Intracellular context affects levels of a chemically dependent destabilizing domain.
Intracellular context affects levels of a chemically dependent destabilizing domain.
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DOI：
10.1371/journal.pone.0043297
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Wandless TJ
中科院分区：
文献类型：
--
作者：
Sellmyer MA;Chen LC;Egeler EL;Rakhit R;Wandless TJ
调节活细胞中蛋白质水平的能力对于理解蛋白质功能至关重要。为了推进蛋白质扰动的工具集，我们开发了一个蛋白质不稳定结构域（DD），可以将其不稳定性赋予感兴趣的融合蛋白。这种不稳定和随后的降解可以通过添加一种针对DD的小分子Shield-1以可逆和剂量依赖的方式恢复。当蛋白质靶向细胞室（如线粒体基质或内质网）时，会遇到不同的局部蛋白质质量控制（QC）机制。这些不同的环境可能对细胞质来源的DD的水平和调节产生深远的影响。在这里，我们表明，在哺乳动物细胞中，细胞质或细胞核中的DD融合物可以有效地降解；然而，即使在没有Shield-1的情况下，靶向线粒体基质或内质网腔的融合也会导致积累。此外，我们用调节蛋白质生产、降解和局部蛋白质质量控制机制的扰动来表征DD的行为。内质网中未折叠蛋白反应的化学诱导导致内质网靶向DD水平降低，表明DD对降解环境的敏感性。这些数据强化了DD是蛋白质扰动的有效工具，表明当地QC机制影响DD的水平，并表明DD可能是监测蛋白质质量控制机制的有用探针。
The ability to regulate protein levels in live cells is crucial to understanding protein function. In the interest of advancing the tool set for protein perturbation, we developed a protein destabilizing domain (DD) that can confer its instability to a fused protein of interest. This destabilization and consequent degradation can be rescued in a reversible and dose-dependent manner with the addition of a small molecule that is specific for the DD, Shield-1. Proteins encounter different local protein quality control (QC) machinery when targeted to cellular compartments such as the mitochondrial matrix or endoplasmic reticulum (ER). These varied environments could have profound effects on the levels and regulation of the cytoplasmically derived DD. Here we show that DD fusions in the cytoplasm or nucleus can be efficiently degraded in mammalian cells; however, targeting fusions to the mitochondrial matrix or ER lumen leads to accumulation even in the absence of Shield-1. Additionally, we characterize the behavior of the DD with perturbants that modulate protein production, degradation, and local protein QC machinery. Chemical induction of the unfolded protein response in the ER results in decreased levels of an ER-targeted DD indicating the sensitivity of the DD to the degradation environment. These data reinforce that DD is an effective tool for protein perturbation, show that the local QC machinery affects levels of the DD, and suggest that the DD may be a useful probe for monitoring protein quality control machinery.
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