In silico and in vivo models for Qatari-specific classical homocystinuria as basis for development of novel therapies.
In silico and in vivo models for Qatari-specific classical homocystinuria as basis for development of novel therapies.
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DOI：
10.1002/humu.23682
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发表时间：
2019-03
期刊：
影响因子：
3.9
通讯作者：
Nasrallah GK
中科院分区：
文献类型：
--
作者：
Ismail HM;Krishnamoorthy N;Al-Dewik N;Zayed H;Mohamed NA;Giacomo VD;Gupta S;Häberle J;Thöny B;Blom HJ;Kruger WD;Ben-Omran T;Nasrallah GK
同型半胱氨酸尿是一种罕见的先天性蛋氨酸代谢错误，由胱氨酸β-合成酶（CBS）缺乏引起。同型半胱氨酸尿在卡塔尔的患病率为1:18 00，主要是由于创始卡塔尔错义突变，c.1006C > T;p.R336C (p.Arg336Cys)。我们对p.R336C突变蛋白的结构-功能关系进行了表征，并利用三种模型（In silico、ΔCBS酵母和CRISPR/Cas9敲入HEK293T和HepG2细胞系）研究了不同的化学伙伴蛋白对恢复p.R336C- cbs活性的影响。蛋白质模型表明p.R336C诱导了严重的构象和结构变化，可能影响了CBS的活性。在互补实验中，野生型CBS，而不是p.R336C突变体，能够恢复ΔCBS缺陷酵母的生长。敲入HEK293T和HepG2细胞的p.R336C降低了CBS的表达水平，降低了其结构稳定性；然而，用甜菜碱（一种化学伴侣）处理p.R336C敲入的HEK293T细胞，恢复了CBS的稳定性和四聚体构象，但没有恢复其活性。综上所述，这些结果表明，p.R336C突变对CBS的结构、稳定性和活性具有有害影响，使用化学伴侣方法治疗可能对恢复p.R336C CBS活性无效。
Homocystinuria is a rare inborn error of methionine metabolism caused by cystathionine β-synthase (CBS) deficiency. The prevalence of homocystinuria in Qatar is 1:1,800 births, mainly due to a founder Qatari missense mutation, c.1006 C>T; p.R336C (p.Arg336Cys). We characterized the structure-function relationship of the p.R336C mutant protein, and investigated the effect of different chemical chaperones to restore p.R336C-CBS activity using three models: In silico, ΔCBS yeast, and CRISPR/Cas9 p.R336C knock-in HEK293T and HepG2 cell lines. Protein modeling suggested that the p.R336C induces severe conformational and structural changes, perhaps influencing CBS activity. Wildtype CBS, but not the p.R336C mutant, was able to restore the yeast growth in ΔCBS deficient yeast in a complementation assay. The p.R336C knock-in HEK293T and HepG2 cells decreased the level of CBS expression and reduce its structural stability; however, treatment of the p.R336C knock-in HEK293T cells with betaine, a chemical chaperone, restored the stability and tetrameric conformation of CBS, but not its activity. Collectively, these results indicate that the p.R336C mutation has a deleterious effect on CBS structure, stability, and activity, and using the chemical chaperones approach for treatment could be ineffective in restoring p.R336C CBS activity.
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影响因子：
5.5
作者：
Hess, Berk;Kutzner, Carsten;Lindahl, Erik
通讯作者：
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