Abstract
Purpose: Our previous studies demonstrated that thermal stress induces the release of Hsp72 from cells by a mechanism independent of the classical protein transport pathway. However, the exact mechanism by which Hsp72, a leaderless protein, gains access to the extracellular milieu remains unknown. Materials and methods: This study is designed to determine the mechanism by which intracellular Hsp72 trafficking and release occurs. The data presented in this study suggests intracellular Hsp72 trafficking using Western blotting and Flow Cytometry. Also, unknown membrane bound proteins were identified doing in-gel digestion and LC-MS/MS. Results: We demonstrate that within 60 minutes after first exposure of cells to heat shock treatment, plasma membrane bound Hsp72 is shed and redistributed into cytosolic compartments. Inhibition of active cell transport by pre-treatment of cells with Cytochalasin B completely abrogated Hsp72 redistribution from the plasma membrane into the cytosol. Cross-linking of plasma membrane bound proteins with Hsp72 followed by Western blot analysis and LC-MS/MS analysis revealed at least seven interacting partners with Hsp72, including nucleolin, Hsp90, gp96, CAP2, TLR2, 4 and 7. Transfection of cells with nucleolin-siRNA completely inhibited baseline and heat shockinduced Hsp72 release. Conclusions: Taken together, this study for the first time demonstrates that the plasma membrane acts as a reservoir for Hsp72 and suggests that nucleolin plays an important role in Hsp72 trafficking and release.
Highlights
The seventy-two kilo-Dalton heat shock protein (Hsp72) belongs to a family of highly conserved group of proteins involved in protein folding, stabilization and cytoplasmic transportation
Taken together, this study for the first time demonstrates that the plasma membrane acts as a reservoir for Hsp72 and suggests that nucleolin plays an important role in Hsp72 trafficking and release
We demonstrate that the plasma membrane acts as a reservoir for Hsp72 and that heat shock stimulates complex formation between Hsp72, Hsp90, nucleolin, gp96, cyclic AMP associated protein-2 (CAP2), TLR2, 4 and 7
Summary
The seventy-two kilo-Dalton heat shock protein (Hsp72) belongs to a family of highly conserved group of proteins involved in protein folding, stabilization and cytoplasmic transportation. The Hsp is expressed within the cytoplasm, and in the plasma membrane of cells, from where it is actively released into the extracellular milieu to bind to and be internalized by professional antigen presenting cells [2,3]. Internalization of extracellular Hsp has been suggested to require endocytosis through binding with lipid components present in plasma membrane [9]. It has been shown that plasma membrane Hsp enhances the plasma membrane density of CD56/CD94 and initiates the cytolytic activity of NK cells [15]. We demonstrate that heat shock induces the shedding and subsequent subcellular redistribution of Hsp from the plasma membrane into cytosolic compartments. We demonstrate that the plasma membrane acts as a reservoir for Hsp and that heat shock stimulates complex formation between Hsp, Hsp, nucleolin, gp, CAP2, TLR2, 4 and 7
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