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Intrinsically disordered proteins are frequent targets for functional regulation through post-translational modification due to their high accessibility to modifying enzymes and the strong influence of changes in primary structure on their chemical properties. While lysine N ε -acetylation was first observed as a common modification of histone tails, proteomic data suggest that lysine acetylation is ubiquitous among both nuclear and cytosolic proteins. However, compared with our biophysical understanding of the other common post-translational modifications, mechanistic studies to document how lysine N ε -acetyl marks are placed, utilized to transduce signals, and eliminated when signals need to be turned off, have not kept pace with proteomic discoveries. Herein we report a nuclear magnetic resonance method to monitor N ε -lysine acetylation through enzymatic installation of a 13 C-acetyl probe on a protein substrate, followed by detection through 13 C direct-detect spectroscopy. We demonstrate the ease and utility of this method using histone H3 tail acetylation as a model. The clearest advantage to this method is that it requires no exogenous tags that would otherwise add steric bulk, change the chemical properties of the modified lysine, or generally interfere with downstream biochemical processes. The non-perturbing nature of this tagging method is beneficial for application in any system where changes to local structure and chemical properties beyond those imparted by lysine modification are unacceptable, including intrinsically disordered proteins, bromodomain containing protein complexes, and lysine deacetylase enzyme assays.
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Quantitative proteomics reveals extensive lysine ubiquitination and transcription factor stability states in Arabidopsis
Abstract Protein activity, abundance, and stability can be regulated by post-translational modification including ubiquitination. Ubiquitination is conserved among eukaryotes and plays a central role in modulating cellular function; yet, we lack comprehensive catalogs of proteins that are modified by ubiquitin in plants. In this study, we describe an antibody-based approach to enrich ubiquitinated peptides coupled with isobaric labeling to enable quantification of up to 18-multiplexed samples. This approach identified 17,940 ubiquitinated lysine sites arising from 6,453 proteins from Arabidopsis (Arabidopsis thaliana) primary roots, seedlings, and rosette leaves. Gene ontology analysis indicated that ubiquitinated proteins are associated with numerous biological processes including hormone signaling, plant defense, protein homeostasis, and metabolism. We determined ubiquitinated lysine residues that directly regulate the stability of three transcription factors, CRYPTOCHROME-INTERACTING BASIC-HELIX-LOOP-HELIX 1 (CIB1), CIB1 LIKE PROTEIN 2 (CIL2), and SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1) using in vivo degradation assays. Furthermore, codon mutation of CIB1 to create a K166R conversion to prevent ubiquitination, via CRISPR/Cas9-derived adenosine base editing, led to an early flowering phenotype and increased expression of FLOWERING LOCUS T (FT). These comprehensive site-level ubiquitinome profiles provide a wealth of data for future functional studies related to modulation of biological processes mediated by this post-translational modification in plants.
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- Award ID(s):
- 2118253
- PAR ID:
- 10596611
- Publisher / Repository:
- The Plant Cell
- Date Published:
- Journal Name:
- The Plant Cell
- Volume:
- 37
- Issue:
- 1
- ISSN:
- 1040-4651
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1093/plcell/koae310
