Mechanism of selective recognition of Lys48-linked polyubiquitin by macrocyclic peptide inhibitors of proteasomal degradation

De novo Semi-Synthetic Platform for Monitoring Protein degradation in Live Cells Mahdi Hasan,[a] Deepanjan Panda,[a] Guy Mann,[a] and Ashraf Brik*[a] We designed a platform for monitoring the degradation of exogenous proteins in live cells. We engineered a semisynthetic platform, which consists of Enhanced Green Fluorescent Protein tagged with SpyCatcher to enable its conjugation to a SpyTag peptide bearing a Von Hippel–Lindau E3 ligand, which was delivered to live cells to promote its degradation. This platform lays the ground for studying the degradation of endogenous proteins equipped with SpyTag and for tracking the degradation of post-translationally modified proteins in live cells. Introduction Depletion or inactivation of proteins is a powerful tool for evaluating their involvement in cellular pathways and medicine. Genetic manipulation such as the use of CRISPR or siRNA are commonly used approaches for such a goal. On the other hand, Inducible degron technologies were also developed to complement the genetic approaches, yet with the possibility to use small molecules to mediate protein degradation.[1] Proteolysis targeting chimera (PROTAC) is promising for both the development of therapeutics and studying protein turnover in biological pathways.[2] This method relies on a heterobifunctional molecule containing ligands for both a E3 ligase and protein of interest (POI) to induce its ubiquitination and subsequent degradation via the ubiquitin proteasome system (UPS). The development of PROTAC depends on a known POI binder and a suitable selection of an E3 recruiter. Although advanced progress has been made in the development of small molecule ligands for several proteins, developing a PROTAC requires significant efforts to optimize such a bifunctional molecule, containing a specific linker.[3] Moreover, for many POIs there is a lack for a specific ligand to be used as part of the PROTAC, and the system requires extensive validation for the biological effect and its phenotype. To overcome the limitation of PROTAC in studying the effect of knockdown of POI, several degron-based technologies have been developed, for example, dTAG,[4] Auxin Inducible Degron,[5] Halo PROTAC[6] and more.[7–9] Engineering degron technologies require a recombinant expression of a degron system, or genetically modifying the DNA to introduce the degrader to the POI.[10] Moreover, these methods are still restricted by the existence of a few ligands for E3 ligases and are still limited to the degradation of endogenously expressed proteins. Recent advances in chemical protein synthesis and semisynthesis enable to site specifically modified proteins with variety of posttranslational modifications (PTMs) for biological and structural studies. However, monitoring the degradation of such modified proteins and therefore dissecting the role of these PTMs in this process inside live cells is still challenging and require huge effort to determine their cross talk with PTMs in aspects of degradation. To overcome these limitations, we thought to design a novel platform that is flexible and systematically can be manipulated as well as can be activated on demand. Here we report our first steps towards building a semi-synthetic platform based on SpyTag/SpyCatcher system that allows tagging the POI with E3 ligand in test-tube, followed by cell delivery to monitor its degradation via the UPS (Figure 1). [a] M. Hasan, Dr. D. Panda, Dr. G. Mann, Prof. Dr. A. Brik Schulich Faculty of Chemistry Technion-Israel Institute of Technology Haifa, 3200008 (Israel) E-mail: [email protected] Supporting information for this article is available on the WWW under https://doi.org/10.1002/cbic.202300731 © 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is noncommercial and no modifications or adaptations are made. Figure 1. Schematic presentation of our strategy to engineer an exogenous EGFP conjugate using the SpyCatcher/SpyTag system and its delivery via bead loading to promote its degradation in live cells. Wiley VCH Donnerstag, 18.01.2024 2403 / 331897 [S. 120/124] 1 ChemBioChem 2024,25, e202300731 (1 of 5) © 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH ChemBioChem www.chembiochem.org Research Article doi.org/10.1002/cbic.202300731 Results and Discussion To design our platform, we thought to take advantage of the SpyTag003/SpyCatcher003[11,12] to link the E3 ligand to the POI. The SpyTag/SpyCatcher elements rapidly form an isopeptide bond between the SpyTag, a short peptide made of 16 residues, and the SpyCatcher, which is a small protein made of 113 residues. Therefore, our design includes first, an expressed POI linked to SpyCatcher while the complementary part will be synthetically made to include the E3 ligand. To execute this design in a simplified system, we chose the Enhanced Green Fluorescent Protein (EGFP) as a model substrate to enable live cell monitoring of its degradation by flow cytometry. Notably, EGFP has been used in many studies to shed light on the different parameters that influence proteasomal degradation.[13,14] This design could be also modified with any synthetic or expressed POI fused to EGFP as a reporter of degradation, which its signal could be easily monitored by flow cytometry. Furthermore, tracking the degradation of the substrate in the absence of EGFP could be achieved via a western blot analysis to avoid tagging the POI with a large protein. Therefore, we expressed and purified EGFP fused to SpyCatcher, as our model system (EGFP-SpyCatcher), which was obtained in a satisfactory amount (Supporting Information Figure S2). The SpyTag peptide was chemically prepared using solid phase peptide synthesis (SPPS) on a Rink amide resin, as described in Scheme 1A. Chemical synthesis of this peptide should allow us to straightforwardly manipulate it, where different E3 ligands can be coupled at different positions on the SpyTag peptide. For a proof of concept, we choose the Von Hippel-Lindau (VHL) ligand, as an E3 recruiter, which has proved as an effective E3 ligand in different PROTACs.[15,16] To investigate the effect of the linker between the EGFPSpyCatcher and the Cand the N-terminus position of E3 ligand, we prepared five analogues of SpyTag peptides linked VHL ligand termed as Spy-V(1-5), (Scheme 1B&C). While the coupling of the VHL to the N-terminus of the SpyTag was straightforward, the attachment to the C-terminus required the incorporation of Alloc-protected Lys to allow after completion of the synthesis to attach the VHL ligand. These peptides were synthesized in very good yields and high purities (Supporting Information Figure S1). With the EGFP-SpyCatcher and the different Spy-V(1-5) peptides in hand, we then attempted the synthesis of the desired final conjugates. Therefore, the EGFPSpyCatcher and the different Spy-V(1-5) were spontaneously reacted at PBS buffer to produce the semisynthetic constructs termed as EGFP(1-5) respectively. Each reaction was monitored by RP-HPLC coupled with MS, indicating a complete reaction within 5 min (Figure 2, Supporting Information Figure S3). To examine whether the EGFP(1-5) analogues undergo ubiquitination, we tested these constructs in a cell free system, using active VHL complex and followed the ubiquitination level of each of them, using western blotting against EGFP. Our results clearly showed that the constructs, after incubation with the VHL complex appeared as a smeared bands on the SDSPAGE, indicated the presence of polyubiquitinated EGFP, (FigScheme 1. Synthetic scheme of SpyTag and Spy-V (1-5) peptides. (A) General synthetic scheme of the SpyTag. (B) synthetic scheme of Spy-V1 and Spy-V (3-5) bearing a VHL ligand at the N-terminal. (C) synthetic scheme of Spy-V2 bearing a VHL ligand at the C-terminal lysine side chain (labeled in red). Figure 2. (A) Schematic presentation of the Synthesis of the EGFP (1-5) conjugates. Represented HPLC and MS analysis of the reaction of the EFGPSpyCatcher with Spy-V1 peptide (B&C). The reaction was monitored at t =0 (B), Peak a corresponds to the Spy-V1 peptide, which peak b corresponds to the EFGP-SpyCatcher and after t=5 min incubation (C), Peak c corresponds to EGFP1 conjugate using RP-HPLC and mass spectrometry (EGFP1, observed mass=42796�4 Da, calculated mass=42793 Da). Wiley VCH Donnerstag, 18.01.2024 2403 / 331897 [S. 121/124] 1 ChemBioChem 2024,25, e202300731 (2 of 5) © 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH ChemBioChem Research Article doi.org/10.1002/cbic.202300731 14397633, 2024, 3, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cbic.202300731 by Technion-Israel Institution Of, Wiley Online Library on [05/11/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License ure 3A, Supporting Information Figure S4). Importantly, when we used the modified EGFP with the SpyTag lacking the E3 ligand (EGFP-ST) we did not observe any ubiquitination bands, supporting the integrity of our platform (Figure 3A, Supporting Information Figure S4). Next, we aimed to examine the behavior of these constructs in live cells and their degradation via the UPS by delivering them to live cells. We chose to deliver the EGFP constructs to live cells via the bead loading approach, which is based on cellbeads collision that generates a stochastic cell‘s disruption on the plasma membrane, allowing for the surrounding cargo to be delivered to adherent cells.[17,18] Notably, using this method has been limited for cell imaging and localization, where inconsistent delivery outcomes is not critical.[19] Recently, we achieved multiplex delivery of four synthetic proteins based on this approach for studying the localization of ubiquitin and SUMO analogues.[20,21] In this study, we optimized the bead loading method by applying on the same culture plates nearly equal amounts of glass beads and simultaneous shaking of all plates to reach homogenous level of the protein delivery for each culture plate. This was verified by measuring the amount of the EGFP signal in the different plates, using flow cytometry and western blot. Using these conditions enabled us to deliver homogeneously the EGFP constructs and monitor their degradation in live cells. We tested the degradation of the EGFP(1-5) analogues and compared the EGFP signal without SpyTag and EGFP-SpyTag without VHL ligand (EGFP-ST). More specifically, after delivery and washing, we monitored the fluorescent intensity after 6 and 16 hours and normalized the intensity mean to the control construct i.e., EGFP-SpyCatcher (Figure 3, Supporting Information Figure S5). Our results showed a significant degradation of EGFP(1-5) compared to the two EGFP control systems. The degradation rates of the constructs were estimated between 55–70% after 16 hours and around 50% after 6 hours according to the flow cytometry results (Figure 3 & Figure S5 in the Supporting Information). These results indicate that longer linkers between the SpyTag and the VHL ligand could not affect the ternary complex of the SpyTag/SpyCatcher with the VHL ligase in this system (Figure 3, Supporting Information FigFigure 3. (A) In vitro ubiquitination assay using VHL complex. (B) WB analyses of the degradation of EGFP constructs in U2OS cells. The EGFP intensity was normalized to the GAPDH intensity. (C) Flow cytometry analyses of the degradation of EGFP constructs, after 16 h incubation in U2OS cells. (D) degradation ratio of EGFP constructs in U2OS cells in the presence of proteasome inhibitor, MG132. Data are presented as normalized fluorescent mean �SEM of three (C) or two (D) independent biological repeats with �10000 cells. n.s indicates for non-significant results, the level of significance between the indicated treatments determined as: *p<=0.05, **p<0.01, ***p <0.001. Wiley VCH Donnerstag, 18.01.2024 2403 / 331897 [S. 122/124] 1 ChemBioChem 2024,25, e202300731 (3 of 5) © 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH ChemBioChem Research Article doi.org/10.1002/cbic.202300731 14397633, 2024, 3, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cbic.202300731 by Technion-Israel Institution Of, Wiley Online Library on [05/11/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License ure S5). Our results indicate that the degradation of the constructs was higher after long incubation time. Our constructs are self-induced for degradation rather than being PROTAC and catalytic. It is possible that the degradation of these constructs has appeared after a longer incubation time due to saturation of the delivered exogenous proteins inside the cell. Furthermore, when cells were treated with the proteasome inhibitor MG132, we observed no degradation of our constructs, further supporting our design of this platform (Figure 3D, Supporting Information Figure S5B). To verify our platform, we attempted to induce the degradation of endogenous proteins, we tested whether the SpyTag linked with E3 ligand can induce the degradation of overexpressed EGFP-SpyCatcher. We prepared stable cell lines that overexpressed EGFP-SpyCatcher. To these cell lines, we delivered Spy-V1 by bead loading and followed the EGFP degradation using flow cytometry, our results indicated degradation of the EGFP, which was estimated at around 25 % degradation of the overexpressed EGFP, after 1, 2, and 4 hours, and 13% degradation after 16 hours compared to the untreated cells. The constant degradation rate after short incubation times is assumed to be because of the noncatalytic nature of our system, moreover, lower degradation rate was observed after 16 hours of incubation, which is due to continued overexpression of the POI (Figure 4). Conclusions In this study, we have developed a first generation of novel platform for monitoring the degradation of exogenous semisynthetic proteins in live cells. We employed SpyCatcher/ SpyTag system to modify EGFP with VHL ligand and used the bead loading approach to deliver it to live cells and monitor its proteasomal degradation. This platform is amenable to straightforward variations as demonstrated by the synthesis of short peptides having the VHL on different positions and linked to the peptide via various linkers. Our platform should allow rapid examination of different E3 ligands, as well as to introduce caged E3 such as the photocleavable VHL and bioorthogonal PROTAC that were recently developed.[22,23] In addition, one can also introduce caged SpyCatcher/SpyTag to control their reactivity in cells. We began using genetic approaches to introduce a specific POI modified with SpyCatcher and trigger its degradation by delivering synthetic SpyTag modified with a desired E3 ligand. We are currently attempting to further improve on relevant proteins with normal expression levels using CRISPR knock-in method. Moreover, Since the Asp-Ala mutant in the SpyTag still exhibits strong non-covalent interaction with the SpyCatcher,[11] this could turn our platform to be catalytic. These efforts and others are currently being examined in our laboratory. Supporting Information The authors have cited additional references within the Supporting Information.[24] Acknowledgements A.B. holds The Jordan and Irene Tark Academic Chair. This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation Program (grant agreement no. 831783). M.H acknowledges the support of the VATAT Scholarship. Some of the figures were prepared with biorender.com. We also thank Yousef Mansour and Dr. Aviv Lutaty for their assistance in the flow cytometry experiments. We are grateful to Prof. Nabieh Ayoub for providing us with the plasmids for the lentivirus system. Conflict of Interests There are no conflicts to declare. Data Availability Statement The data that support the findings of this study are available in the supplementary material of this article. Keywords: Proteasomal degradation ·SpyTag/SpyCatcher · Bead loading delivery ·Peptide synthesis Figure 4. Flow cytometry analysis of the degradation of overexpressed EGFPSpyCatcher U2OS cells after 1, 2, 4, or 16 hours. SpyTag or Spy-V1 were delivered to U2OS via bead loading, the fluorescent intensity was normalized to DMSO treated cells (Control). Data are presented as normalized fluorescent mean �SEM of two independent biological repeats with �10000 cells. The level of significance between the indicated treatments determined as: *p<=0.05, **p <0.01, ***p<0.001. Wiley VCH Donnerstag, 18.01.2024 2403 / 331897 [S. 123/124] 1 ChemBioChem 2024,25, e202300731 (4 of 5) © 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH ChemBioChem Research Article doi.org/10.1002/cbic.202300731 14397633, 2024, 3, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cbic.202300731 by Technion-Israel Institution Of, Wiley Online Library on [05/11/2025]. 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Wang, J. Am. Chem. Soc. 2023,145, 14155–14163. [24] S. Lee, D. Y. Chen, J. S. Humphrey, J. R. Gnarra, W. M. Linehan, R. D. Klausner, Proc. Natl. Acad. Sci. USA 1996,93, 1770–1775. Manuscript received: October 24, 2023 Revised manuscript received: November 26, 2023 Accepted manuscript online: November 30, 2023 Version of record online: December 8, 2023 Wiley VCH Donnerstag, 18.01.2024 2403 / 331897 [S. 124/124] 1 ChemBioChem 2024,25, e202300731 (5 of 5) © 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH ChemBioChem Research Article doi.org/10.1002/cbic.202300731 14397633, 2024, 3, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cbic.202300731 by Technion-Israel Institution Of, Wiley Online Library on [05/11/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License