chaperone-responsive mutants that represent 56 of 713 known patient-derived CBS alleles and may serve as a basis for exploring pharmacological approaches aimed at correcting misfolding in homocystinuria.Electronic supplementary materialThe online version of this article (doi:10.1007/s10545-010-9087-5) contains supplementary material, which is available to authorized users.

Surrogate Genetics and Metabolic Profiling for Characterization of Human Disease Alleles

“…Certain mutations in the heme-binding domain disrupt CBS function in human cells, indicating that heme is critical to protein activity (Janosik et al 2001b). Furthermore, heme increases the activity and dynamics of some CBS alleles (Kopecka et al 2011). Indeed, one of the mutations in our set, H65R, alters a heme-coordinating residue and was not functional in yeast.…”

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.…”

Гендерні Особливості Впливу Гіпергомоцистеїнемії На Метаболізм Сірковмісних Амінокислот Та Гідроген Сульфіду В Печінці

“…Виникає питання щодо факторів, які визна-чають статеві відмінності впливу тіолактонової [13,14]. В дослідженнях in vitro показано, що конкурент-ним інгібітором цистеїндіоксигенази (основного ензиму утилізації цистеїну) є висока концентра-ція гомоцистеїну [15].…”