The molecular role of Sigmar1 in regulating mitochondrial function through mitochondrial localization in cardiomyocytes.
The molecular role of Sigmar1 in regulating mitochondrial function through mitochondrial localization in cardiomyocytes.
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DOI：
10.1016/j.mito.2021.12.002
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发表时间：
2022-01
期刊：
影响因子：
4.4
通讯作者：
Bhuiyan MS
中科院分区：
文献类型：
--
作者：
Abdullah CS;Aishwarya R;Alam S;Remex NS;Morshed M;Nitu S;Miriyala S;Panchatcharam M;Hartman B;King J;Alfrad Nobel Bhuiyan M;Traylor J;Kevil CG;Orr AW;Bhuiyan MS
Sigmar 1是在哺乳动物细胞系统中广泛表达的分子伴侣蛋白。越来越多的研究表明，在临床前啮齿动物心脏损伤模型中，配体激活药理学Sigmar 1具有心脏保护作用。大量的生物化学和免疫电镜研究表明，Sigmar 1的亚细胞定位在很大程度上取决于细胞和器官类型。尽管进行了全面的研究，Sigmar 1在心肌细胞中的直接分子作用仍然难以捉摸。在本研究中，我们使用心肌细胞中的互补显微镜、生化和功能测定来确定Sigmar 1的亚细胞定位、跨膜拓扑结构和功能。量子点在透射电镜下可见Sigmar 1标记的量子点分布于线粒体膜、溶酶体和肌浆网-线粒体界面。心脏细胞裂解物的亚细胞分级证实了Sigmar 1在纯化的线粒体组分和溶酶体组分中的定位。免疫细胞化学证实Sigmar 1与线粒体蛋白在分离的成年小鼠心肌细胞中共定位。Sigmar 1的线粒体定位进一步证实了Sigmar 1与Mito-Tracker在分离的小鼠心脏线粒体中的共定位。一系列的生化实验，包括碱提取和蛋白酶K处理纯化的心脏线粒体，证明Sigmar 1作为一个完整的线粒体膜蛋白。通过在细胞中表达FLAG标记的Sigmar 1片段来确定Sigmar 1对线粒体定位的结构要求。Sigmar 1全长和Sigmar 1的C端缺失片段能够定位于线粒体膜，而N端缺失片段不能掺入线粒体。最后，使用细胞外流量分析仪和高分辨率呼吸测定法的功能测定显示Sigmar 1 siRNA敲低显著改变了心肌细胞中的线粒体呼吸。总的来说，我们发现Sigmar 1定位于线粒体膜，是维持心肌细胞线粒体呼吸稳态不可或缺的。
Sigmar1 is a widely expressed molecular chaperone protein in mammalian cell systems. Accumulating research demonstrated the cardioprotective roles of pharmacologic Sigmar1 activation by ligands in preclinical rodent models of cardiac injury. Extensive biochemical and immuno-electron microscopic research demonstrated Sigmar1’s sub-cellular localization largely depends on cell and organ types. Despite comprehensive studies, Sigmar1’s direct molecular role in cardiomyocytes remains elusive. In the present study, we determined Sigmar1’s subcellular localization, transmembrane topology, and function using complementary microscopy, biochemical, and functional assays in cardiomyocytes. Quantum dots in transmission electron microscopy showed Sigmar1 labeled quantum dots on the mitochondrial membranes, lysosomes, and sarcoplasmic reticulum-mitochondrial interface. Subcellular fractionation of heart cell lysates confirmed Sigmar1’s localization in purified mitochondria fraction and lysosome fraction. Immunocytochemistry confirmed Sigmar1 colocalization with mitochondrial proteins in isolated adult mouse cardiomyocytes. Sigmar1’s mitochondrial localization was further confirmed by Sigmar1 colocalization with Mito-Tracker in isolated mouse heart mitochondria. A series of biochemical experiments, including alkaline extraction and proteinase K treatment of purified heart mitochondria, demonstrated Sigmar1 as an integral mitochondrial membrane protein. Sigmar1’s structural requirement for mitochondrial localization was determined by expressing FLAG-tagged Sigmar1 fragments in cells. Full-length Sigmar1 and Sigmar1’s C terminal-deletion fragments were able to localize to the mitochondrial membrane, whereas N- terminal deletion fragment was unable to incorporate into the mitochondria. Finally, functional assays using extracellular flux analyzer and high-resolution respirometry showed Sigmar1 siRNA knockdown significantly altered mitochondrial respiration in cardiomyocytes. Overall, we found that Sigmar1 localizes to mitochondrial membranes and is indispensable for maintaining mitochondrial respiratory homeostasis in cardiomyocytes.
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DOI：
10.1083/jcb.93.1.97
发表时间：
1982-04
期刊：
The Journal of cell biology
影响因子：
--
作者：
Fujiki Y;Hubbard AL;Fowler S;Lazarow PB
通讯作者：
Lazarow PB
DOI：
10.1126/science.1166127
发表时间：
2009-02-13
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Fontanilla D;Johannessen M;Hajipour AR;Cozzi NV;Jackson MB;Ruoho AE
通讯作者：
Ruoho AE
影响因子：
20.1
作者：
Ackers-Johnson M;Li PY;Holmes AP;O'Brien SM;Pavlovic D;Foo RS
通讯作者：
Foo RS
DOI：
10.1073/pnas.93.15.8072
发表时间：
1996-07-23
影响因子：
11.1
作者：
Hanner, M;Moebius, FF;Glossmann, H
通讯作者：
Glossmann, H
影响因子：
5.8
作者：
Bhuiyan, Md. Shenuarin;Fukunaga, Kohji
通讯作者：
Fukunaga, Kohji