Abstract: TH-PO381

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

Session Information

Category: Cell Biology

  • 201 Cell Signaling, Oxidative Stress

Authors

  • Thongboonkerd, Visith, Siriraj Hospital, Mahidol University, Bangkok, Thailand
  • Manissorn, Juthatip, Siriraj Hospital, Mahidol University, Bangkok, Thailand
Background

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 largely unknown. This study thus aimed to characterize such complex in renal cells by tandem affinity purification (TAP) followed by ultrahigh-resolution tandem mass spectrometry (Qq-TOF MS/MS).

Methods

The full-length HSP90AB1 gene was constructed and subcloned into pGLUE vector, which was designed to express streptavidin- and calmodulin-binding affinity tags at the N-terminus of HSP90, whereas TAP-tag without HSP90 fusion served as the control for TAP purification system. Expression of TAP-tag fusion with HSP90 (TAP-HSP90) in the transfected HEK293T cells was confirmed by immunoblotting. The HSP90-interacting complex was purified by TAP-tag method and subjected to in-solution tryptic digestion and identification by Qq-TOF MS/MS. Functional significance of this complex was then addressed by using small-interfering RNA (siRNA) targeting to HSP90 (siHSP90).

Results

A total of 40 proteins, including four forms of HSP90 and 19 novel HSP90-interacting partners, were successfully identified from this complex using TAP control to subtract non-specific binders. Co-immunoprecipitation followed by immunoblotting and immunofluorescence co-staining confirmed 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 siRNA (siHSP90) caused 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. CaOx crystal-cell adhesion assay revealed that crystal-cell adhesion was significantly decreased in siHSP90-transfected cells as compared to non-transfected control and siControl-transfected cells.

Conclusion

We report herein a number of novel HSP90-interacting proteins in renal cells and demonstrate the potential role of HSP90-interacting complex in kidney stone formation.

Funding

  • Government Support - Non-U.S.