E3-ligase knock down revealed differential titin degradation by autopagy and the ubiquitin proteasome system.
E3-ligase knock down revealed differential titin degradation by autopagy and the ubiquitin proteasome system.
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e3连接酶敲低揭示了自噬和泛素蛋白酶体系统对titin的不同降解。
DOI：
10.1038/s41598-021-00618-7
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发表时间：
2021-10-26
影响因子：
4.6
通讯作者：
Kötter S
中科院分区：
文献类型：
--
作者：
Müller E;Salcan S;Bongardt S;Barbosa DM;Krüger M;Kötter S
肌节蛋白肌联是心肌细胞硬度和心室扩张性的主要决定因素。肌联蛋白的持续机械应力需要良好控制的蛋白质质量控制，其确切机制尚未完全阐明。在这里，我们分析了可能负责心脏肌联蛋白泛素化的 E3 连接酶，并专门研究了自噬体系统在肌联蛋白降解中的参与。对培养的原代大鼠心肌细胞中自噬和蛋白酶体的药理抑制显着提高了肌联蛋白泛素化并增加了肌联蛋白降解。使用体外 Pull Down 测定，我们鉴定了 E3 连接酶 MuRF1-3、CHIP 和 Fbx32 与几个 titin 结构域的结合。免疫荧光分析显示 E3 连接酶的肌节定位。 siRNA 介导的 E3 连接酶 MuRF-1、-3 敲低和 CHIP/Fbx32 组合显着降低了自噬相关肌联蛋白泛素化，而 MuRF-2 和 -3 的敲低则降低了蛋白酶体相关肌联蛋白泛素化。我们证明蛋白酶体和自噬体-溶酶体系统参与肌联丝的降解。我们发现肌联蛋白的泛素化和降解部分受到 MuRF 家族 E3 连接酶的调节。我们进一步鉴定出 CHIP 和 Fbx32 是参与肌联蛋白泛素化的 E3 连接酶。
The sarcomere protein titin is a major determinant of cardiomyocyte stiffness and ventricular distensibility. The constant mechanical stress on titin requires well-controlled protein quality control, the exact mechanisms of which have not yet been fully elucidated. Here, we analyzed E3-ligases potentially responsible for cardiac titin ubiquitination and specifically studied the involvement of the autophagosomal system in titin degradation. Pharmacological inhibition of autophagy and the proteasome in cultured primary rat cardiomyocytes significantly elevated titin ubiquitination and increased titin degradation. Using in-vitro pull down assays we identified binding of E3-ligases MuRF1-3, CHIP and Fbx32 to several titin domains. Immunofluorescence analysis showed sarcomeric localization of the E3-ligases. siRNA-mediated knock-down of the E3-ligases MuRF-1, -3 and a combination of CHIP/Fbx32 significantly reduced autophagy-related titin ubiquitination, whereas knock-down of MuRF-2 and -3 reduced proteasome-related titin ubiquitination. We demonstrated that the proteasomal and the autophagosomal-lysosomal system participate in degradation of the titin filament. We found that ubiquitination and degradation of titin are partially regulated by E3-ligases of the MuRF family. We further identified CHIP and Fbx32 as E3-ligases involved in titin ubiquitination.
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DOI：
10.1016/j.bbagrm.2010.09.001
发表时间：
2011-02
影响因子：
4.7
作者：
Li, Yi-Fan;Wang, Xuejun
通讯作者：
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影响因子：
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发表时间：
2007-03-01
影响因子：
1.9
作者：
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通讯作者：
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影响因子：
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Martin, Andreas