High resolution analysis of proteolytic substrate processing.

Autor:	Schillinger J; Center of Medical Biotechnology, Faculty of Biology, University Duisburg-Essen, Essen, Germany., Koci M; Center of Medical Biotechnology, Faculty of Biology, University Duisburg-Essen, Essen, Germany., Bravo-Rodriguez K; Max-Planck-Institute of Molecular Physiology, Dortmund, Germany., Heilmann G; Center of Medical Biotechnology, Faculty of Biology, University Duisburg-Essen, Essen, Germany., Kaschani F; Center of Medical Biotechnology, Faculty of Biology, University Duisburg-Essen, Essen, Germany., Kaiser M; Center of Medical Biotechnology, Faculty of Biology, University Duisburg-Essen, Essen, Germany., Beuck C; Center of Medical Biotechnology, Faculty of Biology, University Duisburg-Essen, Essen, Germany., Luecke H; Nova School of Science and Technology, Lisbon, Portugal; Department of Biophysics, University of California, Irvine, California, USA., Huber R; Center of Medical Biotechnology, Faculty of Biology, University Duisburg-Essen, Essen, Germany; Max-Planck-Institute for Biochemistry, Martinsried, Germany., Hellerschmied D; Center of Medical Biotechnology, Faculty of Biology, University Duisburg-Essen, Essen, Germany., Burston SG; School of Biochemistry, University of Bristol, Biomedical Sciences Building, Bristol, United Kingdom., Ehrmann M; Center of Medical Biotechnology, Faculty of Biology, University Duisburg-Essen, Essen, Germany. Electronic address: michael.ehrmann@uni-due.de.
Jazyk:	angličtina
Zdroj:	The Journal of biological chemistry [J Biol Chem] 2024 Nov; Vol. 300 (11), pp. 107812. Date of Electronic Publication: 2024 Sep 21.
DOI:	10.1016/j.jbc.2024.107812
Abstrakt:	Members of the widely conserved high temperature requirement A (HtrA) family of serine proteases are involved in multiple aspects of protein quality control. In this context, they have been shown to efficiently degrade misfolded proteins or protein fragments. However, recent reports suggest that folded proteins can also be native substrates. To gain a deeper understanding of how folded proteins are initially processed and subsequently degraded into short peptides by human HTRA1, we established an integrated and quantitative approach using time-resolved mass spectrometry, CD spectroscopy, and bioinformatics. The resulting data provide high-resolution information on up to 178 individual proteolytic sites within folded ANXA1 (consisting of 346 amino acids), the relative frequency of cuts at each proteolytic site, the preferences of the protease for the amino acid sequence surrounding the scissile bond, as well as the degrees of sequential structural relaxation and unfolding of the substrate that occur during progressive degradation. Our workflow provides precise molecular insights into protease-substrate interactions, which could be readily adapted to address other posttranslational modifications such as phosphorylation in dynamic protein complexes. Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article. (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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