Photocatalytic Microenvironment Proteomics of Thiol-Mediated Uptake

Article Article scientifique Article Photocatalytic Microenvironment Proteomics of Thiol-Mediated Uptake eng 2025-07-01 2025-07-28 author Saidjalolov Saidbakhrom Department of Organic Chemistry, University of Geneva, 1211 Geneva, Switzerland.,National Centre of Competence in Research (NCCR) Molecular Systems Engineering, BPR 1095, 4002 Basel, Switzerland. 983995 Chimie organique (groupe 1) (CHOR1) author Wu Yibo 0000-0002-8256-3901 ChemBioMS Proteomics Platform, University of Geneva, 1211 Geneva, Switzerland. 969931 author Renno Giacomo 0000-0003-0514-9974 Department of Organic Chemistry, University of Geneva, 1211 Geneva, Switzerland.,National Centre of Competence in Research (NCCR) Molecular Systems Engineering, BPR 1095, 4002 Basel, Switzerland. 403424 Chimie organique (groupe 1) (CHOR1) author Rose Nicholas Curt Department of Organic Chemistry, University of Geneva, 1211 Geneva, Switzerland.,National Centre of Competence in Research (NCCR) Molecular Systems Engineering, BPR 1095, 4002 Basel, Switzerland. 418844 Chimie organique (groupe 1) (CHOR1) author Gajic Jelena Department of Organic Chemistry, University of Geneva, 1211 Geneva, Switzerland. 922112 author Pologne Bertrand Department of Organic Chemistry, University of Geneva, 1211 Geneva, Switzerland. 919028 Département de chimie organique (CHIOR) author Winssinger Nicolas 0000-0003-1636-7766 Department of Organic Chemistry, University of Geneva, 1211 Geneva, Switzerland. 769664 Département de chimie organique (CHIOR) author Mercier Vincent ACCESS Platform, University of Geneva, 1211 Geneva, Switzerland. 847100 Département de biochimie (CHIBI) author Moreau Dimitri 0000-0002-9282-3324 ACCESS Platform, University of Geneva, 1211 Geneva, Switzerland. 751508 Plateforme ACCESS (ACCESS) author Sakai Naomi 0000-0002-9460-1944 Department of Organic Chemistry, University of Geneva, 1211 Geneva, Switzerland.,National Centre of Competence in Research (NCCR) Molecular Systems Engineering, BPR 1095, 4002 Basel, Switzerland. 131140 Département de chimie organique (CHIOR) author Matile Stefan 0000-0002-8537-8349 Department of Organic Chemistry, University of Geneva, 1211 Geneva, Switzerland.,National Centre of Competence in Research (NCCR) Molecular Systems Engineering, BPR 1095, 4002 Basel, Switzerland. 129654 Département de chimie organique (CHIOR) 3288-3298 10.1021/jacsau.5c00432 40747020 2691-3704 PMC12308380 https://pubs.acs.org/doi/10.1021/jacsau.5c00432 Although facilitated cellular entry of substrates with thiol-reactive motifs has been observed for decades, this so-called thiol-mediated uptake (TMU) remains poorly understood. We have proposed a mechanism of entry involving cellular proteins that form reversible dynamic covalent bonds with thiol-reactive cascade exchangers (CAXs), which is challenging to prove because the substrate-protein bond is transient and constantly shifting. Thus, with conventional proteomics analysis of TMU, continuing exchange during processing should result in labeling of the inert binders rather than the best exchangers, that is, repressors and intracellular targets, instead of the enablers of TMU. Any static covalent bonding to a binding site will also perturb the molecular relay network of interest. The emerging photocatalytic microenvironment mapping (μMap) proteomics, however, promises to catch snapshots of off-equilibrium relay networks without disturbing their flow. Exchange partners that are temporarily within <4 nm radius of photocatalyst-CAX conjugates should be irreversibly biotinylated without systematically interfering with TMU. μMap proteomics of this elusive flow of TMU was explored for three different photocatalyst-CAX conjugates. They were measured against CAX-free photocatalyst controls and dynamic covalent TMU inhibitors. Validated by genetic knockdown, solute carriers (MFSD5, SLC29A2), flippases (ATP11C), and tetraspanins (TSPAN8) are identified as primary exchange partners. This is rewarding because their canonical functions already involve local membrane reorganization. The result is a new understanding of the nature of TMU, which will be helpful to guide future progress toward control over cell penetration for drug delivery and drug discovery. It also highlights the unique potential of photocatalytic proximity labeling proteomics to elucidate off-equilibrium molecular relay networks without disturbing their flow. Cascade exchange Dynamic covalent chemistry Dynamic covalent inhibition Genetic knockdown Off-equilibrium proteomics Photocatalytic microenvironment proteomics Photocatalytic proximity labeling Relay networks Thiol-mediated uptake Dewey 540 Swiss National Science Foundation Chemical biology probes from natural product synthesis, artificial biochemical circuitry and antagomirs 188406 CH Projects Swiss National Science Foundation Translational Dynamic Covalent Exchange Cascades (TIMEUP) 209190 CH Programmes 36 Département de chimie organique 141 false 761 Groupe Matile 762 Groupe Winssinger JACS Au 5 7 f3bee425-9ead-4c35-8b3d-f82067e8444e Article (Published version) Public photocatalytic-microenvironment-pro.pdf 6465793 application/pdf CC BY-4.0 52a988308fc2e32317a418edcd93d4f70b6a0fd2346a8f774638e0a3ef0b3143 openAccess Faculté des sciences / Section de chimie et biochimie / Département de chimie organique

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