Complementary Proteomic and Biochemical Analysis of Peptidases in Lobster Gastric Juice Uncovers the Functional Role of Individual Enzymes in Food Digestion

Bibo-Verdugo, Betsaida; O’Donoghue, Anthony J.; Rojo‐Arreola, Liliana; Craik, Charles S.; García‐Carreño, Fernando

doi:10.1007/s10126-015-9681-5

Cited by 12 publications

(13 citation statements)

References 51 publications

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“…Pepstatin is a potent inhibitor of cathepsin E and other pepsin-type aspartyl proteases (Dunn, 2002). We have successfully used pepstatin-agarose beads to enrich for aspartyl proteases from complex protein mixtures (Bibo-Verdugo et al, 2016). Using a similar approach, we added pepstatin-agarose beads to tumor lysate and then diluted the mixture in either pH 7.5 lysis buffer or pH 3.5 activation buffer.…”

Section: Resultsmentioning

confidence: 99%

Procathepsin E is highly abundant but minimally active in pancreatic ductal adenocarcinoma tumors

O’Donoghue

Ivry

Chaudhury

et al. 2016

Biological Chemistry

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The cathepsin family of lysosomal proteases is increasingly being recognized for their altered expression in cancer and role in facilitating tumor progression. The aspartyl protease cathepsin E is overexpressed in several cancers and has been investigated as a biomarker for pancreatic ductal adenocarcinoma (PDAC). Here we show that cathepsin E expression in mouse PDAC tumors is increased by more than 400-fold when compared to healthy pancreatic tissue. Cathepsin E accumulates over the course of disease progression and accounts for more than 3% of the tumor protein in mice with end-stage disease. Through immunoblot analysis we determined that only procathepsin E exists in mouse PDAC tumors and cell lines derived from these tumors. By decreasing the pH, this procathepsion E is converted to the mature form, resulting in an increase in proteolytic activity. Although active site inhibitors can bind procathepsin E, treatment of PDAC mice with the aspartyl protease inhibitor ritonavir did not decrease tumor burden. Lastly, we used multiplex substrate profiling by mass spectrometry to identify two synthetic peptides that are hydrolyzed by procathepsin E near neutral pH. This work represents a comprehensive analysis of procathepsin E in PDAC and could facilitate the development of improved biomarkers for disease detection.

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Section: Resultsmentioning

confidence: 99%

Procathepsin E is highly abundant but minimally active in pancreatic ductal adenocarcinoma tumors

O’Donoghue

Ivry

Chaudhury

et al. 2016

Biological Chemistry

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“…The preference of some peptidases is affected by substrate amino acid residues in other positions, such as lobster cathepsins L and H (Bibo‐Verdugo et al . ).…”

Section: Peptidasesmentioning

confidence: 97%

“…Protein function is assigned to each EST‐derived sequence, following a BLASTp homology search (Bibo‐Verdugo et al . ).…”

Section: Sampling and Analysing Digestive Peptidasesmentioning

confidence: 97%

“…(d) A pie chart shows the number of cleavage sites sensitive to either E‐64, pepstatin A or both (Bibo‐Verdugo et al . ). Used with permission of Marine Biotechnology.…”

Section: Sampling and Analysing Digestive Peptidasesmentioning

confidence: 97%

“…The technique is named zymoMSP (Bibo‐Verdugo et al . ); Fig. ) and gives clues about the preferences and specificities, of the peptidases in a complex mixture, from site P4 to P4′ and foresee the physiological function according to potential modifications to potential substrates.…”

Section: Sampling and Analysing Digestive Peptidasesmentioning

confidence: 99%

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The toolbox for protein digestion in decapod crustaceans: a review

Toro¹,

García‐Carreño²

2018

Reviews in Aquaculture

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Pancrustacea, including both crustaceans and insects, is the most abundant and diverse clade of Animalia and occupy all types of environments. They are studied because of their ecological, agricultural, health and economic issues, some being delicatessens. Crustaceans evolved during the Cambrian, thanks to adaptations, such as macrophagy, that allowed them to prey on diverse and energy-rich foods, which depended on complex digestive systems that drove evolution. As heterotrophs, crustaceans depend on their ability to obtain nutrients to fulfil their material, mostly carbon and energy requirements. Protein is the most important nutrient; up to 70% dry mass of a crustacean is protein. Hence, food protein is needed to obtain free amino acids to synthesize protein de novo (from scratch), because animals cannot synthesize the ten essential amino acids de novo. The ability to digest protein must have emerged early in evolution, as shown by the finding of trypsin coding genes in eubacterial genomes. Primitive organisms selectively fixed the ability to use foreign protein as a source of amino acids by synthesizing enzymes, now termed peptidases, which were capable of recognizing and hydrolysing peptide bonds in a specific region of a polypeptide chain, based on specificity for the side chains of amino acid residues in the carboxylic side of the site of cleavage. Here, we present a brief history on what and how digestive peptidases in crustaceans became known.

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Engineering of multiple trypsin/chymotrypsin sites in Cry3A to enhance its activity against Monochamus alternatus Hope larvae

Guo

Wang

O’Donoghue

et al. 2020

Pest Management Science

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BACKGROUNDBacillus thuringiensis Cry3 toxins exhibit specific toxicity against several coleopteran larvae. However, owing to its low toxicity to Monochamus alternatus, Cry3A toxin is not useful for managing M. alternatus larvae. Here we assessed the proteolytic activation of Cry3Aa toxin in M. alternatus larval midgut and increased its toxicity by molecular modification.RESULTSOur results indicated that insufficient processing of Cry3Aa protoxin and non‐specific enzymatic digestion of Cry3Aa toxin in the midgut of M. alternatus larvae led to low toxicity. The results of transcriptome analysis, enzymatic assay with fluorogenic substrates, and multiplex substrate profiling by mass spectrometry showed that the main digestive enzymes in M. alternatus larval midgut were trypsin‐like proteases that preferentially cleaved peptides with arginine and lysine residues. Consequently, trypsin recognition sites were introduced into the Domain I of Cry3Aa protoxin in the loop regions between α‐helix 3 and α‐helix 4 to facilitate proteolytic activation. Multiple potential trypsin cleavage sites away from the helix sheet and functional regions in Cry3Aa proteins were also mutated to alanine to prevent non‐specific enzymatic digestion. Bioassays indicated that a modified Cry3Aa‐T toxin (K65A, K70A, K231A, K468A, and K596A) showed a 9.5‐fold (LC50 = 12.3 μg/mL) increase in toxicity to M. alternatus larvae when compared to native Cry3Aa toxin.CONCLUSIONThis study highlights an effective way to increase the toxicity of Cry3Aa toxin to M. alternatus, which may be suitable for managing the resistance of transgenic plants to other pests, including some of the most important pests in agriculture. © 2020 Society of Chemical Industry

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Complementary Proteomic and Biochemical Analysis of Peptidases in Lobster Gastric Juice Uncovers the Functional Role of Individual Enzymes in Food Digestion

Cited by 12 publications

References 51 publications

Procathepsin E is highly abundant but minimally active in pancreatic ductal adenocarcinoma tumors

Procathepsin E is highly abundant but minimally active in pancreatic ductal adenocarcinoma tumors

The toolbox for protein digestion in decapod crustaceans: a review

Engineering of multiple trypsin/chymotrypsin sites in Cry3A to enhance its activity against Monochamus alternatus Hope larvae

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