Mutation of COOH-terminal lysines in overexpressed alpha B-crystallin abrogates ischemic protection in cardiomyocytes.
Mutation of COOH-terminal lysines in overexpressed alpha B-crystallin abrogates ischemic protection in cardiomyocytes.
复制标题
过度表达的 α B-晶状体蛋白中 COOH 末端赖氨酸的突变会消除心肌细胞的缺血保护。
DOI：
10.1152/ajpheart.00512.2001
复制
发表时间：
2002
期刊：
影响因子：
--
通讯作者：
Dillmann,WolfgangH
中科院分区：
文献类型：
--
作者：
Martin,JodyL;Bluhm,WolfgangF;He,Huaping;Mestril,Ruben;Dillmann,WolfgangH
高水平α b -晶体蛋白存在于心肌细胞中，但人们对这种蛋白的功能和重要性知之甚少。像许多其他小的热休克蛋白一样，α b -晶体蛋白形成大的寡聚复合物，其大小可以通过翻译后修饰来调节。这些复合物的大小可以改变蛋白质的功能。自然发生的cooh末端突变对眼睛晶状体有许多有害影响，并改变了寡聚化。因此，我们将α b -晶体蛋白的两个cooh末端赖氨酸突变为甘氨酸（K174/175G），并通过腺病毒载体转染到心肌细胞中。我们分析了这种突变对寡聚化、微管稳定和缺血结果的影响。通过天然PAGE和免疫印迹观察到，突变体的复合物大小下降了近45%。共聚焦分析表明，过表达蛋白不再保护微管蛋白细胞骨架免受缺血应激。与对照组相比，突变体引起缺血时胞质酶释放增加30%，而α b -晶体蛋白过表达减少33%。我们认为α b -晶体蛋白的COOH末端对其正常功能至关重要。
High levels of αB-crystallin are present in the cardiomyocyte, yet little is understood about the function and importance of this protein. Like many other small heat shock proteins, αB-crystallin forms large oligomeric complexes whose size can be regulated by posttranslational modifications. The size of these complexes can modify the function of the protein. A naturally occurring COOH-terminal mutant has many detrimental effects in the lens of the eye and altered oligomerization. Therefore, we mutated the two COOH-terminal lysines of αB-crystallin to glycines (K174/175G) and adenovirally mounted them to transduce cardiomyocytes. We analyzed the effect of this mutation on oligomerization, microtubular stabilization, and ischemic outcome. A nearly 45% downward shift in complex size was observed with the mutant by native PAGE followed by immunoblotting. The overexpressed protein no longer protected the tubulin cytoskeleton against ischemic stress by confocal analysis. The mutant caused a 30% increase in cytosolic enzyme release with ischemia compared with control, whereas a 33% decrease was associated with wild-type αB-crystallin overexpression. We conclude that the COOH terminus of αB-crystallin is crucial to its proper function.