Dataset Information

Shedding light on plant proteolysis: genetically encoded fluorescent sensors as tools for profiling protease activities

ABSTRACT: Proteolysis is a universal process, as proteases play a pivotal role in modulating numerous signaling pathways. By hydrolyzing peptide bonds within their target proteins, proteases control their fate and function. Understanding the temporal and spatial dynamics of proteolytic events, including the proteases that execute them, is crucial for elucidating their particular roles across diverse biological processes. In this study, we developed and characterized a set of genetically encoded Förster resonance energy transfer (FRET)-based reporters for the detection of various proteolytic activities in plants. Our sensors reliably reported the activity of specific proteases, exhibiting performance comparable to established detection systems. In addition, we engineered variants capable of detecting the spatial dynamics of metacaspase-triggered proteolysis after wounding and during programmed cell death in roots. The implementation of these tools in plant research opens new opportunities to explore proteolytic mechanisms with enhanced precision. We demonstrate the feasibility of the FRET-based sensors to detect various activities in vivo with high spatiotemporal resolution. Overall, these biosensors constitute a versatile toolbox for probing protease function within its native cellular context, paving the way for deeper insights into plant biology and signaling. This dataset includes a targeted shotgun proteomics experiment designed to identify the specific endoproteolytic cleavage products generated from the FRET(VRPR) biosensor.

INSTRUMENT(S):

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Rosette

SUBMITTER: Jingjing Huang

LAB HEAD: Frank Van Breusegem

PROVIDER: PXD075809 | Pride | 2026-07-04

REPOSITORIES: Pride

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			Action	DRS
	PRC-7113DDA_UPRC_3702_H11119_2p_IonOpt18_25cm_Breusegem-shotgun-DDA-1.raw	Raw
	PRC-7113DDA_UPRC_3702_H11119_2p_IonOpt18_25cm_Breusegem-shotgun-DDA-1_uncalibrated.mzML	Mzml
	PRC-7113DDA_UPRC_3702_H11119_2p_IonOpt18_25cm_Breusegem-shotgun-DDA-2.raw	Raw
	PRC-7113DDA_UPRC_3702_H11119_2p_IonOpt18_25cm_Breusegem-shotgun-DDA-2_uncalibrated.mzML	Mzml
	checksum.txt	Txt

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Project description:Proteases constitute the largest enzyme gene family in vertebrates with intracellular and secreted proteases having critical roles in cellular and organ physiology. Intestinal tract contains diverse set of proteases mediating digestion, microbial responses, epithelial and immune signaling. Transit of chyme through the intestinal tract results in significant suppression of proteases. Although endogenous protease inhibitors have been identified, the broader mechanisms underlying protease regulation in the intestinal tract remains unclear. The objective of this study was to determine microbial regulation of proteolytic activity in intestinal tract using phenotype of post-infection irritable bowel syndrome, a condition characterized by high fecal proteolytic activity. Proteases of host pancreatic origin (chymotrypsin like pancreatic elastase 2A, 3B and trypsin 2) drove proteolytic activity. Of the 14 differentially abundant taxa, high proteolytic activity state was characterized by complete absence of the commensal Alistipes putredinis. Germ free mice had very high proteolytic activity (10-fold of specific-pathogen free mice) which dropped significantly upon humanization with microbiota from healthy volunteers. In contrast, high proteolytic activity microbiota failed to inhibit it, a defect that corrected with fecal microbiota transplant as well as addition of A. putredinis. These mice also had increased intestinal permeability similar to that seen in patients. Microbiota β-glucuronidases mediate bilirubin deconjugation and unconjugated bilirubin is an inhibitor of serine proteases. We found that high proteolytic activity patients had lower urobilinogen levels, a product of bilirubin deconjugation. Mice colonized with β-glucuronidase overexpressing E. coli demonstrated significant inhibition of proteolytic activity and treatment with β-glucuronidase inhibitors increased it. The findings establish that specific commensal microbiota mediates effective inhibition of host pancreatic proteases and maintains intestinal barrier function through the production of β-glucuronidases. This suggests an important homeostatic role for commensal intestinal microbiota.

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Shedding light on plant proteolysis: genetically encoded fluorescent sensors as tools for profiling protease activities

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