Specificity profiling of deubiquitylases against endogenously generated ubiquitin-protein conjugates.

Valentina Rossio; Joao A Paulo; Xinyue Liu; Steven P Gygi; Randall W King

doi:10.1016/j.chembiol.2024.05.001

Home/Archive/PubMed

PubMed · 38810651

Specificity profiling of deubiquitylases against endogenously generated ubiquitin-protein conjugates.

Valentina Rossio·Joao A Paulo·Xinyue Liu·Steven P Gygi·Randall W King

View original source ↗Export metadata ↓

Abstract

Deubiquitylating enzymes (DUBs) remove ubiquitin from proteins thereby regulating their stability or activity. Our understanding of DUB-substrate specificity is limited because DUBs are typically not compared to each other against many physiological substrates. By broadly inhibiting DUBs in Xenopus egg extract, we generated hundreds of ubiquitylated proteins and compared the ability of 30 DUBs to deubiquitylate them using quantitative proteomics. We identified five high-impact DUBs (USP7, USP9X, USP36, USP15, and USP24) that each reduced ubiquitylation of over 10% of the isolated proteins. Candidate substrates of high-impact DUBs showed substantial overlap and were enriched for disordered regions, suggesting this feature may promote substrate recognition. Other DUBs showed lower impact and non-overlapping specificity, targeting distinct non-disordered proteins including complexes such as the ribosome or the proteasome. Altogether our study identifies candidate DUB substrates and defines patterns of functional redundancy and specificity, revealing substrate characteristics that may influence DUB-substrate recognition.

Explore related subjects

Substrate Specificity Animals Ubiquitin Ubiquitination Deubiquitinating Enzymes Xenopus laevis Xenopus Proteins Xenopus Proteomics Humans Ubiquitin-Specific Proteases DUB substrates TMT-proteomics deubiquitylating enzymes ubiquitin Journal Article Research Support, Non-U.S. Gov't Research Support, N.I.H., Extramural

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Valentina Rossio, Joao A Paulo, Xinyue Liu, Steven P Gygi, Randall W King. 2024-05-28. Specificity profiling of deubiquitylases against endogenously generated ubiquitin-protein conjugates.. https://doi.org/10.1016/j.chembiol.2024.05.001

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2026

Exploring the substrate promiscuity and functional residues of UGT73 family enzymes in Entada phaseoloides.

Flavonoid glycosides and triterpenoid saponins are bioactive plant metabolites with broad applications in food, medicine, and agriculture. These compounds are typically synthesized through glycosylation catalyzed by uridine diphosphate-dependent glycosyltransferases (UGTs). In this study, phylogenetic analysis across multiple species revealed a lineage-specific expansion of the UGT73 family in legumes such as Entada phaseoloides and Glycine max. The genome of the medicinal legume E. phaseoloides was re-annotated using integrated Oxford Nanopore Technologies and Illumina transcriptomic data to identify target genes. Four expanded UGT73 family genes were selected and functionally characterized. UGT73AA6 specifically glycosylates flavonoids, while UGT73CG48 and UGT73CG49 catalyze glycosylation of both flavonoids and pentacyclic triterpenoids. UGT73CG49 exhibits higher catalytic activity for the glucosylation of flavonoids and pentacyclic triterpenes compared to its xylosylation activity. Structural modeling and molecular docking identified key active sites, and site-directed mutagenesis revealed Gly194 as a critical residue enhancing catalytic activity in UGT73CG49. This study provides new insights into the functional evolution and metabolic versatility of the UGT73 family in legumes. The identification and engineering of UGT73 genes from E. phaseoloides lay a foundation for future applications in biosynthetic pathway engineering and the industrial production of high-value glycosides.

Substrate Specificity↗

PubMed2024

Multisubstrate specificity shaped the complex evolution of the aminotransferase family across the tree of life.

Aminotransferases (ATs) are an ancient enzyme family that play central roles in core nitrogen metabolism, essential to all organisms. However, many of the AT enzyme functions remain poorly defined, limiting our fundamental understanding of the nitrogen metabolic networks that exist in different organisms. Here, we traced the deep evolutionary history of the AT family by analyzing AT enzymes from 90 species spanning the tree of life (ToL). We found that each organism has maintained a relatively small and constant number of ATs. Mapping the distribution of ATs across the ToL uncovered that many essential AT reactions are carried out by taxon-specific AT enzymes due to wide-spread nonorthologous gene displacements. This complex evolutionary history explains the difficulty of homology-based AT functional prediction. Biochemical characterization of diverse aromatic ATs further revealed their broad substrate specificity, unlike other core metabolic enzymes that evolved to catalyze specific reactions today. Interestingly, however, we found that these AT enzymes that diverged over billion years share common signatures of multisubstrate specificity by employing different nonconserved active site residues. These findings illustrate that AT family enzymes had leveraged their inherent substrate promiscuity to maintain a small yet distinct set of multifunctional AT enzymes in different taxa. This evolutionary history of versatile ATs likely contributed to the establishment of robust and diverse nitrogen metabolic networks that exist throughout the ToL. The study provides a critical foundation to systematically determine diverse AT functions and underlying nitrogen metabolic networks across the ToL.

Substrate Specificity↗