Mirror-image peptides and proteins are attracting interest as therapeutics, key building blocks for constructing mirror-image life, and as tools to probe the origin of life. Their resistance to proteolytic degradation and unique stereochemistry make D-peptides/proteins particularly appealing for biomedical applications, yet a critical unresolved question is how their intracellular uptake compares with that of natural L-forms. To address this, we systematically investigated the role of cargo chirality in cellular internalization while maintaining a constant delivery vehicle. Three model cargos of increasing size and structural complexity were synthesized in both L- and D-configurations and conjugated to an identical cyclic deca-arginine (cR10) cell-penetrating peptide (CPP). By keeping the CPP scaffold constant, we reduced delivery-related variability and directly assessed the influence of cargo chirality on uptake. Quantitative uptake analysis using flow cytometry, gel analysis, and confocal microscopy across multiple mammalian cell lines reveals that L-cargos are internalized more efficiently than their mirror-image D-counterparts, demonstrating that cargo chirality is a key determinant of uptake efficiency across the chiral biological membrane. Collectively, these findings provide a systematic basis for further exploration of chirality effects in CPP-mediated delivery and may inform the design of mirror-image peptides and proteins for therapeutic or synthetic biology applications.

RNF4, a RING-type E3 ubiquitin ligase, targets polySUMOylated proteins for ubiquitination and subsequent proteasomal degradation. The ability to chemically synthesize RNF4 will enable future studies of its structure and biological function, particularly its role in degrading the oncoprotein PML-RARα in acute promyelocytic leukemia. To achieve this, we performed a total chemical synthesis of RNF4 using sequential native chemical ligation. The presence of nine cysteine residues enables stepwise ligation of five peptide fragments to assemble the full-length protein. Two synthetic strategies were explored: the first employed a convergent C-to-N ligation, while the second used an N-to-C ligation. In the convergent C-to-N approach, cysteine residues were protected with acetamidomethyl groups to prevent side reactions during ligation, although this required multiple deprotection and purification steps. Conversely, the N-to-C synthesis method proceeded efficiently without cysteine protection, thereby simplifying the workflow and reducing the number of purification steps. This research presents a reliable and accessible method for the complete chemical synthesis of RNF4, addressing significant challenges in synthesizing large proteins and opening up new opportunities for future biological research.

Mirror-image peptides and proteins are attracting interest as therapeutics, as key building blocks for constructing mirror-image life, and as tools to probe the origin of life. Their resistance to proteolytic degradation and unique stereochemistry make D-peptides/proteins particularly appealing for biomedical applications, yet a critical unresolved question is how their intracellular uptake compares to that of natural L-forms. To address this, we systematically investigated the role of cargo chirality in cellular internalization while maintaining a constant delivery vehicle. Three model cargos of increasing size and structural complexity were synthesized in both L- and D-configurations and conjugated to an identical cyclic deca-arginine (cR10) cell-penetrating peptide (CPP). By keeping the CPP scaffold constant, we reduced delivery-related variability and directly assessed the influence of the cargo chirality on uptake. Quantitative uptake analysis using flow cytometry, gel analysis, and confocal microscopy across multiple mammalian cell lines reveals that L-cargos are internalized more efficiently than their mirror image D-counterparts, demonstrating that cargo chirality is a key determinant of uptake efficiency across the chiral biological membrane. Collectively, these findings provide a systematic basis for further exploration of chirality effects in CPP-mediated delivery and may inform the design of mirror image peptides and proteins for therapeutic or synthetic biology applications.

Aggressive and therapy-resistant cancers present a significant challenge to treatment and are associated with poor patients’ survival. Identifying molecular pathways and compounds that target these pathways is critical for improving patient outcomes. RNF4, an E3 Ubiquitin ligase, is pivotal for tumorigenesis in part by stabilizing oncoproteins and its role in DNA repair, thereby enhancing cancer cell survival and driving tumorigenesis. Elevated RNF4 levels are associated with poor prognosis in patients with carcinomas, melanoma, and sarcoma. Here, we describe the design and development of R4VPs, dual degrader compounds connecting two E3 ubiquitin ligases; Von Hippel-Lindau protein (VHL) with RNF4. R4VPs promote RNF4 degradation and thereby reduce the levels of its stabilized phosphorylated oncoproteins, while concomitantly eliminating VHL. R4VPs selectively induce ferroptotic cell death in cancer cells, sparing non-tumorigenic and primary cells in part by binding and modifying the anti-ferroptotic selanoproteins GPX4. R4VPs-induced ferroptosis preferentially targeting cells harboring tumor-driving mutations in the EGFR pathway, whereas it does not affect PI3K-transformed cells. As a consequence, R4VPs effectively induce cell death in Receptor Tyrosine Kinase inhibitor-resistant melanoma and primary patient sarcoma cells. Our findings highlight the potential of selective ferroptosis inducers, such as R4VPs, as a therapeutic strategy for hard-to-treat cancers.

The human genome encodes approximately 100 deubiquitinating enzymes (DUBs), but only three are considered proteasome-associated DUBs (pDUBs): PSMD14/Rpn11, USP14, and UCHL5. Among these, only PSMD14 is an integral 19S subunit, whereas USP14 and UCHL5 bind transiently to specific proteasomal subunits. Given the dynamic nature of proteasome composition, we searched for additional pDUBs. USP15 was found to be associated with 26S proteasomes purified from cultured cells. In proteasome preparations from erythrocytes, USP15 was identified as the most abundant transient pDUB. It was even feasible to separate proteasomes containing USP15 from those containing USP14. Although USP15 utilizes an internal ubiquitin-like (UBL) domain for positioning itself at the proteasome, it did not compete with the UBL-containing USP14. USP15 facilitated substrate selection at the proteasome by efficiently disassembling short polyubiquitin (polyUb) chains, while sparing K48-linked tetra-ubiquitin conjugates from deubiquitination. This feature may aid the proteasome in differentiating between substrates to be rescued from those committed to proteolysis. Identification of a fourth pDUB encourages the continued search for additional proteasome-interacting proteins that modulate its substrate specificity in a context-specific manner. ### Competing Interest Statement The authors have declared no competing interest. Israel Science Foundation, https://ror.org/04sazxf24, 2640/23, 755/19, 179/15 European Research Council, https://ror.org/0472cxd90, 831783, 101142726

InfoMetricsFiguresRef. ACS Central ScienceASAPArticle This publication is Open Access under the license indicated. Learn More CiteCitationCitation and abstractCitation and referencesMore citation options ShareShare onFacebookX (Twitter)WeChatLinkedInRedditEmailJump toExpandCollapse First ReactionsJanuary 6, 2025Wash-free Imaging in Live CellsClick to copy article linkArticle link copied!Palladium-mediated arylation enables minimal labeling of peptides and proteins with small fluorogenic amino acids for wash-free imaging.Mahdi HasanMahdi HasanSchulich Faculty of Chemistry Technion-Israel Institute of Technology Haifa, 3200008, IsraelMore by Mahdi HasanAshraf Brik*Ashraf BrikSchulich Faculty of Chemistry Technion-Israel Institute of Technology Haifa, 3200008, Israel*Email: [email protected]More by Ashraf Brikhttps://orcid.org/0000-0001-8745-2250Open PDFACS Central ScienceCite this: ACS Cent. Sci. 2025, XXXX, XXX, XXX-XXXClick to copy citationCitation copied!https://pubs.acs.org/doi/10.1021/acscentsci.4c02083https://doi.org/10.1021/acscentsci.4c02083Published January 6, 2025 Publication History Published online 6 January 2025newsPublished 2025 by American Chemical Society. This publication is licensed under CC-BY 4.0 . License Summary*You are free to share (copy and redistribute) this article in any medium or format and to adapt (remix, transform, and build upon) the material for any purpose, even commercially within the parameters below:Creative Commons (CC): This is a Creative Commons license.Attribution (BY): Credit must be given to the creator.View full license*DisclaimerThis summary highlights only some of the key features and terms of the actual license. It is not a license and has no legal value. Carefully review the actual license before using these materials. This publication is licensed underCC-BY 4.0 . License Summary*You are free to share(copy and redistribute) this article in any medium or format and to adapt(remix, transform, and build upon) the material for any purpose, even commercially within the parameters below: Creative Commons (CC): This is a Creative Commons license. Attribution (BY): Credit must be given to the creator.View full license *DisclaimerThis summary highlights only some of the key features and terms of the actual license. It is not a license and has no legal value. Carefully review the actual license before using these materials. License Summary*You are free to share(copy and redistribute) this article in any medium or format and to adapt(remix, transform, and build upon) the material for any purpose, even commercially within the parameters below: Creative Commons (CC): This is a Creative Commons license. Attribution (BY): Credit must be given to the creator. View full license *DisclaimerThis summary highlights only some of the key features and terms of the actual license. It is not a license and has no legal value. Carefully review the actual license before using these materials. License Summary*You are free to share(copy and redistribute) this article in any medium or format and to adapt(remix, transform, and build upon) the material for any purpose, even commercially within the parameters below: Creative Commons (CC): This is a Creative Commons license. Attribution (BY): Credit must be given to the creator. View full license *DisclaimerThis summary highlights only some of the key features and terms of the actual license. It is not a license and has no legal value. Carefully review the actual license before using these materials. ACS PublicationsPublished 2025 by American Chemical SocietySubjectswhat are subjectsArticle subjects are automatically applied from the ACS Subject Taxonomy and describe the scientific concepts and themes of the article.FluorescenceFluorescence imagingLabelingMonomersPeptides and proteinsIn this issue of ACS Central Science, Jbara, Vendrell, and co-workers report the development of a powerful mini-labeling approach to track the cellular uptake routes of peptides and proteins. (1) Cell delivery of custom-made peptides/proteins and other biomolecules is of great interest for basic research and therapeutic applications. (2) Investigating the entry mechanism and tracking the path of delivered biomolecules to their specific organelle and target are extremely challenging and require tools yet to be further developed and optimized for each target. In this context, fluorescence microscopy has been widely used for studying peptide/protein localization and trafficking with high spatial and temporal resolution. Therefore, great efforts have been invested to find the most effective labeling method that would 1) allow for high-resolution microscopy, also termed super-resolution microscopy (SR); 2) enable wash-free microscopy experiments for real-time tracking; 3) not interfere with the activity of the labeled biomolecule; and 4) not affect the delivery mechanism. Despite some advances in this area and the development of dyes, e.g., cyanines and rhodamines, we still lack a method that can fulfill all requirements. A dye that is neutrally charged and as small as an amino acid side chain will have clear advantages. Regarding the mechanism of action of some of these dyes, point accumulation for imaging in nanoscale topography (PAINT), which has been gaining great interest, utilizes fluorophores that emit light upon binding to targets, with early reports focusing on lipophilic dyes that fluoresce strongly in hydrophobic environments. (3)Investigating the entry mechanism and tracking the path of delivered biomolecules to their specific organelle and target are extremely challenging and require tools yet to be further developed and optimized for each target.To further move this field forward and prepare optimal probes for real-time imaging, the Vendrell group previously developed small unnatural fluorescent amino acids and integrated them into peptides to generate live-cell imaging reporters. The researchers have screened several benzodiazole derivatives, containing C, O, S, or Se in the heterocycle component, attached via S- or N-linkage to the β-carbon of the amino acid. They examined their suitability as reporters for wash-free fluorescence microscopy and incorporation in solid-phase peptide synthesis (SPPS, Figure 1A). These studies have shown that the S-linked amino acid with the thiol-containing heterocycle has the most desirable fluorescence properties, concerning photostability, exhibiting a low background signal in aqueous media and a strong fluorogenic response in a nonpolar environment. Yet when incorporated in a peptide via SPPS, it showed acid lability, rendering it unsuitable for peptide synthesis. (4)Figure 1Figure 1. (A) Cys-linked benzodiazole derivatives for wash-free imaging. (B) Modification of unprotected peptides or proteins via palladium-mediated arylation. (C) Tracking the delivery mechanism and localization of the labeled biomolecule using fluorescence microscopy in live cells.High Resolution ImageDownload MS PowerPoint SlideIn the current issue of ACS Central Science, Jbara, Vendrell, and co-workers presented a creative solution to this problem and were able to efficiently incorporate the Cys-linked benzodiazole as a small fluorescent molecule into peptides and proteins without affecting their bioactivity and their delivery mechanism. The authors developed an organometallic palladium complex bearing a benzodiazole moiety for site-selective insertion of the fluorogenic probe into unprotected peptides or proteins bearing a free Cys residue through palladium-mediated S-arylation, (5) bypassing the exposure of this amino to peptide synthesis conditions (Figure 1B). With this late-stage modification approach, the authors successfully assessed the fluorescence emission of three Cys-benzodiazole derivatives, containing O, S, or Se in the heterocycle component. This study revealed that the S-analog exhibited up to a 27-fold fluorescence increase, with the lowest background signals in water and the strongest response in hydrophobic conditions.In the current issue of ACS Central Science, Jbara, Vendrell, and co-workers were able to efficiently incorporate the Cys-linked benzodiazole as a small fluorescent molecule into peptides and proteins without affecting their bioactivity and their delivery mechanism.The authors demonstrated their method for labeling and tracking different cell-penetrating peptides (CPPs). Specifically, they modified TAT, Penetratin, and sC18 with benzo-2,1,3-thiadiazole. Following wash-free imaging experiments in live cells, they determined the delivery mechanism of the CPPs in spatiotemporal resolution. Once the CPP starts internalizing the cell, the hydrophobic surrounding enables the fluorescence emission of the benzodiazole to rise dramatically, allowing tracking of the delivery mechanisms in live-time imaging (Figure 1C). Their results showed that the TAT peptide internalized the cell via direct translocation, Penetratin via micelle-mediated internalization and endocytic uptake, while the sC18 internalized the cell via endocytic uptake, all of which agree with previous reports. Notably, for the sC18 peptide, the authors performed a fluorescence lifetime imaging microscopy (FLIM) experiment in live cells. They analyzed the florescent lifetime of the peptide in a localization-dependent manner, indicating that sC18 has a longer lifetime when interacting with the plasma membrane and a shorter lifetime in the cytosol and after translocation to the nucleus. The authors observed rapid nuclear localization of the sC18, indicating that it may have a dual internalization mechanism involving cellular endocytosis and direct translocation. Finally, the authors successfully labeled the expressed chemokine protein (mCCL2), bearing two disulfide bonds and a free Cys residue without distributing the native S–S bonds. This enabled mCCL2 tracking in live cells, demonstrating its binding to the chemokine receptor CCR2 and the endocytosis trafficking mechanism.Despite demonstrating the effectiveness of this approach in labeling different peptides, it still has some limitations. Labeling Cys-rich peptides and proteins using palladium-mediated arylation will be challenging and may affect the homogeneity of the product and its function. In principle, this could be overcome by using several technologies for modifying intact proteins and peptides such as π-clump, (6) sortase, (7) coiled-coil PNA labeling, (8) and palladium-mediated S-arylation on metal-binding motifs. (9) Additionally, genetic code expansion could allow precise and selective preparation of modified proteins with benzodiazole. (10)The powerful method developed by Jbara and Vendrell allows for rapidly accessing modified peptides, proteins, and possibly other biomolecules, with minimal labeling.The powerful method developed by Jbara and Vendrell allows for rapidly accessing modified peptides, proteins, and possibly other biomolecules, with minimal labeling. This lays the foundation for 1) investigating delivery and trafficking mechanisms in spatiotemporal resolution; 2) deciphering the role of several cytokines in immune responses and cell signaling; and 3) studying the effect of post-translationally modified proteins on their dynamics in live cells. By further expanding the labeling toolkit of proteins with the benzodiazole moiety, using the above proposed methods, one could access many labeled proteins and address several questions related to their journey in biological processes.Author InformationClick to copy section linkSection link copied!Corresponding AuthorAshraf Brik - Schulich Faculty of Chemistry Technion-Israel Institute of Technology Haifa, 3200008, Israel; https://orcid.org/0000-0001-8745-2250; Email: [email protected]AuthorMahdi Hasan - Schulich Faculty of Chemistry Technion-Israel Institute of Technology Haifa, 3200008, IsraelReferencesClick to copy section linkSection link copied! This article references 10 other publications. 1Nadal-Bufi, F.; Nithun, R. V.; de Moliner, F.; Lin, X.; Habiballah, S.; Jbara, M.; Vendrell, M. Late-Stage Minimal Labeling of Peptides and Proteins for Real-Time Imaging of Cellular Trafficking. ACS Cent Sci. 2024, DOI: 10.1021/acscentsci.4c01249 Google ScholarThere is no corresponding record for this reference.2Mann, G.; Sadhu, P.; Brik, A. Synthetic Proteins behind the Plasma Barrier: Molecular Spies. Acc. Chem. Res. 2022, 55 (15), 2055– 2067, DOI: 10.1021/acs.accounts.2c00236 Google ScholarThere is no corresponding record for this reference.3Oi, C.; Mochrie, S. G. J.; Horrocks, M. H.; Regan, L. PAINT Using Proteins: A New Brush for Super-Resolution Artists.. Protein Sci. 2020, 29, 2142– 2149, DOI: 10.1002/pro.3953 Google ScholarThere is no corresponding record for this reference.4de Moliner, F.; Konieczna, Z.; Mendive-Tapia, L.; Saleeb, R. S.; Morris, K.; Gonzalez-Vera, J. A.; Kaizuka, T.; Grant, S. G. N.; Horrocks, M. H.; Vendrell, M. Small Fluorogenic Amino Acids for Peptide-Guided Background-Free Imaging. Angewandte Chemie - International Edition 2023, 62 (4), e202216231, DOI: 10.1002/anie.202216231 Google ScholarThere is no corresponding record for this reference.5Vinogradova, E. V.; Zhang, C.; Spokoyny, A. M.; Pentelute, B. L.; Buchwald, S. L. Organometallic Palladium Reagents for Cysteine Bioconjugation. Nature 2015, 526 (7575), 687– 691, DOI: 10.1038/nature15739 Google Scholar5Organometallic palladium reagents for cysteine bioconjugationVinogradova, Ekaterina V.; Zhang, Chi; Spokoyny, Alexander M.; Pentelute, Bradley L.; Buchwald, Stephen L.Nature (London, United Kingdom) (2015), 526 (7575), 687-691CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group) Peptides and proteins, contg. cysteine amino acid, were labeled by conjugation of the SH-group with arylpalladium 2-biphenylylphosphine hemilabile reagent, causing arylation the mercapto-group. Reactions based on transition metals have found wide use in org. synthesis, in particular for the functionalization of small mols. However, there are very few reports of using transition-metal-based reactions to modify complex biomols., which is due to the need for stringent reaction conditions (for example, aq. media, low temp. and mild pH) and the existence of multiple reactive functional groups found in biomols. Here we report that palladium(II) complexes can be used for efficient and highly selective cysteine conjugation (bioconjugation) reactions that are rapid and robust under a range of bio-compatible reaction conditions. The straightforward synthesis of the palladium reagents from diverse and easily accessible aryl halide and trifluoromethanesulfonate precursors makes the method highly practical, providing access to a large structural space for protein modification. The resulting aryl bioconjugates are stable towards acids, bases, oxidants and external thiol nucleophiles. The broad utility of the bioconjugation platform was further corroborated by the synthesis of new classes of stapled peptides and antibody-drug conjugates. These palladium complexes show potential as benchtop reagents for diverse bioconjugation applications. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhslCnu7zK&md5=8fbb382c94600af312bb007d31b630566Zhang, C.; Welborn, M.; Zhu, T.; Yang, N. J.; Santos, M. S.; Van Voorhis, T.; Pentelute, B. L. π-Clamp-Mediated Cysteine Conjugation. Nat. Chem. 2016, 8 (2), 120– 128, DOI: 10.1038/nchem.2413 Google Scholar6π-Clamp-mediated cysteine conjugationZhang, Chi; Welborn, Matthew; Zhu, Tianyu; Yang, Nicole J.; Santos, Michael S.; Van Voorhis, Troy; Pentelute, Bradley L.Nature Chemistry (2016), 8 (2), 120-128CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group) Site-selective functionalization of complex mols. is one of the most significant challenges in chem. Typically, protecting groups or catalysts must be used to enable the selective modification of one site among many that are similarly reactive, and general strategies that selectively tune the local chem. environment around a target site are rare. Here, the authors show a four-amino-acid sequence (Phe-Cys-Pro-Phe), which the authors call the π-clamp, that tunes the reactivity of its cysteine thiol for site-selective conjugation with perfluoroarom. reagents. The authors use the π-clamp to selectively modify one cysteine site in proteins contg. multiple endogenous cysteine residues. These examples include antibodies and cysteine-based enzymes that would be difficult to modify selectively using std. cysteine-based methods. Antibodies modified using the π-clamp retained binding affinity to their targets, enabling the synthesis of site-specific antibody-drug conjugates for selective killing of HER2-pos. breast cancer cells. The π-clamp is an unexpected approach to mediate site-selective chem. and provides new avenues to modify biomols. for research and therapeutics. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXitVygurzE&md5=8c3c367f6fd7c64237bd0ba3e9bd40b47Theile, C. S.; Witte, M. D.; Blom, A. E. M.; Kundrat, L.; Ploegh, H. L.; Guimaraes, C. P. Site-Specific N-Terminal Labeling of Proteins Using Sortase-Mediated Reactions. Nat. Protoc 2013, 8 (9), 1800– 1807, DOI: 10.1038/nprot.2013.102 Google Scholar7Site-specific N-terminal labeling of proteins using sortase-mediated reactionsTheile, Christopher S.; Witte, Martin D.; Blom, Annet E. M.; Kundrat, Lenka; Ploegh, Hidde L.; Guimaraes, Carla P.Nature Protocols (2013), 8 (9), 1800-1807, 8 pp.CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group) This protocol describes the use of sortase-mediated reactions to label the N terminus of any given protein of interest. The sortase recognition sequence, LPLPXTG (for Streptococcus aureus sortase A) or LPXTA (for Staphylococcus pyogenes sortase A), can be appended to a variety of probes such as fluorophores, biotin or even to other proteins. The protein to be labeled acts as a nucleophile by attacking the intermediate formed between the probe contg. the LPXTG/A motif and the sortase enzyme. If sortase, the protein of interest and a suitably functionalized label are available, the reactions usually require less than 3 h. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsV2hurrK&md5=3630095942671637c7b047ac2e69c85f8Gavins, G. C.; Gröger, K.; Bartoschek, M. D.; Wolf, P.; Beck-Sickinger, A. G.; Bultmann, S.; Seitz, O. Live Cell PNA Labelling Enables Erasable Fluorescence Imaging of Membrane Proteins. Nat. Chem. 2021, 13 (1), 15– 23, DOI: 10.1038/s41557-020-00584-z Google Scholar8Live cell PNA labelling enables erasable fluorescence imaging of membrane proteinsGavins, Georgina C.; Groeger, Katharina; Bartoschek, Michael D.; Wolf, Philipp; Beck-Sickinger, Annette G.; Bultmann, Sebastian; Seitz, OliverNature Chemistry (2021), 13 (1), 15-23CODEN: NCAHBB; ISSN:1755-4330. (Nature Research) Abstr.: DNA nanotechnol. is an emerging field that promises fascinating opportunities for the manipulation and imaging of proteins on a cell surface. The key to progress is the ability to create a nucleic acid-protein junction in the context of living cells. Here we report a covalent labeling reaction that installs a biostable peptide nucleic acid (PNA) tag. The reaction proceeds within minutes and is specific for proteins carrying a 2 kDa coiled-coil peptide tag. Once installed, the PNA label serves as a generic landing platform that enables the recruitment of fluorescent dyes via nucleic acid hybridization. We demonstrate the versatility of this approach by recruiting different fluorophores, assembling multiple fluorophores for increased brightness and achieving reversible labeling by way of toehold-mediated strand displacement. Addnl., we show that labeling can be carried out using two different coiled-coil systems, with epidermal growth factor receptor and endothelin receptor type B, on both HEK293 and CHO cells. Finally, we apply the method to monitor internalization of epidermal growth factor receptor on CHO cells. [graphic not available: see fulltext]. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXisFaiurbM&md5=7d2f70001ede10fdecfd1dbed58ecd729Willwacher, J.; Raj, R.; Mohammed, S.; Davis, B. G. Selective Metal-Site-Guided Arylation of Proteins. J. Am. Chem. Soc. 2016, 138 (28), 8678– 8681, DOI: 10.1021/jacs.6b04043 Google Scholar9Selective Metal-Site-Guided Arylation of ProteinsWillwacher, Jens; Raj, Ritu; Mohammed, Shabaz; Davis, Benjamin G.Journal of the American Chemical Society (2016), 138 (28), 8678-8681CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society) We describe palladium-mediated S-arylation that exploits natural metal-binding motifs to ensure high site selectivity for a proximal reactive residue. This allows the chem. identification not only of proteins that bind metals but also the environment of the metal-binding site itself through proteomic anal. of arylation sites. The transformation is easy to perform under std. conditions, does not require the isolation of a reactive Ar-Pd complex, is broad in scope, and is applicable in cell lysates as well as to covalent inhibition/modulation of metal-dependent enzymic activity. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVGjtLrF&md5=e4d6d7bb0fb9661c2d562a5a5dd2ae8c10Chin, J. W. Expanding and Reprogramming the Genetic Code.. Nature 2017, 550, 53– 60, DOI: 10.1038/nature24031 Google ScholarThere is no corresponding record for this reference.Cited By Click to copy section linkSection link copied!This article has not yet been cited by other publications.Download PDFFiguresReferences Get e-AlertsGet e-AlertsACS Central ScienceCite this: ACS Cent. Sci. 2025, XXXX, XXX, XXX-XXXClick to copy citationCitation copied!https://doi.org/10.1021/acscentsci.4c02083Published January 6, 2025 Publication History Published online 6 January 2025Published 2025 by American Chemical Society. This publication is licensed under CC-BY 4.0 . License Summary*You are free to share (copy and redistribute) this article in any medium or format and to adapt (remix, transform, and build upon) the material for any purpose, even commercially within the parameters below:Creative Commons (CC): This is a Creative Commons license.Attribution (BY): Credit must be given to the creator.View full license*DisclaimerThis summary highlights only some of the key features and terms of the actual license. It is not a license and has no legal value. Carefully review the actual license before using these materials. Article Views-Altmetric-Citations-Learn about these metrics closeArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated.Recommended Articles FiguresReferencesAbstractHigh Resolution ImageDownload MS PowerPoint SlideFigure 1Figure 1. (A) Cys-linked benzodiazole derivatives for wash-free imaging. (B) Modification of unprotected peptides or proteins via palladium-mediated arylation. (C) Tracking the delivery mechanism and localization of the labeled biomolecule using fluorescence microscopy in live cells.High Resolution ImageDownload MS PowerPoint SlideReferences This article references 10 other publications. 1Nadal-Bufi, F.; Nithun, R. V.; de Moliner, F.; Lin, X.; Habiballah, S.; Jbara, M.; Vendrell, M. Late-Stage Minimal Labeling of Peptides and Proteins for Real-Time Imaging of Cellular Trafficking. ACS Cent Sci. 2024, DOI: 10.1021/acscentsci.4c01249 There is no corresponding record for this reference.2Mann, G.; Sadhu, P.; Brik, A. Synthetic Proteins behind the Plasma Barrier: Molecular Spies. Acc. Chem. Res. 2022, 55 (15), 2055– 2067, DOI: 10.1021/acs.accounts.2c00236 There is no corresponding record for this reference.3Oi, C.; Mochrie, S. G. J.; Horrocks, M. H.; Regan, L. PAINT Using Proteins: A New Brush for Super-Resolution Artists.. Protein Sci. 2020, 29, 2142– 2149, DOI: 10.1002/pro.3953 There is no corresponding record for this reference.4de Moliner, F.; Konieczna, Z.; Mendive-Tapia, L.; Saleeb, R. S.; Morris, K.; Gonzalez-Vera, J. A.; Kaizuka, T.; Grant, S. G. N.; Horrocks, M. H.; Vendrell, M. Small Fluorogenic Amino Acids for Peptide-Guided Background-Free Imaging. Angewandte Chemie - International Edition 2023, 62 (4), e202216231, DOI: 10.1002/anie.202216231 There is no corresponding record for this reference.5Vinogradova, E. V.; Zhang, C.; Spokoyny, A. M.; Pentelute, B. L.; Buchwald, S. L. Organometallic Palladium Reagents for Cysteine Bioconjugation. Nature 2015, 526 (7575), 687– 691, DOI: 10.1038/nature15739 5Organometallic palladium reagents for cysteine bioconjugationVinogradova, Ekaterina V.; Zhang, Chi; Spokoyny, Alexander M.; Pentelute, Bradley L.; Buchwald, Stephen L.Nature (London, United Kingdom) (2015), 526 (7575), 687-691CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group) Peptides and proteins, contg. cysteine amino acid, were labeled by conjugation of the SH-group with arylpalladium 2-biphenylylphosphine hemilabile reagent, causing arylation the mercapto-group. Reactions based on transition metals have found wide use in org. synthesis, in particular for the functionalization of small mols. However, there are very few reports of using transition-metal-based reactions to modify complex biomols., which is due to the need for stringent reaction conditions (for example, aq. media, low temp. and mild pH) and the existence of multiple reactive functional groups found in biomols. Here we report that palladium(II) complexes can be used for efficient and highly selective cysteine conjugation (bioconjugation) reactions that are rapid and robust under a range of bio-compatible reaction conditions. The straightforward synthesis of the palladium reagents from diverse and easily accessible aryl halide and trifluoromethanesulfonate precursors makes the method highly practical, providing access to a large structural space for protein modification. The resulting aryl bioconjugates are stable towards acids, bases, oxidants and external thiol nucleophiles. The broad utility of the bioconjugation platform was further corroborated by the synthesis of new classes of stapled peptides and antibody-drug conjugates. These palladium complexes show potential as benchtop reagents for diverse bioconjugation applications. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhslCnu7zK&md5=8fbb382c94600af312bb007d31b630566Zhang, C.; Welborn, M.; Zhu, T.; Yang, N. J.; Santos, M. S.; Van Voorhis, T.; Pentelute, B. L. π-Clamp-Mediated Cysteine Conjugation. Nat. Chem. 2016, 8 (2), 120– 128, DOI: 10.1038/nchem.2413 6π-Clamp-mediated cysteine conjugationZhang, Chi; Welborn, Matthew; Zhu, Tianyu; Yang, Nicole J.; Santos, Michael S.; Van Voorhis, Troy; Pentelute, Bradley L.Nature Chemistry (2016), 8 (2), 120-128CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group) Site-selective functionalization of complex mols. is one of the most significant challenges in chem. Typically, protecting groups or catalysts must be used to enable the selective modification of one site among many that are similarly reactive, and general strategies that selectively tune the local chem. environment around a target site are rare. Here, the authors show a four-amino-acid sequence (Phe-Cys-Pro-Phe), which the authors call the π-clamp, that tunes the reactivity of its cysteine thiol for site-selective conjugation with perfluoroarom. reagents. The authors use the π-clamp to selectively modify one cysteine site in proteins contg. multiple endogenous cysteine residues. These examples include antibodies and cysteine-based enzymes that would be difficult to modify selectively using std. cysteine-based methods. Antibodies modified using the π-clamp retained binding affinity to their targets, enabling the synthesis of site-specific antibody-drug conjugates for selective killing of HER2-pos. breast cancer cells. The π-clamp is an unexpected approach to mediate site-selective chem. and provides new avenues to modify biomols. for research and therapeutics. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXitVygurzE&md5=8c3c367f6fd7c64237bd0ba3e9bd40b47Theile, C. S.; Witte, M. D.; Blom, A. E. M.; Kundrat, L.; Ploegh, H. L.; Guimaraes, C. P. Site-Specific N-Terminal Labeling of Proteins Using Sortase-Mediated Reactions. Nat. Protoc 2013, 8 (9), 1800– 1807, DOI: 10.1038/nprot.2013.102 7Site-specific N-terminal labeling of proteins using sortase-mediated reactionsTheile, Christopher S.; Witte, Martin D.; Blom, Annet E. M.; Kundrat, Lenka; Ploegh, Hidde L.; Guimaraes, Carla P.Nature Protocols (2013), 8 (9), 1800-1807, 8 pp.CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group) This protocol describes the use of sortase-mediated reactions to label the N terminus of any given protein of interest. The sortase recognition sequence, LPLPXTG (for Streptococcus aureus sortase A) or LPXTA (for Staphylococcus pyogenes sortase A), can be appended to a variety of probes such as fluorophores, biotin or even to other proteins. The protein to be labeled acts as a nucleophile by attacking the intermediate formed between the probe contg. the LPXTG/A motif and the sortase enzyme. If sortase, the protein of interest and a suitably functionalized label are available, the reactions usually require less than 3 h. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsV2hurrK&md5=3630095942671637c7b047ac2e69c85f8Gavins, G. C.; Gröger, K.; Bartoschek, M. D.; Wolf, P.; Beck-Sickinger, A. G.; Bultmann, S.; Seitz, O. Live Cell PNA Labelling Enables Erasable Fluorescence Imaging of Membrane Proteins. Nat. Chem. 2021, 13 (1), 15– 23, DOI: 10.1038/s41557-020-00584-z 8Live cell PNA labelling enables erasable fluorescence imaging of membrane proteinsGavins, Georgina C.; Groeger, Katharina; Bartoschek, Michael D.; Wolf, Philipp; Beck-Sickinger, Annette G.; Bultmann, Sebastian; Seitz, OliverNature Chemistry (2021), 13 (1), 15-23CODEN: NCAHBB; ISSN:1755-4330. (Nature Research) Abstr.: DNA nanotechnol. is an emerging field that promises fascinating opportunities for the manipulation and imaging of proteins on a cell surface. The key to progress is the ability to create a nucleic acid-protein junction in the context of living cells. Here we report a covalent labeling reaction that installs a biostable peptide nucleic acid (PNA) tag. The reaction proceeds within minutes and is specific for proteins carrying a 2 kDa coiled-coil peptide tag. Once installed, the PNA label serves as a generic landing platform that enables the recruitment of fluorescent dyes via nucleic acid hybridization. We demonstrate the versatility of this approach by recruiting different fluorophores, assembling multiple fluorophores for increased brightness and achieving reversible labeling by way of toehold-mediated strand displacement. Addnl., we show that labeling can be carried out using two different coiled-coil systems, with epidermal growth factor receptor and endothelin receptor type B, on both HEK293 and CHO cells. Finally, we apply the method to monitor internalization of epidermal growth factor receptor on CHO cells. [graphic not available: see fulltext]. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXisFaiurbM&md5=7d2f70001ede10fdecfd1dbed58ecd729Willwacher, J.; Raj, R.; Mohammed, S.; Davis, B. G. Selective Metal-Site-Guided Arylation of Proteins. J. Am. Chem. Soc. 2016, 138 (28), 8678– 8681, DOI: 10.1021/jacs.6b04043 9Selective Metal-Site-Guided Arylation of ProteinsWillwacher, Jens; Raj, Ritu; Mohammed, Shabaz; Davis, Benjamin G.Journal of the American Chemical Society (2016), 138 (28), 8678-8681CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society) We describe palladium-mediated S-arylation that exploits natural metal-binding motifs to ensure high site selectivity for a proximal reactive residue. This allows the chem. identification not only of proteins that bind metals but also the environment of the metal-binding site itself through proteomic anal. of arylation sites. The transformation is easy to perform under std. conditions, does not require the isolation of a reactive Ar-Pd complex, is broad in scope, and is applicable in cell lysates as well as to covalent inhibition/modulation of metal-dependent enzymic activity. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVGjtLrF&md5=e4d6d7bb0fb9661c2d562a5a5dd2ae8c10Chin, J. W. Expanding and Reprogramming the Genetic Code.. Nature 2017, 550, 53– 60, DOI: 10.1038/nature24031 There is no corresponding record for this reference.

Ubiquitination significantly influences human health and disease because it plays an essential role in many cellular signaling pathways. To investigate the effects of different types of ubiquitination, various strategies based on synthesis, semisynthesis, or expression have been developed for protein ubiquitination. Here, we introduce a new method for protein ubiquitination using the SpyTag/SpyCatcher system. By combining protein expression with chemical synthesis, we created enhanced green fluorescent protein (eGFP) with ubiquitin chains consisting of 1-4 units linked through Lys48. This allowed us to study how different ubiquitin chains affect proteasomal degradation by the 26S and 20S proteasomes. While the 26S proteasome only trimmed the ubiquitin chain, the 20S proteasome degraded the different ubiquitin variants, highlighting the flexibility of the 20S proteasome in degrading complex ubiquitinated proteins.

Ubiquitination is a critical post-translational modification that regulates key cellular processes such as protein degradation and DNA damage repair. Targeting a specific type of ubiquitin chain (e.g., Lys48 or Lys63-linked ubiquitin chain) via cyclic peptides presents a new strategy to modulate biological processes with therapeutic potential for different diseases. However, such a strategy remains challenging due to the obstacles of cell permeability and bioactivity. Here, we report a new approach to directly examine these parameters by combining palladium-mediated Cys arylation with in situ cell-based screening. Using CP4, a previously identified cyclic peptide modulator of Lys63-linked ubiquitin chains, we generated a focused library of arylated analogues and optimized the Pd-mediated arylation for cell-based screening. We discovered a new analog, CP-P12-ArH, that demonstrated enhanced binding affinity and robust bioactivity, as evidenced by increased γ-H2AX phosphorylation and apoptosis induction in cancer cells. Furthermore, CP-P12- ArH effectively inhibited the in vitro formation of NF-κB essential modulator (NEMO) biomolecular condensates by disrupting the elongation of Lys63-linked ubiquitin chains, offering a novel way to modulate NF-κB signaling. This work establishes a generalizable platform for the rapid optimization of cyclic peptide therapeutics targeting protein-protein interactions.

Ubiquitination is a critical post-translational modification that regulates key cellular processes such as protein degradation and DNA damage repair. Targeting a specific type of ubiquitin chain (e.g., Lys48 or Lys63-linked ubiquitin chain) via cyclic peptides presents a new strategy to modulate biological processes with therapeutic potential for various diseases. However, such a strategy remains challenging due to the obstacles of cell permeability and bioactivity. Here, we present a new method that directly assesses these parameters by integrating palladium-mediated Cys arylation with direct cellular screening. Using CP4, a previously identified cyclic peptide modulator of Lys63-linked ubiquitin chains, we generated a focused library of arylated analogues and optimized the Pd-mediated arylation for direct cellular screening. We discovered a new analog, CP-P12-ArH, that demonstrated enhanced binding affinity and robust bioactivity, as evidenced by increased γ-H2AX phosphorylation and apoptosis induction in cancer cells. Furthermore, CP-P12-ArH effectively inhibited the in vitro formation of NF-κB essential modulator (NEMO) biomolecular condensates by disrupting the elongation of Lys63-linked ubiquitin chains, offering a novel way to modulate NF-κB signaling. This work establishes a generalizable platform for the rapid optimization of cyclic peptide therapeutics targeting protein-protein interactions.

Chemical protein synthesis has emerged as a powerful approach for producing ubiquitin (Ub) and ubiquitin-like modifiers (Ubls) in both their free and conjugated forms, particularly when recombinant or enzymatic strategies are challenging. By providing precise control over the assembly of Ub and Ubls, chemical synthesis enables the generation of complex constructs with site-specific modifications that facilitate detailed functional and structural studies. Ub and Ubls are central regulators of protein homeostasis, regulating a wide range of cellular processes such as cell cycle progression, transcription, DNA repair, and apoptosis. Ubls share an evolutionary link with Ub, resembling its structure and following a parallel conjugation pathway that results in a covalent isopeptide bond with their cellular substrates. Despite their structural similarities and sequence homology, Ub and Ubls exhibit distinct functional differences. Understanding Ubl biology is essential for unraveling how cells maintain their regulatory networks and how disruptions in these pathways contribute to various diseases. In this review, we highlight the chemical methodologies and strategies available for studying Ubls and advancing our comprehensive understanding of the Ubl system in health and disease.

Uniquely modified synthetic proteins are difficult to produce in large quantities, which could limit their use in various in vitro settings and in cellular studies. In this study, we developed a method named “suspension bead loading” (SBL), to deliver protein molecules into suspended living cells using glass beads, which significantly reduces the amount of protein required for effective delivery. We investigated the delivery efficiency of functionally different proteins and evaluated the cytotoxic effect of our method and the chemical and functional integrity of the delivered protein. We utilized SBL to address questions related to ubiquitin-related modifier 1 (URM1). Employing minimal protein quantities, SBL has enabled us to study its behavior within live cells under different redox conditions, including subcellular localization and conjugation patterns. We demonstrate that oxidative stress alters both the localization and conjugation pattern of URM1 in cells, highlighting its possible role in cellular response to such extreme conditions.

The Small Ubiquitin-like Modifier (SUMO) is a crucial post-translational modifier of proteins, playing a key role in various cellular functions. All SUMOs are synthesized as precursor proteins that must be proteolytically processed. However, the maturation process of cleaving the extending C-terminal tail, preceding SUMOylation of substrates, remains poorly understood, especially within cellular environments. Chemical protein synthesis coupled with cell delivery offers great opportunities to prepare SUMO analogues to investigate this process in vitro and in live cells. Applying this unique combination we show that SUMO2 analogues containing the native tail undergo rapid cleavage and nuclear localisation, while a Gly93Ala mutation impairs cleavage and alters localisation. Tail mutations (Val94Glu, Tyr95Ala) affected cleavage rates, highlighting roles in SUMO-SENP protease interactions. In cells, SUMO2 analogues containing tail mutations underwent cleavage and subsequently incorporated into promyelocytic leukemia nuclear bodies (PML-NBs). These findings advance our understanding of SUMO2 maturation and provide a foundation for future studies of this process for different SUMO paralogues in various cell lines and tissues.

Despite the great advances in discovering cyclic peptides against protein targets, their reduced aqueous solubility, cell permeability, and activity of the cyclic peptide restrict its utilization in advanced biological research and therapeutic applications. Here we report on a novel approach of structural alternation of the exocyclic and linker parts that led to a new derivative with significantly improved cell activity allowing us to dissect its mode of action in detail. We have identified an effective cyclic peptide ( CP7 ) that induces approximately a 9-fold increase in DNA damage accumulation and a remarkable increase in apoptotic cancer cell death compared to the reported molecule. Notably, treating cells with CP7 leads to a dramatic decrease in the efficiency of non-homologous end joining (NHEJ) repair of DNA double-strand breaks (DSBs), which is accompanied by an increase in homologous recombination (HR) repair. Interestingly, treating BRCA1-deficient cells with CP7 restores HR integrity, which is accompanied by increased resistance to CP7 . Additionally, CP7 treatment increases the sensitivity of cancer cells to ionizing radiation. Collectively, our findings demonstrate that CP7 is a selective inhibitor of NHEJ, offering a potential strategy to enhance the effectiveness of radiation therapy.

We designed a platform for monitoring the degradation of exogenous proteins in live cells. We engineered a semi-synthetic platform, which consists of Enhanced Green Fluorescent Protein tagged with SpyCatcher to enable its conjugation to a SpyTag peptide bearing a Von Hippel-Lindau E3 ligand, which was delivered to live cells to promote its degradation. This platform lays the ground for studying the degradation of endogenous proteins equipped with SpyTag and for tracking the degradation of post-translationally modified proteins in live cells.

Aggressive and therapy-resistant cancers present a significant challenge to treatment and are associated with poor patients survival. Identifying molecular pathways and compounds that target these pathways is critical for improving patient outcomes. RNF4, an E3-ubiquitin ligase, is pivotal in oncoprotein stabilization and DNA repair, enhancing cancer cell survival driving tumorigenesis. Elevated RNF4 levels are associated with poor prognosis in cancer patients. Here, we describe the development of R4VPs, proteolysis-targeted chimeras-like (PROTACs-like). R4VPs promote RNF4 degradation and reduce the levels of its stabilized oncoproteins. Notably, R4VPs induce ferroptotic cell death selectively in cancer cells, sparing non-tumorigenic and primary cells. Surprisingly, R4VPs-induced ferroptosis is independent of RNF4 but preferentially targets tumor-driving mutations, particularly those in the EGFR pathway, while not affecting PI3K-transformed cells. R4VPs effectively induce cell death in therapy-resistant melanoma and sarcomas including patient-derived sarcoma tumor cells. Our findings highlight the potential of ferroptosis inducers such as R4VPs as a therapeutic strategy for therapy resistance, aggressive, and hard-to-treat cancers. ### Competing Interest Statement AOV, RN, PD, GK AB, and AO are listed as co-inventors in patent filings associated with the technologies described in this manuscript. Ao and AB . are co-inventors of intellectual property that is unrelated to this work (anti-RNF4 mAb). AB is a founder and consultant for Ub-Therapeutics

Understanding the determinants of α-conotoxin (α-CTX) selectivity for different nicotinic acetylcholine receptor (nAChR) subtypes is a requisite for the design of tool compounds to study nAChRs. However, selectivity optimization of these small, disulfide rich peptides is difficult not only because of an absence of α-CTX/nAChR co-structures, but also because it is challenging to predict how a mutation to an α-CTX will alter its potency and selectivity. As a prototypical system to investigate selectivity, we employed the α-CTX LvIA that is 18-fold selective for the α3β2 nAChR over the closely related α3β4 nAChR subtype that is a target for nicotine addiction. Using two-electrode voltage clamp electrophysiology, we identified LvIA[D11R] that is 2-fold selective for the α3β4 nAChR, reversing its subtype preference. This effect is specific to the charge and not shape of LvIA[D11R], as substitution with citrulline retains selectivity for the α3β2 nAChR. Furthermore, LvIA[D11K] shows a stronger reversal, with 4-fold selectivity for the α3β4 nAChR. Motivated by these findings, using site-directed mutagenesis it was found that β2[K79A], but not β2[K78A], largely restores the antagonism of basic mutants at position 11. Finally, to understand the structural basis of this effect we used AlphaFold2 to generate models of LvIA in complex with both nAChR subtypes. Both models confirm the plausibility of an electrostatic mechanism to explain the data and also reproduce a broad range of potency and selectivity structure-activity relationships for LvIA mutants, as measured using free-energy perturbation simulations. Our work highlights how electrostatic interactions can drive α-CTX selectivity and may prove useful as a strategy for optimizing the selectivity of LvIA and other ⍺-CTXs.