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Hsp70 stabilizes lysosomes and reverts Niemann–Pick disease-associated lysosomal pathology

Abstract

Heat shock protein 70 (Hsp70) is an evolutionarily highly conserved molecular chaperone that promotes the survival of stressed cells by inhibiting lysosomal membrane permeabilization1,2,3,4,5, a hallmark of stress-induced cell death6,7,8,9,10. Clues to its molecular mechanism of action may lay in the recently reported stress- and cancer-associated translocation of a small portion of Hsp70 to the lysosomal compartment1,11. Here we show that Hsp70 stabilizes lysosomes by binding to an endolysosomal anionic phospholipid bis(monoacylglycero)phosphate (BMP), an essential co-factor for lysosomal sphingomyelin metabolism12,13,14. In acidic environments Hsp70 binds with high affinity and specificity to BMP, thereby facilitating the BMP binding and activity of acid sphingomyelinase (ASM). The inhibition of the Hsp70–BMP interaction by BMP antibodies or a point mutation in Hsp70 (Trp90Phe), as well as the pharmacological and genetic inhibition of ASM, effectively revert the Hsp70-mediated stabilization of lysosomes. Notably, the reduced ASM activity in cells from patients with Niemann–Pick disease (NPD) A and B—severe lysosomal storage disorders caused by mutations in the sphingomyelin phosphodiesterase 1 gene (SMPD1) encoding for ASM15—is also associated with a marked decrease in lysosomal stability, and this phenotype can be effectively corrected by treatment with recombinant Hsp70. Taken together, these data open exciting possibilities for the development of new treatments for lysosomal storage disorders and cancer with compounds that enter the lysosomal lumen by the endocytic delivery pathway.

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Figure 1: Lysosomal Hsp70 stabilizes lysosomal membranes.
Figure 2: A pH-dependent interaction between Hsp70 and BMP stabilizes lysosomal membranes.
Figure 3: Hsp70 stimulates ASM activity that in turn stabilizes lysosomes.
Figure 4: rHsp70 stimulates ASM activity, stabilizes lysosomes and reverts the lysosomal pathology in NPD fibroblasts.

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Acknowledgements

We thank E. Gulbins and A. Riehle for the NPD A (no. 2) cells, the Charité Berlin and the Johannes Gutenberg-Universität Mainz for the NPD B fibroblasts, C. Ejsing and H. Schulze for advice, and J. Grüenberg, B. Margulis and the Developmental Studies Hybridoma Bank for antibodies. This work was supported by grants from the Danish Cancer Society, the Danish National Research Foundation, the Danish Medical Research Council, the European Commission FP7 (APO-SYS), the Meyer Foundation, the Novo Nordisk Foundation, and the Association for International Cancer Research for M.J., the Finnish Academy and the Sigrid Juselius Foundation for P.K.J.K., Volkswagenstiftung for A.G.R. and C.A., Deutsche Forschungsgemeinschaft (SFB 645, SPP 1267) and EU (LipidomicNet) for K.S., and the Polish Ministry of Science and High Education for A.Z.

Author Contributions T.K. and M.J. designed the study, analysed the data and wrote the paper. T.K. performed experiments for Figs 1a–e, 2b, e, f, 3b, d, f, 4a–c, Supplementary Figs 1, 4 and Supplementary Movies. A.G.R. and C.A. performed experiments for Figs 2d, 3a and Supplementary Fig. 6, and provided NPD cell lines. K.S. contributed to the BIAcore data and provided rASM. N.H.T.P. performed experiments for Fig. 3e and Supplementary Fig. 3. P.K.J.K. designed the biophysical studies on Hsp–lipid interactions. A.K.M. and I.M. performed experiments for Fig. 2a, e. O.D.O. and J.K. performed experiments for Figs 3c, 4d and Supplementary Figs 2 and 5. A.Z. validated the functionality of rHsp70 and its mutants, and provided proteins for control. J.N. provided input to study design. All authors discussed the results and commented on the manuscript.

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Correspondence to Marja Jäättelä.

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Supplementary Information

This file contains Supplementary Figures S1-S6 with Legends and Legends for Supplementary Movies 1 - 2. (PDF 2954 kb)

Supplementary Movie 1

Photo-oxidation-induced lysosomal membrane permeabilization in U-2-OS cells labeled with acridine orange (see Supplementary Information file for full Legend). (MOV 5577 kb)

Supplementary Movie 2

Massive photo-oxidation-induced lysosomal membrane permeabilization in NPDA fibroblasts labeled with acridine orange (see Supplementary Information file for full Legend). (AVI 3804 kb)

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Kirkegaard, T., Roth, A., Petersen, N. et al. Hsp70 stabilizes lysosomes and reverts Niemann–Pick disease-associated lysosomal pathology. Nature 463, 549–553 (2010). https://doi.org/10.1038/nature08710

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