Mehdi Amoura

Centre National de la Recherche Scientifique

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Vincent Aucagne

Centre de Biophysique Moléculaire

A.F. Delmas

Centre de Biophysique Moléculaire

V.P. Terrier

Centre de Biophysique Moléculaire

Skander Abboud

Scripps (United States)

Véronique Piller

Centre de Biophysique Moléculaire

Sandrine Sagan

Sorbonne Université

Vincent Dive

CEA Paris-Saclay

Massimiliano Beltramo

Physiologie de la Reproduction et des Comportements

David Montoir

Cibles et Médicaments des Infections et de l'Immunité

Samir Messaoudi

Centre National de la Recherche Scientifique

Cooperative Institutions

Centre National de la Recherche Scientifique

Université Paris-Saclay

Commissariat à l'Énergie Atomique et aux Énergies Alternatives

Sorbonne Université

CEA Paris-Saclay

Centre de Biophysique Moléculaire

Laboratoire des Biomolécules

Université Paris Sciences et Lettres

Université Paris Cité

École Normale Supérieure - PSL

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Enzyme‐Cleavable Linkers for Protein Chemical Synthesis through Solid‐Phase Ligations

Angewandte Chemie International Edition (2021)

Skander Abboud Mehdi Amoura Jean‐Baptiste Madinier Brigitte Renoux Sébastien Papot

The total synthesis of long proteins requires the assembly of multiple fragments through successive ligations. The need for intermediate purification steps is a strong limitation, particularly in terms of overall yield. One solution to this problem would be solid-supported chemical ligation (SPCL), for which a first peptide segment must be immobilized on a SPCL-compatible solid support through a linker that can be cleaved under very mild conditions to release the assembled protein. The cleavage of SPCL linkers has previously required chemical conditions sometimes incompatible with sensitive protein targets. Herein, we describe an alternative enzymatic approach to trigger cleavage under extremely mild and selective conditions. Optimization of the linker structure and use of a small enzyme able to diffuse into the solid support were key to the success of the strategy. We demonstrated its utility by the assembly of three peptide segments on the basis of native chemical ligation to afford a 15 kDa polypeptide.

Linker

Native chemical ligation

Cleavage (geology)

Chemical Ligation

Solid-Phase Synthesis

Peptide Synthesis

Chemical modification

10.1002/anie.202103768

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Citations (9)

An improved synthesis of N-Hnb-Cys peptide crypto-thioesters ‒studies on the epimerization of cysteine during solid-phase reductive amination

HAL (Le Centre pour la Communication Scientifique Directe) (2019)

Skander Abboud Mehdi Amoura V.P. Terrier A.F. Delmas Vincent Aucagne

Epimer

Reductive amination

Peptide Synthesis

Source

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Citations (0)

Simple Pseudo-dipeptides with a P2′ Glutamate

Journal of Biological Chemistry (2012)

Laurent Devel Fabrice Beau Mehdi Amoura Laura Vera E. Cassar-Lajeunesse

Moiety

Acetohydroxamic acid

Side chain

Hydroxamic acid

10.1074/jbc.m112.380782

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Citations (35)

Second generation N-2-hydroxybenzyl-cysteine peptide crypto-thioesters for native chemical ligation

HAL (Le Centre pour la Communication Scientifique Directe) (2018)

Mehdi Amoura V.P. Terrier A.F. Delmas Vincent Aucagne

Native chemical ligation

Source

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Citations (0)

Head to tail cyclisation of cell-penetrating peptides: impact on GAG-dependent internalisation and direct translocation

Chemical Communications (2019)

Mehdi Amoura Françoise Illien Alain Joliot Karine Guitot John Offer

Small cationic lipidated cyclic cell-penetrating peptides enter efficiently by GAG-dependent endocytosis, expanding the macrocycle favours direct translocation.

10.1039/c9cc01265f

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Citations (26)

N-2-hydroxybenzyl-cysteine peptide crypto-thioesters for native chemical ligation

HAL (Le Centre pour la Communication Scientifique Directe) (2018)

Mehdi Amoura Daad Sarraf Lionel Nauton V.P. Terrier Dominique Lelièvre

Native chemical ligation

Chemical Ligation

Source

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Citations (0)

Halogen Bonding Controls Selectivity of FRET Substrate Probes for MMP-9

Chemistry & Biology (2014)

Isabelle Tranchant Laura Vera Bertrand Czarny Mehdi Amoura Evelyne Cassar

Halogen bond

10.1016/j.chembiol.2014.01.008

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Citations (27)

Design and optimization of alternative thioester precursors to simplify protein synthesis by native chemical ligation

HAL (Le Centre pour la Communication Scientifique Directe) (2018)

Mehdi Amoura V.P. Terrier A.F. Delmas Vincent Aucagne

Thioester

Native chemical ligation

Source

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Citations (0)

Enzyme‐Cleavable Linkers for Protein Chemical Synthesis through Solid‐Phase Ligations

Angewandte Chemie (2021)

Skander Abboud Mehdi Amoura Jean‐Baptiste Madinier Brigitte Renoux Sébastien Papot

Abstract The total synthesis of long proteins requires the assembly of multiple fragments through successive ligations. The need for intermediate purification steps is a strong limitation, particularly in terms of overall yield. One solution to this problem would be solid‐supported chemical ligation (SPCL), for which a first peptide segment must be immobilized on a SPCL‐compatible solid support through a linker that can be cleaved under very mild conditions to release the assembled protein. The cleavage of SPCL linkers has previously required chemical conditions sometimes incompatible with sensitive protein targets. Herein, we describe an alternative enzymatic approach to trigger cleavage under extremely mild and selective conditions. Optimization of the linker structure and use of a small enzyme able to diffuse into the solid support were key to the success of the strategy. We demonstrated its utility by the assembly of three peptide segments on the basis of native chemical ligation to afford a 15 kDa polypeptide.

Linker

Native chemical ligation

Solid-Phase Synthesis

Cleavage (geology)

Chemical Ligation

Peptide Synthesis

Chemical modification

10.1002/ange.202103768

Cite

Citations (1)

Quantitative fluorescence spectroscopy and flow cytometry analyses of cell-penetrating peptides internalization pathways: optimization, pitfalls, comparison with mass spectrometry quantification

Scientific Reports (2016)

Françoise Illien Nicolás Rodríguez Mehdi Amoura Alain Joliot Pallerla Manjula

Abstract The mechanism of cell-penetrating peptides entry into cells is unclear, preventing the development of more efficient vectors for biotechnological or therapeutic purposes. Here, we developed a protocol relying on fluorometry to distinguish endocytosis from direct membrane translocation, using Penetratin, TAT and R9. The quantities of internalized CPPs measured by fluorometry in cell lysates converge with those obtained by our previously reported mass spectrometry quantification method. By contrast, flow cytometry quantification faces several limitations due to fluorescence quenching processes that depend on the cell line and occur at peptide/cell ratio >6.10 8 for CF-Penetratin. The analysis of cellular internalization of a doubly labeled fluorescent and biotinylated Penetratin analogue by the two independent techniques, fluorometry and mass spectrometry, gave consistent results at the quantitative and qualitative levels. Both techniques revealed the use of two alternative translocation and endocytosis pathways, whose relative efficacy depends on cell-surface sugars and peptide concentration. We confirmed that Penetratin translocates at low concentration and uses endocytosis at high μM concentrations. We further demonstrate that the hydrophobic/hydrophilic nature of the N -terminal extremity impacts on the internalization efficiency of CPPs. We expect these results and the associated protocols to help unraveling the translocation pathway to the cytosol of cells.

Internalization

Mass cytometry

Cell-penetrating peptide

Single-Cell Analysis

Cell membrane

10.1038/srep36938

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Citations (104)