Cytoskeletal Drugs Modulate Off-Target Protein Folding Landscapes Inside Cells
Cytoskeletal Drugs Modulate Off-Target Protein Folding Landscapes Inside Cells
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DOI：
10.1021/acs.biochem.0c00299
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发表时间：
2020-07-21
期刊：
影响因子：
2.9
通讯作者：
Gruebele, Martin
中科院分区：
文献类型：
--
作者：
Davis, Caitlin M.;Gruebele, Martin
药物可以分解由微管和肌动蛋白细丝组成的动态细胞骨架网络。细胞骨架药物的工作原理是扰乱单体-聚合物的平衡，从而改变细胞内大分子聚集物的大小和数量。细胞骨架解体后的拥挤和非特异性表面相互作用(“粘着”)的变化可以直接或间接地影响蛋白质的稳定性、结构和功能，这是通过改变细胞质的流动性和改变细胞质中其他大分子的拥挤和粘着来实现的。细胞骨架分解对细胞内蛋白质能量格局的影响还有待观察。在这里，我们测量了几种细胞骨架药物对两种对大分子拥挤具有不同体外敏感性的FRET标记蛋白质折叠能量谱的影响。磷酸甘油酸激酶(PGK)以前被证明对拥挤更敏感，而可变主要蛋白样序列表达(VlsE)以前被证明对粘着更敏感。比较了解聚肌动蛋白细丝(细胞松弛素D和长春花素B)或微管(诺可达唑和长春花碱)的药物在细胞内的效果。拥挤感应器蛋白CrH2-FRET证实，细胞骨架药物减少了细胞内的拥挤程度，同时也减少了细胞的总体积。PGK致密性和折叠稳定性的降低可以通过这些药物引起的拥挤程度的减少来解释。VlsE对药物的相反反应表明，细胞骨架的解聚也改变了细胞环境中的粘连。我们的结果表明，单体-聚合物细胞骨架平衡的扰动，例如，在自然细胞迁移或药物治疗的压力下，对细胞中蛋白质的能量格局有非靶向的影响。
The dynamic cytoskeletal network of microtubules and actin filaments can be disassembled by drugs. Cytoskeletal drugs work by perturbing the monomer-polymer equilibrium, thus changing the size and number of macromolecular crowders inside cells. Changes in both crowding and nonspecific surface interactions ("sticking") following cytoskeleton disassembly can affect the protein stability, structure, and function directly or indirectly by changing the fluidity of the cytoplasm and altering the crowding and sticking of other macro-molecules in the cytoplasm. The effect of cytoskeleton disassembly on protein energy landscapes inside cells has yet to be observed. Here we have measured the effect of several cytoskeletal drugs on the folding energy landscape of two FRET-labeled proteins with different in vitro sensitivities to macromolecular crowding. Phosphoglycerate kinase (PGK) was previously shown to be more sensitive to crowding, whereas variable major protein-like sequence expressed (VlsE) was previously shown to be more sensitive to sticking. The in-cell effects of drugs that depolymerize either actin filaments (cytochalasin D and latrunculin B) or microtubules (nocodazole and vinblastine) were compared. The crowding sensor protein CrH2-FRET verified that cytoskeletal drugs decrease the extent of crowding inside cells despite also reducing the overall cell volume. The decreased compactness and folding stability of PGK could be explained by the decreased extent of crowding induced by these drugs. VlsE's opposite response to the drugs shows that depolymerization of the cytoskeleton also changes sticking in the cellular milieu. Our results demonstrate that perturbation of the monomer-polymer cytoskeletal equilibrium, for example, during natural cell migration or stresses from drug treatment, has off-target effects on the energy landscapes of proteins in the cell.