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Site-specific ubiquitination affects protein energetics and proteasomal degradation

Abstract

Changes in the cellular environment modulate protein energy landscapes to drive important biology, with consequences for signaling, allostery and other vital processes. The effects of ubiquitination are particularly important because of their potential influence on degradation by the 26S proteasome. Moreover, proteasomal engagement requires unstructured initiation regions that many known proteasome substrates lack. To assess the energetic effects of ubiquitination and how these manifest at the proteasome, we developed a generalizable strategy to produce isopeptide-linked ubiquitin within structured regions of a protein. The effects on the energy landscape vary from negligible to dramatic, depending on the protein and site of ubiquitination. Ubiquitination at sensitive sites destabilizes the native structure and increases the rate of proteasomal degradation. In well-folded proteins, ubiquitination can even induce the requisite unstructured regions needed for proteasomal engagement. Our results indicate a biophysical role of site-specific ubiquitination as a potential regulatory mechanism for energy-dependent substrate degradation.

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Fig. 1: Generation of substrates with isopeptide-linked ubiquitin in structured regions and equilibrium unfolding studies.
Fig. 2: Native-state proteolysis demonstrates the effects of mono-ubiquitination on the energetics of partial unfolding.
Fig. 3: Mono-ubiquitin-mediated substrate destabilization directly modulates degradation rate.
Fig. 4: Ubiquitin-mediated destabilization of barstar is sufficient to expose a proteasome-engageable unstructured region.
Fig. 5: Model for the consequences of site-specific, ubiquitin-induced substrate energy landscape modulation on proteasomal degradation.

Data availability

The data that support the findings of this study are available within the manuscript and its supplementary information or from the corresponding author upon reasonable request. All constructs generated for this study are also available from the corresponding author upon reasonable request. Source data for Figs. 24 are presented with the paper.

References

  1. Raschke, T. M., Kho, J. & Marqusee, S. Confirmation of the hierarchical folding of RNase H: a protein engineering study. Nat. Struct. Biol. 6, 825–830 (1999).

    CAS  PubMed  Google Scholar 

  2. Kenniston, J. A., Burton, R. E., Siddiqui, S. M., Baker, T. A. & Sauer, R. T. Effects of local protein stability and the geometric position of the substrate degradation tag on the efficiency of ClpXP denaturation and degradation. J. Struct. Biol. 146, 130–140 (2004).

    CAS  PubMed  Google Scholar 

  3. Liu, T., Whitten, S. T. & Hilser, V. J. Ensemble-based signatures of energy propagation in proteins: a new view of an old phenomenon. Proteins Struct. Funct. Genet 62, 728–738 (2006).

    CAS  PubMed  Google Scholar 

  4. Martin, A., Baker, T. A. & Sauer, R. T. Protein unfolding by a AAA+ protease is dependent on ATP-hydrolysis rates and substrate energy landscapes. Nat. Struct. Mol. Biol. 15, 139–145 (2008).

    CAS  PubMed  Google Scholar 

  5. Xin, F. & Radivojac, P. Post-translational modifications induce significant yet not extreme changes to protein structure. Bioinformatics 28, 2905–2913 (2012).

    CAS  PubMed  Google Scholar 

  6. Swatek, K. N. & Komander, D. Ubiquitin modifications. Cell Res. 26, 399–422 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Prakash, S., Tian, L., Ratliff, K. S., Lehotzky, R. E. & Matouschek, A. An unstructured initiation site is required for efficient proteasome-mediated degradation. Nat. Struct. Mol. Biol. 11, 830–837 (2004).

    CAS  PubMed  Google Scholar 

  8. Yu, H. & Matouschek, A. Recognition of client proteins by the proteasome. Annu. Rev. Biophys. 46, 149–173 (2017).

    CAS  PubMed  Google Scholar 

  9. Hagai, T., Azia, A., Tóth-Petróczy, Á. & Levy, Y. Intrinsic disorder in ubiquitination substrates. J. Mol. Biol. 412, 319–324 (2011).

    CAS  PubMed  Google Scholar 

  10. Godderz, D. et al. Cdc48-independent proteasomal degradation coincides with a reduced need for ubiquitylation. Sci. Rep. 5, 1–8 (2015).

    Google Scholar 

  11. Tsuchiya, H. et al. In vivo ubiquitin linkage-type analysis reveals that the Cdc48-Rad23/Dsk2 axis contributes to K48-linked chain specificity of the proteasome. Mol. Cell 66, 488–502.e7 (2017).

    CAS  PubMed  Google Scholar 

  12. Olszewski, M. M., Williams, C., Dong, K. C. & Martin, A. The Cdc48 unfoldase prepares well-folded protein substrates for degradation by the 26S proteasome. Commun. Biol 2, 29 (2019).

    PubMed  PubMed Central  Google Scholar 

  13. Hagai, T. & Levy, Y. Ubiquitin not only serves as a tag but also assists degradation by inducing protein unfolding. Proc. Natl Acad. Sci. USA 107, 2001–2006 (2010).

    CAS  PubMed  Google Scholar 

  14. Gavrilov, Y., Hagai, T. & Levy, Y. Nonspecific yet decisive: ubiquitination can affect the native-state dynamics of the modified protein. Protein Sci. 24, 1580–1592 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Faggiano, S. & Pastore, A. The challenge of producing ubiquitinated proteins for structural studies. Cells 3, 639–656 (2014).

    PubMed  PubMed Central  Google Scholar 

  16. Morimoto, D., Walinda, E., Fukada, H., Sugase, K. & Shirakawa, M. Ubiquitylation directly induces fold destabilization of proteins. Sci. Rep. 6, 39453 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Cundiff, M. D. et al. Ubiquitin receptors are required for substrate-mediated activation of the proteasome’s unfolding ability. Sci. Rep. 9, 14506 (2019).

    PubMed  PubMed Central  Google Scholar 

  18. Saeki, Y., Isono, E. & Toh-E, A. Preparation of ubiquitinated substrates by the PY motif-insertion method for monitoring 26S proteasome activity. Methods Enzymol. 399, 215–227 (2005).

    CAS  PubMed  Google Scholar 

  19. Kim, H. C., Steffen, A. M., Oldham, M. L., Chen, J. & Huibregtse, J. M. Structure and function of a HECT domain ubiquitin-binding site. EMBO Rep. 12, 334–341 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Kamadurai, H. B. et al. Mechanism of ubiquitin ligation and lysine prioritization by a HECT E3. eLife 2013, 1–26 (2013).

    Google Scholar 

  21. Khurana, Ritu, Hate, AnitaT., Nath, Utpal & Udgaonkar, J. B. pH dependence of the stability of barstar to chemical and thermal denaturation. Protein Sci. 4, 1133–1144 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Myers, J. K., Pace, C. N. & Scholtz, J. M. Denaturant m values and heat capacity changes: relation to changes in accessible surface areas of protein unfolding. Protein Sci. 4, 2138–2148 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Nolting, B. et al. The folding pathway of a protein at high resolution from microseconds to seconds. Proc. Natl Acad. Sci. USA 94, 826–830 (1997).

    CAS  PubMed  Google Scholar 

  24. Zaidi, F. N., Nath, U. & Udgaonkar, J. B. Multiple intermediates and transition states during protein unfolding. Nat. Struct. Biol. 4, 1016–1024 (1997).

    CAS  PubMed  Google Scholar 

  25. Park, C. & Marqusee, S. Probing the high energy states in proteins by proteolysis. J. Mol. Biol. 343, 1467–1476 (2004).

    CAS  PubMed  Google Scholar 

  26. Park, C. Probing transient partial unfolding in proteins by native‐state proteolysis.Bio. Des.3, 117–128.

  27. Bard, J. A. M., Bashore, C., Dong, K. C. & Martin, A. The 26S proteasome utilizes a kinetic gateway to prioritize substrate degradation. Cell 177, 286–298.e15 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Bashore, C. et al. Ubp6 deubiquitinase controls conformational dynamics and substrate degradation of the 26S proteasome. Nat. Struct. Mol. Biol. 22, 1–10 (2015).

    Google Scholar 

  29. Chojnacki, M. et al. Polyubiquitin-photoactivatable crosslinking reagents for mapping ubiquitin interactome identify Rpn1 as a proteasome ubiquitin-associating subunit. Cell Chem. Biol. 24, 443–457.e6 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Lee, C., Schwartz, M. P., Prakash, S., Iwakura, M. & Matouschek, A. ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal. Mol. Cell 7, 627–637 (2001).

    CAS  PubMed  Google Scholar 

  31. Twomey, E. C. et al. Substrate processing by the Cdc48 ATPase complex is initiated by ubiquitin unfolding. Science 365, eaax1033 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. De la Peña, A. H., Goodall, E. A., Gates, S. N., Lander, G. C. & Martin, A. Substrate-engaged 26S proteasome structures reveal mechanisms for ATP-hydrolysis–driven translocation. Science 362, eaav0725 (2018).

  33. Worden, E. J., Dong, K. C. & Martin, A. An AAA motor-driven mechanical switch in Rpn11 Controls Deubiquitination at the 26S Proteasome. Mol. Cell 67, 799–811.e8 (2017).

    CAS  PubMed  Google Scholar 

  34. Greene, E. R. et al. Specific lid-base contacts in the 26S proteasome control the conformational switching required for substrate engagement and degradation. eLife 8, https://doi.org/10.1101/687921 (2019).

  35. Reichard, E. L. et al. Substrate ubiquitination controls the unfolding ability of the proteasome. J. Biol. Chem. 291, jbc.M116.720151 (2016).

    Google Scholar 

  36. Guo, Q. et al. In situ structure of neuronal C9orf72 poly-GA aggregates reveals proteasome recruitment. Cell 172, 696–705.e12 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Komander, D. & Rape, M. The ubiquitin code. Annu. Rev. Biochem. 81, 203–229 (2012).

    CAS  PubMed  Google Scholar 

  38. Sakamoto, K. M. et al. Protacs: chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation. Proc. Natl Acad. Sci. USA 98, 8554–8559 (2001).

    CAS  PubMed  Google Scholar 

  39. Nowak, R. P. et al. Plasticity in binding confers selectivity in ligand-induced protein degradation article. Nat. Chem. Biol. 14, 706–714 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Smith, B. E. et al. Differential PROTAC substrate specificity dictated by orientation of recruited E3 ligase. Nat. Commun. 10, 1–13 (2019).

    Google Scholar 

  41. Huang, H. T. et al. A chemoproteomic approach to query the degradable kinome using a multi-kinase degrader. Cell Chem. Biol. 25, 88–99.e6 (2018).

    CAS  PubMed  Google Scholar 

  42. Bondeson, D. P. et al. Lessons in PROTAC design from selective degradation with a promiscuous warhead. Cell Chem. Biol. 25, 78–87.e5 (2018).

    CAS  PubMed  Google Scholar 

  43. Batey, S., Nickson, A. A. & Clarke, J. Studying the folding of multidomain proteins. HFSP J. 2, 365–377 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Carrion-Vazquez, M. et al. The mechanical stability of ubiquitin is linkage dependent. Nat. Struct. Biol. 10, 738–743 (2003).

    CAS  PubMed  Google Scholar 

  45. Morimoto, D. et al. The unexpected role of polyubiquitin chains in the formation of fibrillar aggregates. Nat. Commun. 6, 6116 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Sousa, R. & Lafer, E. M. The physics of entropic pulling: a novel model for the Hsp70 motor mechanism. Int. J. Mol. Sci. 20, 2334 (2019).

    CAS  PubMed Central  Google Scholar 

  47. Freudenthal, B. D., Gakhar, L., Ramaswamy, S. & Washington, M. T. Structure of monoubiquitinated PCNA and implications for translesion synthesis and DNA polymerase exchange. Nat. Struct. Mol. Biol. 17, 479–484 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Varadan, R., Walker, O., Pickart, C. & Fushman, D. Structural properties of polyubiquitin chains in solution. J. Mol. Biol. 324, 637–647 (2002).

    CAS  PubMed  Google Scholar 

  49. Eddins, M. J., Varadan, R., Fushman, D., Pickart, C. M. & Wolberger, C. Crystal structure and solution NMR studies of Lys48-linked tetraubiquitin at neutral pH. J. Mol. Biol. 367, 204–211 (2007).

    CAS  PubMed  Google Scholar 

  50. Debelouchina, G. T., Gerecht, K. & Muir, T. W. Ubiquitin utilizes an acidic surface patch to alter chromatin structure. Nat. Chem. Biol. 13, 105–110 (2017).

    CAS  PubMed  Google Scholar 

  51. Beckwith, R., Estrin, E., Worden, E. J. & Martin, A. Reconstitution of the 26S proteasome reveals functional asymmetries in its AAA+ unfoldase. Nat. Struct. Mol. Biol. 20, 1164–1172 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Matyskiela, M. E., Lander, G. C. & Martin, A. Conformational switching of the 26S proteasome enables substrate degradation. Nat. Struct. Mol. Biol. 20, 781–788 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Pollard, T. D. MBOC technical perspective: a guide to simple and informative binding assays. Mol. Biol. Cell 21, 4061–4067 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank all members of the Marqusee and Martin laboratories for helpful discussions. We also thank B. Maguire and K. Dong for assistance with protein purification and troubleshooting expertise. We acknowledge support from the US National Institutes of Health: grant nos. R01-GM050945 (S.M.) and R01-GM094497 (A.M.). S.M. is a Chan Zuckerberg Biohub investigator. A.M. is an investigator of the Howard Hughes Medical Institute.

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E.C.C. and E.R.G. performed the experiments and analyzed data. E.C.C., E.R.G., A.M. and S.M. contributed to experimental design, data interpretation and manuscript preparation.

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Correspondence to Andreas Martin or Susan Marqusee.

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Source Data Fig. 2

Full uncropped gels from Fig. 2

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Full uncropped gels from Fig. 3

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Full uncropped gels from Fig. 4

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Carroll, E.C., Greene, E.R., Martin, A. et al. Site-specific ubiquitination affects protein energetics and proteasomal degradation. Nat Chem Biol 16, 866–875 (2020). https://doi.org/10.1038/s41589-020-0556-3

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