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Heat shock protein 90 targets a chaperoned peptide to the static early endosome for efficient cross-presentation by human dendritic cells.

Tanaka T, Okuya K, Kutomi G, Takaya A, Kajiwara T, Kanaseki T, Tsukahara T, Hirohashi Y, Torigoe T, Hirata K, Okamoto Y, Sato N, Tamura Y - Cancer Sci. (2014)

Bottom Line: In this study, we found that an Hsp90-cancer antigen peptide complex was efficiently cross-presented by human monocyte-derived DCs and induced peptide-specific CTLs.Furthermore, we observed that the internalized Hsp90-peptide complex was strictly sorted to the Rab5(+), EEA1(+) static early endosome and the Hsp90-chaperoned peptide was processed and bound to MHC class I molecules through an endosome-recycling pathway.Our data indicate that targeting of the antigen to a "static" early endosome by Hsp90 is essential for efficient cross-presentation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Sapporo Medical University School of Medicine, Sapporo, Japan; United Graduate School of Veterinary Sciences, Yamaguchi University, Yamaguchi, Japan.

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Heat shock protein 90 (Hsp90)–survivin-2B75-93 precursor peptide complex localized to static early endosomes within human monocyte-derived dendritic cells (Mo-DCs). (a) Human Mo-DCs were incubated at 37°C with Alexa 594-labeled Hsp90–survivin-2B75-93 peptide complex for 1 h and then washed and fixed. Organelles were stained with an anti-EEA1 mAb for early endosomes, anti-Rab5 polyclonal antibody for early endosomes, and anti-LAMP-1 polyclonal antibody for late endosomes/lysosomes followed by Alexa 488-conjugated goat anti-rabbit IgG or anti-mouse IgG and were visualized with confocal laser microscopy. Arrowheads indicate colocalization of the internalized Hsp90–survivin-2B75-93 peptide complex and each organelle. (b) To quantify the percentage of colocalization, a single z-plane of one cell was evaluated. For each protein and organelle combination, a total of 150 cells (50 cells from three independent experiments) were analyzed. Data are shown as means + SEM of three independent experiments. *P < 0.01.
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fig04: Heat shock protein 90 (Hsp90)–survivin-2B75-93 precursor peptide complex localized to static early endosomes within human monocyte-derived dendritic cells (Mo-DCs). (a) Human Mo-DCs were incubated at 37°C with Alexa 594-labeled Hsp90–survivin-2B75-93 peptide complex for 1 h and then washed and fixed. Organelles were stained with an anti-EEA1 mAb for early endosomes, anti-Rab5 polyclonal antibody for early endosomes, and anti-LAMP-1 polyclonal antibody for late endosomes/lysosomes followed by Alexa 488-conjugated goat anti-rabbit IgG or anti-mouse IgG and were visualized with confocal laser microscopy. Arrowheads indicate colocalization of the internalized Hsp90–survivin-2B75-93 peptide complex and each organelle. (b) To quantify the percentage of colocalization, a single z-plane of one cell was evaluated. For each protein and organelle combination, a total of 150 cells (50 cells from three independent experiments) were analyzed. Data are shown as means + SEM of three independent experiments. *P < 0.01.

Mentions: For further support of the above-described results, we investigated the intracellular routing of Hsp90 after uptake of it in DCs, using confocal laser microscopy. The Mo-DCs were incubated with Alexa 594-labeled Hsp90–survivin2B75-93 peptide complex for 1 h. Following incubation, the cells were fixed and stained with antibodies against markers for organelle structures including EEA1, Rab5, and LAMP-1. Alexa 594-labeled Hsp90–peptide complex was detected in EEA1+ and Rab5+-early endosomes but not in lysosomes (Fig.4a). Quantitative analysis of the colocalization between the exogenous Hsp90–peptide complex and Rab5, EEA1, and LAMP1 revealed average colocalization incidences of 78.0%, 88.7%, and 7.3%, respectively, providing further evidence that the exogenous Hsp90–peptide complex was delivered to the endosome-recycling pathway (Fig.4b). We also examined the dynamics of Alexa 594-labeled LDL as a positive control protein for the dynamic early endosomal pathway (Fig.5). Alexa594-labeled soluble LDL localized to the Rab5+-early endosome as well as the LAMP-1+-late endosome/lysosome, but not to the EEA1+-compartment, thus indicating the dynamic endosomal pathway. These results indicated that the Hsp90–peptide complex was sorted into the static endosomal pathway, not the dynamic endosomal pathway, within human Mo-DCs. In contrast, the soluble LDL protein, which underwent degradation, was translocated to the dynamic endosomal pathway. These results suggested that targeting to the “static” early endosome was required for efficient cross-presentation by Mo-DCs.


Heat shock protein 90 targets a chaperoned peptide to the static early endosome for efficient cross-presentation by human dendritic cells.

Tanaka T, Okuya K, Kutomi G, Takaya A, Kajiwara T, Kanaseki T, Tsukahara T, Hirohashi Y, Torigoe T, Hirata K, Okamoto Y, Sato N, Tamura Y - Cancer Sci. (2014)

Heat shock protein 90 (Hsp90)–survivin-2B75-93 precursor peptide complex localized to static early endosomes within human monocyte-derived dendritic cells (Mo-DCs). (a) Human Mo-DCs were incubated at 37°C with Alexa 594-labeled Hsp90–survivin-2B75-93 peptide complex for 1 h and then washed and fixed. Organelles were stained with an anti-EEA1 mAb for early endosomes, anti-Rab5 polyclonal antibody for early endosomes, and anti-LAMP-1 polyclonal antibody for late endosomes/lysosomes followed by Alexa 488-conjugated goat anti-rabbit IgG or anti-mouse IgG and were visualized with confocal laser microscopy. Arrowheads indicate colocalization of the internalized Hsp90–survivin-2B75-93 peptide complex and each organelle. (b) To quantify the percentage of colocalization, a single z-plane of one cell was evaluated. For each protein and organelle combination, a total of 150 cells (50 cells from three independent experiments) were analyzed. Data are shown as means + SEM of three independent experiments. *P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4317773&req=5

fig04: Heat shock protein 90 (Hsp90)–survivin-2B75-93 precursor peptide complex localized to static early endosomes within human monocyte-derived dendritic cells (Mo-DCs). (a) Human Mo-DCs were incubated at 37°C with Alexa 594-labeled Hsp90–survivin-2B75-93 peptide complex for 1 h and then washed and fixed. Organelles were stained with an anti-EEA1 mAb for early endosomes, anti-Rab5 polyclonal antibody for early endosomes, and anti-LAMP-1 polyclonal antibody for late endosomes/lysosomes followed by Alexa 488-conjugated goat anti-rabbit IgG or anti-mouse IgG and were visualized with confocal laser microscopy. Arrowheads indicate colocalization of the internalized Hsp90–survivin-2B75-93 peptide complex and each organelle. (b) To quantify the percentage of colocalization, a single z-plane of one cell was evaluated. For each protein and organelle combination, a total of 150 cells (50 cells from three independent experiments) were analyzed. Data are shown as means + SEM of three independent experiments. *P < 0.01.
Mentions: For further support of the above-described results, we investigated the intracellular routing of Hsp90 after uptake of it in DCs, using confocal laser microscopy. The Mo-DCs were incubated with Alexa 594-labeled Hsp90–survivin2B75-93 peptide complex for 1 h. Following incubation, the cells were fixed and stained with antibodies against markers for organelle structures including EEA1, Rab5, and LAMP-1. Alexa 594-labeled Hsp90–peptide complex was detected in EEA1+ and Rab5+-early endosomes but not in lysosomes (Fig.4a). Quantitative analysis of the colocalization between the exogenous Hsp90–peptide complex and Rab5, EEA1, and LAMP1 revealed average colocalization incidences of 78.0%, 88.7%, and 7.3%, respectively, providing further evidence that the exogenous Hsp90–peptide complex was delivered to the endosome-recycling pathway (Fig.4b). We also examined the dynamics of Alexa 594-labeled LDL as a positive control protein for the dynamic early endosomal pathway (Fig.5). Alexa594-labeled soluble LDL localized to the Rab5+-early endosome as well as the LAMP-1+-late endosome/lysosome, but not to the EEA1+-compartment, thus indicating the dynamic endosomal pathway. These results indicated that the Hsp90–peptide complex was sorted into the static endosomal pathway, not the dynamic endosomal pathway, within human Mo-DCs. In contrast, the soluble LDL protein, which underwent degradation, was translocated to the dynamic endosomal pathway. These results suggested that targeting to the “static” early endosome was required for efficient cross-presentation by Mo-DCs.

Bottom Line: In this study, we found that an Hsp90-cancer antigen peptide complex was efficiently cross-presented by human monocyte-derived DCs and induced peptide-specific CTLs.Furthermore, we observed that the internalized Hsp90-peptide complex was strictly sorted to the Rab5(+), EEA1(+) static early endosome and the Hsp90-chaperoned peptide was processed and bound to MHC class I molecules through an endosome-recycling pathway.Our data indicate that targeting of the antigen to a "static" early endosome by Hsp90 is essential for efficient cross-presentation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Sapporo Medical University School of Medicine, Sapporo, Japan; United Graduate School of Veterinary Sciences, Yamaguchi University, Yamaguchi, Japan.

Show MeSH
Related in: MedlinePlus