Activity-based protein profiling of rice (Oryza sativa L.) bran serine hydrolases

Abstract

Rice bran is an underutilized agricultural by-product with economic importance. The unique phytochemicals and fatty acid compositions of bran have been targeted for nutraceutical development. The endogenous lipases and hydrolases are responsible for the rapid deterioration of rice bran. Hence, we attempted to provide the first comprehensive profiling of active serine hydrolases (SHs) present in rice bran proteome by activity-based protein profiling (ABPP) strategy. The active site-directed fluorophosphonate probe (rhodamine and biotin-conjugated) was used for the detection and identification of active SHs. ABPP revealed 55 uncharacterized active-SHs and are representing five different known enzyme families. Based on motif and domain analyses, one of the uncharacterized and miss annotated SHs (Os12Ssp, storage protein) was selected for biochemical characterization by overexpressing in yeast. The purified recombinant protein authenticated the serine protease activity in time and protein-dependent studies. Os12Ssp exhibited the maximum activity at a pH between 7.0 and 8.0. The protease activity was inhibited by the covalent serine protease inhibitor, which suggests that the ABPP approach is indeed reliable than the sequence-based annotations. Collectively, the comprehensive knowledge generated from this study would be useful in expanding the current understanding of rice bran SHs and paves the way for better utilization/stabilization of rice bran.

Development of Oxadiazolone Activity-Based Probes Targeting FphE for Specific Detection of Staphylococcus aureus Infections

“…Because serine hydrolases all use a highly nucleophilic active site serine, it is possible to use reactive electrophiles such as fluorophosphonates (FPs) to develop activity-based probes (ABPs) to label and track their activity in live cells and even whole organisms . FP-based probes enable global profiling of serine hydrolases across various organisms by a method known as activity-based protein profiling (ABPP). − Using the broadly reactive fluorphosphonate probe, FP tetramethylrhodamine (FP-TMR), we recently identified a family of metabolic serine hydrolases in S. aureus that we named fluorophosphonate-binding hydrolases (Fphs; FphA–J) .…”

Development of Oxadiazolone Activity-Based Probes Targeting FphE for Specific Detection ofS. aureusInfections

“…13 FP-based probes enable global profiling of serine hydrolases across various organisms by a method known as activity-based protein profiling (ABPP). [18][19][20][21][22][23][24][25] Using the broadly reactive fluorphosphonate probe, FP tetramethylrhodamine (FP-TMR), we recently identified a family of metabolic serine hydrolases in S. aureus that we named fluorophosphonate-binding hydrolases (Fphs; FphA-J). 26 Among the Fphs, we found that the lipid esterase FphB is likely a virulence factor important for infection in the heart and liver in vivo.…”