Reversible surface modifications of functional proteins for accelerated cytosolic delivery via cell-penetrating peptide clusters

reversible-surface-modifications-of-functional-proteins-for-accelerated-cytosolic-delivery-via-cell-penetrating-peptide-clusters
Reversible surface modifications of functional proteins for accelerated cytosolic delivery via cell-penetrating peptide clusters

References

  1. Carter, P. J. & Lazar, G. A. Next generation antibody drugs: pursuit of the ‘high-hanging fruit’. Nat. Rev. Drug Discov. 17, 197–223 (2018).

    Google Scholar 

  2. Schneider, A. F. L., Benz, L. S., Lehmann, M. & Hackenberger, C. P. R. Cell-permeable nanobodies allow dual-color super-resolution microscopy in untransfected living cells. Angew. Chem. Int. Ed. 60, 22075–22080 (2021).

    Google Scholar 

  3. Mann, G., Sadhu, P. & Brik, A. Synthetic proteins behind the plasma barrier: molecular spies. Acc. Chem. Res. 55, 2055–2067 (2022).

    Google Scholar 

  4. Stewart, M. P. et al. In vitro and ex vivo strategies for intracellular delivery. Nature 538, 183–192 (2016).

    Google Scholar 

  5. Saha, A. et al. Suspension bead loading (SBL): An economical protein delivery platform to study URM1’s behavior in live cells. Angew. Chem. Int. Ed. 63, e202410135 (2024).

    Google Scholar 

  6. Lawrence, M. S., Phillips, K. J. & Liu, D. R. Supercharging proteins can impart unusual resilience. J. Am. Chem. Soc. 129, 10110–10112 (2007).

    Google Scholar 

  7. Mandal, S., Mann, G., Satish, G. & Brik, A. Enhanced live-cell delivery of synthetic proteins assisted by cell-penetrating peptides fused to DABCYL. Angew. Chem. Int. Ed. 60, 7333–7343 (2021).

    Google Scholar 

  8. Dougherty, P. G., Sahni, A. & Pei, D. Understanding cell penetration of cyclic peptides. Chem. Rev. 119, 10241–10287 (2019).

    Google Scholar 

  9. Nischan, N. et al. Covalent attachment of cyclic TAT peptides to GFP results in protein delivery into live cells with immediate bioavailability. Angew. Chem. Int. Ed. 54, 1950–1953 (2015).

    Google Scholar 

  10. Herce, H. D. et al. Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells. Nat. Chem. 9, 762–771 (2017).

    Google Scholar 

  11. Schneider, A. F. L., Kithil, M., Cardoso, M. C., Lehmann, M. & Hackenberger, C. P. R. Cellular uptake of large biomolecules enabled by cell-surface-reactive cell-penetrating peptide additives. Nat. Chem. 13, 530–539 (2021).

    Google Scholar 

  12. Erazo-Oliveras, A. et al. Protein delivery into live cells by incubation with an endosomolytic agent. Nat. Methods 11, 861–867 (2014).

    Google Scholar 

  13. Akishiba, M. et al. Cytosolic antibody delivery by lipid-sensitive endosomolytic peptide. Nat. Chem. 9, 751–761 (2017).

    Google Scholar 

  14. Tietz, O., Cortezon-Tamarit, F., Chalk, R., Able, S. & Vallis, K. A. Tricyclic cell-penetrating peptides for efficient delivery of functional antibodies into cancer cells. Nat. Chem. 14, 284–293 (2022).

    Google Scholar 

  15. Meledin, R., Mali, S. M., Kleifeld, O. & Brik, A. Activity-based probes developed by applying a sequential dehydroalanine formation strategy to expressed proteins reveal a potential α-Globin-modulating deubiquitinase. Angew. Chem. Int. Ed. 57, 5645–5649 (2018).

    Google Scholar 

  16. Mishra, V. et al. IL-1β turnover by the UBE2L3 ubiquitin conjugating enzyme and HECT E3 ligases limits inflammation. Nat. Commun. 14, 4385 (2023).

    Google Scholar 

  17. Chen, J., Ai, Y., Wang, J., Haracska, L. & Zhuang, Z. Chemically ubiquitylated PCNA as a probe for eukaryotic translesion DNA synthesis. Nat. Chem. Biol. 6, 270–272 (2010).

    Google Scholar 

  18. Brock, D. J. et al. Efficient cell delivery mediated by lipid-specific endosomal escape of supercharged branched peptides. Traffic 19, 421–435 (2018).

    Google Scholar 

  19. Liu, Z. et al. The antioxidant activity and genotoxicity of isogarcinol. Food Chem 253, 5–12 (2018).

    Google Scholar 

  20. Pei, D. & Buyanova, M. Overcoming endosomal entrapment in drug delivery. Bioconjugate Chem 30, 273–283 (2019).

    Google Scholar 

  21. Giustarini, D., Dalle-Donne, I., Milzani, A., Fanti, P. & Rossi, R. Analysis of GSH and GSSG after derivatization with N-ethylmaleimide. Nat. Protoc. 8, 1660–1669 (2013).

    Google Scholar 

  22. Angeles-Boza, A. M., Erazo-Oliveras, A., Lee, Y.-J. & Pellois, J.-P. Generation of endosomolytic reagents by branching of cell-penetrating peptides: tools for the delivery of bioactive compounds to live cells in cis or trans. Bioconjugate Chem 21, 2164–2167 (2010).

    Google Scholar 

  23. Costes, S. V. et al. Automatic and quantitative measurement of protein-protein colocalization in live cells. Biophys. J. 86, 3993–4003 (2004).

    Google Scholar 

  24. Teo, S. L. Y. et al. Unravelling cytosolic delivery of cell penetrating peptides with a quantitative endosomal escape assay. Nat. Commun. 12, 3721 (2021).

    Google Scholar 

  25. Goldenthal, K. L., Pastan, I. & Willingham, M. C. Initial steps in receptor-mediated endocytosis. The influence of temperature on the shape and distribution of plasma membrane clathrin-coated pits in cultured mammalian cells. Exp. Cell Res. 152, 558–564 (1984).

    Google Scholar 

  26. Hunt, L. et al. Low-temperature pausing of cultivated mammalian cells. Biotechnol. Bioeng. 89, 157–163 (2005).

    Google Scholar 

  27. Hirose, H. et al. Transient focal membrane deformation induced by arginine-rich peptides leads to their direct penetration into cells. Mol. Ther. 20, 984–993 (2012).

    Google Scholar 

  28. Sun, Y. et al. Phase-separating peptides for direct cytosolic delivery and redox-activated release of macromolecular therapeutics. Nat. Chem. 14, 274–283 (2022).

    Google Scholar 

  29. Sangsuwan, R., Tachachartvanich, P. & Francis, M. B. Cytosolic delivery of proteins using amphiphilic polymers with 2-pyridinecarboxaldehyde groups for site-selective attachment. J. Am. Chem. Soc. 141, 2376–2383 (2019).

    Google Scholar 

  30. Lin, Q. et al. Imaging the cytosolic drug delivery mechanism of HDL-like nanoparticles. Pharm. Res. 31, 1438–1449 (2014).

    Google Scholar 

  31. Panja, P. & Jana, N. R. Lipid-raft-mediated direct cytosolic delivery of polymer-coated soft nanoparticles. J. Phys. Chem. B 124, 5323–5333 (2020).

    Google Scholar 

  32. Los, D. A., Mironov, K. S. & Allakhverdiev, S. I. Regulatory role of membrane fluidity in gene expression and physiological functions. Photosynth. Res. 116, 489–509 (2013).

    Google Scholar 

  33. Singh, R., Kats, L., Blättler, W. A. & Lambert, J. M. Formation of N-substituted 2-iminothiolanes when amino groups in proteins and peptides are modified by 2-iminothiolane. Anal. Biochem. 236, 114–125 (1996).

    Google Scholar 

  34. Pjura, P. E., Grzeskowiak, K. & Dickerson, R. E. Binding of Hoechst 33258 to the minor groove of B-DNA. J. Mol. Biol. 197, 257–271 (1987).

    Google Scholar 

  35. Riedl, J. et al. Lifeact: a versatile marker to visualize F-actin. Nat. Methods 5, 605–607 (2008).

    Google Scholar 

  36. Ren, L., Lv, J., Wang, H. & Cheng, Y. A coordinative dendrimer achieves excellent efficiency in cytosolic protein and peptide delivery. Angew. Chem. Int. Ed. 59, 4711–4719 (2020).

    Google Scholar 

  37. Zhang, S. et al. Robust reversible cross-linking strategy for intracellular protein delivery with excellent serum tolerance. Nano Lett 22, 8233–8240 (2022).

    Google Scholar 

  38. Le, Z. et al. Direct cytosolic delivery of proteins and CRISPR-Cas9 genome editing by gemini amphiphiles via non-endocytic translocation pathways. ACS Cent. Sci. 9, 1313–1326 (2023).

    Google Scholar 

  39. Chuah, J.-A. & Numata, K. Stimulus-responsive peptide for effective delivery and release of DNA in plants. Biomacromolecules 19, 1154–1163 (2018).

    Google Scholar 

  40. Wu, H., Santana, I., Dansie, J. & Giraldo, J. P. In vivo delivery of nanoparticles into plant leaves. Curr. Protoc. Chem. Biol. 9, 269–284 (2017).

    Google Scholar 

  41. David, Y., Vila-Perelló, M., Verma, S. & Muir, T. W. Chemical tagging and customizing of cellular chromatin states using ultrafast trans-splicing inteins. Nat. Chem. 7, 394–402 (2015).

    Google Scholar 

  42. Demirer, G. S. et al. Carbon nanocarriers deliver siRNA to intact plant cells for efficient gene knockdown. Sci. Adv. 6, eaaz0495 (2020).

    Google Scholar 

  43. Arafiles, J. V. V. et al. Cell-surface-retained peptide additives for the cytosolic delivery of functional proteins. J. Am. Chem. Soc. 145, 24535–24548 (2023).

    Google Scholar 

  44. Deshaies, R. J. & Joazeiro, C. A. RING domain E3 ubiquitin ligases. Annu. Rev. Biochem. 78, 399–434 (2009).

    Google Scholar 

  45. Mathur, S., Fletcher, A. J., Branigan, E., Hay, R. T. & Virdee, S. Photocrosslinking activity-based probes for ubiquitin RING E3 Ligases. Cell Chem. Biol. 27, 74–82 (2020).

    Google Scholar 

  46. Dove, K. K. et al. Structural studies of HHARI/UbcH7Ub reveal unique E2Ub conformational restriction by RBR RING1. Structure 25, 890–900 (2017).

    Google Scholar 

  47. Pan, M. et al. Quasi-racemic X-ray structures of K27-linked ubiquitin chains prepared by total chemical synthesis. J. Am. Chem. Soc. 138, 7429–7435 (2016).

    Google Scholar 

  48. Ai, H., Pan, M. & Liu, L. Chemical synthesis of human proteoforms and application in biomedicine. ACS Cent. Sci. 10, 1442–1459 (2024).

    Google Scholar 

  49. Chu, G.-C. et al. Ferricyanide-promoted oxidative activation and ligation of protein thioacids in neutral aqueous media. CCS Chem 6, 2031–2043 (2024).

    Google Scholar 

  50. Lechtenberg, B. C. et al. Structure of a HOIP/E2~ubiquitin complex reveals RBR E3 ligase mechanism and regulation. Nature 529, 546–550 (2016).

    Google Scholar 

  51. Hua, F., Hao, W., Wang, L. & Li, S. Linear ubiquitination mediates EGFR-induced NF-κB pathway and tumor development. Int. J. Mol. Sci. 22, 11875 (2021).

    Google Scholar 

  52. Mix, K. A., Lomax, J. E. & Raines, R. T. Cytosolic delivery of proteins by bioreversible esterification. J. Am. Chem. Soc. 139, 14396–14398 (2017).

    Google Scholar 

  53. Dong, S. et al. Recent advances in chemical protein synthesis: method developments and biological applications. Sci. China Chem. 67, 1060–1096 (2024).

    Google Scholar 

  54. Yang, Z. et al. Advances in the chemical synthesis of human proteoforms. Sci. China Life Sci. 68, 2515–2549 (2025).

    Google Scholar 

  55. Du, Y. et al. Mechanistic insights into the stimulation of the histone H3K9 methyltransferase Clr4 by proximal H3K14 ubiquitination. Sci. Adv. 11, eadu1864 (2025).

    Google Scholar 

Download references