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

Domain: chemistry
Published: 2026-03-02
Editorial outcome: major_revision

Abstract
A long-standing goal in biomedical research is to label and manipulate intracellular targets, which could be achieved through the cytosolic delivery of exogenous functional proteins. The development of Tat clusters has advanced the nontoxic intracellular delivery of functional antibodies at low concentrations, but the variety of proteins that can be successfully delivered remains limited. Here, we find that by simply reversibly modifying the surface of functional proteins with anionic peptide patches, various protein cargoes (which are normally difficult to deliver) can be delivered into living cells by synergetic electrostatic interactions with the cationic cell-penetrating peptide clusters TAT3. To demonstrate the applicability of this approach, we successfully deliver functional proteins with widely varying molecular weights (∼1.5 kDa to 430 kDa) and isoelectric points (less than 5 to greater than 9) into the cytosol of cells. By exploiting this method, we also achieve protein delivery in plant tissues, which is more challenging due to the presence of intact plant cell walls. This strategy is further applied for the cytosolic delivery of synthetic protein probes carrying posttranslational modifications (PTMs), which can aid in in situ mapping of the intracellular PTM-mediated interactome. Overall, this strategy is expected to enrich cytosolic protein delivery technology and help to repurpose a wide range of customized and therapeutic proteins for emerging intracellular applications.

Introduction
The ability to deliver exogenous proteins into living cells is important for a wide range of applications in biomedical research, e.g., profiling intracellular protein‒protein interactions (PPIs) using synthetic protein probes equipped with photoreactive warheads, visualizing and targeting organelles using fluorescent protein-labeled cargoes, and detecting and localizing antigens in cells and tissues using exogenous monoclonal antibodies1-3. To overcome the poor cell membrane permeability of proteins, several ingenious strategies have been explored for delivering proteins into the cytosol of cells4-6; among these systems, cell-penetrating peptide (CPP)-mediated delivery has emerged as a prominent carrier system7,8. CPPs can deliver a wide range of protein cargoes, such as functional enzymes, small antibody fragments (nanobodies) and fluorescent proteins, into living cells9-11. However, cargo proteins must be added at relatively high concentrations (>10 μM) to guarantee delivery efficiency, which is either inconvenient or impossible due to solubility or toxicity10,12,13.

A recent landmark finding was that the strategic assembly of the highly positively charged CPP Tat into multimeric clusters greatly improved its cellular uptake efficiency. Building upon this finding, Tietz et al. developed a family of trimeric Tat that enables the nontoxic intracellular delivery of functional antibodies and antibody fragments at low concentrations (e.g., 1-2 μM) by simple incubation of Tat clusters with protein cargoes14. Encouraged by these important discoveries, we became interested in the cytosolic delivery of other types of functional proteins, such as fluorescent proteins for targeted labeling, E2-ubiquitin (Ub) conjugates for in situ mapping of PPIs of Ub ligases, and bioactive enzymes that can degrade intracellular RNA. However, we and other researchers have found that after co-incubation with trimeric Tat, only a small variety of proteins can be delivered into cells at low concentrations14; that is, a wide range of protein cargoes with different isoelectric points and molecular weights are difficult to deliver via the Tat cluster strategy. One possible reason is that these protein cargoes lack favorable interactions with trimeric Tat.

Here, we found that by simply modifying the surface of functional proteins with anionic peptide patches, protein cargoes that are otherwise difficult to deliver could be readily delivered into living cells at ~1.5 μM by synergetic electrostatic interactions with the cationic CPP TAT3. Motivated by these observations, we developed a reversible anionic patch modification-based approach to enable the intracellular delivery of a broad variety of functional proteins, including antibodies, synthetic protein probes bearing posttranslational modifications (PTMs), fluorescent proteins, and functional enzymes, whose molecular weights range from ∼1.5 kDa to 430 kDa and isoelectric points range from less than 5 to greater than 9 (Fig.1). Upon cellular uptake, the anionic patch spontaneously dissociates from the protein, thereby releasing native cargoes. This strategy was also applied for the cytosolic delivery of protein cargoes into intact plants, which is more challenging to deliver due to the presence of plant cell walls. Finally, the utility of this strategy was exemplified by the cytosolic delivery of synthetic protein probes, such as the E2-Ub photoaffinity probe, which helps in in situ mapping of the PTM-mediated intracellular interactome.

Results
Anionic surface modification achieves cellular uptake of UbcH786C

Owing to our enduring interest in the use of E2 probes to identify their partner E3s (Ub E3 ligases),15 we began this work with the cytosolic delivery of a commonly used E2 enzyme, UbcH7 (pI: 8.87, MW: 18.7 kDa).16 The CPP cR10 was selected for conjugation17 to UbcH786C -T (a UbcH7 mutant retaining only the catalytic cysteine at position 86 (Supplementary Fig. 3a), which was coupled to fluorophore NHS-TAMRA (T)) via a disulfide bond. However, we observed significant precipitation during the cR10 conjugation (Supplementary Fig. 3b) disfavoring its further implementation. We then turn to the co-incubation strategy with TATE peptides and test three types of TAT: trimeric cyclic TAT (tri-cTat, developed by Tietz et al14), trimeric linear TAT (TAT3, derived from Brock et al18) and mono-TAT (commonly used CPP) (Supplementary Fig. 1a-1c and 3c). These TATs were each mixed with 1.5 µM UbcH786C -T and then incubated with HepG2 cells for 30 minutes. Confocal laser scanning microscopy (CLSM) analysis revealed no detectable diffuse red fluorescence in cells treated with 1 µM or 3 µM TAT trimers (tri-cTat and TAT3) or equivalent molar concentrations of mono-TAT (3 µM and 9 µM) (Supplementary Fig. 3d-3f). Therefore, both previous approaches, covalent conjugation with cR10 and coincubation with TAT, failed to deliver UbcH786C into cells.

One plausible explanation for the failed delivery of UbcH786C by co-incubation with TAT may due to its positive charge at physiological pH (pI: 8.87), which counteracts with the positively charged TAT peptides. To reduce the positive charge of UbcH7, we designed and synthesized an acidic amino acid-rich peptide E3D2 (sequence: PEDEDELAC) (Supplementary Fig. 1d). The cysteine residue in E3D2 was first activated by 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) reagent (Supplementary Fig. 1e), followed by a disulfide exchange reaction with UbcH7 in phosphate-buffered saline (PBS, pH 7.4) (Fig. 2a). Mass spectrometry analysis verified the almost complete conversion of UbcH786C to the mono-E3D2 modified adduct (UbcH786C E3D2+) within 1 hour (Fig. 2b), which was subsequently purified by fast protein liquid chromatography (FPLC). The UbcH786C E3D2+ conjugate was fluorescently labeled with NHS-TAMRA, and the labeled product (UbcH786C E3D2+-T) was mixed with three different TATs and then co-incubated with HepG2 cells for 30 minutes. Diffuse TAMRA red fluorescence was observed in cells treated with 1.5 µM UbcH786C E3D2+-T and 3 µM TAT3 (Fig. 2c). Z-stack imaging further confirmed the cytoplasmic and nuclear distribution of the fluorescence (Supplementary Fig. 4a). Nuclear mean fluorescence intensity (MFI) was used here because it can distinguish proteins that have successfully escaped into the cytosol and those that are trapped within endosomes.11 MFI analysis revealed that the MFI of HepG2 cells co-incubated with UbcH786C E3D2+-T and TAT3 was as high as 16 compared to the unmodified UbcH786C -T (MFI = 1) (Fig. 2d). Cytotoxicity assessment (CCK-8 assay)19 showed >95% cell viability at TAT3 concentrations between 1-3 µM (Fig. 2e). These results demonstrate that E3D2 surface modification enables efficient intracellular delivery of UbcH786C when mixed with TAT3 with minimal cytotoxicity in HepG2 cells. In comparison, when the same concentration of UbcH786C E3D2+-T was delivered, although some punctate red fluorescence20 was observed in cells co-incubated with 3 µM tri-cTat, the cells had simultaneously exhibited significant membrane rupture, whereas little red fluorescence was observed in cells co-incubated with 9 µM mono-TAT (Supplementary Fig. 4b).

Reversible surface modification enables efficient cellular uptake of UbcH786C with minimal cytotoxicity

To investigate the effect of the number of acidic amino acids in peptide sequences on UbcH786C delivery efficiency, we designed and synthesized five peptides with different numbers of acidic amino acids (Supplementary Fig. 1d, 1f-1i), which were subsequently conjugated to UbcH786C using the above protocol (Fig. 2a). Fluorescence imaging and quantitative analysis demonstrated the successful intracellular delivery of all modified cargoes by co-incubation with 3 µM TAT3 (Fig. 3a and Supplementary Fig. 5), in which UbcH786C E4D3+-T exhibited the highest nuclear MFI. Despite containing more acidic amino acids in their sequences, UbcH786C E6D5+-T and UbcH786C E5D4+-T showed approximately 50% reduction in nuclear MFI compared to UbcH786C E4D3+-T (Fig. 3b). Based on these results, we selected the E4D3 sequence (PEDEDEDELAC) for the surface modification of UbcH786C (the adduct was designated UbcH786C+).

Further concentration gradient experiments revealed that UbcH786C +-T in the concentration range from 0.5 to 9 µM could be delivered into cells by co-incubation with 3 µM TAT3 (Supplementary Fig. 6a), with nuclear MFI showing a linear correlation with incubation concentrations (Fig. 3c). Cell viability remained >95% at all concentrations tested (Supplementary Fig. 6b). We then prepared E4D3-modified UbcH7 by disulfide bonds at either the N-terminus (UbcH717C) or C-terminus (UbcH7137C). Both cargoes showed comparable intracellular delivery with UbcH786C +-T in HepG2 cells (Fig. 3d and Supplementary Fig. 6c), indicating that the surface modification site does not significantly affect the delivery efficiency. Notably, serum-containing conditions were found to decrease delivery efficiency (Supplementary Fig. 6d), consistent with previous reports.11

To verify the dissociation of E4D3 from UbcH7 in the cell, we labeled E4D3 with TAMRA (designated T+) (Supplementary Fig. 1J), and conjugated it via disulfide bond to nuclear-localized UbcH786C (NLS-UbcH786C -FITC). After co-incubation of the adduct (NLS-UbcH786C T+-FITC) and TAT3 with HepG2 cells, CLSM images revealed that the green fluorescence of NLS-UbcH786C was predominantly localized in the nucleus, whereas the red fluorescence of TAMRA-E4D3 was diffusely distributed in both the cytosol and the nucleus (Fig. 3e and 3f). This spatial separation demonstrates that the disulfide-linked NLS-UbcH786C T+-FITC undergoes reductive cleavage in the cytosol,21 releasing free NLS-UbcH786C -FITC and E4D3-TAMRA. Complementarily, the circular dichroism (CD) spectrum of 1 mM glutathione (GSH)-pretreated UbcH786C + showed very similar absorption to native UbcH786C (Fig. 3g), indicating the reversion to an identical secondary structure.

Finally, we quantified the delivery efficiency by flow cytometry under optimized conditions (3 µM TAT3 and 1.5 µM UbcH786C +-T), and found that approximately 72.4% of the cells exhibited fluorescence signals (Fig. 3h-i). The HepG2 cells were then lysed and analyzed by fluorescence SDS-PAGE, which revealed the presence of UbcH786C +-T in the cell lysate (Supplementary Fig. 7), confirming that UbcH7 was successfully delivered into living cells with minimal cytotoxicity by anionic surface modification.

UbcH786C + is internalized via macropinocytosis

To probe the interaction between UbcH786C +-T and TAT3, we performed native gel electrophoresis analysis12. As showed in Fig. 4a, co-incubation of UbcH786C +-T with TAT3 generated a band shift above the original UbcH786C +-T. This shifted band exhibited concentration-dependent attenuation with increasing heparin concentrations, accompanied by a concomitant intensification of the original band. The complete disappearance of the band shift occurred when the concentration of heparin exceeded 2 µM, demonstrating that the non-covalent complex formation between UbcH786C +-T and TAT3 is mediated by electrostatic interactions.14

We next investigated the delivery mechanisms of UbcH786C +-T and TAT3, starting with 70 kDa dextran-FITC, a well-established marker of macropinocytosis. HepG2 cells co-incubated with dextran-FITC and TAT3 showed diffuse green fluorescence in the cytoplasm (Fig. 4b and Supplementary Fig. 8a) as reported in previous work.13,22 Further experiments co-incubating cells with dextran-Cy5, UbcH786C +-T and TAT3 showed simultaneous red fluorescence of UbcH786C +-T and purple fluorescence of dextran-Cy5 in the cells (Fig. 4c and Supplementary Fig. 8b), indicating that UbcH786C +-T was delivered into the cells via the macropinocytosis in the same way as 70 kDa dextran. Co-localization analysis using the LysoTracker showed that less than 40% of UbcH786C +-T signals co-localized23 with the LysoTracker (Fig. 4d), suggesting that the majority of UbcH786C +-T escapes from endosomes.24

We also explored the role of transduction in the cellular uptake of UbcH786C +-T. Unlike macropinocytosis, transduction can occur at 4 °C25,26 and it has been reported that the accumulation of CPPs at the cell membrane by transduction would result in a local membrane flip-flop27. Our observations showed no detectable red fluorescence in cells co-incubated with UbcH786C +-T and TAT3 at 4 °C (Supplementary Fig. 8c). Furthermore, using annexin V, a specific binding protein for membrane phosphatidylserine, we did not observe any enrichment of phosphatidylserine during UbcH786C +-T internalization (Supplementary Fig. 8d). These results suggest that transduction is not the primary pathway for the delivery of UbcH786C +-T and TAT3.

Finally, we quantified the delivery efficiency of UbcH786C +-T in the presence of various pathway inhibitors28 by flow cytometry. Inhibitors, including chlorpromazine (CPM, an inhibitor of clathrin-mediated endocytosis), amiloride (AM, an inhibitor of macropinocytosis), methyl-beta-dextrin (MβCD, a cholesterol depleting substance), were used as described in previous studies.29-32 As shown in Fig. 4e and Supplementary Fig. 8e, the most pronounced fluorescence attenuation (reaching only 20% of the control group) was observed under AM treatment. Taken together, these results demonstrate that the E4D3-modified UbcH786C can form electrostatic complexes with TAT3, and then be internalized via macropinocytosis and reach the cytosol by escaping from endosomes.

Synergizing anionic surface modification and electrostatic interaction with TAT3 enables cytosolic delivery of various cargoes into living cells

We then attempted to deliver a broad palette of proteins with different molecular weights (MWs) and isoelectric points (pIs) into cells using the anionic surface modification strategy (Fig. 5a). For the preparation of E4D3-modified protein cargoes, proteins containing free cysteine residues (e.g., NLS-UbcH7, BFP, RFP, Lifeact, β-Gal, and BSA) were directly conjugated to DTNB-activated E4D3 via disulfide bond as described above. For proteins lacking free cysteines (including RNase A, IgG, HRP, and Ub), we implemented a thiolation protocol using a widely adopted reagent 2-iminothiolane33 (25 equiv.) to generate free thiol, followed by E4D3 conjugation via disulfide bond to prepare E4D3-modified adduct (cargo+) (Fig. 5b). Mass spectrometric analysis confirmed the successful introduction of up to four free thiol groups per protein molecule after 2-iminothiolane treatment, and finally installed one to two E4D3 per protein molecule (Supplementary Fig. 9a and 9b).

We first evaluated the cellular delivery of the positively charged protein NLS-UbcH7 (pI: 9.4, MW: 19.6 kDa). Co-incubation of NLS-UbcH7+-FITC and TAT3 resulted in distinct nuclear green fluorescence in treated cells (Fig. 5c). Quantitative analysis showed that approximately 86% of the FITC signals co-localized with Hoechst34, and 85% of the Hoechst signal colocalized with FITC (Supplementary Fig. 9c). In stark contrast, non-modified NLS-UbcH7-FITC was not delivered into cells when co-incubated with TAT3 (Supplementary Fig. 10a). We then investigated the delivery of neutrally charged red fluorescent protein (RFP, pI: 7.3, MW: 26.9 kDa), which was very difficult to deliver intracellularly when co-incubation with tri-cTat in previous studies.14 Both confocal microscopy and Z-stack imaging revealed diffuse red fluorescence in HepG2 cells co-incubated with RFP+ and TAT3, whereas control groups (cells incubated with RFP and TAT3) showed minimal fluorescence (Fig. 5c, Supplementary Fig. 9d and 10b). Further experiments extended to proteins with lower pIs including blue fluorescent protein (BFP, pI: 6.6) and bovine serum albumin (BSA, pI: 4.7, MW: 69.3 kDa), only E4D3-modified adducts showed successful intracellular delivery when co-incubated with TAT3 (Fig. 5c, Supplementary Fig. 10c and 10d).

We next investigated intracellular delivery of the Lifeact peptide (pI: 4.9, MW: 1.9 kDa), which can target filamentous actin (F-actin) in living cells.35 CLSM revealed distinct green fluorescence in cells co-incubated with Lifeact+-FITC and TAT3 (Fig. 5c and Supplementary Fig. 10e). Quantitative analysis showed a high degree of colocalization between Lifeact+-FITC signals and the F-actin marker SiR700-Actin (M1 = 0.589, M2 = 0.528) (Supplementary Fig. 9e). Moreover, after quantified by image-based analytical method14, we found that all fluorescence labeled proteins exhibited transduction efficiencies of over 55% (Fig. 5d). We also examined the cellular delivery of two enzyme cargoes: ribonuclease A (RNase A, pI: 8.6, MW: 13.7 kDa) - known to induce cell death,36 and horseradish peroxidase (HRP, pI: 7.0, MW: 40 kDa) - capable of catalyzing the conversion of the colorless substrate tetramethylbenzidine (TMB) to a blue product in the presence of hydrogen peroxide.37 HepG2 cells treated with RNase A (up to 3 µM), TAT3 alone, or their mixture maintained ≥90% cell viability. In contrast, co-incubation with RNase A+ and TAT3 resulted in concentration-dependent cell death (Fig. 5e). In parallel, functional assessment of HRP delivery showed that HepG2 cells co-incubated with TAT3 and HRP+ exhibited the most intense blue color in TMB assays (Fig. 5f). These functional assays confirm successful intracellular delivery of biologically active RNase A+ and HRP+ into target cells.

Finally, we explored the molecular weight capacity of our strategy for protein delivery, starting with full-length immunoglobulin G antibody (IgG, MW: 150 kDa). CLSM showed diffuse green fluorescence in cells incubated with IgG+-FITC and TAT3 (Fig. 5c and Supplementary Fig. 10f), indicating successful intracellular delivery of IgG+-FITC. β-Gal (MW: 430 kDa), a tetrameric complex enzyme capable of catalyzing the hydrolysis of colorless 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-Gal) to form blue products,38 was then tested. HepG2 cells incubated with β-Gal+ and TAT3 showed blue staining after treatment with the X-Gal kit (Fig. 5g). As a control, direct conjugation of β-Gal with R10 resulted in significantly weaker intracellular blue signals under the same conditions (Fig. 5g). These results demonstrate the applicability of the strategy of synergizing anionic surface modification and electrostatic interaction for the cytosolic delivery of different cargoes in the bioactive form.

Cytosolic delivery of proteins in different mammalian cell lines and plants

Next, we investigated the efficacy of the strategy in different cell lines. Co-incubation of UbcH786C +-T and TAT3 in BEAS-2B (human bronchial epithelium), HeLa (human cervical adenocarcinoma) and Jurkat (human T lymphocyte) cells all resulted in diffuse red fluorescence similar to HepG2 cells (Fig. 6a and Supplementary Fig. 11a), demonstrating the applicability of the strategy in multiple mammalian cells.

In contrast to mammalian cells, plant cells possess a structurally dense cell wall outside the plasma membrane (Fig. 6b), which imposes additional constraints on the cellular delivery of exogenous biomolecules.39 Herein, Arabidopsis thaliana (A. thaliana) and Nicotiana tabacum (N. tabacum) were chosen as model plants. Leaves were infiltrated40 with a mixture of UbcH786C +-T and TAT3 and incubated for 1 hour, then the leaves were re-infiltrated in situ with QSY21-NHS (N-hydroxysuccinimide linked fluorescence quencher moiety)41 to quench the extracellular fluorescence of TAMRA. The plant leaves were then cut and washed with water to remove protein samples adsorbed on the leaf surface and subjected to CLSM. As shown in Fig. 6c, obvious red fluorescence was observed in the epidermal cells and was distributed around the cell contour, similar to previous work.42 In contrast, leaves co-incubated with UbcH786C -T and TAT3 showed negligible red fluorescence under identical conditions. Furthermore, we tested the delivery of the negatively charged protein Ub. Distinct red fluorescence was only observed in plant leaves when co-incubated with Ub+-T and TAT3 (Fig. 6c). These results demonstrate the successful delivery of both positively charged (UbcH786C) and negatively charged (Ub) proteins to A. thaliana and N. tabacum leaves using the strategy of synergizing anionic surface modification and electrostatic interaction. In addition, we evaluated the delivery efficiency of disulfide-linked cR10-conjugated Ub-TAMRA (cR10-Ub-T), but observed only weak fluorescence signals, no enhanced fluorescence was detected even when TNB-R10-ILFF43 was added (Supplementary Fig. 11b). Taken together, these data confirm the broad applicability of our strategy for the delivery of protein cargoes to mammalian cell lines and plant cells.

Chemical synthesized surface modified E2-Ub probes empowers the exploration of E2-E3 interactome in living cells

Encouraged by the successful delivery of UbcH786C into various cell lines, we next attempted to deliver E2-Ub probes to profile their partner E3s in living cells, with the goal of mapping the E2-E3 network in physiological processes.44 Inspired by the biotinylated E2-Ub photocrosslinking probe (UBE2D3-Ub Bpa) reported by Mathur et al,45 we modified its surface with E3D4 and applied it to capture RING E3s (the largest E3 subfamily) in living cells. For this end, the E2 mutant (UBE2D3 C85K S22R) was recombinantly expressed in which the catalytic cysteine was mutated to lysine to allow the formation of an isopeptide bond with ubiquitin (Ub), an isopeptide bond that was demonstrated by structural analysis to be an acceptable structural mimetic of the native thioester.46 Moreover, serine was replaced by arginine to disrupt the self-association of the probe. The biotinylated Ub Bpa31 (substitution of benzophenone (Bpa) at position 31 of ubiquitin), obtained by Fmoc-based solid-phase peptide synthesis (SPPS),47-49 was then conjugated to the E2 mutant with an E1-activating enzyme to form isopeptide bonds.50 Finally, the E4D3 peptide was conjugated via disulfide bonds and fluorescently labeled with NHS-TAMRA to form the UBE2D3-Ub Bpa probe+-T (Fig. 7a). The CLSM images revealed diffuse red fluorescence in HeLa cells with small number of puncta after incubation with probe+-T and TAT3 (Fig. 7b), which is similar to the previous studies on delivering large-molecular-weight proteins such as Fab fragments14.

We next investigated the ability of probe+ to profile endogenous RING E3 in a proteome-wide manner in response to epidermal growth factor (EGF)51 stimulation. HeLa cells were first preincubated with MG132 and bafilomycin for 6 hours and then stimulated with EGF. The experimental cells (A) were then incubated with probe+ and TAT3, while the control cells (B) were incubated with probe+ alone, then both cells were resuspended in cold PBS and irradiated with 365 nm UV on ice to activate Bpa.45 Finally, the cell lysates were collected, and biotinylated proteins were enriched using streptavidin magnetic beads for LC-tandem MS analysis (Fig. 7c). Among the total proteins identified by MS, 42 were RING E3s, 20 of which were UBE2D3 partners (Supplementary Table 1). We then filtered the detected E3s using criteria (fold change > 2 and P-value < 0.05) to ensure that the detected E3 signals correlated with E3 activity or abundance. Our results showed that 21 RING E3s were significantly enriched, and 11 of these E3s have been reported to catalyze protein ubiquitination in cooperation with UBE2D3. Furthermore, another 7 RING E3s were reported to catalyze protein ubiquitination in cooperation with other E2s belonging to the UBE2D family (Fig. 7d and Supplementary Table 2). Notably, the UBE2D3-Ub Bpa probe was previously used to profile RING E3s in EGF-stimulated HEK293T cell lysates.45 A comparison of these two data sets revealed that 10 partner E3s were labeled in both living cells and lysates. We identified additional E3s, such as UHRF1, TRIM32, ZNF598, RNF168 and RNF181, and Mathur et al. 45 also reported different E3s, including BRCA1, BRE1A, BRE1B, HLTF and PCGF6. Together, these findings collectively show the complementarity between the two approaches, offering valuable tools for studying E2–E3 pairing relationships in physiological or pathological processes.

Methods
General materials and methods

Fmoc-amino acids were purchased from GL Biochem (China). Resins were purchased from Tianjin Nankai HECHENG S&T Co., Ltd (China). Coupling reagents were purchased from CSBio Ltd. (China). Dichloromethane (DCM) and N, N-Dimethylformamide (DMF) were purchased from J&K Scientific Ltd (China). Ni-NTA resin was purchased from LABLEAD (China). Chemically competent BL21 (DE3) cells were purchased from TransGen Biotech ltd (China). Inorganic salts were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai). Yeast extract and Tryptone were purchased from Sigma-Aldrich. DMEM, RPMI-1640, PBS and penicillin/streptomycin were purchased from Gibco. FBS was purchased from Lonsera. 35mm glass-bottom dishes were purchased from Biosharp. Metabolic inhibitors and fluorescent molecules were purchased from MedChemExpresss (USA). Living cell dyes such as Hoechst 33258, LysoTracker™ Deep Red were purchased from Thermo Fisher Scientific (USA), and SiR700-Actin were purchased from Spirochrome (Switzerland). TMB, X-Gal staining kit, CCK8 kit and Calcein/PI cytotoxicity assay kit were purchased from BeyotimPe (China). RNase A (Ribonuclease A) was purchased from Yeasen Biotechnology Co., Ltd. (China). Goat anti-Rabbit IgG (Immunoglobulin G) and BSA (bovine serum albumin) was purchased from Sangon Biotech (China). HRP (Horseradish Peroxidase) was purchased from Aladdin (China). β-Gal (β-Galactosidase) was purchased from Solarbio (China). Peptide were analyzed or purified with RP-HPLC (SHIMADZU, Prominence LC 20AT). For analysis, analytical Welch XB-C18 columns (4.6 × 250 mm, 5 μm, 120 Å, 1.0 mL/min) were used. For purification, semi-preparative Welch XB-C18 (250 × 10 mm, 5 μm, 120 Å, flow rate 4.0 mL/min) were used. Analysis and purification were monitored at 214 and 254 nm. Solvents were sonicated for 30 min before use. Analysis and purification condition: a linear gradient of 5%-80% acetonitrile (with 0.08% v/v TFA) in water (with 0.1% v/v TFA) over 30 min. Fast Protein Liquid Chromatography (FPLC) was performed on an AKTA purifier (GE Healthcare Life Science) with column of Superdex TM 75 Increase 10/300 GL. Phosphate buffer (PBS, pH 7.4) was filtered with 0.22 μm filter paper, sonicated for 30 min and precooled for 10 min before use. Purification was monitored at 280 nm wavelength. ESI-MS were measured on a Shimadzu LC/MS-2020 system. High resolution mass spectra were measured on a Q Exactive Plus mass spectrometer coupled to an Easy-nLC1200 system running on 50% acetonitrile with 0.1% formic acid using the Xcalibur software. Protein spectra were devonvoluted using the UniDec tool. Automated Fmoc-based microwave peptide synthesis was performed on a Liberty Blue 2.0™ automated microwave peptide synthesizer (CEM Corporation, North Carolina, USA).

Experimental model

For bacteria cultures, all recombinant expressed protein genes were synthesized by Genscript (Nanjing) and cloned into pET-28a vector. Protein (RFP, BFP, UbcH7 mutants, and UBE2D3 C85KS22R) plasmids were transfected into E. coli BL21 (DE3) cells and cultured in Luria-Bertani (LB) medium at 18 °C. For mammalian cell cultures, HepG2(CL-0103), HeLa (CL-0101) and Jurkat (CL-0129) cells were purchased from Procell, BEAS-2B was a generous gift from Guiran Xiao Lab (Hefei University of Technology, Hefei). HepG2, HeLa and BEAS-2B cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 100 units/mL penicillin and 0.1 mg/mL streptomycin at 37 °C in an atmosphere of 5% CO2, while Jurkat cells were cultured in RPMI-1640 supplemented with 10% FBS and penicillin/streptomycin at 37 °C in an atmosphere of 5% CO2. For plant cultures, take several Nicotiana tabacum or Arabidopsis thaliana seeds and spread them evenly over the surface of the soil. Cover the soil with cling film to keep the soil moist and leave a few small holes for gas exchange with the outside. After one week of incubation at a constant temperature and humidity, the seedlings were transferred to individual pots (one plant per pot) and the nutrient solution was replenished every three days. Plants for fluorescence imaging were grown for 4-5 weeks under a 16 hours light/14 hours dark regimen at 22 °C/20 °C.

Protein expression and purification

Protein (RFP, BFP, UbcH7 mutants and UBE2D3 C85KS22R) plasmids were transfected into E. coli BL21 (DE3) cells. Cells was grown in LB containing 0.1 mg/mL Kanamycin at 37 °C to an OD600 of 0.8. IPTG was then added to a final concentration of 0.4 mM and the mixture was incubated for 12 hours at 16 °C. Cell precipitates were collected by centrifugation at 4 × g, 8 °C for 15 minutes, and then resuspended in lysis buffer (20 mM Tris, 150 mM NaCl, pH 7.4) and sonicated in an ice bath. The suspension was centrifuged at 4 °C, 10 × g for 30 minutes to obtain the supernatant. All proteins fused to the His6 tag were purified on Ni-NTA columns. Proteins were washed with wash buffer (20 mM Tris, 150 mM NaCl, 40 mM imidazole, pH 7.4) and eluted with elution buffer (20 mM Tris, 150 mM NaCl, 250 mM imidazole, pH 7.4). After concentration by 3K MWCO tubes, the proteins were further purified on a Superdex 75 10/300 GL column. Purified proteins are used immediately for experiments or stored at -80 °C until use.

Synthesis of peptides

All peptides used in this study were obtained via the standard Fluorenyl-methoxycarbonyl (Fmoc) based solid phase peptide synthesis (SPPS) on Rink amide resin (0.29 mmol/g) or Wang resin (0.44 mmol/g). Fmoc-based SPPS was performed using a Liberty Blue 2.0™ automated microwave peptide synthesizer (CEM Corporation, North Carolina, USA). Fmoc deprotection: The Fmoc group was removed using a 20% solution of piperidine in DMF at 90 ℃ (1 × 1 minute, 5 mL). The resins were then washed with DMF (Dimethylformamide, 4 × 5 mL). Amino acid couplings: The amino acids (10.0 equiv. to resin loading), oxyma (Ethyl cyanoglyoxylate-2-oxime, 10.0 equiv.) and DIC (N,N'-Diisopropylcarbodiimide, 10.0 equiv.) were dissolved in DMF, with amino acid final concentration at 0.2 mol/L. Afterward, the solution was added to the resins and the reaction for 2 minutes at 90 ℃. The resins were then washed with DMF (2 × 5 mL). Resin cleavage: A cleavage cocktail (TFA / Phenol / Thioanisole / 1, 2 ethanedithiol / water / Triisopropylsilane, 83/5/5/2.5/2.5/2, v/v/v/v/v/v) was added to dried resin, after which the mixture was shaken at 37 ºC for 3 hours. The resin was then filtered and the resulting cleavage solutions were volatilized under a stream of nitrogen. Cold ether was added to the residue and the mixture was thoroughly mixed and centrifuged. The supernatant was discarded to afford crude peptide.

TAT3 peptides was synthesized by incorporation of lysine that was orthogonally protected with Mtt (methyltrityl) group. Specifically, Fmoc-Lys(Mtt)-OH was used in solid-phase peptide synthesis to give resins loaded with linear peptides. After completion of the linear peptide synthesis, the N-terminal amino blocked by blocking solution (Ac2O/DIEA/DMF, 1/1/8, v/v/v) at 37 °C (2 × 5 minutes). Next, the Mtt group was removed using 2% TFA (v/v) with 1% TIPS (Triisopropylsilane, v/v) in DCM (6 × 5 minutes). Finally, an automated microwave peptide synthesizer was used to couple the TAT sequence onto the amino of the lysine side-chain. Fmoc was removed by 20% solution of piperidine in DMF at 90 °C (1 × 1 minutes), and then the resins were washed by DMF (4 × 10 mL). Amino acids (16.7 equiv. to resin loading), oxyma (16.7 equiv.) and DIC (16.7 equiv.) were dissolved in DMF and added to the resins at 90 °C (2 × 2 minutes). Resin cleavage process is the same as general resin cleavage.47

For the synthesis of tri-cTat, pentaerythritol (0.5 g, 3.67 mmol, 1 equiv.) was dissolved in anhydrous DMF (20 mL). Then sodium hydride (1.76 g, 44.04 mmol, 12 equiv., 60% in oil) was added under anhydrous and anaerobic conditions at 0 °C. The mixture was allowed to react for 30 minutes. Propargyl bromide (2.4 g, 20.1 mmol, 5.5 equiv.) was subsequently added to the mixture and reacted at 0 °C for 30 min before being heated to 50 °C and reacted for 16 hours. The reaction mixture was separated between EtOAc and H2O. The organic layer washed with H2O (x3) and brine (x3), then dried over anhydrous Na2SO4. The solvent was then removed in vacuo, and the product was purified by preparative TLC using 10 % EtOAc/hexane mixture. The tetrakis(2-propynyloxymethyl) methane was obtained as a white solid (890.9 mg, 3.09 mmol, 84.2 % yield). The synthesis of the azidolysine-cyclicTat peptide was completed on the Rink Amide-AM resins by introducing Fmoc-Lys(N3)-OH and Fmoc-Lys(Mtt)-OH. The azidolysine-cyclicTat was then conjugated to the tetrakis(2-propynyloxymethyl) methane scaffold via copper-catalyzed azide alkyne cycloaddition (CuAAC) reactions. The following were dissolved in 30 % DMSO / PBS: Tetrakis(2-propynyloxymethyl)methane (0.45 mg, 1.56 μmol, 1 equiv.), and azidolysine-cyclicTat (10 mg, 6.23 μmol, 4 equiv.), and CuSO4 (1.56 mg, 6.23 μmol, 4 equiv.), and sodium L-ascorbate (2.5 mg, 12.64 μmol, 8 equiv.), and Tris((1-hydroxy-propyl-1H-1,2,3-triazol-4-yl)methyl)amine (THPTA) (13.6 mg, 31.2 μmol, 20 equiv.), and aminoguanidine hydrochloride (0.52 mg, 4.68 μmol, 3 equiv.) and heated with stirring at 50 ℃ for 30 minutes. The product was purified by HPLC (1.3 mg, 0.255 μM, 16.3% yield).

For the synthesis of biotinylated Ub-Bpa, the C-terminal glycine was coupled to Wang resin using Fmoc-Gly-OH (10 equiv.), oxyma (10.0 equiv.), DIC (10.0 equiv.), and DMAP (4-Dimethylaminopyridine, 1 equiv.) in DMF overnight at 37 °C with shaking. Bpa coupling was achieved by incubating Fmoc-BPA-OH (4 equiv.), PyAOP (4 equiv.), HOAT (4 equiv.) and NMM (N-Methylmorpholine, 8 equiv.) in DMF overnight at 37 °C with shaking. Biotin coupling was completed by incubating Biotin(5-((3aS,4S,6aR)-2-Oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl) pentanoic acid, 10 equiv.), oxyma (10 equiv.), DIC (10 equiv.) in DMSO for 30 minutes at 75 °C with shaking. The remaining amino acids and AEEA were condensed using the Liberty Blue 2.0™ automated microwave peptide synthesizer.

Preparation of cR10-UbcH787C -T

UbcH7 (0.5 mg/mL) was first reacted with NHS-TAMRA (8 equiv.) in PBS (pH 7.4) buffer to generate UbcH787C -TAMRA (UbcH787C -T). This fluorescent conjugate was subsequently reacted with cR10 (3 equiv.) via a disulfide bond exchange to produce the final product (cR10-UbcH787C -T), and then purified by FPLC on a Superdex 75 10/300 GL column.

Preparation of anionic surface modified protein cargoes

The anionic peptides were first activated with 5 equiv. DTNB in reaction buffer (6 M Gn-HCl, 0.2 M NaH2PO4, pH 7.4) at room temperature stirring for 30 minutes. The product was then purified by RP-HPLC and lyophilized until use. Anionic peptide modified proteins with free cysteines are obtained using similar procedure, and we take the preparation of UbcH7E3D2+-T as an example for the description. Purified UbcH7 was diluted with PBS (pH 7.4) to 0.5 mg/mL. Subsequently, 3 equiv. E3D2-TNB was added and incubated gently in a shaker at room temperature for 1 hour, followed by fluorescent labeling with 8 equiv. NHS-TAMRA for a further 1 hour. The UbcH7E3D2+-T product was purified by FPLC on a Superdex 75 10/300 GL column. Proteins without free cysteines such as IgG and HRP, were first thiolated with 2-iminothiolane,33 followed by disulfide bond exchange reactions to obtain the E4D3-modified proteins.

Cellular uptake in mammalian cells

We added the final concentration of TAT3 and protein cargoes directly to the cells without pre-incubation. The incubation volume of the medium is 100 µL for CLSM detection. In detail, the protein cargoes were diluted to the final concentration using DMEM, followed by the addition of TAT3, then the samples were mixed and left at room temperature for 5 minutes. The cells were subsequently washed twice with PBS and then incubated with the samples at 37 °C for 30 minutes. After incubation, all samples were washed with PBS containing 25 µg/mL heparin for imaging. For live-cell confocal laser scanning microscopy (CLSM) experiments, cells were plated onto sterile 35 mm glass-bottom dishes and grown for 24 hours. Subsequently cells were incubated with corresponding samples, followed by staining with the dyes according to manufacturer’s instructions and washing with PBS containing heparin for imaging.

CLSM images were taken with 63X/1.4 Oil DIC M27 lens on a Zeiss LSM 980 Axio Observer confocal laser scanning microscope. The cells were imaged using argon laser (405 nm) for visualizing Hoechst 33258 and BFP, argon laser (488 nm) for visualizing FITC, and argon laser (543 nm) for visualizing TAMRA and RFP, argon laser (639 nm) for visualizing LysoTracker™ Deep Red and SiR700-Actin. All treatments were performed in three separate experiments. All image analysis was performed using FiJi software. We used the nuclear mean fluorescence intensity (MFI) to assess the efficiency of delivery by identify individual cells and the cytosolic compartment based on Hoechst nuclear staining. The nuclear MFI is presented as the mean ± standard deviation of n = 3 independent biological replicates calculated by one-way ANOVA with Tukey’s test correction. The quantification of the co-localization parameters (Pearson’s and Manders’ coefficients) was evaluated using the JACoP plugin in the FiJi software. In the example shown in Fig. 4d, the magnitude of the Pearson’s correlation coefficient is attributed to the local concentration of UbcH786C +-T and LysoTracker™ Deep Red in the cells, which eventually results in intensity differences between the two fluorophores. The Manders’ coefficient M1 represents the percentage of UbcH786C +-T that overlaps with LysoTracker™ Deep Red, while the Manders’ coefficient M2 represents the percentage of LysoTracker™ Deep Red that overlaps with UbcH786C +-T.

Internalization mechanism study

HepG2 cells were seeded onto 6-well plates and grown for 24 hours. To investigate the internalization mechanisms, the cells were preincubated with various metabolic inhibitors, including 20 µM chlorpromazine (CPM), 50 µM amiloride (AM), 2.5 mM methyl-beta-dextrin (MβCD) for 30 minutes, respectively, followed by incubation with 1.5 µM UbcH786C +-T and 3 µM TAT3 for 30 minutes, then the cells were washed three times with PBS containing heparin. Cellular uptake was quantified by flow cytometry (CytoFLEX, Beckman) and analyzed by FlowJo software.

Intracellular enzyme activity assay

For the RNase A activity assay, HepG2 cells were seeded in 96-well plates at 4000 cells per well for 24 hours, and followed by incubation with different concentrations of RNase A+ and RNase A in the presence of 3 µM TAT3 for 30 minutes. Subsequently the cells were incubated in fresh culture medium for another 24 hours, after which CCK-8 solution was added to the wells for 2 hours and the absorbance was measured at 450 nm. For the HRP activity assay, HepG2 cells were seeded in 96-well plates at 5000 cells per well for 24 hours, and followed by incubation with 0.5 µM HRP+ and HRP in the presence of 3 µM TAT3 for 30 minutes. Cellular HRP activities were then detected by TMB according to manufacturer’s instructions. For the β-Gal activity assay, HepG2 cells were plated onto sterile 35 mm glass-bottom dishes and grown for 24 hours followed by incubation with corresponding samples and staining with X-Gal staining kit according to manufacturer’s instructions.

Cell viability analysis

For Cell Counting Kit-8 (CCK-8) assay, HepG2 cells were seeded in 96-well plates at 4000 cells per well for 24 hours and then treated with different concentrations of TAT3 for 30 minutes. 0.64% phenol treatment was used as a control. Subsequently cells were incubated in fresh culture medium for another 24 hours and then CCK-8 solution was added to the wells for 2 hours and the absorbance was measured at 450 nm.

Fluorescent experiments in plants

3 µM UbcH786C +-T or Ub+-T was mixed with 3 µM TAT3 and then loaded into a 1 mL needleless plastic syringe. Place the tip of the syringe against the back of the leaf and press the tip of the syringe lightly against the leaf and slowly depress the syringe plunger while applying counter-pressure from the other side with the index finger. Successful infiltration is observed when the water-soaked area on the leaf blade expands, and after incubation for 1 hour, the plant leaves were infiltrated with 90 µM QSY21-NHS (N-hydroxysuccinimide linked fluorescence quencher moiety) and incubated for a further 1 hour. QSY21-NHS can be used to label the primary amines (R-NH2) of extracellular UbcH786C +-T or TAT3 to quench the extracellular fluorescence of TAMRA. The plant leaves were then cut and washed with water to remove protein samples adsorbed on the leaf surface. The leaf epidermis was torn off to allow clearer CLSM observation.

Preparation of E2-Ub photocrosslinking probes

Biotinylated Ub31 Bpa was prepared by automated Fmoc-based microwave peptide synthesis on Wang resin and refolded in reaction buffer (20 mM Tris, 150 mM NaCl, pH 7.4). The UBE2D3 C85K S22R mutant was obtained by E. coli expression and purified with FPLC. To generate the E2-Ub photocrosslinking probes, the UBE2D3 mutant (200 µM) was incubated with biotinylated Ub31 Bpa (100 µM) and Uba1 (1 µM) in reaction buffer (20 mM Tris, 150 mM NaCl, 3 mM ATP, 5 mM MgCl2, 1 mM TCEP, pH 8.5) at 37 °C for 24 hours. The E2-UbBpa conjugate was applied onto a Superdex TM 75 Increase 10/300 GL column (PBS, pH 7.4) for purification. The purified probes were then conjugated with the E4D3 peptide to give E2UBD3-Ub Bpa probe+.

Proteomic profiling of EGF-stimulated living HeLa cells

HeLa cells were first pre-incubated with MG132 and bafilomycin for 6 hours to prevent potential degradation of E3s and then stimulated with EGF. Experimental cells (A) were incubated with 8 µM probe+ and 3 µM TAT3 for 30 minutes, while control cells (B) were incubated with 8 µM probe+ only. After three times washes with heparin to remove membrane-bound probes, the cells were resuspended in cold PBS and irradiated with 365 nm UV for 30 minutes at a distance of ~3 cm in cold PBS on ice to activate the probe+. Finally, the cells were lysed with RIPA lysis buffer (containing 1mM PMSF). The lysate was centrifuged at 12 × g for 30 minutes at 4 °C, and the collected supernatant was used for subsequent proteomic analysis.

Mass spectrometry data collection and analysis

The identification process for the E2-Ub probe was as follows: Experimental group A: 8 µM probe+ and 3 µM TAT3 for 30 minutes, control group B: 8 µM probe+ only. Each group was repeated three times (biological replicates). The collected supernatants were first incubated with streptavidin agarose beads (Promega) at 4 °C overnight, then washed with four buffers in the following order: the buffer I (25 mM tris, 150 mM NaCl, 1 mM DTT, 0.5% v/v NP-40, pH = 7.6) once, the buffer II (PBS containing 0.5% w/w SDS) twice, the buffer III (PBS containing 1 M NaCl) twice, and finally the buffer IV (50 mM NH4HCO3, pH=7.6) for ten times. The enriched proteins on the beads were then eluted with loading buffer at 95 °C for ten minutes, followed by SDS-PAGE separation. The proteins in the gel were then sequentially treated with 5 mM DTT and 11 mM IAA. In-gel digestion was then performed overnight using trypsin in a 50 mM NH4HCO3 buffer. The peptides were extracted using buffer V (a 1:1 CH₃CN/H2O buffer containing 0.1% TFA) and then concentrated.

A Thermo-Dionex Ultimate 3000 high-performance liquid chromatography system was used for gradient elution at a flow rate of 0.3 µL/min for 120 minutes. The system was connected directly to a Thermo Orbitrap Fusion 480 mass spectrometer. Separation was achieved using a custom-made fused-quartz capillary column (Upchurch, Oak Harbor, WA) with an inner diameter of 75 µm and a length of 150 mm. The column was packed with 5 µm C-18 resin (Varian, Lexington, MA) with a particle size of 300 Å. Mobile phase 1 was an aqueous solution containing 0.1% formic acid and mobile phase 2 was an acetonitrile solution containing 0.1% formic acid. Data processing utilized the Sequest HT algorithm within Proteome Discoverer software (PD, version 2.5). HeLa cell samples were analyzed using the Human Protein Database as the query source. The search parameters were set as follows: full trypsin specificity, allowing up to two uncut sites. Methionine oxidation (M) was treated as a variable modification and cysteine carbamoylation (C) as a fixed modification. The precursor ion mass tolerance was set to 20 ppm for MS scanning and 20 mmu for MS/MS spectra. The false discovery rate (FDR) for peptides was calculated using the Percolator function in the PD software with a threshold of 1% based on a decoy database.

Statistics and reproducibility

All statistical analyses were performed using GraphPad Prism (v10.1.2). Numbers (n) of samples or replicates are indicated in the figures or figure legends. All fluorescence imaging experiments were repeated independently at least three times on different days with similar results. Control groups were included in all experiments in this study. All statistical tests performed in this study were two-sided. Statistical significance between two groups was analyzed using an unpaired Student's t-test. For comparisons among multiple groups, one-way analysis of variance (ANOVA) with Tukey’s test correction was used.

Data availability

The mass spectrometry proteomics data generated in this study have been deposited in the ProteomeXchange [http://proteomecentral.proteomexchange.org] database under accession code PXD059494. Source data are provided with this paper. Data supporting the findings of this study are available from the corresponding author Yi-Ming Li upon request.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this study, the authors developed a platform for delivering diverse protein cargoes into mammalian and plant cells through a combination of anionic peptide engineering and CPP-mediated delivery. The authors are motivated by their interest in the use of E2 probes to identify their partner E3s (Ub E3 ligases). This is a very relevant scientific challenge that would have significant implications for understanding ubiquitin biology.\nBy conjugating proteins to a custom-designed anionic peptide (E4D3) via a cleavable disulfide bond and co-incubating with the cationic cell-penetrating peptide TAT3, they achieved efficient cytosolic delivery of a wide range of proteins with molecular weights from ~2 kDa to 430 kDa and isoelectric points from ~4 to 10. The system operates at low micromolar concentrations, avoids toxicity, and facilitates the intracellular release of native proteins upon disulfide cleavage, providing a practical and scalable strategy for functional protein delivery in both animal and plant systems. The authors have conducted a very good study, spanning development to applications. Therefore, the manuscript is suitable for Nat Com following these comments.\nComments:\n1. Clarification is needed on the conjugation strategy used for attaching the cR10 CPP to the UbcH786C-T mutant, particularly since the available cysteine residue was also used to link TAMRA via a disulfide bond. However, since this protein contains only a single cysteine residue, it is unclear how both modifications were achieved simultaneously.\n2. The central rationale of this study is that increasing the negative charge of protein cargo using the anionic E4D3 tag enhances electrostatic interactions with the cationic CPP TAT3, thereby promoting cellular uptake. However, if this mechanism holds, then proteins that are already highly negatively charged (i.e., low pI) should, in theory, also be internalized when co-incubated with TAT3 alone. Could the authors clarify this apparent inconsistency and explain why inherently anionic proteins failed to show delivery in the absence of the E4D3 tag? The authors are aware of the work of Liu on supercharged proteins https://doi.org/10.1021/ja071641y\n3.\n4. Raines and Cowrker have done a nice study on masking temporarily negative charges to make proteins permeable. What do the authors think of this in relation to their work (https://doi.org/10.1021/jacs.7b06597)?\n5. The manuscript emphasizes delivery at “low micromolar concentrations”; however, the concentration used throughout (1.5 µM) is generally considered moderate for CPP-based delivery, as supported by several existing studies. It would be more accurate to contextualize this concentration with relevant literature, including prior studies where these authors have shown protein delivery below one micromolar (see, for example, Angewandte Chemie International Edition, 2022, e2022075 and Ref. 11).\n6. In this study, the anionic peptide is linked to the protein cargo via a disulfide bond, which limits this method to proteins that naturally contain cysteine residues. When cysteines are absent, thiolation is employed to introduce sulfhydryl groups; however, this chemical modification may alter the protein’s structure and folding. Consequently, this approach has limitations and may not be widely applicable to all proteins. This should be discussed.\n7. The authors are overlooking previous research regarding the delivery of synthetic proteins and probes. Brik et al. have made significant contributions to the field of synthetic protein delivery, particularly in the context of the Ub and Ubl systems (e.g., Chemical Communications, 2022, 58, 8782-8785, ChemBioChem, 2022, 23, e202200122, Chemical Communications, 2021, 57, 9438 – 9441, Angewandte Chemie International Edition, 2021, 13, 60,7333 –7343, Angewandte Chemie International Edition, 2024, 63, e202410135.). In particular, some similar problems like those of Ub were used in this study to compare the delivery of different proteins and have also been reported in some of the above refs. Including the work of others not only recognizes additional contributions but also provides valuable context for readers concerning the broader landscape of protein delivery approaches and applications, as discussed in Accounts of Chemical Research (2023, 56, 14, 1953–1965). The latter reference is particularly relevant in the introduction section of the manuscript among refs 1-3 that directly refer to what is stated in the first sentences.\n8. The authors should avoid overstating their findings, given that the field is already crowded with methods. Attempting to present this method as a transformative breakthrough is, at the very least, inappropriate. We have learned that every protein can yield surprises, even with minimal changes made to these proteins.\n9. What is the percentage of cells that are transfected with the different proteins?\nOther comments:\n1. In the results section, the subtitle “UbcH786C+ is internalized via micropinocytosis” states that the internalization mechanism is via micropinocytosis; however, the authors subsequently demonstrate that macropinocytosis is the actual mode of entry. This appears to be a typographical error that should be corrected.\n2. In Figure 2c, the x-axis lacks a clear label, making it unclear what the presented values represent.\n3. On page 8, under the Discussion section, line 12, there appears to be a minor typographical error where “viaa” is written instead of “via.”\n4. The title should exclude the concentration “ Synergizing anionic chemical engineering with electrostatic interactions to empower cytosolic delivery of diverse proteins.”", "reviewer_2": "This is an interesting and timely contribution to the field of intracellular delivery of large biologics. The authors describe an approach to engineer the surfaces of proteins with anionic peptides, which create noncovalent electrostatic interactions with Tat clusters, and thereby deliver various protein cargoes into cells at low concentrations.\nBoth the use of trimeric CPP clusters and the use of non-covalent association of CPP and cargo have been demonstrated previously. Particularly, the Tat3 which the author use as their lead peptide for the delivery of cargos into cells has been described and studied previously as 3Tat (Brock DJ, Kustigian L, Jiang M, Graham K, Wang TY, Erazo‐Oliveras A, Najjar K, Zhang J, Rye H, Pellois JP. Efficient cell delivery mediated by lipid‐specific endosomal escape of supercharged branched peptides. Traffic. 2018 Jun;19(6):421-35.) This article is not currently cited in the manuscript and should be include with similarities / inspiration of current work / designs clearly stated & discussed. In general, the manuscript should be amended to clarify which innovation and agents in this paper are novel, and which have been described and studied elsewhere to support the claim of novelty in this paper.\nThe contribution of this article to the field is the discreet engineering of proteins with anionic peptides in combination with the use of trimeric peptide clusters. This article represents an important advance of the field, and is broad in scope, exemplifying a variety of use cases which are of interest to the field of intracellular delivery.\nI have several concerns about this article which should be addressed prior to publication in Nature Communications.\n1) The authors mention their “standing interest in the use of E2 probes” and cite one paper [15] which appears to have originated from a different lab. Similar to general comment above, some clearer attribution of previous is essential in revised version.\n2) The description and characterization of peptide agents developed / used in this study is insufficient for reproduction – synthetic details should be added. MS analysis of peptides is very difficult to interpret, and chemical identity is hard to verify because the size of spectra is very small and m/z values cannot be discerned. Please add full size spectra.\n3) Why are there cysteines in the Tat3? The original version of these peptides (Brock et al. – reference above) excludes the GC motif between the Tat sequence and the KGKGKG backbone used here. Is there a particular reason why these cysteines were added? These could form disulphide bonds among themselves or with cargos – this could fundamentally change the mechanism of cargo-CPP interaction proposed here. Also – is the C-terminal of the backbone peptide a ketone or is that a typo?\n4) The structure of the tri-cTat CPP is different from the agent disclosed in the literature. Is that an intentional change? Compared to original, there is a CH2 group missing in the central scaffold. Synthetic method should be added to manuscript (particularly if different from original molecule).\n5) Manuscript requires further information on quantification. N numbers of individual cells analysed should be quoted for all analysis; the microscopy data in the majority of extended figures has not by analysed / quantified.\n6) Quantification of delivery – Nuclear MFI – is this the most appropriate measure of efficacy given that the proposed mechanism of delivery is that anionic surface modification and a CPP detach from cargo upon delivery into the cytosol. Do protein cargos traffic into the nucleus without CPP?\n7) All optimization of this technology was done on one specific enzyme. The paper makes a claim for this approach as generally applicable technology; while the delivery of other cargos is presented in Figure 4, it is not possible to establish the efficacy of delivery from the data presented. Efficacy of uptake data should be added for these cargos or claim at generality revised and discussed appropriately in the Discussion section.", "reviewer_3": "Major Concerns\no The manuscript presents the biotinylated E2~Ub photocrosslinking probe as if it were an innovation of the authors. However, the precise probe architecture has previously been reported (Mathur et al.). Proper scholarly practice requires the authors to cite this prior work explicitly at the appropriate point in the manuscript and clarify that their contribution is the synthesis of a variant carrying the E4D3 tag.\no Details on probe synthesis are insufficient. It is unclear how biotin was introduced into the probe and the precise chemical structure of the biotin + linker region probe is not described. Moreover, no mass spectrometry data are provided to confirm the integrity and identity of the final construct.\no The authors claim successful probe delivery into cells, but the images suggest that the probe does not distribute diffusely in all cells. Instead, it accumulates in subcellular puncta in some cells. This observation must be clarified, as it has important implications for probe E3 accessibility, target engagement, and interpretation of the results.\no The experiment replicating Mathur et al., using lysates, with EGF treatment lacks a minus-EGF control. I am not suggesting the experiment be repeated, but the purpose of EGF treatment in Mathur et al. was to demonstrate activation of CBL. PJA2 was also detected upon EGF stimulation, but neither are in the current study highlighting a shortcoming with the approach for studying these EGF responsive E3s. This should be discussed and reconciled. A likely explanation is the subcellular location of the probe is restricted, as suggested by the microscopy.\no The manuscript misrepresents prior work by implying that only 7 RING E3 ligases were detected by Mathur et al. when in fact 25 were reported. It states that 4 of the 7 are unique to Mathur et al. This is incorrect as the number is far higher.\no Mathur et al. restricted their analysis to proteins annotated with the Pfam term “RING”. This excluded cullin scaffolds, substrate receptors, the exchange factor CAND1, and UBR4. When this is considered, the number of detected RING E3s in the authors study is ~26. The an accurate comparison, the available raw data from Mathur might need to be subjected to be re filtering because the pram domain database would have been updated since that study.\no Taken together, the current comparative analysis is flawed. The reality is that the in-cell and lysate-based approaches are complementary with a similar number of uniquely detected RING E3s, and partial overlap. This must be revised.\nSummary\nThe manuscript requires significant revision for scholarly accuracy, methodological transparency, and fair contextualisation.\nSpecifically:\n• Cite prior work correctly and position the present study as a variant approach.\n• Provide clear methodological details (biotin incorporation, probe structure, mass spec validation).\n• Address conclusions drawn about probe subcellular localisation.\n• Correct the comparative analysis to reflect that the two approaches are complementary rather with the careful reanalysis of the unique and overlapping E3s after filtering for RING E3s."}, "author_response": "Response: We thank for the positive comments.\nComments:\n1. Clarification is needed on the conjugation strategy used for attaching the cR10 CPP to the UbcH786C-T mutant, particularly since the available cysteine residue was also used to link TAMRA via a disulfide bond. However, since this protein contains only a single cysteine residue, it is unclear how both modifications were achieved simultaneously.\nResponse: As shown in Figure 1 below, UbcH7 was first labeled with NHS-TAMRA via a nucleophilic 87C substitution reaction to generate UbcH7 -TAMRA (UbcH7 -T). This fluorescent conjugate was subsequently 87C 87C reacted with cR10 via a disulfide bond exchange to produce the final product, cR -UbcH7 -T. We have 10 87C provided a detailed description of the sample preparation process in the revised manuscript (P20, line 471-475) and in Extended Data Fig. 1B.\nFigure 1. Preparation flowchart of cR -UbcH7 -T.\n10 87C\n2. The central rationale of this study is that increasing the negative charge of protein cargo using the anionic E4D3 tag enhances electrostatic interactions with the cationic CPP TAT3, thereby promoting cellular uptake. However, if this mechanism holds, then proteins that are already highly negatively charged (i.e., low pI) should, in theory, also be internalized when co-incubated with TAT3 alone. Could the authors clarify this apparent inconsistency and explain why inherently anionic proteins failed to show delivery in the absence of the E4D3 tag? The authors are aware of the work of Liu on supercharged proteins https://doi.org/10.1021/ja071641y.\nResponse: The central idea of this work is to carry out “localized charge engineering” on protein surfaces, thereby artificially inducing electrostatic interactions with cationic cell-penetrating peptides (CPPs) to achieve efficient cytosolic protein delivery. Although the isoelectric points (pIs) of proteins such as bovine serum albumin (BSA) and blue fluorescent protein (BFP) are relatively low — approximately 4.7 for BSA and 6.6 for BFP — the pIs value reflects the average charge density of the entire protein structure. This average negative charge distribution may not be sufficient to form high-density local negative charge \"patches\" capable of generating electrostatic interactions with TAT . Our experimental data (Figure 2) also support this explanation, 3 that is, neither native BSA nor BFP alone can be successfully delivered into cells via co-incubation with TAT . 3 In contrast, the E4D3 modification introduces a high-density anionic peptide segment at specific sites on the protein surface, thereby creating a localized negatively charged microenvironment. This design allows for strong electrostatic association with the positively charged TAT , which is crucial for the successful delivery in this 3 study (Page 10, lines 287-292).\nWe thank the reviewer for drawing our attention to the work of David R. Liu’s team on supercharged proteins (J. Am. Chem. Soc. 2007, 129, 10110). Compared with the strategy of regulating the overall net charge of proteins in that work, our method focuses more on engineering local charge density. This provides a valuable approach for delivering proteins that are inherently difficult to convert into a \"supercharged\" state. We have now cited this important Ref. in the \"Introduction\" section (Ref. 6) of the revised manuscript.\nFigure 2. Cellular uptake of BSA and BFP using TAT .\n3\n3. Raines and Coworker have done a nice study on masking temporarily negative charges to make proteins permeable. What do the authors think of this in relation to their work (https://doi.org/10.1021/jacs.7b06597)?\nResponse: Although both our and Raines’s strategy focus on \"charge engineering\" on the protein surfaces, the Raines’s approach employs multi-site chemical modification to mask the native negative charges on protein cargos, such as GFP (Figure 3). This increases their net positive charge, enabling autonomous cellular uptake.\nIn contrast, our strategy involves site-specific conjugation of the E4D3 peptide to increase local negative charge density. This facilitates localized electrostatic interactions with the cationic CPP TAT , thereby achieving TAT - 3 3 mediated transmembrane delivery.\nWe thank the reviewer for this valuable Ref. and have cited it in the relevant section of the manuscript (see Ref. 51).\nFigure 3. Comparison of these two approaches\n5. The manuscript emphasizes delivery at \"low micromolar concentrations\"; however, the concentration used throughout (1.5 µM) is generally considered moderate for CPP-based delivery, as supported by several existing studies. It would be more accurate to contextualize this concentration with relevant literature, including prior studies where these authors have shown protein delivery below one micromolar (see, for example, Angewandte Chemie International Edition, 2022, e2022075 and Ref. 11).\nResponse: We agree with the reviewer's comment and have replaced the term \"low concentration\" with the specific value of \"1.5 µM\" throughout the manuscript.\n6. In this study, the anionic peptide is linked to the protein cargo via a disulfide bond, which limits this method to proteins that naturally contain cysteine residues. When cysteines are absent, thiolation is employed to introduce sulfhydryl groups; however, this chemical modification may alter the protein's structure and folding. Consequently, this approach has limitations and may not be widely applicable to all proteins. This should be discussed.\nResponse: We thank this suggestion. Although previous studies (Nat. Chem. 2021, 13, 530) have indicated that introducing sulfhydryl groups through thiolation may not affect the function of certain proteins, we agree with the reviewer that such chemical modifications could potentially alter the structure and bioactivity of other proteins. Accordingly, we have included additional analysis addressing this point in the \"Discussion\" section (Page 10, lines 276-278) of the revised manuscript.\n7. The authors are overlooking previous research regarding the delivery of synthetic proteins and probes. Brik et al. have made significant contributions to the field of synthetic protein delivery, particularly in the context of the Ub and Ubl systems (e.g., Chemical Communications, 2022, 58, 8782-8785, ChemBioChem, 2022, 23, e202200122, Chemical Communications, 2021, 57, 9438 - 9441, Angewandte Chemie International Edition, 2021, 13, 60,7333 -7343, Angewandte Chemie International Edition, 2024, 63, e202410135.)\nIn particular, some similar problems like those of Ub were used in this study to compare the delivery of different proteins and have also been reported in some of the above refs. Including the work of others not only recognizes additional contributions but also provides valuable context for readers concerning the broader landscape of protein delivery approaches and applications, as discussed in Accounts of Chemical Research (2023, 56, 14, 1953–1965). The latter Ref. is particularly relevant in the introduction section of the manuscript among refs 1-3 that directly refer to what is stated in the first sentences.\nResponse: We apologize for the missing of these significant contributions from Brik’s group. We have now incorporated key publications from the Brik group on ubiquitin delivery (Angew. Chem. Int. Ed. 2021, 60, 7333-7343. See Ref. 7; Angew. Chem. Int. Ed. 2024, 63, e202410135. See Ref. 5), as well as a highly relevant review article (Acc. Chem. Res. 2023, 56, 1953-1965. See Ref. 3) in revised manuscript.\n8. The authors should avoid overstating their findings, given that the field is already crowded with methods. Attempting to present this method as a transformative breakthrough is, at the very least, inappropriate. We have learned that every protein can yield surprises, even with minimal changes made to these proteins.\nResponse: We have checked the entire manuscript thoroughly and removed the overstated claims.\n9. What is the percentage of cells that are transfected with the different proteins?\nResponse: The transduction efficiency of proteins was quantified using a combination of flow cytometry and confocal fluorescence imaging. Flow cytometric analysis of UbcH7 -T showed a transduction efficiency of 86C 72.4% in HepG2 cells when incubated with of 1.5 μM UbcH7 -T and 3 μM TAT (Figure 4A). To validate 86C 3 this finding, we further analyzed fluorescence images from three independent experiments using the statistical method described in the literature (Nat. Chem. 2022, 14, 284-293) (Figure 4B), which yielded a closely matched transduction efficiency of 77.8% (Figure 4C).\nWe then used the image-based analytical approach to evaluate the delivery efficiency of other fluorescently labeled proteins. As summarized in Figure 4C, all of the proteins exhibited consistent transduction efficiencies exceeding 55%. These quantitative efficiency data are now presented as a new bar graph in Fig. 4D in the manuscript, and representative fluorescence images for these experiments can be found in the Supporting Information (Fig. S4).\nFor non-fluorescent functional proteins (e.g., RNase A and HRP) whose transduction efficiency cannot be directly measured via flow cytometry or imaging, their successful delivery has also been demonstrated through intracellular functional assays (see Fig. 4E-G in the revised manuscript). We have strengthened the analysis and discussion of transduction efficiencies across different protein types in the revised manuscript.\nFigure 4. (A) Flow cytometry analysis of the HepG2 cells treated with 1.5 µM UbcH7 +-T in the presence of 86C TAT for 30 minutes. (B) Quantification methods of the percentage of transduced cells in the literature (Nat. 3 Chem. 2022, 14, 284-293). (C) Quantification of the percentage of transduced cells in the manuscript.\nOther comments:\n1. In the results section, the subtitle \"UbcH786C+ is internalized via micropinocytosis\" states that the internalization mechanism is via micropinocytosis; however, the authors subsequently demonstrate that macropinocytosis is the actual mode of entry. This appears to be a typographical error that should be corrected.\nResponse: We have corrected \"micropinocytosis\" to \"macropinocytosis\" throughout the manuscript.\n2. In Figure 2c, the x-axis lacks a clear label, making it unclear what the presented values represent.\nResponse: We have added a label above the graph in Figure 2c in the revised manuscript.\n3. On page 8, under the Discussion section, line 12, there appears to be a minor typographical error where \"viaa\" is written instead of \"via.\"\nResponse: We have corrected \"viaa\" to \"via\" in the manuscript.\n4. The title should exclude the concentration \" Synergizing anionic chemical engineering with electrostatic interactions to empower cytosolic delivery of diverse proteins.\"\nResponse: In accordance with the reviewer's suggestion, we have revised the title of the manuscript, especially removing the descriptor \"low micromolar concentrations.\"\nReviewer 2:\nThis is an interesting and timely contribution to the field of intracellular delivery of large biologics. The authors describe an approach to engineer the surfaces of proteins with anionic peptides, which create noncovalent electrostatic interactions with Tat clusters, and thereby deliver various protein cargoes into cells at low concentrations.\nBoth the use of trimeric CPP clusters and the use of non-covalent association of CPP and cargo have been demonstrated previously. Particularly, the Tat3 which the author use as their lead peptide for the delivery of cargos into cells has been described and studied previously as 3Tat (Brock DJ, Kustigian L, Jiang M, Graham K, Wang TY, Erazo‐Oliveras A, Najjar K, Zhang J, Rye H, Pellois JP. Efficient cell delivery mediated by lipid‐specific endosomal escape of supercharged branched peptides. Traffic. 2018 Jun;19(6):421-35.)\nThis article is not currently cited in the manuscript and should be include with similarities / inspiration of current work / designs clearly stated & discussed. In general, the manuscript should be amended to clarify which innovation and agents in this paper are novel, and which have been described and studied elsewhere to support the claim of novelty in this paper.\nThe contribution of this article to the field is the discreet engineering of proteins with anionic peptides in combination with the use of trimeric peptide clusters. This article represents an important advance of the field, and is broad in scope, exemplifying a variety of use cases which are of interest to the field of intracellular delivery.\nResponse: We thank the reviewer for their positive comments. We have carefully revised the manuscript according to suggestions, with specific revision and clarifications provided below:\n(1) In the section of TAT synthesis, we have now cited the foundational work by Brock et al. (Traffic. 2018 3 Jun;19(6):421-3; see Ref. 18) and clarified that the TAT used in this study is based on the supercharged branched 3 peptides originally reported by Brock et al (Page 4, lines 73-75).\n(2) As rightly noted by the reviewer, the core innovation of our work not lies in TAT itself, but in the 3 introduction of a novel strategy that enables the efficient and general cytosolic protein delivery by engineering protein cargos with anionic motifs and using trimeric peptide clusters as carriers. In this context, E4D3 is a newly designed peptide. Conjugation of this peptide to protein cargos in a site-specific manner creates a localized, high- density negative surface charge, which significantly enhances electrostatic interaction with TAT . This 3 ultimately enables the efficient delivery of a broad range of proteins that were previously difficult to transport.\nWe have now articulated this innovative aspect in both the \"Results\" and \"Discussion\" sections (Page 10, lines 271-274) more clearly to better distinguish our contribution from prior studies.\nI have several concerns about this article which should be addressed prior to publication in Nature Communications.\nComments:\n1. The authors mention their “standing interest in the use of E2 probes” and cite one paper [15] which appears to have originated from a different lab. Similar to general comment above, some clearer attribution of previous is essential in revised version.\nResponse: We thank the reviewer for this observation. A long-standing interest of our laboratory is to develop E2-based probes to elucidate the functions of their E3 partners, as demonstrated by our previous work (Chem. Commun. 2019, 55, 7109; Angew. Chem. Int. Ed. 2021, 60, 17171; Sci. China Chem., 2025, 68. https://doi.org/10.1007/s11426-025-2697-6). In revised manuscript, we have updated the Ref. 15 where we describe this \"standing interest\" (Angew. Chem. Int. Ed. 2021, 60, 17171).\n2. The description and characterization of peptide agents developed / used in this study is insufficient for reproduction – synthetic details should be added. MS analysis of peptides is very difficult to interpret, and chemical identity is hard to verify because the size of spectra is very small and m/z values cannot be discerned. Please add full size spectra.\nResponse: We have added the synthetic methods and purification procedures for all peptides in \"Methods\" section. We also have provided the high-resolution mass spectrometry data in Fig. S1, where all m/z values are clearly labeled.\n3. Why are there cysteines in the Tat3? The original version of these peptides (Brock et al. – Ref. above) excludes the GC motif between the Tat sequence and the KGKGKG backbone used here. Is there a particular reason why these cysteines were added? These could form disulphide bonds among themselves or with cargos – this could fundamentally change the mechanism of cargo-CPP interaction proposed here. Also – is the C-terminal of the backbone peptide a ketone or is that a typo?\nResponse: In our initial approach to synthesize TAT , we used native chemical ligation (NCL) for a two- 3 fragment ligation (Figure 5A). Specifically, cysteine was conjugated to the lysine side chains of a KGKGKG backbone, which was then ligated with GRKKRRQRRRG-hydrazide via NCL. However, this method yielded an efficiency of only 12.2%. Following systematic optimization, we found that performing linear solid-phase peptide synthesis of TAT using Rink Amide Resin could achieve a notably higher yield of 55.2%. As a result, 3 the C-terminus of the resulting TAT is an amide group (–CONH₂).\nFigure 5. (A) Synthesis route of TAT . (B) Chromatographic and mass spectrometric characterisation of TAT . 3 3 (C) Stability evaluation of mixture of UbcH7 E4D3+ and TAT . The mixture was preserved at room temperature 86C 3 and detected by LC-MS for 16 days. (D) Coomassie blue staining and fluorescence images of UbcH7 +-T and 86C TAT samples in the presence of different concentrations of heparin.\n3\n4. The structure of the tri-cTat CPP is different from the agent disclosed in the literature. Is that an intentional change? Compared to original, there is a CH2 group missing in the central scaffold. Synthetic method should be added to manuscript (particularly if different from original molecule).\nResponse: We thank the reviewer for pointing out this mistake. The tri-cTat molecule used in our study is exactly the same as that reported in the literature (Nat. Chem. 2022, 14, 284). We have corrected the molecular structure in Fig. S1B of the revised manuscript and added a detailed description of the synthetic procedure in the \"Methods\" section (Page 19, lines 429-446).\n5. Manuscript requires further information on quantification. N numbers of individual cells analysed should be quoted for all analysis; the microscopy data in the majority of extended figures has not by analysed / quantified.\nResponse: We have revised all figure captions involving quantitative analysis of microscopy images, explicitly quoted the individual cell number for all analysis. Corresponding statistical details have also been updated in the \"Methods\" section.\nThe key image data in the Extended Data Figures has now been analyzed quantitatively and integrated into the main text. Specifically, the quantification of Extended Data Fig. 3A corresponds to Fig. 2C, the quantification of Extended Data Fig. 3C corresponds to Fig. 2D, and quantification of Extended Data Fig. 8A is now presented in Extended Data Fig. 8B. Quantitative analysis of some earlier images (e.g., Extended Data Fig. 1D, 1E, 1F, and 2B) from preliminary CPP screening experiments, quantitative analysis was deemed unreliable due to generally weak fluorescence signals and low signal-to-noise ratios. These images are retained as qualitative examples, and their captions have been updated to clarify their illustrative purpose and the reason why they have not been quantified (due to unsuitable signal characteristics).\n6. Quantification of delivery – Nuclear MFI – is this the most appropriate measure of efficacy given that the proposed mechanism of delivery is that anionic surface modification and a CPP detach from cargo upon delivery into the cytosol. Do protein cargos traffic into the nucleus without CPP?\nResponse: (1) We selected nuclear MFI as a quantitative metric because it can distinguish between proteins that have successfully escaped into the cytosol and those that are trapped within endosomes. Only soluble proteins that have completed endosomal escape and exhibit diffuse distribution throughout the cell (such as UbcH7 86C used in this manuscript) can freely diffuse into the nucleus (see, for example, the ubiquitin delivery in Figure 6A of Angew. Chem. Int. Ed. 2021, 60, 7333). This approach thus provides a more accurate measure of cytosolic delivery efficiency (Figure 6B, Nat. Chem. 2021, 13, 530). This quantification method has been adopted in previous studies (Nat. Chem. 2021, 13, 530; J. Am. Chem. Soc. 2023, 145, 24535), and we have now clarified its rationale in the revised manuscript (Page 4, lines 92-93).\n(2) It has been reported that UbcH7 itself can freely diffuse through nuclear pores independently of CPPs and exhibits a diffuse distribution throughout the entire cell (Figure 6C, J. Biol. Chem. 2001, 276, 19640; J. Endocrinol. 2006, 190, 621). Our experimental results are consistent with this: red fluorescently labeled UbcH7 exhibited a diffuse distribution throughout the entire cell (Figure 6D), whereas green fluorescently 86C labelled TAT accumulated in the nucleoli (Figure 6E). This spatial segregation suggests that UbcH7 3 86C dissociates from TAT in the cytosol. Therefore, the nuclear fluorescence signal of UbcH7 is derived from 3 86C the dissociated UbcH7 that has diffused freely into the nucleus.\n86C\nFigure 6 (A) Nuclear MFI of ubiquitin in the literature (Angew. Chem. Int. Ed. 2021, 60, 7333-7343). (B) Nuclear MFI of NLS-mCherry in the literature (Nat. Chem. 2021, 13, 530-539). (C) Fluorescence images of COS-7 cells transfected with UbcH7-GFP in the literature (J. Biol. Chem. 2001, 276, 19640). (D) Quantification of the UbcH7 -T fluorescence intensity along the white lines shown in the left-hand fluorescence images. The 86C gray box denotes the nuclear region. (E) Quantification of the FITC-TAT fluorescence intensity along the white 3 lines shown in the left-hand fluorescence images. The gray box denotes the nuclear region.\n7. All optimization of this technology was done on one specific enzyme. The paper makes a claim for this approach as generally applicable technology; while the delivery of other cargos is presented in Figure 4, it is not possible to establish the efficacy of delivery from the data presented. Efficacy of uptake data should be added for these cargos or claim at generality revised and discussed appropriately in the Discussion section.\nResponse: Following the established statistical approach from Nat. Chem. 2022, 14, 284 (Figure 7A), we quantified the transduction efficiency of all fluorescently labeled protein cargos by analyzing fluorescence images from three independent biological replicates. As shown in Figure 7B, the calculated transduction efficiency for UbcH7 -T was 77.8%, which closely matches the 72.4% positive cell rate determined by flow 86C cytometry, confirming the reliability of our measurements.\nUsing this image-based quantification method, we found that the delivery efficiencies of other fluorescently labeled proteins (Lifeact, NLS‑UbcH7, BFP, IgG, RFP, BSA) all exceeded 55% (Figure 7B). These quantitative efficiency data are now presented as a new bar graph in Fig. 4D in the manuscript (Page 7, lines 192-193), and representative fluorescence images for these experiments can be found in the Supporting Information (Fig. S4).\nFor non-fluorescent functional proteins (e.g., RNase A and HRP) whose transduction efficiency cannot be directly measured via flow cytometry or imaging, their successful delivery has also been demonstrated through intracellular functional assays (see Fig. 4E-G in the revised manuscript).\nIn addition, we have carefully moderated the claims regarding \"generality\" throughout the revised manuscript, and focused on the demonstrated capacity to deliver multiple distinct protein cargos in the discussion section.\nFigure 7 (A) Quantification methods of the percentage of transduced cells in the literature (Nat. Chem. 2022, 14, 284-293). (B) Quantification of the percentage of transduced cells in the manuscript.\nReviewer #3 (Remarks to the Author):\nMajor Concerns\n1. The manuscript presents the biotinylated E2~Ub photocrosslinking probe as if it were an innovation of the authors. However, the precise probe architecture has previously been reported (Mathur et al.). Proper scholarly practice requires the authors to cite this prior work explicitly at the appropriate point in the manuscript and clarify that their contribution is the synthesis of a variant carrying the E4D3 tag.\nResponse: We agree with the reviewer's opinion. The structure of the biotinylated E2-Ub probe used in our study originates from the work of Mathur et al. (Cell Chem. Biol. 2020, 27, 74-82. e6.). Our contribution is chemically conjugating an E4D3 tag to this probe, which enables its delivery into living cells. We have now cited this foundational work in the section of probe synthesis and reclassified our study as a complementary approach based on their design (Page 9, lines 239-240).\n2. Details on probe synthesis are insufficient. It is unclear how biotin was introduced into the probe and the precise chemical structure of the biotin + linker region probe is not described. Moreover, no mass spectrometry data are provided to confirm the integrity and identity of the final construct.\nResponse: The biotinylated Ub-Bpa was prepared via linear solid-phase peptide synthesis, with two AEEA groups incorporated between the biotin and ubiquitin sequences (Figure 8A, according to JACS Au 2023, 3, 2873). We have provided mass spectrometry of the final product E2-Ub+ probe (Figure 8B, also see Fig. S2 in the revised manuscript), and the detailed synthetic procedure has been updated in the \"Methods\" section.\nFigure 8 (A) Sequence and chemical structure of biotinylated Ub-Bpa. (B) Mass spectrometric of E2-Ub+ probe.\nDue to the presence of three free cysteine residues (C21, C107 and C111) on UBE2D3-Ub, the molecular weight of 30,762 corresponds to the final product E2-Ub+ probe (UBE2D3-Ub conjugated with three E4D3 tags). Molecular weights of 28,240 Da and 29,501 Da correspond to minor products featuring conjugation with one or two E4D3 tags, respectively.\n3. The authors claim successful probe delivery into cells, but the images suggest that the probe does not distribute diffusely in all cells. Instead, it accumulates in subcellular puncta in some cells. This observation must be clarified, as it has important implications for probe E3 accessibility, target engagement, and interpretation of the results.\nResponse: We agree with the reviewer that punctate fluorescence patterns were indeed observed in some cells, indicating probe accumulation within the endosome. Colocalization analysis using LysoTracker revealed that more than 70% of the internalized probe successfully reached the cytosol via endosomal escape pathways (Figure 9A). This finding is consistent with the previous reports demonstrating that a fraction of CPP-delivered protein cargos, particularly larger macromolecules such as mCherry and Fab fragments (Nat. Chem. 2021, 13, 530; Nat. Chem. 2022, 14, 284), typically remain sequestered within lysosomes and appear as bright puncta (Figure 9B).\nMeanwhile, proteomic analysis further confirms that the probe remains functionally active when delivered into live cells, as demonstrated by its ability to capture known UBE2D3 partner E3s (Fig. 6D in the revised manuscript).\nFigure 9 (A) Whole cell co-localization analysis of Probe+-T and LysoTracker signal. Co-localization images from the left: red channel (Probe+-T); purple channel (LysoTracker). (B) Representative images of cytosolic delivery of mCherry (Nat. Chem. 2021, 13, 530-539) ang Fab (Nat. Chem. 2022, 14, 284-293.).\n4. The experiment replicating Mathur et al., using lysates, with EGF treatment lacks a minus-EGF control. I am not suggesting the experiment be repeated, but the purpose of EGF treatment in Mathur et al. was to demonstrate activation of CBL. PJA2 was also detected upon EGF stimulation, but neither are in the current study highlighting a shortcoming with the approach for studying these EGF responsive E3s. This should be discussed and reconciled. A likely explanation is the subcellular location of the probe is restricted, as suggested by the microscopy.\nResponse: Upon careful re-examining our proteomics dataset, we noted that the two E3s (CBL and PJA2) mentioned by the reviewer were also identified in our experiments (Table 1). However, based on our predefined thresholds, they were not classified as significantly enriched (proteins with a fold change > 2 and a p-value < 0.05 were considered as significantly enriched). As Fig. 6D in the main text only displays significantly enriched proteins, CBL and PJA2 were excluded.\nWe also agree with the reviewer's opinion that the partial entrapment of the delivered probes within endosomal compartments may have restricted their access to certain E3s spatially, thereby limiting their capture. As suggested, we have now added a discussion of this point in the corresponding section (Page 9, lines 248-250).\nC: Gene Abundances: Abundances: Abundances: Abundances: Abundances: Abundances:\nAccession name C1 Sample C2 Sample C3 Sample S1 Sample S2 Sample S3: Sample\nP22681 CBL 266996 634300 292309 337656 1241400 261477\nO43164 PJA2 377155 121890 279441 180775 175399 214645\nTable 1 Raw data of CBL and PJA2 identified in this manuscript.\n5. The manuscript misrepresents prior work by implying that only 7 RING E3 ligases were detected by Mathur et al. when in fact 25 were reported. It states that 4 of the 7 are unique to Mathur et al. This is incorrect as the number is far higher.\nResponse: In the original manuscript, our statement was: \"Notably, the UBE2D3-Ub Bpa probe was previously used to profile RING E3s in EGF-stimulated HEK293T cell lysates. A comparison of these two data sets (Table S3) revealed that 7 partner E3s were labeled in both living cells and lysates,\". This intended to indicate that seven UBE2D3 partner E3s identified by our method overlapped with those identified by Mathur et al., rather than that only 7 RING E3s were detected in their work. To prevent any potential misinterpretation, we have deleted this sentence from the revised manuscript.\n6. Mathur et al. restricted their analysis to proteins annotated with the Pfam term “RING”. This excluded cullin scaffolds, substrate receptors, the exchange factor CAND1, and UBR4. When this is considered, the number of detected RING E3s in the authors study is ~26. The an accurate comparison, the available raw data from Mathur might need to be subjected to be re filtering because the pram domain database would have been updated since that study.\nTaken together, the current comparative analysis is flawed. The reality is that the in-cell and lysate-based approaches are complementary with a similar number of uniquely detected RING E3s, and partial overlap. This must be revised.\nResponse: As requested by the reviewer, we carefully analyzed our experimental data. After excluding the cullin scaffolds, substrate receptors, the exchange factor CAND1, and UBR4, we identified 42 RING-type E3 ligases totally, among which 21 were significantly enriched (fold change > 2 and p-value < 0.05).\nComparing this dataset with that of Mathur et al., we found that 10 of these E3 ligases were identified by both methods: BRAP, CBL, PJA2, MIB1, RAD18, RING1, RNF168, RNF10, RNF114, and RNF126. We also identified additional E3s, such as UHRF1, TRIM32, ZNF598, RNF168 and RNF181, and Mathur et al. also reported different E3s, including BRCA1, BRE1A, BRE1B, HLTF and PCGF6. Together, these findings collectively show the complementarity between the two approaches, offering valuable tools for studying E2–E3 pairing relationships.\nWe have updated Figure 6D, Table S1 and Table S2 in the manuscript accordingly, and revised the corresponding descriptions and analyses in the main text (Page 9, lines 263-267).\nSummary\nThe manuscript requires significant revision for scholarly accuracy, methodological transparency, and fair contextualisation. Specifically:\n• Cite prior work correctly and position the present study as a variant approach.\n• Provide clear methodological details (biotin incorporation, probe structure, mass spec validation).\n• Address conclusions drawn about probe subcellular localisation.\n• Correct the comparative analysis to reflect that the two approaches are complementary rather with the careful reanalysis of the unique and overlapping E3s after filtering for RING E3s.\nResponse: We have made revision to the four aspects of suggestions by the reviewers in combination with each of the above specific issues.\nWe greatly thank these referees for the insightful suggestions that greatly help us improve the quality of the paper.\nWith my best wishes,\nLei Liu, Ph.D."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have done very good work on revising the current MS. Just one note that the cited account of chemical research appears in Ref 3, should be the following one and not the one that appears in this version:\nCite this: Acc. Chem. Res. 2022, 55, 15, 2055–2067\nhttps://doi.org/10.1021/acs.accounts.2c00236\nAssessment of authors' responses to comments of Reviewer 2:\nOverall, the revision is excellent. However, the following correction should be done before recommending publication.\nComment # 1: The author should cite Angewante Chemie, 2018, International Edition, 57, 5645-5649, as the original work on the sequential DHA formation, not the cited paper in ref 15.", "reviewer_3": "The authors have addressed my concerns."}, "author_response": ""}]

Decision letter