Wheat Fusarium Protease Specificity and Effect on Dough Properties

Fusarium infection is a worldwide agricultural problem of billion dollar proportions globally, and it has increasingly threatened entire regional food supplies. In addition to the toxin deoxynivalenol (DON), Fusarium species express digestive enzymes that degrade starch and protein, affecting the quality of infected grains, especially wheat processing performance which depends largely on gluten proteins. In this study, the impact of Fusarium protease on the functionality of Canada Western Red Spring (CWRS) wheat was assessed by adding Fusarium-damaged kernels (FDK) to a FDK-free base wheat sample. Digestion of beta-casein by extracts of flours, milled from sound and FDK-spiked wheat samples, demonstrated elevated cleavage in FDK-spiked flour extracts as follows: N-terminal to lysine (eight-fold), N- and C-terminal to isoleucine (four-fold and three-fold, respectively), N-terminal to tyrosine (three-fold) and C-terminal to arginine at P1′ (five-fold). Comparison of abbreviated (45 min) and standard (135 min) extensigraph test results indicated that desirable increases in dough resistance to extension (Rmax) due to gluten re-polymerization after longer resting were partially to completely counteracted in FDK-spiked flours in a dose-dependent manner. Baking tests confirmed that while loaf volume is similar, proofed dough from FDK-spiked samples caused detectable loaf collapse at 3% FDK. Extensigraph Rmax and Fusarium protease levels were inversely related, and effected by both the extent and severity of infection. While the current FDK tolerances for grading Canadian wheat can effectively control protease damage, prevalence of deoxynivalenol (DON) weak- and non-producing Fusarium strains/species (e.g., F. avenaceum) in some growing regions must be considered to protect functionality if grading is solely based on DON content.

Low cytoplasmic magnesium increases the specificity of the Lon and ClpAP proteases

Proteolysis is a fundamental property of all living cells. In the bacterium Salmonella enterica serovar Typhimurium ( S. Typhimurium), the HspQ protein controls the specificities of the Lon and ClpAP proteases. Upon acetylation, HspQ stops being a Lon substrate and no longer enhances proteolysis of the Lon substrate Hha. The accumulated HspQ protein binds to the protease adaptor ClpS, hindering proteolysis of ClpS-dependent substrates of ClpAP, such as Oat, a promoter of antibiotic persistence. HspQ is acetylated by the protein acetyl transferase Pat from acetyl-CoA bound to the acetyl-CoA binding protein Qad. We now report that low cytoplasmic Mg 2+ promotes qad expression, which protects substrates of Lon and ClpSAP by furthering HspQ amounts. The qad promoter is activated by PhoP, a regulatory protein highly activated in low cytoplasmic Mg 2+ that also represses clpS transcription. Both the qad gene and PhoP repression of the clpS promoter are necessary for antibiotic persistence. PhoP promotes qad transcription also in Escherichia coli , which shares a similar PhoP box in the qad promoter region with S. Typhimurium, S. bongori , and Enterobacter cloacae . Our findings identify cytoplasmic Mg 2+ and the PhoP protein as critical regulators of protease specificity in multiple enteric bacteria. Importance The bacterium Salmonella enterica serovar Typhimurium narrows down the spectrum of substrates degraded by the proteases Lon and ClpAP in response to low cytoplasmic Mg 2+ , a condition that decreases protein synthesis. This control is exerted by PhoP, a transcriptional regulator activated in low cytoplasmic Mg 2+ that governs proteostasis and is conserved in enteric bacteria. The uncovered mechanism enables bacteria to control the abundance of pre-existing proteins.

Quantitative profiling of protease specificity

Proteases are an important class of enzymes, whose activity is central to many physiologic and pathologic processes. Detailed knowledge of protease specificity is key to understanding their function. Although many methods have been developed to profile specificities of proteases, few have the diversity and quantitative grasp necessary to fully define specificity of a protease, both in terms of substrate numbers and their catalytic efficiencies. We have developed a concept of “selectome”; the set of substrate amino acid sequences that uniquely represent the specificity of a protease. We applied it to two closely related members of the Matrixin family–MMP-2 and MMP-9 by using substrate phage display coupled with Next Generation Sequencing and information theory-based data analysis. We have also derived a quantitative measure of substrate specificity, which accounts for both the number of substrates and their relative catalytic efficiencies. Using these advances greatly facilitates elucidation of substrate selectivity between closely related members of a protease family. The study also provides insight into the degree to which the catalytic cleft defines substrate recognition, thus providing basis for overcoming two of the major challenges in the field of proteolysis: 1) development of highly selective activity probes for studying proteases with overlapping specificities, and 2) distinguishing targeted proteolysis from bystander proteolytic events.

Quantitative profiling of protease specificity

Proteases comprise an important class of enzymes, whose activity is central to many physiologic and pathologic processes. Detailed knowledge of protease specificity is key to understanding their function. Although many methodologies have been developed to profile specificities of proteases, few have the diversity and quantitative grasp necessary to fully define specificity of a protease, both in terms of substrate numbers and their catalytic efficiencies. We have developed a concept of “selectome”, which defines the set of substrates that uniquely represents specificity of a protease. We applied it to two closely related members of the Matrixin family – MMP-2 and MMP-9 by using substrate phage display coupled with Next Generation Sequencing and information theory-based data analysis. We have also derived a quantitative measure of substrate specificity, which accounts for both the numbers and relative catalytic efficiencies of substrates. Using these advances greatly facilitates uncovering selectivity between closely related members of protease families and provides insight into to the degree of contribution of catalytic cleft specificity to protein substrate recognition, thus providing basis to overcoming two of the major challenges in the field of proteolysis: 1) development of highly selective activity probes and inhibitors for studying proteases with overlapping specificities, and 2) distinguishing targeted proteolysis from bystander proteolytic events.

A Review on Therapeutic Potential of Protease Inhibitors from Medicinal Plants

Most of the currently available therapeutic agents, particularly for cardiovascular disorders and cancers are very expensive and induce some serious side effects. Some of these drugs have also become less effective due to the emergence of antibiotic resistance. There is a necessity and great demand for the development of novel efficacious plant-based agents that are of pharmacologically effective. In this connection, this review focuses on therapeutic potential of plant protease inhibitors. Protease inhibitors are of a particular concern at present due to their potent ability to inhibit protease enzymes that are involved in pathogenesis of various human diseases. In addition to their function as protein-degrading enzymes, protease inhibitors are now well-known for their capability to involve in many biological activities as signaling molecules. Plant protease inhibitors are also engaged in several physiological and pathological processes, such as blood clotting, inflammation, immune regulation, apoptosis and carcinogenesis. Therefore, isolation of plant protease inhibitors and evaluation of their therapeutic capacity against chronic human diseases have become a major research interest. Nevertheless, protease inhibitor content and protease specificity vary significantly even in the same plant species depending on the geographical location and environmental factors. Consequently, it is important to identify potent therapeutic potential of each plant protease inhibitor on human health individually.