双分子荧光互补测定法评估细胞中 HSP90-客户蛋白相互作用。

Bimolecular Fluorescence Complementation Assay to Evaluate HSP90-Client Protein Interactions in Cells.

机构信息

Biomedical Biotechnology Research Unit (BioBRU), Department of Biochemistry and Microbiology, Rhodes University, Makhanda, South Africa.

Biomedical Research and Drug Discovery Research Group, Faculty of Health Sciences, Higher Colleges of Technology, Sharjah, United Arab Emirates.

出版信息

Methods Mol Biol. 2023;2693:95-103. doi: 10.1007/978-1-0716-3342-7_8.

DOI:10.1007/978-1-0716-3342-7_8

PMID:37540429

Abstract

Protein-protein interactions (PPI) in cells play a pivotal role in cellular function and dynamics. Cellular proteostasis is maintained by PPI networks between molecular chaperones, co-chaperones, and client proteins. Consequently, strategies to visualize and analyze PPI in cells are useful in understanding protein homeostasis regulation. The Bimolecular Fluorescence Complementation (BiFC) assay has emerged as a useful tool for studying PPI between proteins in live or fixed cells. BiFC is based on the detection of fluorescence generated when interacting protein pairs, produced as fusion proteins with either the N- or C-terminal fragment of a fluorescent protein, are in sufficient proximity to permit reconstitution of the split fluorophore. Here, we describe the application of the BiFC assay to a model of chaperone-client interactions using Hsp90 and the validated client protein CDK4. This assay allows for the distribution and spatiotemporal analysis of HSP90-CDK4 complexes in live or fixed cells and is amenable to studying the effects of inhibitors and mutations on chaperone-client protein networks.

摘要

细胞中的蛋白质-蛋白质相互作用（PPI）在细胞功能和动态中起着关键作用。分子伴侣、共伴侣和客户蛋白之间的 PPI 网络维持着细胞的蛋白质稳态。因此，可视化和分析细胞中 PPI 的策略有助于理解蛋白质动态平衡的调节。双分子荧光互补（BiFC）测定法已成为研究活细胞或固定细胞中蛋白质之间相互作用的有用工具。BiFC 基于当相互作用的蛋白质对，作为融合蛋白产生，具有荧光蛋白的 N-或 C-末端片段，在足够接近的距离以允许分裂荧光团的重组时产生的荧光的检测。在这里，我们描述了使用 Hsp90 和经过验证的客户蛋白 CDK4 对伴侣-客户相互作用模型应用 BiFC 测定法。该测定法允许在活细胞或固定细胞中对 HSP90-CDK4 复合物进行分布和时空分析，并且适用于研究抑制剂和突变对伴侣-客户蛋白网络的影响。