Characterizations of HSP90-Interacting Complex in Renal Cells Using Tandem Affinity Purification and Its Potential Role in Kidney Stone Formation

• Main Authors: Juthatip Manissorn, Nilubon Singhto, Visith Thongboonkerd
• Other Authors: Faculty of Medicine, Siriraj Hospital, Mahidol University
• Format: Article
• Published: 2019
• Subjects: Biochemistry, Genetics and Molecular Biology
• Online Access: https://repository.li.mahidol.ac.th/handle/123456789/44962
• Institution: Mahidol University

© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 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, α-tubulin, and β-actin) and novel (vimentin, calpain-1, and importin-β1) partners. Knockdown of HSP90 by small-interfering RNA (siHSP90) causes significant changes in levels of HSP70, α-tubulin, β-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.