van den IJssel Paul, Wheelock Robert, Prescott Alan, Russell Paul, Quinlan Roy A
School of Life Sciences, Medical Science Institute, The University, Dundee DD1 5EH, UK.
Exp Cell Res. 2003 Jul 15;287(2):249-61. doi: 10.1016/s0014-4827(03)00092-2.
In this study, the small heat shock protein (sHSP) chaperones, alpha B-crystallin and HSP27, are identified as nuclear speckle components in unstressed cells in tissue culture. This new finding suggests a constitutive function for these sHSP chaperones in the nucleus and suggests a new perspective on the cardiomyopathy-causing mutation for alpha B-crystallin that could involve transcriptional splicing effects. Both alpha B-crystallin and HSP27 were immunolocalised to nuclear speckles (interchromatin granule clusters). While alpha B-crystallin was preferentially localised to speckles as shown by colocalisation with non-snRNP, SC35, as well as the snRNP components Sm and U1A, HSP27 was also seen associated with the nucleolar compartment, indicating a subtle difference between these closely related sHSPs. Actinomycin D treatment caused the relocalisation of alpha B-crystallin along with Sm and SC35 to a smaller number of more distinct spots, suggesting a link between speckle localisation and the transcriptional status of the cells. We then examined several transformed, immortalised, and primary cells expressing endogenous alpha B-crystallin as well as some cells with ectopic alpha B-crystallin expression. All consistently showed alpha B-crystallin in nuclear speckles. The nuclear localisation of the sHSPs was also confirmed biochemically and 2D gel electrophoresis revealed that there was only one major nuclear alpha B-crystallin isoform. This suggested that phosphorylation was not required for nuclear localisation of alpha B-crystallin. This was confirmed by the transient transfection of HeLa cells with a phosphorylation-defective alpha B-crystallin. In contrast, the transfection of R120G alpha B-crystallin, the mutation that causes cardiomyopathy, inhibited the nuclear speckle localisation of alpha B-crystallin. These data suggest that the cardiomyopathy-causing mutation for alpha B-crystallin has nuclear as well as cytoplasmic consequences, suggesting an explanation for the difference in severity of the desmin and alpha B-crystallin transgenic models of their respective cardiomyopathies.
在本研究中，小热休克蛋白（sHSP）伴侣蛋白αB-晶状体蛋白和HSP27被鉴定为组织培养中未受应激细胞的核斑点成分。这一新发现表明这些sHSP伴侣蛋白在细胞核中具有组成性功能，并为αB-晶状体蛋白导致心肌病的突变提出了一个新视角，该突变可能涉及转录剪接效应。αB-晶状体蛋白和HSP27都通过免疫定位定位于核斑点（染色质间颗粒簇）。虽然αB-晶状体蛋白优先定位于斑点，这通过与非snRNP、SC35以及snRNP成分Sm和U1A的共定位得以显示，但HSP27也可见与核仁区室相关，表明这些密切相关的sHSP之间存在细微差异。放线菌素D处理导致αB-晶状体蛋白以及Sm和SC35重新定位于数量更少、更明显的斑点，这表明斑点定位与细胞的转录状态之间存在联系。然后我们检查了几种表达内源性αB-晶状体蛋白的转化细胞、永生化细胞和原代细胞，以及一些异位表达αB-晶状体蛋白的细胞。所有细胞均一致显示αB-晶状体蛋白存在于核斑点中。sHSP的核定位也通过生化方法得以证实，二维凝胶电泳显示只有一种主要的核αB-晶状体蛋白同工型。这表明αB-晶状体蛋白的核定位不需要磷酸化。用磷酸化缺陷型αB-晶状体蛋白瞬时转染HeLa细胞证实了这一点。相反，导致心肌病的R120GαB-晶状体蛋白突变转染抑制了αB-晶状体蛋白的核斑点定位。这些数据表明，αB-晶状体蛋白导致心肌病的突变在细胞核和细胞质中均有后果，这为结蛋白和αB-晶状体蛋白各自心肌病转基因模型严重程度差异提供了解释。
