Singh Laishram R, Chen Xulin, Kozich Viktor, Kruger Warren D
Division of Population Science, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA.
Mol Genet Metab. 2007 Aug;91(4):335-42. doi: 10.1016/j.ymgme.2007.04.011. Epub 2007 May 30.
Missense mutations in the cystathionine beta-synthase (CBS) gene, such as I278T, are responsible for CBS deficiency, the most common inherited disorder in sulfur metabolism. Expression of human mutant CBS proteins in Saccharomyces cerevisiae reveals that most disease causing mutations severely inhibit enzyme activity and cannot support growth of yeast on cysteine-free media. Here, we show that the osmolyte chemical chaperones glycerol, trimethylamine-N-oxide, dimethylsulfoxide, proline or sorbitol, when added to yeast media, allows growth on cysteine-free media and causes increased enzyme activity from I278T and three other mutant CBS proteins. Rescuable mutants are ones that are predicted to cause a decrease in solvent accessible surface area. The increase in enzyme activity is associated with stabilization of the tetramer form of the enzyme. This effect is not specific to yeast, as addition of the chaperone glycerol resulted in increased I278T activity when the enzyme is produced either in Escherichia coli or in a coupled in vitro transcription/translation reaction. However, no stimulation of specific activity was observed when chaperones were added directly to purified I278T indicating that the presence of chemical chaperones is required during translation. We also found that by mixing different chaperones we could achieve rescue at significantly lower chaperone concentrations. Taken together, our data show that chemical chaperones present during the initial folding process can facilitate proper folding of several mutant CBS proteins and suggest it may be possible to treat some inborn errors of metabolism with agents that enhance proper protein folding.
胱硫醚β-合酶（CBS）基因中的错义突变，如I278T，是CBS缺乏症的病因，CBS缺乏症是硫代谢中最常见的遗传性疾病。在酿酒酵母中表达人类突变型CBS蛋白表明，大多数致病突变会严重抑制酶活性，并且无法支持酵母在无半胱氨酸培养基上生长。在此，我们表明，当向酵母培养基中添加渗透溶质化学伴侣甘油、三甲胺-N-氧化物、二甲基亚砜、脯氨酸或山梨醇时，酵母能够在无半胱氨酸培养基上生长，并使I278T及其他三种突变型CBS蛋白的酶活性增加。可挽救的突变体是那些预计会导致溶剂可及表面积减少的突变体。酶活性的增加与酶的四聚体形式的稳定有关。这种效应并非酵母所特有，因为当在大肠杆菌中或在体外转录/翻译偶联反应中产生该酶时，添加伴侣甘油会导致I278T活性增加。然而，当将伴侣直接添加到纯化的I278T中时，未观察到比活性的刺激，这表明在翻译过程中需要化学伴侣的存在。我们还发现，通过混合不同的伴侣，我们可以在显著更低的伴侣浓度下实现挽救。综上所述，我们的数据表明，在初始折叠过程中存在的化学伴侣可以促进几种突变型CBS蛋白的正确折叠，并表明有可能用增强蛋白质正确折叠的药物治疗一些先天性代谢缺陷。
