Department of Chemical and Systems Biology, Stanford University, Stanford, California, United States of America.
PLoS One. 2012;7(9):e43297. doi: 10.1371/journal.pone.0043297. Epub 2012 Sep 12.
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.
在活细胞中调节蛋白质水平的能力对于理解蛋白质功能至关重要。为了推进蛋白质干扰的工具集，我们开发了一种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。这种失稳和随后的降解可以通过添加小分子 Shield-1 以可逆和剂量依赖的方式进行挽救，小分子 Shield-1 特异性针对 DD。当靶向细胞区室（如线粒体基质或内质网（ER））时，蛋白质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影响。在这里，我们表明，细胞质或核中的 DD 融合可以在哺乳动物细胞中有效降解；然而，将融合靶向到线粒体基质或 ER 腔中会导致即使没有 Shield-1 也会积累。此外，我们还描述了 DD 与调节蛋白质产生、降解和局部蛋白质 QC 机制的扰动剂的行为。内质网中未折叠蛋白反应的化学诱导导致靶向 ER 的 DD 水平降低，表明 DD 对降解环境敏感。这些数据不仅强化了 DD 是一种有效的蛋白质干扰工具，还表明局部 QC 机制会影响 DD 的水平，并提示 DD 可能是监测蛋白质质量控制机制的有用探针。
