Subunit‐selective proteasome activity profiling uncovers uncoupled proteasome subunit activities during bacterial infections

Abstract

The proteasome is a nuclear-cytoplasmic proteolytic complex involved in nearly all regulatory pathways in plant cells. The three different catalytic activities of the proteasome can have different functions, but tools to monitor and control these subunits selectively are not yet available in plant science. Here, we introduce subunit-selective inhibitors and dual-color fluorescent activity-based probes for studying two of the three active catalytic subunits of the plant proteasome. We validate these tools in two model plants and use this to study the proteasome during plant-microbe interactions. Our data reveal that Nicotiana benthamiana incorporates two different paralogs of each catalytic subunit into active proteasomes. Interestingly, both β1 and β5 activities are significantly increased upon infection with pathogenic Pseudomonas syringae pv. tomato DC3000 lacking hopQ1-1 [PtoDC3000(ΔhQ)] whilst the activity profile of the β1 subunit changes. Infection with wild-type PtoDC3000 causes proteasome activities that range from strongly induced β1 and β5 activities to strongly suppressed β5 activities, revealing that β1 and β5 activities can be uncoupled during bacterial infection. These selective probes and inhibitors are now available to the plant science community, and can be widely and easily applied to study the activity and role of the different catalytic subunits of the proteasome in different plant species.

Comparative study of salt stress-induced physiological and molecular responses in tomato (Solanum lycopersicum L.)

“…As they have different specifies and their excitation and emission wavelength are different, LW124 and MVB127 can be used by co-labelling (Misas-Villamil et al, 2017). LW124 labeling does not display significant differences upon salt treatment (Fig.33A), consistent with earlier observations that β1 labeling is unaffected.…”

“…Therefore, subunit-selective inhibitors were used to identify the upper signal. N3β1 is an epoxyketone-based inhibitor that specifically inhibits the β1 catalytic subunit of the proteasome, whereas N3β5 is a vinyl-sulfone-based inhibitor targeting the β5 catalytic subunit of the proteasome (Verdoes et al, 2010;Misas-Villamil et al, 2017). Pre-incubation with N3β1 caused reduction in the fluorescence of the middle-and the lower signal in both of 0-, and 250 mM NaCl treated samples whereas preincubation with N3β5 reduced the signal intensity of the upper signal in the 250 mM NaCl treated sample (Fig.31C), suggesting that the upper differential signal is caused by a β5 subunit.…”

“…To confirm the identity of the shifting of the catalytic subunits, the root extract was labelled with subunit-specific activity-based probes, LW124 and MVB127 (Li et al, 2013;Misas-Villamil et al, 2017). LW124 is specific for β1 and has an epoxyketone reactive group and a fluorophore with excitation and emission wavelength at 470 nm and 530 nm, respectively.…”

Chemical Proteomics of Host–Microbe Interactions

“…The van der Hoorn group, who have established the use of many ABPs in plant science, employed two probes ( 5 and 6 , Figure D) developed by Li et al . to analyze activity of the β1 and β5 subunits in Nicotiana benthamiana plants infected with the pathogen Pseudomonas syringae . Interestingly, they observed a variable response, with upregulation or suppression of the β5 subunit in response to infection, and uncoupling of β1 and β5 activities.…”

On the Promising Role of Enzyme Activity Assay in Interpreting Comparative Proteomic Data in Plants

“…The increasing application of comparative ABPP in plants was reported for measuring various enzymes, especially proteases, α‐glycosidases, and other hydrolases during seed germination, fruit metabolism, plant–microbe interaction in Arabidopsis , tomato, and other species . By comparative ABPP, labeled proteomes are compared to display activity differences in a compared condition, thus allowing to detect differential labeling of single proteins .…”