Structural Basis for Specificity of Propeptide-Enzyme Interaction in Barley C1A Cysteine Peptidases

Abstract

C1A cysteine peptidases are synthesized as inactive proenzymes. Activation takes place by proteolysis cleaving off the inhibitory propeptide. The inhibitory capacity of propeptides from barley cathepsin L and B-like peptidases towards commercial and barley cathepsins has been characterized. Differences in selectivity have been found for propeptides from L-cathepsins against their cognate and non cognate enzymes. Besides, the propeptide from barley cathepsin B was not able to inhibit bovine cathepsin B. Modelling of their three-dimensional structures suggests that most propeptide inhibitory properties can be explained from the interaction between the propeptide and the mature cathepsin structures. Their potential use as biotechnological tools is discussed.

Inhibitory Properties of Cysteine Protease Pro-Peptides from Barley Confer Resistance to Spider Mite Feeding

“…In previous reports, the in vitro inhibitory properties of several recombinant pro-peptides from barley C1A cathepsin L-, B- and F-like against their own enzymes or/and towards commercial proteases were analysed and kinetic assays revealed that all pro-peptides exhibited a competitive inhibition [ 5 , 23 ]. Now, to obtain further insights on the protective role of these C1A pro-sequences, we selected phytophagous coleopteran and acari species, whose CysProt are the main digestive enzymes in their guts [ 33 , 34 , 41 – 43 ].…”

“…The pro-peptides contain the non-contiguous ERFNIN signature found in cathepsin L- and H-like or the ERFNAQ variant in cathepsin F-like, while cathepsin B-like proteases lack this motif [ 7 , 12 ]. Interestingly, C1A pro-peptides from different species, including plants, have the capacity to inhibit several exogenous C1A peptidases [ 21 – 23 ]. Molecular modelling of three-dimensional protein structures has shown that most of the specific inhibitory properties of barley pro-peptides are determined from their interactions with the mature proteases [ 23 ].…”

“…Interestingly, C1A pro-peptides from different species, including plants, have the capacity to inhibit several exogenous C1A peptidases [ 21 – 23 ]. Molecular modelling of three-dimensional protein structures has shown that most of the specific inhibitory properties of barley pro-peptides are determined from their interactions with the mature proteases [ 23 ]. In vitro assays and artificial diets using the recombinant pro-region of the papaya proteinase IV have demonstrated a selective inhibition of digestive CysProt from several phytophagous beetles [ 24 ].…”

Development of wheat genotypes expressing a glutamine-specific endoprotease from barley and a prolyl endopeptidase from Flavobacterium meningosepticum or Pyrococcus furiosus as a potential remedy to celiac disease

“…Both criteria are met in the established transgenic lines. EPHvB2 that belong to the C1A cysteine protease family is encoded as a proenzyme, where the propeptide or the prosegment serves as both inhibitor and chaperone to respectively facilitate spatiotemporal regulation of the proteolytic activity and proper folding of the protease (Cambra et al 2012; Schilling et al 2009; Bethune et al 2006; Cappetta et al 2002). Both of these properties have immense importance in the present research, as the propeptide keeps the enzyme in an inactive state in grains and likely facilitates its refolding after thermal denaturation.…”

Roles of C1A peptidases during barley leaf senescence mediated by abiotic stresses

“…Although proteases involved in drought may differ from those specifically expressed in senescence (Khanna-Chopra et al, 1999;Beyene et al, 2006), experimental evidences suggest that drought-sensitive plants have higher proteolytic activity compared to resistant ones (Simova-Stoilova et al, 2010). In this scenario, it is essential a fine-tuning regulation of protease activities mediated by specific inhibitors, cofactors and the activation of zymogens in response to specific environmental conditions (Cambra et al, 2012a;Martinez et al, 2012). Protease inhibitory proteins are the main regulators of protein degradation caused by proteases.…”