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

Juan Luo, Yao Yu, Ke Wang, Sizhe He, Longjie Wang, Fangfang Liang, Jason W. Chin, Shan Tang

Published: 2024-06-11 DOI: 10.1038/s41596-024-01006-x

acyl–enzyme intermediates

genetically encoded 2,3-diaminopropionic acid

hydrolase substrates

mass spectrometry

substrate capture

AI 解读

Genetic manipulation and immortalized culture of ex vivo primary human germinal center B cells

The cell-line-derived subcutaneous tumor model in preclinical cancer research

Subcellular patch-clamp techniques for single-bouton stimulation and simultaneous pre- and postsynaptic recording at cortical synapses

NovoSpaRc: flexible spatial reconstruction of single-cell gene expression with optimal transport

Genome-wide pooled CRISPR screening in neurospheres

High-throughput cultivation and identification of bacteria from the plant root microbiota

Viral crosslinking and solid-phase purification enables discovery of ribonucleoprotein complexes on incoming RNA virus genomes

Mitochondrial single-cell ATAC-seq for high-throughput multi-omic detection of mitochondrial genotypes and chromatin accessibility

Addendum: Micro-CT acquisition and image processing to track and characterize pulmonary nodules in mice

Multiplexed single-cell proteomics using SCoPE2

查看全部

Sections

Figures

References

Introduction
Materials
Procedure 1: DAP-mediated substrate trapping in mammalian cell lysate
Procedure 2: DAP-mediated substrate trapping in adherent mammalian cells
- Part 1: preparation of DAP-containing UL36USP in HEK293T cells
- Part 2: DAP-mediated substrate trapping in HEK293T cells
Procedure 3: DAP-mediated substrate trapping in suspension mammalian cells
- Part 1: preparation of DAP-containing UL36USP in Expi293 cells
- Part 2: DAP-mediated substrate trapping in Expi293 cells
Procedure 4: substrate identification and validation
- Large-scale preparation of hydrolase–substrate conjugates
- Sample preparation for LC–MS/MS analysis
Troubleshooting
Timing
Anticipated results
- Reporting summary
Acknowledgements

Tang, S. et al. Mechanism-based traps enable protease and hydrolase substrate discovery. Nature 602, 701–707 (2022).
Bond, J. S. Proteases: history, discovery, and roles in health and disease. J. Biol. Chem. 294, 1643–1651 (2019).
Turk, B. Targeting proteases: successes, failures and future prospects. Nat. Rev. Drug Discov. 5, 785–799 (2006).
Santos, R. et al. A comprehensive map of molecular drug targets. Nat. Rev. Drug Discov. 16, 19–34 (2017).
Ahrén, B. DPP-4 inhibition and the path to clinical proof. Front. Endocrinol. 10, 376 (2019).
Fricker, L. D. Proteasome inhibitor drugs. Annu. Rev. Pharmacol. Toxicol. 60, 457–476 (2020).
Mahase, E. Covid-19: Pfizer’s paxlovid is 89% effective in patients at risk of serious illness, company reports. BMJ 375, n2713 (2021).
Pérez-Silva, J. G., Español, Y., Velasco, G. & Quesada, V. The Degradome database: expanding roles of mammalian proteases in life and disease. Nucleic Acids Res. 44, D351–D355 (2016).
Gomez-Fabra Gala, M. & Vögtle, F. N. Mitochondrial proteases in human diseases. FEBS Lett. 595, 1205–1222 (2021).
Vizovisek, M., Ristanovic, D., Menghini, S., Christiansen, M. G. & Schuerle, S. The tumor proteolytic landscape: a challenging frontier in cancer diagnosis and therapy. Int. J. Mol. Sci. 22, 2514 (2021).
Huguenin-Dezot, N. et al. Trapping biosynthetic acyl-enzyme intermediates with encoded 2,3-diaminopropionic acid. Nature 565, 112–117 (2019).
Liang, L. J. et al. Chemical synthesis of activity-based E2-ubiquitin probes for the structural analysis of E3 ligase-catalyzed transthiolation. Angew. Chem. Int. Ed. 60, 17171–17177 (2021).
Squair, D. R. & Virdee, S. A new dawn beyond lysine ubiquitination. Nat. Chem. Biol. 18, 802–811 (2022).
Wang, Y. & Tang, S. Capturing covalent catalytic intermediates by enzyme mutants: recent advances in methodologies and applications. ChemBioChem 24, e202300036 (2023).
Eiamthong, B. et al. Discovery and genetic code expansion of a polyethylene terephthalate (PET) Hydrolase from the human saliva metagenome for the degradation and bio-functionalization of PET. Angew. Chem. Int. Ed. 61, e202203061 (2022).
López-Otín, C. & Overall, C. M. Protease degradomics: a new challenge for proteomics. Nat. Rev. Mol. Cell Biol. 3, 509–519 (2002).
Luo, S. Y., Araya, L. E. & Julien, O. Protease substrate identification using N-terminomics. ACS Chem. Biol. 14, 2361–2371 (2019).
Vande Walle, L. et al. Proteome-wide identification of HtrA2/Omi substrates. J. Proteome Res. 6, 1006–1015 (2007).
Crawford, E. D. & Wells, J. A. Caspase substrates and cellular remodeling. Annu. Rev. Biochem. 80, 1055–1087 (2011).
Saita, S. et al. PARL mediates Smac proteolytic maturation in mitochondria to promote apoptosis. Nat. Cell Biol. 19, 318–328 (2017).
Liao, R. J. & van Wijk, K. J. Discovery of AAA+ protease substrates through trapping approaches. Trends Biochem. Sci. 44, 528–545 (2019).
Knopf, J. D. et al. Intramembrane protease RHBDL4 cleaves oligosaccharyltransferase subunits to target them for ER-associated degradation. J. Cell Sci. 133, jcs243790 (2020).
Beard, H. A., Barniol-Xicota, M., Yang, J. & Verhelst, S. H. L. Discovery of cellular roles of intramembrane proteases. ACS Chem. Biol. 14, 2372–2388 (2019).
Rawlings, N. D. et al. The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database. Nucleic Acids Res. 46, D624–D632 (2018).
Tinzl, M. & Hilvert, D. Trapping transient protein species by genetic code expansion. ChemBioChem 22, 92–99 (2021).
Kattenhorn, L. M., Korbel, G. A., Kessler, B. M., Spooner, E. & Ploegh, H. L. A deubiquitinating enzyme encoded by HSV-1 belongs to a family of cysteine proteases that is conserved across the family Herpesviridae. Mol. Cell 19, 547–557 (2005).
Frankenfield, A. M., Ni, J. W., Ahmed, M. & Hao, L. Protein contaminants matter: building universal protein contaminant libraries for DDA and DIA proteomics. J. Proteome Res. 21, 2104–2113 (2022).
Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794–1805 (2011).
Tyanova, S. et al. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat. Methods 13, 731–740 (2016).

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

Supplementary information

推荐阅读