Targeting the interaction of AIMP2-DX2 with HSP70 suppresses cancer development

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Abstract

A tumorigenic factor, AIMP2 lacking exon 2 (AIMP2-DX2), is often upregulated in many cancers. However, how its cellular level is determined is not understood. Here, we report heat-shock protein HSP70 as a critical determinant for the level of AIMP2-DX2. Interaction of the two factors was identified by interactome analysis and structurally determined by X-ray crystallography and NMR analyses. HSP70 recognizes the amino (N)-terminal flexible region, as well as the glutathione S-transferase domain of AIMP2-DX2, via its substrate-binding domain, thus blocking the Siah1-dependent ubiquitination of AIMP2-DX2. AIMP2-DX2-induced cell transformation and cancer progression in vivo was further augmented by HSP70. A positive correlation between HSP70 and AIMP2-DX2 levels was shown in various lung cancer cell lines and patient tissues. Chemical intervention in the AIMP2-DX2–HSP70 interaction suppressed cancer cell growth in vitro and in vivo. Thus, this work demonstrates the importance of the interaction between AIMP2-DX2 and HSP70 on tumor progression and its therapeutic potential against cancer.

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Fig. 1: Interaction between DX2 and HSP70.
Fig. 2: Significance of HSP70 for stabilization of DX2.
Fig. 3: Structural determination of the interaction between DX2 and HSP70.
Fig. 4: HSP70-mediated protection of DX2 from Siah1-mediated degradation.
Fig. 5: Effect of the protein–protein interaction inhibitor, BC-DXI-495, on DX2-mediated proliferation.
Fig. 6: Mode of action of BC-DXI-495 on DX2.

Data availability

All data generated or analyzed during this study are included in this published article or are available from the corresponding author upon request. The structure coordinates have been deposited in the Protein Data Bank under codes 6JPV for HSP70 395-537-MYRLPNVHG and 6K39 for HSP70 395-537-YRLPNVHG. NMR assignment data for 13C- and 15N-labeled DX251-251-C136S-C222S have been deposited at the Biological Magnetic Resonance Bank (BMRB) with accession code 27914.

References

  1. 1.

    Hartl, F. U., Bracher, A. & Hayer-Hartl, M. Molecular chaperones in protein folding and proteostasis. Nature 475, 324–332 (2011).

  2. 2.

    Mayer, M. P. Hsp70 chaperone dynamics and molecular mechanism. Trends Biochem. Sci. 38, 507–514 (2013).

  3. 3.

    Feder, M. E. & Hofmann, G. E. Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology. Annu. Rev. Physiol. 61, 243–282 (1999).

  4. 4.

    Kumar, S. et al. Targeting Hsp70: a possible therapy for cancer. Cancer Lett. 374, 156–166 (2016).

  5. 5.

    Goloudina, A. R., Demidov, O. N. & Garrido, C. Inhibition of HSP70: a challenging anti-cancer strategy. Cancer Lett. 325, 117–124 (2012).

  6. 6.

    Calderwood, S. K. & Gong, J. Heat shock proteins promote cancer: it’s a protection racket. Trends Biochem. Sci. 41, 311–323 (2016).

  7. 7.

    Wu, J. et al. Heat shock proteins and cancer. Trends Pharmacol. Sci. 38, 226–256 (2017).

  8. 8.

    Sherman, M. Y. & Gabai, V. L. Hsp70 in cancer: back to the future. Oncogene 34, 4153–4161 (2015).

  9. 9.

    Alderson, T. R., Kim, J. H. & Markley, J. L. Dynamical structures of Hsp70 and Hsp70-Hsp40 complexes. Structure 24, 1014–1030 (2016).

  10. 10.

    Flaherty, K. M., DeLuca-Flaherty, C. & McKay, D. B. Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein. Nature 346, 623–628 (1990).

  11. 11.

    Zhu, X. et al. Structural analysis of substrate binding by the molecular chaperone DnaK. Science 272, 1606–1614 (1996).

  12. 12.

    Sekhar, A., Rosenzweig, R., Bouvignies, G. & Kay, L. E. Hsp70 biases the folding pathways of client proteins. Proc. Natl Acad. Sci. USA 113, E2794–E2801 (2016).

  13. 13.

    Mayer, M. P. & Gierasch, L. M. Recent advances in the structural and mechanistic aspects of Hsp70 molecular chaperones. J. Biol. Chem. 294, 2085–2097 (2019).

  14. 14.

    Sekhar, A., Rosenzweig, R., Bouvignies, G. & Kay, L. E. Mapping the conformation of a client protein through the Hsp70 functional cycle. Proc. Natl Acad. Sci. USA 112, 10395–10400 (2015).

  15. 15.

    Alvira, S. et al. Structural characterization of the substrate transfer mechanism in Hsp70/Hsp90 folding machinery mediated by Hop. Nat. Commun. 5, 5484 (2014).

  16. 16.

    Nillegoda, N. B., Wentink, A. S. & Bukau, B. Protein disaggregation in multicellular organisms. Trends Biochem. Sci. 43, 285–300 (2018).

  17. 17.

    Mashaghi, A. et al. Alternative modes of client binding enable functional plasticity of Hsp70. Nature 539, 448–451 (2016).

  18. 18.

    Fernandez-Fernandez, M. R., Gragera, M., Ochoa-Ibarrola, L., Quintana-Gallardo, L. & Valpuesta, J. M. Hsp70 - a master regulator in protein degradation. FEBS Lett. 591, 2648–2660 (2017).

  19. 19.

    Kim, S., You, S. & Hwang, D. Aminoacyl-tRNA synthetases and tumorigenesis: more than housekeeping. Nat. Rev. Cancer 11, 708–718 (2011).

  20. 20.

    Park, S. G., Ewalt, K. L. & Kim, S. Functional expansion of aminoacyl-tRNA synthetases and their interacting factors: new perspectives on housekeepers. Trends Biochem. Sci. 30, 569–574 (2005).

  21. 21.

    Han, J. M. et al. AIMP2/p38, the scaffold for the multi-tRNA synthetase complex, responds to genotoxic stresses via p53. Proc. Natl Acad. Sci. USA 105, 11206–11211 (2008).

  22. 22.

    Kim, M. J. et al. Downregulation of FUSE-binding protein and c-myc by tRNA synthetase cofactor p38 is required for lung cell differentiation. Nat. Genet. 34, 330–336 (2003).

  23. 23.

    Kim, D. G. et al. Oncogenic mutation of AIMP2/p38 inhibits its tumor-suppressive interaction with Smurf2. Cancer Res. 76, 3422–3436 (2016).

  24. 24.

    Choi, J. W. et al. AIMP2 promotes TNFα-dependent apoptosis via ubiquitin-mediated degradation of TRAF2. J. Cell Sci. 122, 2710–2715 (2009).

  25. 25.

    Yum, M. K. et al. AIMP2 controls intestinal stem cell compartments and tumorigenesis by modulating Wnt/β-catenin signaling. Cancer Res. 76, 4559–4568 (2016).

  26. 26.

    Choi, J. W. et al. Cancer-associated splicing variant of tumor suppressor AIMP2/p38: pathological implication in tumorigenesis. PLoS Genet. 7, e1001351 (2011).

  27. 27.

    Choi, J. W. et al. Splicing variant of AIMP2 as an effective target against chemoresistant ovarian cancer. J. Mol. Cell Biol. 4, 164–173 (2012).

  28. 28.

    Jung, J. Y. et al. Ratio of autoantibodies of tumor suppressor AIMP2 and its oncogenic variant is associated with clinical outcome in lung cancer. J. Cancer 8, 1347–1354 (2017).

  29. 29.

    Lee, H. S. et al. Chemical suppression of an oncogenic splicing variant of AIMP2 induces tumour regression. Biochem. J. 454, 411–416 (2013).

  30. 30.

    Won, Y. S. & Lee, S. W. Selective regression of cancer cells expressing a splicing variant of AIMP2 through targeted RNA replacement by trans-splicing ribozyme. J. Biotechnol. 158, 44–49 (2012).

  31. 31.

    Rohde, M. et al. Members of the heat-shock protein 70 family promote cancer cell growth by distinct mechanisms. Genes Dev. 19, 570–582 (2005).

  32. 32.

    Cesa, L. C. et al. X-linked inhibitor of apoptosis protein (XIAP) is a client of heat shock protein 70 (Hsp70) and a biomarker of its inhibition. J. Biol. Chem. 293, 2370–2380 (2018).

  33. 33.

    Clerico, E. M., Tilitsky, J. M., Meng, W. & Gierasch, L. M. How hsp70 molecular machines interact with their substrates to mediate diverse physiological functions. J. Mol. Biol. 427, 1575–1588 (2015).

  34. 34.

    Mayer, M. P. & Bukau, B. Hsp70 chaperones: cellular functions and molecular mechanism. Cell. Mol. Life Sci. 62, 670–684 (2005).

  35. 35.

    Liebscher, M. & Roujeinikova, A. Allosteric coupling between the lid and interdomain linker in DnaK revealed by inhibitor binding studies. J. Bacteriol. 191, 1456–1462 (2009).

  36. 36.

    Zhang, P., Leu, J. I., Murphy, M. E., George, D. L. & Marmorstein, R. Crystal structure of the stress-inducible human heat shock protein 70 substrate-binding domain in complex with peptide substrate. PLoS ONE 9, e103518 (2014).

  37. 37.

    Xu, W. et al. Surface charge and hydrophobicity determine ErbB2 binding to the Hsp90 chaperone complex. Nat. Struct. Mol. Biol. 12, 120–126 (2005).

  38. 38.

    Oh, A. Y. et al. Inhibiting DX2-p14/ARF interaction exerts antitumor effects in lung cancer and delays tumor progression. Cancer Res. 76, 4791–4804 (2016).

  39. 39.

    Sharma, S. V., Bell, D. W., Settleman, J. & Haber, D. A. Epidermal growth factor receptor mutations in lung cancer. Nat. Rev. Cancer 7, 169–181 (2007).

  40. 40.

    Kwon, N. H., Fox P. L. & Kim, S. Aminoacyl-tRNA synthetases as therapeutic targets. Nat. Rev. Drug Discov. 18, 629–650 (2019).

  41. 41.

    Song, J. S. et al. Preclinical pharmacokinetics of PDE-310, a novel PDE4 inhibitor. Drug Metab. Pharmacokinet. 26, 192–200 (2011).

  42. 42.

    Verchot, J. Cellular chaperones and folding enzymes are vital contributors to membrane bound replication and movement complexes during plant RNA virus infection. Front. Plant Sci. 3, 275 (2012).

  43. 43.

    Taniguchi, M. et al. Pyrrhocoricin, a proline-rich antimicrobial peptide derived from insect, inhibits the translation process in the cell-free Escherichia coli protein synthesis system. J. Biosci. Bioeng. 121, 591–598 (2016).

  44. 44.

    Meng, W., Clerico, E. M., McArthur, N. & Gierasch, L. M. Allosteric landscapes of eukaryotic cytoplasmic Hsp70s are shaped by evolutionary tuning of key interfaces. Proc. Natl Acad. Sci. USA 115, 11970–11975 (2018).

  45. 45.

    Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

  46. 46.

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

  47. 47.

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).

  48. 48.

    Hassan, A. Q. et al. The novolactone natural product disrupts the allosteric regulation of Hsp70. Chem. Biol. 22, 87–97 (2015).

  49. 49.

    Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011).

  50. 50.

    Dominguez, C., Boelens, R. & Bonvin, A. M. HADDOCK: a protein-protein docking approach based on biochemical or biophysical information. J. Am. Chem. Soc. 125, 1731–1737 (2003).

  51. 51.

    Morris, G. M. et al. AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J. Comput. Chem. 30, 2785–2791 (2009).

  52. 52.

    Alexander, N., Woetzel, N. & Meiler, J. bcl::Cluster: a method for clustering biological molecules coupled with visualization in the Pymol molecular graphics system. IEEE Int. Conf. Comput. Adv. Bio. Med. Sci. 2011, 13–18 (2011).

  53. 53.

    Gulcin, I. & Taslimi, P. Sulfonamide inhibitors: a patent review 2013–present. Expert Opin. Ther. Pat. 28, 541–549 (2018).

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Acknowledgements

This work was supported by the Global Frontier Project grant (no. NRF-M3A6A4-2010-0029785) and the IMRCTR grant (no. NRF-2018R1A5A2023127) of the National Research Foundation funded by the Ministry of Science and ICT of Korea. We thank T. Otomo of The Scripps Research Institute for helpful discussions. X-ray diffraction data were collected at Pohang Accelerator Laboratory beamlines 5C, 7A and 11C and Photon Factory beamline 1A. We used the NMR instruments of the Protein Structure Group at the Korea Basic Science Institute.

Author information

S.L., H.Y.C., D.G.K. and S.K. conceived the study. S.L., H.Y.C., D.G.K., M.H.K., K.L., Y.H.J. and S.K. designed all the experiments. S.L., D.G.K. and Y.R. performed and analyzed most experiments including cell and molecular biological experiment and in vivo analysis. H.Y.C., S.Y.S. and A.U.M. performed and analyzed X-ray crystallography and NMR experiments. M.K., D.B. and A.S. synthesized all the BC-DXI compounds. Y.L. performed and analyzed the in vitro pull-down assay between DX2 and HSP70 or HSP90. J.L. and W.S.Y. performed mass spectrometry analysis. H.K.K. took the required fluorescence images. M.H.K., K.L., Y.H.J. and S.K. reviewed and discussed the data. S.L., H.Y.C., D.G.K. and S.K. wrote the manuscript. All authors edited the manuscript.

Correspondence to Sunghoon Kim.

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Synthetic procedures.

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Lim, S., Cho, H.Y., Kim, D.G. et al. Targeting the interaction of AIMP2-DX2 with HSP70 suppresses cancer development. Nat Chem Biol 16, 31–41 (2020) doi:10.1038/s41589-019-0415-2

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