A Disease-causing Mutation in the Active Site of Serine Palmitoyltransferase Causes Catalytic Promiscuity
A Disease-causing Mutation in the Active Site of Serine Palmitoyltransferase Causes Catalytic Promiscuity
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DOI：
10.1074/jbc.m110.122259
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发表时间：
2010-07-23
影响因子：
4.8
通讯作者：
Dunn, Teresa M.
中科院分区：
文献类型：
--
作者：
Gable, Kenneth;Gupta, Sita D.;Dunn, Teresa M.
常染色体显性外周感觉神经病HSAN 1是由丝氨酸棕榈酰转移酶（SPT）的LCB 1亚基突变引起的。来自患者和表达致病突变（C133 W）的转基因小鼠的血清含有升高水平的1-脱氧鞘氨醇（1-deoxySa），这可能是由于丙氨酸与棕榈酰辅酶A的不适当缩合引起的。突变的异二聚体SPT是无催化活性的。然而，突变体异源三聚体SPT具有与野生型活性的10-20%相似的活性，并且支持缺乏内源性SPT的酵母细胞的生长。此外，长链碱基分析显示，在酵母和哺乳动物细胞中表达异源三聚体突变体酶的1-脱氧Sa的合成显著多于表达野生型酶的细胞。野生型和突变型酶对丝氨酸具有相似的亲和力。令人惊讶的是，这些酶对丙氨酸也具有相似的亲和力，表明C133 W突变的主要影响是增强丙氨酸与酰基辅酶A底物缩合的活化。在体内合成的1-deoxySa的突变体酶是成比例的丙氨酸丝氨酸的生长培养基中的比例，这表明该比例可用于调节鞘氨醇和1-deoxySa的相对合成。通过将SPT表达为单链融合蛋白以确保所有三个亚基的化学计量表达，我们发现GADD 153，内质网应激的标志物，在表达突变异源三聚体的细胞中显著升高。GADD 153在用1-deoxySa处理的细胞中也升高。总之，这些数据表明，HSAN 1突变扰乱SPT的活性位点，导致HSAN 1表型的功能获得。
The autosomal dominant peripheral sensory neuropathy HSAN1 results from mutations in the LCB1 subunit of serine palmitoyltransferase (SPT). Serum from patients and transgenic mice expressing a disease-causing mutation (C133W) contain elevated levels of 1-deoxysphinganine (1-deoxySa), which presumably arise from inappropriate condensation of alanine with palmitoyl-CoA. Mutant heterodimeric SPT is catalytically inactive. However, mutant heterotrimeric SPT has similar to 10-20% of wild-type activity and supports growth of yeast cells lacking endogenous SPT. In addition, long chain base profiling revealed the synthesis of significantly more 1-deoxySa in yeast and mammalian cells expressing the heterotrimeric mutant enzyme than in cells expressing wild-type enzyme. Wild-type and mutant enzymes had similar affinities for serine. Surprisingly, the enzymes also had similar affinities for alanine, indicating that the major affect of the C133W mutation is to enhance activation of alanine for condensation with the acyl-CoA substrate. In vivo synthesis of 1-deoxySa by the mutant enzyme was proportional to the ratio of alanine to serine in the growth media, suggesting that this ratio can be used to modulate the relative synthesis of sphinganine and 1-deoxySa. By expressing SPT as a single-chain fusion protein to ensure stoichiometric expression of all three subunits, we showed that GADD153, a marker for endoplasmic reticulum stress, was significantly elevated in cells expressing mutant heterotrimers. GADD153 was also elevated in cells treated with 1-deoxySa. Taken together, these data indicate that the HSAN1 mutations perturb the active site of SPT resulting in a gain of function that is responsible for the HSAN1 phenotype.