由于胱硫醚β-合酶(CBS)缺乏引起的高半胱氨酸尿症(HCU)是硫氨基酸代谢最常见的先天性错误。最近的工作表明,无论其拓扑结构如何,错义致病突变都会导致C端调节域的不稳定性,这可能会转化为CBS的错误折叠,受损装配,功能的丧失。然而,目前尚不清楚调节域的不稳定性如何转化为细胞CBS更新以及哪些降解途径参与CBS蛋白抑制。这里,我们开发了一种基于人类HEK293的细胞模型,该模型缺乏内在CBS并稳定过表达野生型(WT)CBS或其10个最常见的错义HCU突变体。我们发现HCU突变体,除了I278T变体,表达与CBSWT相似或更好,其中一些显示低聚受损,活性和对变构激活剂S-腺苷甲硫氨酸的反应。所有HCU突变体的细胞稳定性,除了P49L和A114V,显著低于CBSWT的稳定性,表明他们的退化增加了。对CBSWT和两个代表性CBS突变体(T191M和I278T)的泛素化分析显示,蛋白酶体降解是CBS处置的主要途径,HCU突变体的溶酶体-自噬和内质网相关降解(ERAD)途径的轻微参与。蛋白酶体抑制显著增加T191M和I278TCBS突变体的半衰期和活性。溶酶体和ERAD抑制对CBS周转只有很小的影响,但是ERAD抑制拯救了T191M和I278TCBS突变体的活性与蛋白酶体抑制相似。总之,本研究为CBS在HCU中的蛋白质稳定提供了新的见解。
Homocystinuria (HCU) due to cystathionine beta-synthase (CBS) deficiency is the most common inborn error of sulfur amino acid metabolism. Recent work suggests that missense pathogenic mutations-regardless of their topology-cause instability of the C-terminal regulatory domain, which likely translates into CBS misfolding, impaired assembly, and loss of function. However, it is unknown how instability of the regulatory domain translates into cellular CBS turnover and which degradation pathways are involved in CBS proteostasis. Here, we developed a human HEK293-based cellular model lacking intrinsic CBS and stably overexpressing wild-type (WT) CBS or its 10 most common missense HCU mutants. We found that HCU mutants, except the I278T variant, expressed similarly or better than CBS WT, with some of them showing impaired oligomerization, activity and response to allosteric activator S-adenosylmethionine. Cellular stability of all HCU mutants, except P49L and A114V, was significantly lower than the stability of CBS WT, suggesting their increased degradation. Ubiquitination analysis of CBS WT and two representative CBS mutants (T191M and I278T) showed that proteasomal degradation is the major pathway for CBS disposal, with a minor involvement of lysosomal-autophagic and endoplasmic reticulum-associated degradation (ERAD) pathways for HCU mutants. Proteasomal inhibition significantly increased the half-life and activity of T191M and I278T CBS mutants. Lysosomal and ERAD inhibition had only a minor impact on CBS turnover, but ERAD inhibition rescued the activity of T191M and I278T CBS mutants similarly as proteasomal inhibition. In conclusion, the present study provides new insights into proteostasis of CBS in HCU.