期刊
PROTEOMICS
卷 18, 期 24, 页码 -出版社
WILEY
DOI: 10.1002/pmic.201800004
关键词
chaperone; heat shock protein 90; HEK293T; kidney stone; siRNA; TAP tag
资金
- Mahidol University research grant
- Thailand Research Fund [IRN60W0004, IRG5980006]
- Faculty of Medicine Siriraj Hospital
- Royal Golden Jubilee PhD Program [PHD/0182/2554]
Heat shock protein 90 (HSP90) is a highly abundant molecular chaperone that interacts with many other intracellular proteins to regulate various cellular processes. However, compositions of the HSP90-interacting complex remain underinvestigated. This study thus aims to characterize such complex in human embryonic kidney (HEK293T) cells under normal physiologic state using tandem affinity purification (TAP) followed by protein identification using an ultrahigh-resolution tandem mass spectrometer (Qq-TOF MS/MS). A total of 32 proteins, including four forms of HSP90 and 16 novel HSP90-interacting partners, are successfully identified from this complex using TAP control to subtract nonspecific binders. Co-immunoprecipitation followed by immunoblotting and immunofluorescence co-staining confirms the association of HSP90 with known (HSP70, alpha-tubulin, and beta-actin) and novel (vimentin, calpain-1, and importin-beta 1) partners. Knockdown of HSP90 by small-interfering RNA (siHSP90) causes significant changes in levels of HSP70, alpha-tubulin, beta-actin, vimentin, and calpain-1, all of which are calcium oxalate (CaOx) crystal-binding proteins that play significant roles in kidney stone formation. Moreover, crystal-binding capability is significantly decreased in siHSP90-transfected cells as compared to non-transfected control and siControl-transfected cells. In summary, herein, a number of novel HSP90-interacting proteins in renal cells is reported and the potential role of HSP90-interacting complex in kidney stone formation is demonstrated.
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