A Minimal-Tag Bioorthogonal Labelling Approach to Protein Uptake, Traffic and Delivery

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Controlled in‐cell generation of active Palladium(0) species for bioorthogonal decaging

Publication . Konč, Juraj; Sabatino, Valerio; Jiménez‐Moreno, Ester; Latocheski, Eloah; Pérez, Laura Rodríguez; Day, Jason; Domingos, Josiel B.; Bernardes, Gonçalo J. L.

Owing to their bioorthogonality, transition metals have become very popular in the development of biocompatible bond-cleavage reactions. However, many approaches require design and synthesis of complex ligands or formulation of nanoparticles which often perform poorly in living cells. This work reports on a method for the generation of an active palladium species that triggers bond-cleaving reactions inside living cells. We utilized the water-soluble Na2 PdCl4 as a simple source of PdII which can be intracellularly reduced by sodium ascorbate to the active Pd0 species. Once generated, Pd0 triggers the cleavage of allyl ether and carbamate caging groups leading to the release of biologically active molecules. These findings do not only expand the toolbox of available bioorthogonal dissociative reactions but also provide an additional strategy for controlling the reactivity of Pd species involved in Pd-mediated bioorthogonal reactions.

2022Journal article

Restricted access

A water-bridged cysteine-cysteine redox regulation mechanism in bacterial protein tyrosine phosphatases

Publication . Bertoldo, Jean B.; Rodrigues, Tiago; Dunsmore, Lavinia; Aprile, Francesco A.; Marques, Marta C.; Rosado, Leonardo A.; Boutureira, Omar; Steinbrecher, Thomas B.; Sherman, Woody; Corzana, Francisco; Terenzi, Hernán; Bernardes, Gonçalo J. L.

The emergence of multidrug-resistant Mycobacterium tuberculosis (Mtb) strains highlights the need to develop more efficacious and potent drugs. However, this goal is dependent on a comprehensive understanding of Mtb virulence protein effectors at the molecular level. Here, we used a post-expression cysteine (Cys)-to-dehydrolanine (Dha) chemical editing strategy to identify a water-mediated motif that modulates accessibility of the protein tyrosine phosphatase A (PtpA) catalytic pocket. Importantly, this water-mediated Cys-Cys non-covalent motif is also present in the phosphatase SptpA from Staphylococcus aureus, which suggests a potentially preserved structural feature among bacterial tyrosine phosphatases. The identification of this structural water provides insight into the known resistance of Mtb PtpA to the oxidative conditions that prevail within an infected host macrophage. This strategy could be applied to extend the understanding of the dynamics and function(s) of proteins in their native state and ultimately aid in the design of small-molecule modulators.

2017Journal article

Open access

Structure-based design of potent tumor-associated antigens: modulation of peptide presentation by single-atom O/S or O/SE substitutions at the glycosidic linkage

Publication . Compañón, Ismael; Guerreiro, Ana; Mangini, Vincenzo; Castro-López, Jorge; Escudero-Casao, Margarita; Avenoza, Alberto; Busto, Jesús H.; Castillón, Sergio; Jiménez-Barbero, Jesús; Asensio, Juan L.; Jiménez-Osés, Gonzalo; Boutureira, Omar; Peregrina, Jesús M.; Hurtado-Guerrero, Ramón; Fiammengo, Roberto; Bernardes, Gonçalo J. L.; Corzana, Francisco

GalNAc-glycopeptides derived from mucin MUC1 are an important class of tumor-associated antigens. α- O-glycosylation forces the peptide to adopt an extended conformation in solution, which is far from the structure observed in complexes with a model anti-MUC1 antibody. Herein, we propose a new strategy for designing potent antigen mimics based on modulating peptide/carbohydrate interactions by means of O → S/Se replacement at the glycosidic linkage. These minimal chemical modifications bring about two key structural changes to the glycopeptide. They increase the carbohydrate-peptide distance and change the orientation and dynamics of the glycosidic linkage. As a result, the peptide acquires a preorganized and optimal structure suited for antibody binding. Accordingly, these new glycopeptides display improved binding toward a representative anti-MUC1 antibody relative to the native antigens. To prove the potential of these glycopeptides as tumor-associated MUC1 antigen mimics, the derivative bearing the S-glycosidic linkage was conjugated to gold nanoparticles and tested as an immunogenic formulation in mice without any adjuvant, which resulted in a significant humoral immune response. Importantly, the mice antisera recognize cancer cells in biopsies of breast cancer patients with high selectivity. This finding demonstrates that the antibodies elicited against the mimetic antigen indeed recognize the naturally occurring antigen in its physiological context. Clinically, the exploitation of tumor-associated antigen mimics may contribute to the development of cancer vaccines and to the improvement of cancer diagnosis based on anti-MUC1 antibodies. The methodology presented here is of general interest for applications because it may be extended to modulate the affinity of biologically relevant glycopeptides toward their receptors.

2019Journal article

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A fluorogenic probe for cell surface phosphatidylserine using an intramolecular indicator displacement sensing mechanism

Publication . Zwicker, Vincent E.; Oliveira, Bruno; Yeo, Jia Hao; Fraser, Stuart T.; Bernardes, Gonçalo J. L.; New, Elizabeth J.; Jolliffe, Katrina A.

The detection of externalized phosphatidylserine (PS) on the cell surface is commonly used to distinguish between living, apoptotic, and necrotic cells. The tools of choice for many researchers to study apoptosis are annexin V-fluorophore conjugates. However, the use of this 35 kDa protein is associated with several drawbacks, including temperature sensitivity, Ca2+ dependence, and slow binding kinetics. Herein, a fluorogenic probe for cell surface PS, P-IID, is described, which operates by an intramolecular indicator displacement (IID) mechanism. An intramolecularly bound coumarin indicator is released in the presence of cell surface PS, leading to a fluorescence "turn-on" response. P-IID demonstrates superior performance when compared to annexin V, for both fluorescence imaging and flow cytometry. This allows P-IID to be used in time-lapse imaging of apoptosis using confocal laser scanning microscopy and demonstrates the utility of the IID mechanism in live cells.

2018Journal article

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De novo design of potent and selective mimics of IL-2 and IL-15

Publication . Silva, Daniel-Adriano; Yu, Shawn; Ulge, Umut Y.; Spangler, Jamie B.; Jude, Kevin M.; Labão-Almeida, Carlos; Ali, Lestat R.; Quijano-Rubio, Alfredo; Ruterbusch, Mikel; Leung, Isabel; Biary, Tamara; Crowley, Stephanie J.; Marcos, Enrique; Walkey, Carl D.; Weitzner, Brian D.; Pardo-Avila, Fátima; Castellanos, Javier; Carter, Lauren; Stewart, Lance; Riddell, Stanley R.; Pepper, Marion; Bernardes, Gonçalo J. L.; Dougan, Michael; Garcia, K. Christopher; Baker, David

We describe a de novo computational approach for designing proteins that recapitulate the binding sites of natural cytokines, but are otherwise unrelated in topology or amino acid sequence. We use this strategy to design mimics of the central immune cytokine interleukin-2 (IL-2) that bind to the IL-2 receptor βγc heterodimer (IL-2Rβγc) but have no binding site for IL-2Rα (also called CD25) or IL-15Rα (also known as CD215). The designs are hyper-stable, bind human and mouse IL-2Rβγc with higher affinity than the natural cytokines, and elicit downstream cell signalling independently of IL-2Rα and IL-15Rα. Crystal structures of the optimized design neoleukin-2/15 (Neo-2/15), both alone and in complex with IL-2Rβγc, are very similar to the designed model. Neo-2/15 has superior therapeutic activity to IL-2 in mouse models of melanoma and colon cancer, with reduced toxicity and undetectable immunogenicity. Our strategy for building hyper-stable de novo mimetics could be applied generally to signalling proteins, enabling the creation of superior therapeutic candidates.

2019Journal article

Restricted access