Comprehensive analysis of expression and function of 51 sarco(endo) plasmic reticulum Ca2+-ATPase mutants associated with Darier disease
Comprehensive analysis of expression and function of 51 sarco(endo) plasmic reticulum Ca2+-ATPase mutants associated with Darier disease
复制标题
DOI：
10.1074/jbc.m601966200
复制
发表时间：
2006-08-11
影响因子：
4.8
通讯作者：
Iizuka, Hajime
中科院分区：
文献类型：
--
作者：
Miyauchi, Yuki;Daiho, Takashi;Iizuka, Hajime
我们检测了Darier病（DD）家系中发现的肌浆网Ca ~（2+）-ATP酶2b（SERCA 2b）的51个突变，即迄今为止报道的大多数残基替换和缺失突变。用各突变cDNA转染COS-1细胞，用从细胞制备的微粒体分析SERCA 2b蛋白的表达和功能，并与野生型进行比较。15个突变体表现出明显降低的表达。在36个突变体中，29个突变体的Ca ~（2+）-ATP酶活性完全消失或受到强烈抑制，7个突变体的Ca ~（2+）-ATP酶活性较高或正常。在上述7个突变体中的4个中，Ca 2+转运活性显著降低或几乎完全丧失，因此与ATP水解解偶联。另外三个是例外情况，因为它们在蛋白质表达和Ca 2+转运功能方面似乎是正常的，但是发现具有由三个突变改变的动力学性质的异常，这恰好在我们先前发现的三个DD谱系中（Sato，K.，Yamasaki，K.，Daiho，T.，Miyauchi，Y.，高桥，H.，Ishida-Yamamoto，A.，中村，S.，饭冢，H.，和Suzuki，H.（2004）J.Biol.Chem.279，35595 - 35603）。总的来说，我们的研究结果表明，在大多数情况下（48 51）DD突变导致严重破坏的Ca 2+稳态的蛋白质表达和/或运输功能的缺陷，因此DD，但即使是轻微的稳态干扰将导致疾病。我们的研究结果也提供了进一步的洞察SERCA的结构与功能的关系，并揭示了关键区域和残基的酶。
We examined possible defects of sarco(endo) plasmic reticulum Ca2+-ATPase 2b (SERCA2b) associated with its 51 mutations found in Darier disease (DD) pedigrees, i.e. most of the substitution and deletion mutations of residues reported so far. COS-1 cells were transfected with each of the mutant cDNAs, and the expression and function of the SERCA2b protein was analyzed with microsomes prepared from the cells and compared with those of the wild type. Fifteen mutants showed markedly reduced expression. Among the other 36, 29 mutants exhibited completely abolished or strongly inhibited Ca2+-ATPase activity, whereas the other seven possessed fairly high or normal ATPase activity. In four of the aforementioned seven mutants, Ca2+ transport activity was significantly reduced or almost completely lost, therefore uncoupled from ATP hydrolysis. The other three were exceptional cases as they were seemingly normal in protein expression and Ca2+ transport function, but were found to have abnormalities in the kinetic properties altered by the three mutations, which happened to be in the three DD pedigrees found by us previously (Sato, K., Yamasaki, K., Daiho, T., Miyauchi, Y., Takahashi, H., Ishida-Yamamoto, A., Nakamura, S., Iizuka, H., and Suzuki, H. (2004) J. Biol. Chem. 279, 35595 - 35603). Collectively, our results indicated that in most cases (48 of 51) DD mutations cause severe disruption of Ca2+ homeostasis by the defects in protein expression and/or transport function and hence DD, but even a slight disturbance of the homeostasis will result in the disease. Our results also provided further insight into the structure-function relationship of SERCAs and revealed critical regions and residues of the enzyme.