Capturing acyl-enzyme intermediates with genetically encoded 2,3-diaminopropionic acid for hydrolase substrate identification.

Journal

Nature protocols
ISSN: 1750-2799
Titre abrégé: Nat Protoc
Pays: England
ID NLM: 101284307

Informations de publication

Date de publication:
12 Jun 2024
Historique:
received: 25 08 2023
accepted: 29 03 2024
medline: 13 6 2024
pubmed: 13 6 2024
entrez: 12 6 2024
Statut: aheadofprint

Résumé

Catalytic mechanism-based, light-activated traps have recently been developed to identify the substrates of cysteine or serine hydrolases. These traps are hydrolase mutants whose catalytic cysteine or serine are replaced with genetically encoded 2,3-diaminopropionic acid (DAP). DAP-containing hydrolases specifically capture the transient thioester- or ester-linked acyl-enzyme intermediates resulting from the first step of the proteolytic reaction as their stable amide analogs. The trapped substrate fragments allow the downstream identification of hydrolase substrates by mass spectrometry and immunoblotting. In this protocol, we provide a detailed step-by-step guide for substrate capture and identification of the peptidase domain of the large tegument protein deneddylase (UL36

Identifiants

DOI: 10.1038/s41596-024-01006-x PMID: 38867073
pubmed: 38867073
doi: 10.1038/s41596-024-01006-x
pii: 10.1038/s41596-024-01006-x
doi:

Types de publication

Journal Article Review

Langues

eng

Sous-ensembles de citation

IM

Informations de copyright

© 2024. Springer Nature Limited.

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Auteurs

Juan Luo (J)

Department of Oncology, The First Affiliated Hospital of USTC, Key Laboratory of Immune Response and Immunotherapy, Centre for Advanced Interdisciplinary Science and Biomedicine of IHM, MOE Key Laboratory for Membraneless Organelles & Cellular Dynamics, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, China.

Yao Yu (Y)

Department of Oncology, The First Affiliated Hospital of USTC, Key Laboratory of Immune Response and Immunotherapy, Centre for Advanced Interdisciplinary Science and Biomedicine of IHM, MOE Key Laboratory for Membraneless Organelles & Cellular Dynamics, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, China.

Ke Wang (K)

Department of Oncology, The First Affiliated Hospital of USTC, Key Laboratory of Immune Response and Immunotherapy, Centre for Advanced Interdisciplinary Science and Biomedicine of IHM, MOE Key Laboratory for Membraneless Organelles & Cellular Dynamics, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, China.

Sizhe He (S)

Department of Oncology, The First Affiliated Hospital of USTC, Key Laboratory of Immune Response and Immunotherapy, Centre for Advanced Interdisciplinary Science and Biomedicine of IHM, MOE Key Laboratory for Membraneless Organelles & Cellular Dynamics, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, China.
ORCID: 0009-0002-1077-2648

Longjie Wang (L)

Department of Oncology, The First Affiliated Hospital of USTC, Key Laboratory of Immune Response and Immunotherapy, Centre for Advanced Interdisciplinary Science and Biomedicine of IHM, MOE Key Laboratory for Membraneless Organelles & Cellular Dynamics, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, China.
ORCID: 0009-0000-7722-6284

Fangfang Liang (F)

Department of Medical Oncology, Guangxi Medical University First Affiliated Hospital, Nanning, Guangxi Zhuang Autonomous Region, China.

Jason W Chin (JW)

Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.
ORCID: 0000-0003-1219-4757

Shan Tang (S)

Department of Oncology, The First Affiliated Hospital of USTC, Key Laboratory of Immune Response and Immunotherapy, Centre for Advanced Interdisciplinary Science and Biomedicine of IHM, MOE Key Laboratory for Membraneless Organelles & Cellular Dynamics, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, China. stang@ustc.edu.cn.
ORCID: 0000-0003-4483-1108

Classifications MeSH