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Sieber, S.A.* ; Gronauer, T.F. ; Eck, L.K.* ; Ludwig, C.*

A photocrosslinking probe to capture the substrates of caseinolytic protease P.

Angew. Chem.-Int. Edit.:e202409220 (2024)
Verlagsversion Postprint DOI PMC
Open Access Hybrid
Creative Commons Lizenzvertrag
Protein homeostasis in bacteria is regulated by proteases such as the tetradecameric caseinolytic protease P (ClpP). Although substrates of ClpP have been successfully deciphered in genetically engineered cells, methods which directly trap processed proteins within native cells remain elusive. Here, we introduce an in situ trapping strategy which utilizes trifunctional probes that bind to the active site serine of ClpP and capture adjacent substrates with an attached photocrosslinking moiety. After enrichment using an alkyne handle, substrate deconvolution by mass spectrometry (MS) is performed. We show that our two traps bind substoichiometrically to ClpP, retain protease activity, exhibit unprecedented selectivity for Staphylococcus aureus ClpP in living cells and capture numerous known and novel substrates. The exemplary validation of trapped hits using a targeted proteomics approach confirmed the fidelity of this technology. In conclusion, we provide a novel chemical platform suited for the discovery of serine protease substrates beyond genetic engineering.
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Publikationstyp Artikel: Journalartikel
Dokumenttyp Wissenschaftlicher Artikel
Schlagwörter Caseinolytic Protease P (clpp) ; Chemical Proteomics ; Chemical Biology ; Substrate Traps ; Targeted Proteomics; Aureus Stress Tolerance; Staphylococcus-aureus; Clpp Protease; Computational Platform; Alpha-toxin; Virulence; Roles; Degradation
Sprache englisch
Veröffentlichungsjahr 2024
HGF-Berichtsjahr 2024
ISSN (print) / ISBN 1433-7851
e-ISSN 1521-3773
Zeitschrift Angewandte Chemie - Internationale Edition
Quellenangaben Band: , Heft: , Seiten: , Artikelnummer: e202409220 Supplement: ,
Verlag Wiley
Verlagsort Weinheim
Begutachtungsstatus Peer reviewed
Institut(e) CF Metabolomics & Proteomics (CF-MPC)
POF Topic(s) 30203 - Molecular Targets and Therapies
PSP-Element(e) A-630700-001
Förderungen Projekt DEAL
German Research Foundation DFG, Sonderforschungsbereich
DOI 10.1002/anie.202409220
WOS ID 001327069200001
Scopus ID 85205540308
PubMed ID 39073273
Erfassungsdatum 2024-08-01
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