Abstract P6-08-08: Nucleolin as an Intracellular Transporter of Hsp72

Abstract

Abstract Background: 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: In this study we use flow cytometry to measure the expression of surface bound Hsp72 in THP1 human monocytic cells. Sucrose gradient ultracentrifugation was also used to isolate and recover intracellular fractions. Western blot analysis was used to probe for intracellular fractions expressing Hsp72, Hsp90, Grp96, CAP2, TLR2, 4, or 7. Baculovirus expression vector system (BEVS) was used to generate highly purified endotoxin free Hsp72. Plasma membrane cross-linking assay was used to identify the interacting proteins of Hsp72 on cell plasma membrane. Laser scanning confocal microscopy was used to demonstrate the plasma membrane localization and internalization of Hsp72 with other interacting proteins. In-gel digestion and LC-mass spectrometry was used to identify unique proteins colocalizing with Hsp72. Results: The data presented in this study suggest that Hsp72 trafficking within the cell and its release is assisted in part by, nucleolin. We further demonstrate that within 60 minutes after first exposure of cells to heat shock treatment, plasma membrane bound Hsp72 is internalized 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, in-gel digestion and LC-MS/MS analysis revealed seven interacting partners with Hsp72, including Hsp90, nucleolin, gp96, CAP2, TLR2, 4 and 7. Transfection of cells with nucleolin-siRNA completely inhibited baseline and heat shock-induced Hsp72 release. Discussion: Our study for the first time demonstrates that the plasma membrane acts as a reservoir for Hsp72 and confirms that nucleolin plays an important role in Hsp72 trafficking and release. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-08-08.