Abstract
ATE1 is a conserved enzyme that catalyzes the covalent addition of arginine to proteins bearing N-terminal or mid-chain Asp and Glu residues. N-terminal (Nt) arginylation can also occur on Cys, Asn, and Gln following enzymatic conversion, often marking proteins for degradation. Essential for development, this pathway contributes to protein quality control and stress responses. Despite growing insight into ATE1 structure and function, the mechanisms governing its substrate selectivity and coordination with upstream oxygenase and deamidase remain poorly defined. Here, we reconstitute the human processing cascades that generate Nt-arginylated proteins in E. coli, enabling step-resolved analysis of arginylation outcomes in a cellular context. By co-expressing human ADO, NTAN1, or NTAQ1 with ATE1 in a modular system, we achieved efficient conversion of Nt-Cys, Asn, and Gln into arginylation-permissive forms, recapitulating key features of upstream processing. Using this platform, we demonstrated that N-terminal processing is efficient and that ATE1 preferentially modifies protein N-termini over internal acidic residues. Mid-chain arginylation of α-synuclein was detectable but occurred at low frequency, with no major differences in site selectivity observed across the ATE1 isoforms tested. Together, this bacterial reconstitution system provides a scalable experimental platform for quantitative, protein-level analysis of ATE1 substrate specificity under defined conditions.
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Acknowledgements
We thank Dr. Anna Kashia at the University of Pennsylvania for sharing mouse ATE1 expression constructs. We are grateful to Dr. Ebmeier at the University of Colorado Boulder Mass Spectrometry Facility for help with initial pilot experiments.
Funding
This work was supported by grants from NIH R35 GM150678 to Y.Z., NIH R21 CA292191 to Z.L., NIH R01 HL177113 to Z.L. and B.A.G., Research Education Component (REC) through an NIA grant P30AG066444 to Z.L., Case Comprehensive Cancer Center (P30CA043703 to Y.Z. and T32CA059366 to T.A.), and summer research scholarships provided by CWRU SOURCE. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.
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Lan, X., Searfoss, R.M., Lee, D. et al. Reconstitution of protein arginylation pathways in bacteria for robust identification and quantification. Commun Biol (2026). https://doi.org/10.1038/s42003-026-10275-z
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DOI: https://doi.org/10.1038/s42003-026-10275-z
