Abstract
Chaperone Hsp70 can cross the plasma membrane of living cells using mechanisms that so far have not received much research attention. Searching the part of the molecule that is responsible for transport ability of Hsp70, we found a cationic sequence composed of 20 amino acid residues on its surface, KST peptide, which was used in further experiments. We showed that KST peptide enters living cells of various origins with the same efficiency as the full-length chaperone. KST peptide is capable of carrying cargo with a molecular weight 30 times greater than its own into cells. When we compared the membrane-crossing activity of KST peptide in complex with Avidin (KST–Av complex) with that of similarly linked canonical TAT peptide, we found that TAT peptide penetrated SK-N-SH human neuroblastoma cells at a similar rate and efficiency as the KST peptide. Furthermore, KST peptide can carry protein complexes consisting of a specific antibody coupled to the peptide through the Avidin bridge. An antibody to Hsp70 delivered to SK-N-SH cells with high expression level of Hsp70 reduced the protective power of the chaperone and sensitized the cells to the pro-apoptotic effect of staurosporine. We studied the mechanisms of penetration of KST–Av and full-length Hsp70 inside human neuroblastoma SK-N-SH and human erythroleukemia K-562 cells and found that both used an active intracellular transport mechanism that included vesicular structures and negatively charged lipid membrane domains. Competition analysis of intracellular transport showed that the chaperone reduced intracellular penetration of KST peptide and conversely KST peptide prevented Hsp70 transport in a dose-dependent manner.
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Acknowledgments
We thank Dr. N. Fusenig (DKFZ, Germany) for HaCaT cells; Dr. Marja Jäättelä for providing us with hsp70 plasmid; Dr. Oleg Demidov (INSERM, France) for rab-5, rab-7, and mito-PAGFP plasmids; Mr. Michael Vorobyev and Dr. Gregory Shtein (Institute of Cytology of RAS, Russia) for the help with confocal microscopy experiments; and Ms. Ekaterina Fedorova for technical assistance. This work was supported by grants from the Russian Foundation of Basic Research 14-08-00164 and 13-04-01299 and of the Molecular and Cell Biology Program of the Russian Academy of Sciences.
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Komarova, E.Y., Meshalkina, D.A., Aksenov, N.D. et al. The discovery of Hsp70 domain with cell-penetrating activity. Cell Stress and Chaperones 20, 343–354 (2015). https://doi.org/10.1007/s12192-014-0554-z
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DOI: https://doi.org/10.1007/s12192-014-0554-z