The discovery of Hsp70 domain with cell-penetrating activity

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.

Role of membrane Hsp70 in radiation sensitivity of tumor cells

“…Potential lipid candidates which have been proven to interact with recombinant Hsp70 are phosphatidylserine PS and the lipid raft component globoyltriaosylceramide Gb3 [ 35 , 51 ]. Furthermore, Hsp70 and peptides derived thereof have been found to be able to cross membranes of living tumor cells by the involvement of lipid rafts and endocytosis-dependent/independent mechanisms [ 16 , 52 , 53 ]. Therefore, it was assumed that after interaction of cytosolic Hsp70 with lipid vesicles containing PS or Gb3, Hsp70 can interact with these vesicles in the cytosol.…”

The remarkable multivalency of the Hsp70 chaperones

“…It is believed that the interactions of HSPA8 with membranes involve changes in the conformations and selfassociation of the protein (Armijo et al 2014). Recently, Guzhova and coworkers (Komarova et al 2015) provided evidence of a cationic amino acid sequence of 20 amino acids (amino acids 493-512, between SBD and LID) on HSPA1A that has cell-penetrating activity similar to the full-size protein. This peptide termed KST can carry into cells cargo having a molecular mass 30 times greater than its own.…”

Intracellular antigens as targets for antibody based immunotherapy of malignant diseases

“…Potential lipid candidates which have been proven to interact with recombinant HSP70 are phosphatidylserine PS and the lipid raft component globotriaosylceramide Gb3 (Gehrmann et al., 2008; Schilling et al., 2009). Furthermore, HSP70 and peptides derived thereof have been found to be able to cross membranes of living tumor cells by the involvement of lipid rafts and endocytosis‐dependent/independent mechanisms (Komarova et al., 2015; Shevtsov et al., 2014; Stangl et al., 2014). Therefore, it is assumed that after interaction of cytosolic HSP70 with lipid vesicles containing PS or Gb3, HSP70 can interact with these vesicles in the cytosol.…”