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Structure and Function of the Region Near Phosphorylation Site of Sarcoplasmic Reticulum Calcium Pump

肌浆网钙泵磷酸化位点附近区域的结构与功能

基本信息

批准号：
10680576
负责人：
DAIHO Takashi
金额：
$ 2.05万
依托单位：
ASAHIKAWA MEDICAL COLLEGE
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

The author previously suggested that ArgィイD1198ィエD1 and HisィイD15ィエD1 of the NHィイD22ィエD2-terminal region in sarcoplasmic reticulum CaィイD12+ィエD1-ATPase (SERCA 1a) are located near the phosphorylation site.The functional role of ArgィイD1198ィエD1 was investigated by site-directed mutagenesis. The turnover rate of the mutant R198K was almost the same as that of the wild type, whereas the tumover rates of the mutants R198Q, R198A, and R198I were substantially lower than that of the wild type. The turnover rate was further reduced in the mutant R198E. The rate of dephosphorylation of the ADP-insensitive phosphoenzyme was reduced substantially in the mutant R198Q, more strongly in the mutants R198A and R198I and most strongly in the mutant R198E, but to a much lesser extent in the R198K. These results indicate that the positive charge and high hydrophilicity of ArgィイD1198ィエD1 are critical for rapid hydrolysis of the ADP-insensitive phosphoenzyme.Amino acid residues in the NHィイD22ィエD2-terrninal r … More egion (GluィイD12ィエD1-AlaィイD114ィエD1) of SERCA1a were deleted or substituted, and the mutants were expressed in COS-1 cells. Deletion of any single residue in the AlaィイD13ィエD1-SerィイD16ィエD1 region, deletion of two or more consecutive residues in the AlaィイD13ィエD1-ThrィイD19ィエD1 region, or substitution of A4K, A4D, and H5K caused strongly reduced expression. Deletion of any single residue in the AlaィイD13ィエD1-SerィイD16ィエD1 region caused only a small decrease in the specific CaィイD12+ィエD1. Transport rate, whereas other mutants showing low expression levels had greatly reduced specific CaィイD12+ィエD1 transport rates. Translation, transcription, and integration into the microsomal membranes were not impaired in the mutants which showed very low expression levels in COS-1 cells. Degradation of these mutants was substantially faster than that of the wild type. Lactacystin, a specific inhibitor of proteasome, inhibited the degradation accelerated by single-residue deletion of AlaィイD13ィエD1. These results suggest that the NHィイD22ィエD2-terminal region (AlaィイD13ィエD1-ThrィイD19ィエD1) of SERCA 1a is sensitive to the endoplasmic reticulum-mediated quality control and is thus critical for either correct folding of this protein or stabilization of the correctly folded SERCA 1a protein or both. Less

作者曾提出肌浆网Ca ~（2+）D_1-ATP酶（SERCA 1a）NH_（22）D_2端的Arg_（1198）D_1和His_（15）D_1位于磷酸化位点附近，本研究通过定点突变研究了Arg_（1198）D_1的功能作用。突变体R198 K的周转率与野生型几乎相同，而突变体R198 Q、R198 A和R198 I的周转率显著低于野生型。在突变体R198 E中，周转率进一步降低。ADP不敏感的磷酸酶的去磷酸化速率在突变体R198 Q中显著降低，在突变体R198 A和R198 I中更强烈，在突变体R198 E中最强烈，但在R198 K中程度要小得多。这些结果表明，精氨酸双酯酶D1198双酯酶D1的正电荷和高亲水性是ADP不敏感的磷酸酶快速水解的关键。 ...更多信息将SERCA 1a基因的Glu突变体D12突变体D1-Ala突变体D114突变体D1进行缺失或置换，并在COS-1细胞中表达。在Ala外显子D13外显子D1-Ser外显子D16外显子D1区域中缺失任何单个残基，在Ala外显子D13外显子D1-Thr外显子D19外显子D1区域中缺失两个或多个连续残基，或A4 K、A4 D和H5 K的取代引起表达强烈降低。在丙氨酸氨基转移酶D13氨基转移酶D1-丝氨酸氨基转移酶D16氨基转移酶D1区域中任何单个残基的缺失仅引起特异性Ca氨基转移酶D12+氨基转移酶D1的小幅度降低。转运速率，而其他表现出低表达水平的突变体大大降低了特异性Ca ~（2+）D12+ Ca ~（2+）D1转运速率。在COS-1细胞中表达水平非常低的突变体中，翻译、转录和整合到微粒体膜中没有受损。这些突变体的降解速度大大快于野生型。蛋白酶体特异性抑制剂Lactacystin可抑制Ala修饰的D13蛋白酶D1单残基缺失所加速的降解。这些结果表明，SERCA 1a的NH3-D22-D2-末端区域（Ala-D13-D1-Thr-D19-D1）对内质网介导的质量控制敏感，因此对于该蛋白的正确折叠或正确折叠的SERCA 1a蛋白的稳定或两者都是至关重要的。少