Antimicrobial activity of an aspartic protease from Salpichroa origanifolia fruits

Abstract: Plant proteases play a fundamental role in several processes like growth, development and in response to biotic and abiotic stress. In particular, aspartic proteases (AP) are expressed in different plant organs and have antimicrobial activity. Previously, we purified an AP from Salpichroa origanifolia fruits called salpichroin. The aim of this work was to determine the cytotoxic activity of this enzyme on selected plant and human pathogens. For this purpose, the growth of the selected pathogens was analysed after exposure to different concentrations of salpichroin. The results showed that the enzyme was capable of inhibiting Fusarium solani and Staphylococcus aureus in a dose-dependent manner. It was determined that 1·2 μmol l−1 of salpichroin was necessary to inhibit 50% of conidial germination, and the minimal bactericidal concentration was between 1·9 and 2·5 μmol l−1. Using SYTOX Green dye we were able to demonstrate that salpichroin cause membrane permeabilization. Moreover, the enzyme treated with its specific inhibitor pepstatin A did not lose its antibacterial activity. This finding demonstrates that the cytotoxic activity of salpichroin is due to the alteration of the cell plasma membrane barrier but not due to its proteolytic activity. Antimicrobial activity of the AP could represent a potential alternative for the control of pathogens that affect humans or crops of economic interest. Significance and Impact of the Study: This study provides insights into the antimicrobial activity of an aspartic protease isolated from Salpichroa origanifolia fruits on plant and human pathogens. The proteinase inhibited Fusarium solani and Staphylococcus aureus in a dose-dependent manner due to the alteration of the cell plasma membrane barrier but not due to its proteolytic activity. Antimicrobial activity of salpichroin suggests its potential applications as an important tool for the control of pathogenic micro-organisms affecting humans and crops of economic interest. Therefore, it would represent a new alternative to avoid the problems of environmental pollution and antimicrobial resistance.Fil: Díaz, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Luján; ArgentinaFil: Rocha, Gabriela Fernanda. Universidad Nacional de Luján; ArgentinaFil: Kise, Francisco. Universidad Nacional de Luján; ArgentinaFil: Rosso, A. M.. Universidad Nacional de Luján; ArgentinaFil: Guevara, Maria Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Parisi, M.G.. Universidad Nacional de Luján; Argentin

Crystal structure of peptide-bound neprilysin reveals key binding interactions

Neprilysin (NEP) is a promiscuous zinc metalloprotease with broad substrate specificity and cleaves a remarkable diversity of substrates through endopeptidase action. Two of these – amyloid-β and natriuretic peptides – implicate the enzyme in both Alzheimer’s disease and cardiovascular disease, respectively. Here, we report the creation of a catalytically inactive NEP (E584D) to determine the first peptide-bound crystal structure at 2.6 Å resolution. The structure reveals key interactions involved in substrate binding which we have identified to be conserved in other known zinc metalloproteases. In addition, the structure provides evidence for a potential exosite within the central cavity that may play a critical role in substrate positioning. Together, these results contribute to our understanding of the molecular function of NEP.</p

Haplotype-resolved genome of heterozygous African cassava cultivar TMEB117 (Manihot esculenta)

Cassava (Manihot esculenta Crantz) is a vital tropical root crop providing essential dietary energy to over 800 million people in tropical and subtropical regions. As a climate-resilient crop, its significance grows as the human population expands. However, yield improvement faces challenges from biotic and abiotic stress and limited breeding. Advanced sequencing and assembly techniques enabled the generation of a highly accurate, nearly complete, haplotype-resolved genome of the African cassava cultivar TMEB117. It is the most accurate cassava genome sequence to date with a base-level accuracy of QV > 64, N50 > 35 Mbp, and 98.9% BUSCO completeness. Over 60% of the genome comprises repetitive elements. We predicted over 45,000 gene models for both haplotypes. This achievement offers valuable insights into the heterozygosity genome organization of the cassava genome, with improved accuracy, completeness, and phased genomes. Due to its high susceptibility to African Cassava Mosaic Virus (ACMV) infections compared to other cassava varieties, TMEB117 provides an ideal reference for studying virus resistance mechanisms, including epigenetic variations and smallRNA expressions

Crystal structure of peptide-bound neprilysin reveals key binding interactions

Neprilysin (NEP) is a promiscuous zinc metalloprotease with broad substrate specificity and cleaves a remarkable diversity of substrates through endopeptidase action. Two of these – amyloid-β and natriuretic peptides – implicate the enzyme in both Alzheimer’s disease and cardiovascular disease, respectively. Here, we report the creation of a catalytically inactive NEP (E584D) to determine the first peptide-bound crystal structure at 2.6 Å resolution. The structure reveals key interactions involved in substrate binding which we have identified to be conserved in other known zinc metalloproteases. In addition, the structure provides evidence for a potential exosite within the central cavity that may play a critical role in substrate positioning. Together, these results contribute to our understanding of the molecular function of NEP.</p

Multiplex enzyme activity imaging by MALDI-IMS of substrate library conversions

Enzymes are fundamental to biological processes and involved in most pathologies. Here we demonstrate the concept of simultaneously mapping multiple enzyme activities (EA) by applying enzyme substrate libraries to tissue sections and analyzing their conversion by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). To that end, we spray-applied a solution of 20 naturally derived peptides that are known substrates for proteases, kinases, and phosphatases to zinc-fixed paraffin tissue sections of mouse kidneys. After enzyme conversion for 5 to 120 min at 37 °C and matrix application, the tissue sections were imaged by MALDI-IMS. We could image incubation time-dependently 16 of the applied substrates with differing signal intensities and 12 masses of expected products. Utilizing inherent enzyme amplification, EA-IMS can become a powerful tool to locally study multiple, potentially even lowly expressed, enzyme activities, networks, and their pharmaceutical modulation. Differences in the substrate detectability highlight the need for future optimizations

SARS-CoV-2 Proteases: Role and Potential as Drug Target

The coronavirus disease of 2019 (COVID-19) has become a long global pandemic caused by a transmitted and pathogenic virus called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Even though WHO has retracted the global emergency status of COVID-19, it remains a threat. Various antiviral treatments are being devised and developed due to the coronavirus's high rate of mutation and need to create more effective treatments for infections. Protease is an important part of the life cycle of SARS CoV-2 hence it is intended as an antiviral target. Several protease inhibitor candidates have been identified, but there is still much to learn, including the structure and mechanism by which these inhibitors inhibit protease. This article investigates the function of proteases in the SARS CoV-2 life cycle and the mechanism of protease inhibition. Past and present research on the protease inhibitor mechanism of action was evaluated in order to generate this literature review. Here we found that the main protease (Mpro), one of SARS-CoV's proteases, is highly conserved among coronaviruses and has no human homolog. As a result, numerous Mpro inhibitors have been developed in an effort to treat COVID-19. PAXLOVID, an Mpro inhibitor, is already approved by FDA for emergency use

Discrimination of Methionine Sulfoxide and Sulfone by Human Neutrophil Elastase

Human neutrophil elastase (HNE) is a uniquely destructive serine protease with the ability to unleash a wave of proteolytic activity by destroying the inhibitors of other proteases. Although this phenomenon forms an important part of the innate immune response to invading pathogens, it is responsible for the collateral host tissue damage observed in chronic conditions such as chronic obstructive pulmonary disease (COPD), and in more acute disorders such as the lung injuries associated with COVID-19 infection. Previously, a combinatorially selected activity-based probe revealed an unexpected substrate preference for oxidised methionine, which suggests a link to oxida-tive pathogen clearance by neutrophils. Here we use oxidised model substrates and inhibitors to confirm this observation and to show that neutrophil elastase is specifically selective for the di-oxygenated methionine sulfone rather than the mono-oxygenated methionine sulfoxide. We also posit a critical role for ordered solvent in the mechanism of HNE discrimination between the two oxidised forms methionine residue. Preference for the sulfone form of oxidised methionine is especially significant. While both host and pathogens have the ability to reduce methionine sulfoxide back to methionine, a biological pathway to reduce methionine sulfone is not known. Taken to-gether, these data suggest that the oxidative activity of neutrophils may create rapidly cleaved elas-tase “super substrates” that directly damage tissue, while initiating a cycle of neutrophil oxidation that increases elastase tissue damage and further neutrophil recruitment

A novel type 1 cystatin involved in the regulation of Rhipicephalus microplus midgut cysteine proteases

Rhipicephalus microplus is a cattle ectoparasite found in tropical and subtropical regions around the world with great impact on livestock production. R. microplus can also harbor pathogens, such as Babesia sp. and Anaplasma sp. which further compromise cattle production. Blood meal acquisition and digestion are key steps for tick development. In ticks, digestion takes place inside midgut cells and is mediated by aspartic and cysteine peptidases and, therefore, regulated by their inhibitors. Cystatins are a family of cysteine peptidases inhibitors found in several organisms and have been associated in ticks with blood acquisition, blood digestion, modulation of host immune response and tick immunity. In this work, we characterized a novel R. microplus type 1 cystatin, named Rmcystatin-1b. The inhibitor transcripts were found to be highly expressed in the midgut of partially and fully engorged females and they appear to be modulated at different days post-detachment. Purified recombinant Rmcystatin-1b displayed inhibitory activity towards typical cysteine peptidases with high affinity. Moreover, rRmcystatin-1b was able to inhibit native R. microplus cysteine peptidases and RNAi-mediated knockdown of the cystatin transcripts resulted in increased proteolytic activity. Moreover, rRmcystatin-1b was able to interfere with B. bovis growth in vitro. Taken together our data strongly suggest that Rmcystatin-1b is a regulator of blood digestion in R. microplus midgut.Fil: Lu, Stephen. Universidade Federal de Sao Paulo; BrasilFil: da Rocha, Leticia A.. Universidade Federal de Sao Paulo; BrasilFil: Torquato, Ricardo J.S.. Universidade Federal de Sao Paulo; BrasilFil: da Silva Vaz Junior, Itabajara. Universidade Federal do Rio Grande do Sul; BrasilFil: Jacobsen, Monica Ofelia. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Patobiologia Veterinaria. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Patobiologia Veterinaria.; ArgentinaFil: Tanaka, Aparecida S.. Universidade Federal de Sao Paulo; Brasi