Targeting endogenous nuclear antigens by electrotransfer of monoclonal antibodies in living cells

Freund, Guillaume; Sibler, Annie-Paule; Desplancq, Dominique; Oulad‐Abdelghani, Mustapha; Vigneron, Marc; Gannon, Julian; Regenmortel, M.H.V. Van; Weiss, Étienne

doi:10.4161/mabs.25084

Cited by 47 publications

(77 citation statements)

References 20 publications

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“…4C), the transduced anti-E6 mAb can diffuse within the nucleus only after cell division. 26 By contrast, the electro-transferred uThr112 mAb was partially detected in the nucleus after 24h but exhibited a predominant nuclear staining pattern 72h post-treatment, indistinguishable from the pattern provided by the conventional ATF7 in vitro immuno-staining (Fig. 4C).…”

Section: Resultsmentioning

confidence: 71%

“…We used an intra-cytoplasmic delivery method of mAbs into living cells 26 to assess the capacity of the uThr112 mAb to bind to its endogenous epitope in a cellular context. The uThr112 and the E6 mAbs, as control, were electro-transferred into HeLa cells.…”

Section: Resultsmentioning

confidence: 99%

“…To be re-localized into the nucleus, the uThr112 mAb was conjugated with a peptide corresponding to SV40 nuclear localization signal (uThr112-NLS) as previously described. 26 HeLa cells were transduced with either the uThr112 or the uThr112-NLS antibodies. After 24h, cells were treated or not with CSK-Triton buffer and fixed for immunofluorescence.…”

Section: Resultsmentioning

confidence: 99%

“…Using an intra-cytoplasmic antibody delivery, 26 we could show that the uThr112 mAb remains fully active and is brought to the nucleus by binding to the endogenous ATF7 epitope. Moreover, this antibody is able to specifically inhibit Thr112 phosphorylation of endogenous ATF7 protein in mitotic cells.…”

Section: Discussionmentioning

confidence: 99%

“…8,26,49 Electroporation of mAbs in living cells and antibody-NLS conjugates were performed as previously described. 26 In vitro kinase assay and DNA affinity precipitation assay (DAPA)…”

Section: Fluorescence Microscopy and Fluorescence-activated Cell Sortmentioning

confidence: 99%

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ATF7 is stabilized during mitosis in a CDK1-dependent manner and contributes to cyclin D1 expression

et al. 2015

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The transcription factor ATF7 undergoes multiple post-translational modifications, each of which has distinct effects upon ATF7 function. Here, we show that ATF7 phosphorylation on residue Thr112 exclusively occurs during mitosis, and that ATF7 is excluded from the condensed chromatin. Both processes are CDK1/cyclin B dependent. Using a transduced neutralizing monoclonal antibody directed against the Thr112 epitope in living cells, we could demonstrate that Thr112 phosphorylation protects endogenous ATF7 protein from degradation, while it has no effect on the displacement of ATF7 from the condensed chromatin. The crucial role of Thr112 phosphorylation in stabilizing ATF7 protein during mitosis was confirmed using phospho-mimetic and phospho-deficient mutants. Finally, silencing ATF7 by CRISPR/Cas9 technology leads to a decrease of cyclin D1 protein expression levels. We propose that mitotic stabilized ATF7 protein re-localizes onto chromatin at the end of telophase and contributes to induce the cyclin D1 gene expression.

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Section: Resultsmentioning

confidence: 71%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…8,26,49 Electroporation of mAbs in living cells and antibody-NLS conjugates were performed as previously described. 26 In vitro kinase assay and DNA affinity precipitation assay (DAPA)…”

Section: Fluorescence Microscopy and Fluorescence-activated Cell Sortmentioning

confidence: 99%

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ATF7 is stabilized during mitosis in a CDK1-dependent manner and contributes to cyclin D1 expression

et al. 2015

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Transduction Methods for Cytosolic Delivery of Proteins and Bioconjugates into Living Cells

Chiper

Niederreither

Zuber

2017

Adv Healthcare Materials

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The human organism and its constituting cells rely on interplay between multiple proteins exerting specific functions. Progress in molecular biotechnologies has facilitated the production of recombinant proteins. When administrated to patients, recombinant proteins can provide important healthcare benefits. To date, most therapeutic proteins must act from the extracellular environment, with their targets being secreted modulators or extracellular receptors. This is because proteins cannot passively diffuse across the plasma membrane into the cytosol. To expand the scope of action of proteins for cytosolic targets (representing more than 40% of the genome) effective methods assisting protein cytosolic entry are being developed. To date, direct protein delivery is extremely tedious and inefficient in cultured cells, even more so in animal models of pathology. Novel techniques are changing this limitation, as recently developed in vitro methods can robustly convey large amount of proteins into cell cultures. Moreover, advances in protein formulation or protein conjugates are slowly, but surely demonstrating efficiency for targeted cytosolic entry of functional protein in vivo in tumor xenograft models. In this review, various methods and recently developed techniques for protein transport into cells are summarized. They are put into perspective to address the challenges encountered during delivery.

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Nanobodys: Strategien zur chemischen Funktionalisierung und intrazelluläre Anwendungen

et al. 2018

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Nanobodys sind ein modernes Mittel zur Erkennung und Modulation von Antigenen, die für herkömmliche Antikörper unzugänglich sind. Aufgrund ihrer kompakten Form und ihrer hohen Stabilität werden sie häufig in der Grundlagenforschung verwendet. In diesem Aufsatz werden zentrale Aspekte der Funktionalisierung von Nanobodys nebst ausgewählten Anwendungen in der Molekularbiologie dargestellt. Während ältere Konjugationsstrategien auf der zufälligen Modifikation natürlicher Aminosäuren beruhen, basieren neuere Methoden auf ortsspezifischer Modifikation mit funktionellen Einheiten. Solche Techniken umfassen chemoenzymatische Ansätze, Ligation exprimierter Proteine und die Unterdrückung des Amber‐Codons in Kombination mit bioorthogonalen Modifikationsstrategien. Mit einer ständig wachsenden Auswahl an Methoden aus der Proteinsynthese und ‐konjugation sind auch die Anwendungen auf dem Vormarsch. Diese reichen von hochentwickelter Bildgebung und Massenspektrometrie bis hin zum Einschleusen von Nanobodys in lebende Zellen, um intrazelluläre Antigene zu visualisieren und zu manipulieren.

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Targeting endogenous nuclear antigens by electrotransfer of monoclonal antibodies in living cells

Cited by 47 publications

References 20 publications

ATF7 is stabilized during mitosis in a CDK1-dependent manner and contributes to cyclin D1 expression

ATF7 is stabilized during mitosis in a CDK1-dependent manner and contributes to cyclin D1 expression

Transduction Methods for Cytosolic Delivery of Proteins and Bioconjugates into Living Cells

Nanobodys: Strategien zur chemischen Funktionalisierung und intrazelluläre Anwendungen

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