Mendes Marisa I S, Smith Desirée E C, Vicente João B, Tavares De Almeida Isabel, Ben-Omran Tawfeg, Salomons Gajja S, Rivera Isabel A, Leandro Paula, Blom Henk J
Metabolism and Genetics Group, Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Metabolic Unit, Department of Clinical Chemistry, VU University Medical Centre, Amsterdam 1081 HV, The Netherlands and.
Metabolic Unit, Department of Clinical Chemistry, VU University Medical Centre, Amsterdam 1081 HV, The Netherlands and.
Hum Mol Genet. 2015 Dec 20;24(25):7339-48. doi: 10.1093/hmg/ddv431. Epub 2015 Oct 12.
The key regulatory point of L-methionine (Met) and L-homocysteine (Hcy) degradation is catalyzed by cystathionine beta-synthase (CBS). CBS deficiency is caused by mutations in CBS gene, often resulting in protein misfolding. The prevalence of CBS deficiency in Qatar is 1/1800, ∼200-fold higher than the worldwide prevalence of 1/344 000. Almost all patients bear the CBS p.R336C variant. More than 20 years ago, it was shown in vitro that two unrelated protein variants with a substitution of an arginine (Arg) residue by cysteine (Cys) could be rescued by cysteamine (mercaptoethylamine), likely via formation of a disulfide between Cys and cysteamine, functionally mimicking the wild-type (WT) Arg side-chain. Based on these findings, we aimed to study whether cysteamine was able to improve the function of p.R336C CBS variant. Additionally, we tested the effect of mercaptoethylguanidine (MEG), a compound with a guanidino and a thiol function that may resemble Arg structure better than cysteamine. Three purified recombinant CBS proteins (p.R336C, p.R336H and WT) were pre-incubated with cysteamine, MEG or Cys (as negative control), and CBS activity and stability were measured. Pre-incubation with cysteamine and MEG increased the enzymatic activity of the p.R336C protein, which was absent upon pre-incubation with Cys. The WT and the p.R336H variant enzyme activity presented no increase with any of the tested compounds. Our results show that cysteamine and MEG are able to specifically improve the function of the CBS p.R336C variant, suggesting that any Arg-to-Cys substitution accessible to these small molecules may be converted back to a moiety resembling Arg.
L-蛋氨酸（Met）和L-同型半胱氨酸（Hcy）降解的关键调控点由胱硫醚β-合酶（CBS）催化。CBS缺乏症由CBS基因突变引起，常导致蛋白质错误折叠。卡塔尔CBS缺乏症的患病率为1/1800，比全球患病率1/344000高出约200倍。几乎所有患者都携带CBS p.R336C变体。20多年前，体外实验表明，两种不相关的蛋白质变体，其中精氨酸（Arg）残基被半胱氨酸（Cys）取代，可被半胱胺（巯基乙胺）挽救，可能是通过Cys与半胱胺之间形成二硫键，在功能上模拟野生型（WT）Arg侧链。基于这些发现，我们旨在研究半胱胺是否能够改善p.R336C CBS变体的功能。此外，我们测试了巯基乙基胍（MEG）的效果，MEG是一种具有胍基和硫醇功能的化合物，其可能比半胱胺更类似于Arg结构。三种纯化的重组CBS蛋白（p.R336C、p.R336H和WT）与半胱胺、MEG或Cys（作为阴性对照）进行预孵育，然后测量CBS活性和稳定性。与半胱胺和MEG预孵育可增加p.R336C蛋白的酶活性，而与Cys预孵育则无此活性。WT和p.R336H变体酶活性在任何测试化合物作用下均未增加。我们的结果表明，半胱胺和MEG能够特异性改善CBS p.R336C变体的功能，这表明这些小分子可作用的任何Arg-to-Cys取代都可能被转化回类似于Arg的部分。
