Suspension Bead Loading (SBL): An Economical Protein Delivery Platform to Study URM1's Behavior in Live Cells

Chemical Biology Suspension Bead Loading (SBL): An Economical Protein Delivery Platform to Study URM1’s Behavior in Live Cells Abhishek Saha+, Reem Mousa+, Yam Alalouf, Pradeep Sadhu, Mahdi Hasan, Shaswati Mandal, Guy Mann, and Ashraf Brik* Abstract: Uniquely modified synthetic proteins are difficult to produce in large quantities, which could limit their use in various in vitro settings and in cellular studies. In this study, we developed a method named “suspension bead loading” (SBL), to deliver protein molecules into suspended living cells using glass beads, which significantly reduces the amount of protein required for effective delivery. We investigated the delivery efficiency of functionally different proteins and evaluated the cytotoxic effect of our method and the chemical and functional integrity of the delivered protein. We utilized SBL to address questions related to ubiquitin-related modifier 1 (URM1). Employing minimal protein quantities, SBL has enabled us to study its behavior within live cells under different redox conditions, including subcellular localization and conjugation patterns. We demonstrate that oxidative stress alters both the localization and conjugation pattern of URM1 in cells, highlighting its possible role in cellular response to such extreme conditions. Introduction Uniquely modified synthetic proteins are designed to answer fundamental biological questions by incorporating elements that are usually inaccessible or difficult to achieve via recombinant expression.[1–4] Chemical protein synthesis allows variations in protein‘s chemical composition, including side chain and backbone modifications. It allows access to site-specific post-translationally modified proteins, as well as activity-based probes. However, obtaining large quantities of uniquely modified proteins via chemical synthesis is challenging, which could limit their applications in structural, biochemical, and functional studies. The latter requires the delivery of these costume-made proteins into cells which is often challenging due to their large size and polar nature; hence, utilization of a delivery method is necessary to cross the plasma membrane.[5–8] An efficient delivery approach is transiently attaching a protein cargo to a cell-penetrating peptide (CPP),[9,10] However, the efficiency may vary from one protein cargo to another depending on their physiochemical properties.[11,12] Additionally, several delivery methods, including CPPs, could produce background noise during cell imaging due to endosomal entrapment of cargo molecules, limiting their applicability in fundamental biological studies.[13] Therefore, developing a general method, applicable to various protein cargos, regardless of their type and size, that enables studies in different biological systems, while using a minimal amount of protein, especially synthetic protein, is highly desirable. Cell delivery using physical methods (e.g., microinjection, electroporation, sonoporation) offers a great alternative,[14] as it does not require chemical manipulations of the cargo molecule and is less prone to endosomal entrapment. In this regard, bead loading (BL) is an excellent platform for research as it induces minimal cellular stress, requires no specific instruments, and can be applied to various cell types and cargoes.[15–17] Recently, we reported the use of a BL approach for the cellular delivery of multiple synthetic proteins at once. Using multiplexed bead loading (MBL), we were able to deliver up to four functionally different proteins simultaneously into living cells and monitor their cellular localizations.[7,18,19] However, the delivery of functional, uniquely modified synthetic proteins to adherent cells using MBL requires large protein quantities (Figure 1), which can sometimes be particularly challenging to obtain using chemical protein synthesis. Therefore, we aimed to develop a more economical delivery approach that requires significantly lower amounts of synthetic protein for cellular studies, such as gel analysis, flow cytometry, localization, proteomics, etc. Motivated by this we aimed to deliver functional proteins in suspension using physical disruption. Here we report the “suspension bead loading” (SBL) approach, utilizing glass beads to induce physical disruption [*] Dr. A. Saha+ Birla Instandte of Technology and Science, Pilani Hyderabad Campus, Jawaharnagar Kapra Mandal, Medchal District 500078 Hyderabad, Telangana, India Dr. R. Mousa,+Y. Alalouf, P. Sadhu, M. Hasan, S. Mandal, G. Mann, Prof. A. Brik Schulich Faculty of Chemistry, Technion-Israel Institute of Technology, 3200008 Haifa (Israel) E-mail: [email protected] [+] These authors contributed equally to this work. © 2024 The Author(s). Angewandte Chemie International Edition published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. Angewandte Chemie Research Article www.angewandte.org How to cite: Angew. Chem. Int. Ed. 2024,63, e202410135 doi.org/10.1002/anie.202410135 Angew. Chem. Int. Ed. 2024,63, e202410135 (1 of 9) © 2024 The Author(s). Angewandte Chemie International Edition published by Wiley-VCH GmbH to suspended cells thus promoting protein delivery (Figure 1). Even though adherent cells are not often handled in suspension for long periods, the developed method shows no toxicity to cells and offers an acceptable proportion of loaded cells. We examined the applicability of SBL on various mammalian cell lines using functionally different proteins to study their cellular uptake and sub-cellular localization. Additionally, ubiquitin (Ub) activity-based probe (ABP) delivered by SBL was able to capture known deubiquitinating enzymes (DUBs) in living cells. Finally, we applied this method to study, for the first time, the cellular behavior of chemically synthesized ubiquitin-related modifier-1(URM1), under normal and oxidative stress conditions. Results and Discussion To examine the effectiveness of SBL, we used chemically synthesized Ub and SUMO2 analogs (1–5, Figure 2), and the commercially available enhanced green fluorescent protein (EGFP, 6). The syntheses of Ub and SUMO2 were performed using Fmoc solid phase peptide synthesis (SPPS)[5,18] and carried on 2-chlorotrityl chloride (2-CTC) and rink amide resins (Supporting Information section 4, 5, Figure S1–S5), where the synthetic proteins were also covalently modified with a fluorophore (Supporting Information section 11). Initially, we aimed to optimize the SBL method to achieve efficient cellular delivery of TAMRA-Ub (1). We applied SBL to suspended U2OS using different amounts of beads, incubation times, cargo concentrations, and solution volumes, in PBS buffer at pH 7.5, containing 0.1% non-ionic surfactant Pluronic™ F-68. Quantification of the loaded U2OS cells by flow cytometry analysis after attempting different conditions (Supporting Information section 9), showed that an amount of 1 mg of glass beads for ~106cells (10-fold cell pellet size (v/v)), solution of 4 μM of 1and 5 s vortexing followed by 10s centrifugation, were found to be critical for protein delivery (Supporting Information section 6, 7 and Figure S12). Delivery under these conditions followed by 4 h incubation at 37°C in CO2(5%) to allow for cell adherence, showed a maximum amount of cargo loading, where it reached up to ~45% loading (Figure 3a). Using the same conditions, we have also examined the delivery of 1to HeLa and HEK293T cell lines, where it showed up to ~50% and ~65% loading, respectively (Figure 3b–c). These results show that our developed SBL method produces a considerable number of loaded cells containing the protein cargo. Next, we monitored the treated cells using a laser scanning confocal microscope (LSCM) to investigate the sub-cellular localization of the delivered protein cargo. We simultaneously delivered Cy5-Ub (2) and TAMRA-SUMO2 (3), into U2OS cells using SBL. Delivery was followed by 4 h incubation at 37°C to allow for cell adherence, followed by Hoechst staining before imaging. The LSCM images showed that 2was localized both in the cytosol and nucleus, which is consistent with its known behavior[18] (Supporting Information section 7, 8 and Figure S13). Moreover, to show the applicability of multiplexed bead loading in suspension, we loaded U2OS cells with a mixture of TAMRA-SUMO2 (3) and EGFP (6) (1:1 v/v) and analyzed via LSCM. The images showed successful cell delivery of both proteins (Figure 4). As expected, SUMO2 is mainly localized in the nucleus, and inside the PML nuclear condensates (PMLNBs),[20] while EGFP was distributed throughout the cells and is excluded from PML-NBs. These findings are consistent with our earlier report applying MBL.[18] Since most physical delivery methods affect cell viability,[13,18] we investigated the cytotoxic effect of the SBL approach. We performed a conventional MTT cell-proliferation assay on U2OS cells after delivering 1(Supporting Information section 25). Results showed that >85% of the cells remained viable after performing our delivery procedure (Figure 5a). The propidium iodide (PI) based cell cycle assay (Supporting Information section 10) further supported these results and demonstrated the impact on the different Figure 1. Schematic representation of intracellular protein delivery by multiplexed bead loading (MBL) and the newly developed suspension bead loading (SBL), where glass beads are utilized to induce physical disruption to suspended cells and promote intracellular delivery of a protein of interest (POI). Created with BioRender.com. Figure 2. The synthetic proteins (1–5) employed in our study of developing SBL cell delivery, (for synthesis and analysis see Figure S1– 5). Figure 3. Cellular uptake of TAMRA-Ub (1) applying our optimized SBL conditions and quantified by standard flow cytometry analysis in (a) U2OS, (b) HeLa, and (c) HEK293T cell lines. w/o refers to without beads. Data is presented as mean values�SD, for n=3 repetitions. Angewandte Chemie Research Article Angew. Chem. Int. Ed. 2024,63, e202410135 (2 of 9) © 2024 The Author(s). Angewandte Chemie International Edition published by Wiley-VCH GmbH 15213773, 2024, 48, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/anie.202410135 by Technion-Israel Institution Of, Wiley Online Library on [06/11/2025]. 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 cell cycle phases (G1, S, and G2/M). We have compared the treated and the untreated U2OS cells at similar delivery conditions, where the quantification of cells at various cell cycle stages (G1, S, and G2/M) showed that our method did not induce a significant toxic effect on living cells (Figure 5b). The successful protein delivery along with the minimal cytotoxic effect of the SBL platform prompted us to investigate the fate of our proteins in cells. Therefore, we synthesized Bicyclo[6.1.0]non-4-yn-9-ylmethyl-4- (cyanoethynyl)phenyl)carbamate (APN-BCN) conjugated Ub (4), where we have taken 4as a model protein for strain promoted click reaction (SPAAc).[22] We incubated the cell lysate after delivering 4with 1 eq. of commercial CalFluor647-azide and measured fluorescence intensity (671 nm) using a plate reader, after excitation at 647 nm. We observed a significant increase in the fluorescence intensity of CalFluor647, indicating that 4a was formed, (Figure 6a, b). These results emphasize the competence of the click Figure 4. Live cell LSCM images of U2OS cells loaded with SUMO2 (3) (red: b, g) and EGFP (6) (green: c, h). Hoechst was used as a nuclear stain (white grey: a, f, k, p). Merged red and green channels (d, I, n, s) show the difference in cellular localization between 3 and 6. Scale bar 20 μm (Full-view: a–e, k–o) and 5 μm (zoom-view: f–j, p–t). BF stands for brightfield. Figure 5. Cytotoxicity studies for SBL using the optimized cell delivery conditions in U2OS cell lines. (a) MTT-cell proliferation assay after SBL delivery of TAMRA-Ub (1). MG132 was taken as a control. Data is presented as mean values�SD for n=3 independent experiments, each experiment was performed in sextet. (b) Cell cycle studies of U2OS cell lines after SBL delivery of 1. Data is presented as mean values�SD for n=3. Angewandte Chemie Research Article Angew. Chem. Int. Ed. 2024,63, e202410135 (3 of 9) © 2024 The Author(s). Angewandte Chemie International Edition published by Wiley-VCH GmbH 15213773, 2024, 48, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/anie.202410135 by Technion-Israel Institution Of, Wiley Online Library on [06/11/2025]. 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 reaction between the probe and the dye and confirm the integrity of 4in live cells and the potential application of the method in pull-down studies. Finally, to validate the functionality of proteins delivered to cells, we examined the conjugation of 1to endogenous Ub chains. Hence, we delivered 1to U2OS cells followed by subsequent treatment with DMSO or MG-132 that inhibits proteasomal degradation of Ub-conjugated proteins in mammalian cells. The fluorescent gel analysis shows higher Ub-conjugation of 1with MG-132 treatment. The yellow bands in Figure 6c demonstrate the expected behavior of 1, where various Ub chains were formed, affirming its cellular functionality. To further support that our method does not affect the function of the delivered protein in cells, we prepared a propargylamine (PA) ABP. DUBs catalyze the removal of Ub from target proteins and are also involved in Ub maturation, recycling, and editing.[22] Previous reports show that most of the DUBs in a human genome are cysteine proteases and conjugate to the PA functionalized Ub in cells.[23,24] To demonstrate the applicability of our SBL method in the labeling of DUBs by a specific Ub-ABP, we used TAMRA-Ub-PA (5) and delivered it to U2OS cells employing SBL. The fluorescent SDS-PAGE gel of cell lysates (Figure 6d) shows that cells treated with 5formed distinct fluorescent bands when compared to the negative controls (1), and buffer (UT), suggesting that DUBs were captured by ABP 5. To further support this observation, we prepared biotinylated Ub-PA for SBL delivery and pulldown enrichment using streptavidin beads. Proteomics analysis indicated that our synthetic probe captured various DUBs in live cells (Figure 6e & Figure S20, Section 27). This example of the physical delivery of ABP and capturing DUBs in live cells represents an interesting advancement in the field since previous studies were mainly performed on cell lysates. These findings also demonstrate the applicability of our approach to deliver different synthetic probes to live cells to interrogate cellular components. Next, we aimed to apply our method to shed light on the cellular behavior of URM1, a small protein characterized by the conserved β-grasp-fold and the C-terminal di-glycine motif (GG).[26] While URM1 shares structural features with Ub and other UBLs,[27] it is activated by an unusual mechanism that yields a unique thiocarboxylate intermediate (COSH). URM1 proceeds with the urmylation pathway via an unidentified mechanism that utilizes the E1 enzyme, yet without evidence of the involvement of E2 and E3 enzymes,[28] forming an isopeptide bond with a Lys residue of the target protein.[29] The thiocarboxylated URM1 is generated via adenylation of the carboxylate (COOH) by an ATP-dependent E1 (Uba4 in yeast and MOCS in human), which subsequently generates acyl-disulfide bond, resulting in the formation of the thiocarboxylated URM1.[30] Recent in vitro studies showed that URM1 conjugation depends solely on its thiocarboxylated C-terminus and a target protein’s redox-active cysteine, occurring even on residues beyond Lys such as Ser and Thr, regardless of the presence or absence of the characteristic GG motif.[28] Despite efforts to fill the gap in our understanding of URM1’s cellular function,[31] various key questions remain unanswered. These include its sub-cellular localization and response to oxidative stress, the impact of C-terminus functionality on its function and localization, and URM1’s ability to conjugate under normal and oxidative stress conditions. Aiming to learn more about URM1, we have designed three TAMRA-labeled URM1 analogs, where the C-terminus has different modifications: carboxylic acid (URM1-COOH), hydrazide moiety (URM1-CONHNH2) and deleted glycine at position 101 (URM1-ΔG-COOH), to study their localization and conjugation pattern under normal and oxidative stress conditions. We began our study by chemically synthesizing the 101 amino acids human URM1 via SPPS and native chemical ligation (NCL).[1,32] Since URM1’s sequence lacks a Cys residue, and its Ala residues are not strategically positioned for ligation combined with desulfurization, we decided to use a thiol-modified Asp residue for NCL combined with desulfurization.[33] Hence, Asp at position 61 was replaced by β-mercapto-Asp to promote NCL at Gly60-Asp61. Unfortunately, both segments URM1(1–60)-thioester and URM1(61–101) were challenging to synthesize, probably due to their hydrophobic nature. Additionally, difficulties arose during ligation, and the conditions of the desulfurization step led to protein aggregation and backbone cleavages. We then adopted an alternative approach where we replaced Gln at position 32 with Cys residue (Figure 7a). The thiol side chain was alkylated with bromoacetamide to obtain pseudo-glutamine,(Ψ-Gln) which differs from Gln by a single atom.[34–36] The URM1(1–31) bearing a C-terminus Figure 6. Chemical and functional characterization of protein cargoes delivered using SBL. (a) Schematic SPAAc reaction of 4 with CalFluor647-Azide through click chemistry. (b) Fluorescent intensity analysis of CalFluor647-azide before and after SPAAc reaction with the delivered 4. (c) Fluorescent gel analysis of the U2OS lysate after delivery of 1 in the presence and absence of MG-132. (d) Fluorescent gel analysis of the DUB captured by 5 against 1 as negative control. (e) Volcano-plot illustrating enriched proteins captured by Biotin-UbPA. X-axis depicts log 2-fold change and the y-axis depicts Log10 p value. The proteins highlighted in blue represent statistically significant results for Biotin-Ub-PA compared to Biotin-Ub-OH. Angewandte Chemie Research Article Angew. Chem. Int. Ed. 2024,63, e202410135 (4 of 9) © 2024 The Author(s). Angewandte Chemie International Edition published by Wiley-VCH GmbH 15213773, 2024, 48, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/anie.202410135 by Technion-Israel Institution Of, Wiley Online Library on [06/11/2025]. 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 thioester surrogate was prepared using Fmoc-SPPS on a Rink amide resin functionalized with an N-methyl diaminobenzoic acid (NMe-Dbz)[37] linker, and TAMRA with a PEG spacer at its N-terminus. The C-terminal URM1(32– 101) was also prepared by Fmoc-SPPS carried on CTC resin, resulting in URM1-COOH, and on hydrazide-CTC resin to produce URM1-CONHNH2.[38] The hydrazide functionality meant to serve as a synthetic precursor to the C-terminal thiocarboxylate moiety.[39] Having these fragments in hand, the URM1-COOH was prepared by Cys-NCL in phosphate buffer at pH ~7 for 18 h, followed by alkylation with bromoacetamide (Figure 7b). We purified the alkylated product and characterized it using HPLC and mass spectrometry (Figure 7b–c). We further characterized our synthetic URM1-COOH by circular dichroism (CD) where the spectrum displayed the characteristic secondary structure features common for UBLs (Figure 7d). Additionally, the synthetic URM1COOH was recognized by the anti-URM1 antibody (Figure 7e). All other analogs were prepared and characterized in a similar fashion utilizing the appropriate precursors (Supporting Information section 17–21). Upon subjecting URM1-CONHNH2to thioacid conversion through sodium nitrate activation followed by thiolysis with sodium sulfide,[39] the protein precipitated completely, possibly due to its hydrophobic nature and the pH changes necessary for this reaction. We tried to overcome this by generating URM1 bearing a bis(2-sulfanylethyl)amido (SEA) linker at its C-terminal, for thiolysis under neutral pH.[40] However, employing the known thiolysis conditions did not yield the desired product, leading to unidentified masses. Alternatively, we attempted to prepare the thioacid analog through subjecting only the C-terminus segment (32– 101)-CONHNH2to thiolysis before ligation, without success, despite repeated trials. At this stage, we decided to proceed with the available analogs to cellular studies, particularly since we have the carboxylic acid derivative, which serves as the natural precursor for URM1-thioacid in cells.[26,41] With these analogs in hand, we attempted the delivery of each using SBL. Under normal conditions, we delivered 20 μM of URM1-COOH into U2OS cells, following the conditions described above. For cell adherence post-delivery, we incubated the cells for 4 h at 37°C and tracked URM1-COOH localization by LSCM. Interestingly, URM1COOH was mainly localized to the nucleolar compartment (Figure 8a). We next examined whether oxidative stress affects URM1’s localization. Therefore, we pre-treated cells with an established stressor, diamide {1,1-Azobis(N,N-dimethylformamide)},[29] for 10 mins followed by a washing step and delivery. The changes in cell morphology upon stress induction served as an indication of oxidative stress phenotype. Notably, under these conditions, URM1 mostly diffused from the nucleolus to the nucleus and the cytosol (Figure 8b). Subsequently, URM1-CONHNH2and URM1-ΔGCOOH were individually introduced to U2OS cells using SBL under normal and oxidative stress conditions. Interestingly, LSCM for both analogs exhibited similar behavior to URM1-COOH, localizing in the nucleoli under normal conditions and diffusing out during oxidative stress (Figure S14, S15). These results indicate that in terms of localization, the C-terminal modification or Gly deletion does not affect the cellular protein distribution (Figure S15). We next investigated the conjugation pattern of URM1 analogs to understand their fate after delivery under normal and oxidative stress conditions. We delivered the proteins to U2OS cells under normal conditions or following an overnight treatment with diamide. Subsequently, cells were lysed[42] and analyzed with florescent gel. Specifically, we tracked our fluorescent-labeled proteins using the TAMRA channel. Interestingly, we found that all URM1 analogs were conjugated to give higher molecular weight conjugates. Notably, this pattern’s intensity was significantly increased under oxidative stress conditions, (Figure 9). To validate the subcellular localization and conjugation pattern obtained by SBL, we compared these results with those obtained using the BL approach.[18] Therefore, we delivered all analogs to adherent U2OS cells following the protocol detailed in Supporting Information. The results revealed consistent nucleoli localization under normal conditions (Figure 10, Figure S16–17). Furthermore, under oxidative stress, both URM1-COOH (Figure 10) and URM1-ΔG-COOH (Figure S16) exhibited a pattern resembling SBL, where the protein diffused out of the nucleoli. We also reassessed the conjugation pattern for URM1COOH under both normal and oxidative stress conditions with BL, yielding similar results to those obtained using SBL (Figure S19). Notably, while we noticed improved delivery efficiency for BL compared to SBL in LSCM, this was not the case with the gel analysis results, in which we Figure 7. (a) General Scheme showing the sequence of URM1, with the ligation site highlighted, and the synthetic approach for the preparation of URM1. (b) HPLC analysis for one-pot NCL and alkylation reactions for URM1-COOH (c) ESI-MS analysis of URM1-COOH (observed mass 11948.0�1.8, expected mass 11947.25) (d) CD spectra of URM1 (e) WB analysis using anti-URM1. Angewandte Chemie Research Article Angew. Chem. Int. Ed. 2024,63, e202410135 (5 of 9) © 2024 The Author(s). Angewandte Chemie International Edition published by Wiley-VCH GmbH 15213773, 2024, 48, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/anie.202410135 by Technion-Israel Institution Of, Wiley Online Library on [06/11/2025]. 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 observed nearly identical intensity. This can be explained by the different handling of cells in both methods. In the confocal microscopy study using BL, all treated cells are visualized, yet in SBL this is not the case, since only a fraction of cells are seeded into plates for scanning. Yet, in the lysis assays in both methods, all the treated cells are included in the analysis. Our results from both SBL and BL further emphasize the involvement of URM1 as a protein modifier in oxidative stress response. Contrary to our findings, Jentsch and coworkers previously reported that URM1 does not form conjugates under normal conditions.[29] To further support our results, we examined the conjugation status of the endogenous URM1 in response to oxidative stress via Figure 8. Live cell LSCM images of U2OS cells loaded with 20 μM of URM1-COOH using the SBL delivery approach under (a) normal conditions and (b) oxidative stress conditions where cells were treated with 400 μM of Diamide for 10 min. The scale bar is 20 μm. Full-view and zoomed images are presented. Hoechst was used as a nuclear stain (blue). Experiments were repeated three times. Figure 9. Fluorescent gel analysis of U2OS cells treated with (a) URM1COOH, (b) URM1-ΔG-COOH and (c) URM1-CONHNH2. Under normal (N) and oxidative stress (S), UT represents the untreated sample. Angewandte Chemie Research Article Angew. Chem. Int. Ed. 2024,63, e202410135 (6 of 9) © 2024 The Author(s). Angewandte Chemie International Edition published by Wiley-VCH GmbH 15213773, 2024, 48, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/anie.202410135 by Technion-Israel Institution Of, Wiley Online Library on [06/11/2025]. 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 western blot (WB) with the anti-URM1 antibody. We observed increased conjugation under oxidative stress conditions compared to non-stressed ones, (Figure S18). An additional interesting finding that was observed during our data analysis is related to the deleted Gly analog, which showed similar behavior. This also differs from the findings of Jentsch and co-workers but aligns well with a recent study published by Glatt and co-workers showing that URM1 could conjugate in vitro even without the characteristic GG motif. Regarding the hydrazide analog which also showed similar behavior, we hypothesize that this moiety undergoes hydrolysis, via yet undiscovered enzyme, to form a carboxylic acid in cells, akin to Ub behavior with C-terminus adducts that are removed by UCH-L1 to generate Ub-COOH.[43] Finally, examining the published list of protein substrates undergoing urmylation under oxidative conditions revealed that many of these proteins are not confined to the nucleolus but also exist in the nucleoplasm, vesicles, and cytosol. This may elucidate the role of URM1 under oxidative stress, where it diffuses out of the nucleolus to modify substrates out of the nucleus, presumably protecting them from oxidative damage. The observed localization under normal conditions versus under stress could emphasize the dual role of URM1 as a sulfur carrier for tRNA thiolation in the nucleolus and as a protein modifier for substrates not necessarily located in the nucleolus.[44] This URM1’s behavior may be attributed to one of its substrates, Nucleoporin (Nup160),[44] known as part of the nuclear pore complex and responsible for the translocation of macromolecules across the nucleus membrane. It is plausible that this substrate, which is in close proximity to URM1 undergoes oxidative stress-dependent interaction, leading to URM1’s migration out of the nucleus. Conclusion In summary, we have developed SBL as a new noncytotoxic method that was applied to the delivery of different functional proteins, in suspension phase, which requires small amounts of protein(s). Our method can serve as a general delivery approach of recombinant as well as synthetic protein cargo and ABP to various cell lines. SBL is also economical and suitable for multiplexed loading of proteins without requiring any special machinery or equipment. Using SBL, we successfully studied aspects of URM1’s cellular properties using a small quantity of its synthetic Figure 10. Live cell LSCM images of U2OS cells loaded with URM1-COOH using BL under normal and oxidative stress conditions where the cells were treated with 400 μM of Diamide for 10 min. Scale bar is 20 μm. Hoechst was used as a nuclear stain (blue). Experiments were repeated three times. Angewandte Chemie Research Article Angew. Chem. Int. Ed. 2024,63, e202410135 (7 of 9) © 2024 The Author(s). Angewandte Chemie International Edition published by Wiley-VCH GmbH 15213773, 2024, 48, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/anie.202410135 by Technion-Israel Institution Of, Wiley Online Library on [06/11/2025]. 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 analogs, compared to BL (100 μl vs. 1 ml of protein solution). As URM1 was challenging to synthesize, which limits biological studies, SBL came to address these obstacles and enabled a straightforward investigation despite the limited quantities of the proteins. We successfully addressed the behavior of URM1 where it localizes in the nucleoli under normal conditions and diffuses out under oxidative stress. This result further emphasizes the role of URM1 and its involvement in stress damage response. We also found that regardless of its C-terminal functionality, URM1 forms fewer conjugates under normal conditions compared to oxidative stress. Along with the recent in vitro studies,[28] our results further support that the characteristic GG motif is not required for URM1’s conjugation. With these tools in hand, we are currently further investigating other aspects of urmylation pathways. 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). R. M. acknowledges the support of the Fine scholarship for post-doctoral research fellow. M. H acknowledges the support of the VATAT scholarship. Some of the Figures were created with BioRender.com. We thank Yousef Mansour and Dr. Aviv Lutaty for their assistance in the flow cytometry experiments. We also thank Dr. Tamar Ziv and her colleagues from the Smoler Proteomics Center for the proteomics analysis. Conflict of Interest The authors declare no conflict of interest. Data Availability Statement The data that support the findings of this study are available in the supplementary material of this article. Keywords: Protein delivery ·Suspension Bead loading (SBL) · Chemical protein synthesis ·Ubiquitin related modifier-1 (URM1) ·Ubiquitin like modifiers [1] S. B. H. Kent, Chem. Soc. Rev. 2009,38, 338–351. [2] S. Bondalapati, M. Jbara, A. Brik, Nat. Chem. 2016,8, 407–418. [3] S. S. Kulkarni, J. Sayers, B. Premdjee, R. J. Payne, Nat. Chem. Rev. 2020,2, 1–17. [4] V. Agouridas, O. El Mahdi, J. C. M. Diemer, M. Cargoët, M. Monbaliu, O. Melnyk, Chem. Rev. 2019,119, 7328–7443. [5] S. Mandal, G. Mann, G. Satish, A. Brik, Angew. Chem. Int. Ed. 2021,60, 7333–7343. [6] Y. David, M. Vila-Perelló, S. Verma, T. W. Muir, Nat. Chem. 2015,7, 394402. [7] G. Mann, P. Sadhu, A. Brik, Acc. Chem. Res. 2022,55, 2055– 2067. [8] A. E. Rabideau, B. L. Pentelute, ACS Chem. Biol. 2016,11, 490–15. [9] A. T. Jones, E. J. Sayers, J. Controlled Release 2012,161, 582– 591. [10] A. F. L. Schneider, M. Kithil, M. C. Cardoso, M. Lehmann, C. P. R. Hackenberger, Nat. Chem. 2021,13, 530–539. [11] J. R. Maiolo, M. Ferrer, E. A. Ottinger, Biochim. Biophys. Acta Biomembr. 2005,1712, 161–172. [12] K. Deprey, L. Becker, J. Kritzer, A. Plückthun, Bioconjugate Chem. 2019,30, 1006–1027. [13] A. F. L. Schneider, L. S. Benz, M. Lehmann, C. P. R. Hackenberger, Angew. Chem. Int. Ed. 2021,60, 22075–22080. [14] S. Du, S. S. Liew, L. Li, S. Q. Yao, J. Am. Chem. Soc. 2018, 140, 15986–15996. [15] P. L. McNeil, E. Warder, J. Cell Sci. 1988,88, 669–678. [16] M. P. Stewart, R. Langer, K. F. Jensen, Chem. Rev. 2018,18, 7409–7531. [17] C. A. Cialek, G. Galindo, A. L. Koch, M. N. Saxton, T. J. Stasevich, J. Visualization 2021,172, 1–16. [18] G. Mann, P. Sadhu, A. Brik, ChemBioChem 2022,23, e202200122. [19] M. Hasan, D. Panda, G. Mann, A. Brik, ChemBioChem 2024, 25, e20230073. [20] J. Keiten-Schmitz, L. Röder, E. Hornstein, M. Müller-McNicoll, S. Müller, Front. Mol. Biosci. 2021,8, 673038. [21] N. J. Agard, J. A. Prescher, C. R. Bertozzi, J. Am. Chem. Soc. 2004,126, 5046–15047. [22] F. E. Reyes-Turcu, K. D. Wilkinson, Chem. Rev. 2009,109, 1495–1508. [23] W. Gui, P. Paudel, Z. Zhuang, in Compr. Nat. Prod. III,2020, pp. 589–602. [24] P. Gopinath, S. Ohayon, M. Nawatha, A. Brik, Chem. Soc. Rev. 2016,45, 4171–4198. [25] M. Li, C. L. Brooks, N. Kon, W. Gu, Mol. Cell 2004,13, 879– 886. [26] X. Zhang, X. L. Chen, Cell. Signalling 2021,81, 1–7. [27] K. Furukawa, N. Mizushima, T. Noda, Y. Ohsumi, J. Biol. Chem. 2000,275, 7462–7465. [28] K. E. Ravichandran, L. Kaduhr, B. Skupien-Rabian, E. Shvetsova, M. Sokołowski, R. Krutyhołowa, D. Kwasna, C. Brachmann, S. Lin, S. Guzman Perez, P. Wilk, M. Kösters, P. Grudnik, U. Jankowska, S. A. Leidel, R. Schaffrath, S. Glatt, EMBO J. 2022,41, 1–23. [29] A. G. Van Der Veen, K. Schorpp, C. Schlieker, L. Buti, J. R. Damon, E. Spooner, H. L. Ploegh, S. Jentsch, Proc. Natl. Acad. Sci. USA 2011,108, 1763–1770. [30] J. Schmitz, M. M. Chowdhury, P. Hänzelmann, M. Nimtz, E. Y. Lee, H. Schindelin, S. Leimkühler, Biochemistry 2008,47, 6479–6489. [31] C. Brachmann, L. Kaduhr, A. Jüdes, K. E. Ravichandran, J. D. West, S. Glatt, R. Schaffrath, Redox Biol. 2020,30, 1–11. [32] P. Dawson, T. Muir, I. Clark-Lewis, S. Kent, Science 1994,266, 776–779. [33] R. E. Thompson, B. Chan, L. Radom, K. A. Jolliffe, R. J. Payne, Angew. Chem. Int. Ed. 2013. [34] S. Rajagopal, S. B. H. Kent, Protein Sci. 2007,16, 2056–64. [35] V. Y. Torbeev, S. B. H. Kent, Angew. Chem. Int. Ed. 2007,46, 1667–1670. [36] R. Boehringer, B. Kieffer, V. Torbeev, Chem. Sci. 2018,16, 2056–2064. [37] J. B. Blanco-Canosa, B. Nardone, F. Albericio, P. E. Dawson, J. Am. Chem. Soc. 2015,137, 7197–7209. [38] G. M. Fang, Y. M. Li, F. Shen, Y. C. Huang, J. Bin Li, Y. Lin, H. K. Cui, L. Liu, Angew. Chem. Int. Ed. 2011,50, 7645–7649. Angewandte Chemie Research Article Angew. Chem. Int. Ed. 2024,63, e202410135 (8 of 9) © 2024 The Author(s). Angewandte Chemie International Edition published by Wiley-VCH GmbH 15213773, 2024, 48, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/anie.202410135 by Technion-Israel Institution Of, Wiley Online Library on [06/11/2025]. 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 [39] X. H. Tan, X. Zhang, R. Yang, C. F. Liu, ChemBioChem 2008, 9, 1052–1056. [40] S. L. Pira, E. Boll, O. Melnyk, Org. Lett. 2013,15, 5346–5349. [41] F. Wang, M. Liu, R. Qiu, C. Ji, Protein Cell 2011,2, 612–619. [42] M. H. Tatham, M. S. Rodriguez, D. P. Xirodimas, R. T. Hay, Nat. Protoc. 2009,4, 1363–1371. [43] C. M. Pickart, I. A. Rose, J. Biol. Chem. 1985,260, 7903–7910. [44] S. Leidel, P. G. A. Pedrioli, T. Bucher, R. Brost, M. Costanzo, A. Schmidt, R. Aebersold, C. Boone, K. Hofmann, M. Peter, Nature 2009,458, 228–232. Manuscript received: May 29, 2024 Accepted manuscript online: September 9, 2024 Version of record online: October 29, 2024 Angewandte Chemie Research Article Angew. Chem. Int. Ed. 2024,63, e202410135 (9 of 9) © 2024 The Author(s). Angewandte Chemie International Edition published by Wiley-VCH GmbH 15213773, 2024, 48, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/anie.202410135 by Technion-Israel Institution Of, Wiley Online Library on [06/11/2025]. 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