Carangoides bartholomaei (Cuvier, 1833) stomach: a source of aspartic proteases for industrial and biotechnological applications

The viscera and other residues from fish processing are commonly discarded by the fishing industry. These by-products can be a source of digestive enzymes with industrial and biotechnological potential. In this study, we aimed at the extraction, characterization, and application of acidic proteases from the stomach of Carangoides bartholomaei (Cuvier, 1833). A crude extract from the stomachs was obtained and submitted to a partial purification process by salting-out, which obtained a Purified Extract (PE) with a specific proteolytic activity of 54.0 U⋅mg-1. A purification of 1.9 fold and a yield of 41% were obtained. The PE presents two isoforms of acidic proteases and a maximum proteolytic activity at 45 °C and pH 2.0. The PE acidic proteolytic activity was stable in the pH range of 1.5 to 7.0 and temperature from 25 °C to 50 °C. Purified Extract kept 35% of its proteolytic activity at the presence of NaCl 15% (m/v) but was totally inhibited by pepstatin A. Purified Extract aspartic proteases presented high activity in the presence of heavy metals such as Cd2+, Hg2+, Pb2+, Al3+, and Cu2+. The utilization of PE as an enzymatic addictive in the collagen extraction from Nile tilapia scales has doubled the process yield. The results indicate the potential of these aspartic proteases for industrial and biotechnological applications.

Obtainment and characterization of digestive aspartic proteases from the fish Caranx hippos (Linnaeus, 1766)

This work aimed to obtain aspartic proteases of industrial and biotechnological interest from the stomach of the crevalle jack fish (Caranx hippos). In order to do so, a crude extract (CE) of the stomach was obtained and subjected to a partial purification by salting-out, which resulted in the enzyme extract (EE) obtainment. EE proteases were characterized physicochemically and by means of zymogram. In addition, the effect of chemical agents on their activity was also assessed. By means of salting-out it was possible to obtain a purification of 1.6 times with a yield of 49.4%. Two acid proteases present in the EE were observed in zymogram. The optimum temperature and thermal stability for EE acidic proteases were 55 ºC and 45 °C, respectively. The optimum pH and pH stability found for these enzymes were pH 1.5 and 7.0, respectively. Total inhibition of EE acid proteolytic activity was observed in the presence of pepstatin A. dithiothreitol (DTT) and Ca2+ did not promote a significant effect on enzyme activity. In the presence of heavy metals, such as Al3+, Cd2+ and Hg2+, EE acidic proteases showed more than 70% of their enzymatic activity. The results show that it is possible to obtain, from the stomach of C. hippos, aspartic proteases with high proteolytic activity and characteristics that demonstrate potential for industrial and biotechnological applications.

Effects of Dietary Inclusion of Clostridium autoethanogenum Protein on the Growth Performance and Liver Health of Largemouth Bass (Micropterus salmoides)

Clostridium autoethanogenum protein (CAP) is a novel protein source for aqua-feeds. The present study aimed to investigate the effects of dietary CAP on growth performance, immunity, and liver health status of juvenile largemouth bass (Micropterus salmoides). Four isonitrogenous and isolipid experimental diets were formulated to replace 0% (D1, control), 25% (D2), 50% (D3), and 75% (D4) of fish meal by CAP. Fish (15.05 ± 0.08 g) were randomly fed one of four experimental diets for 8 weeks. The results showed that weight gain (WG), specific growth rate (SGR), feeding rate (FR), viscerosomatic index (VSI), and hepatosomatic index (HSI) of the D4 group were significantly lower than D1, D2, and D3 groups (P < 0.05). With the increase of substitution level, the total antioxidant capacity (T-AOC) of liver tissue was significantly decreased, while the plasma alkaline phosphatase (AKP) activity was significantly increased (P < 0.05). Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were significantly higher in D3 and D4 groups than in D1 and D2 groups (P < 0.05). Replacing 50 or 75% fish meal by CAP significantly induced the transcription level of apoptosis-promoting genes (bcl-2-associated death protein [bad] and bcl-2-assoxicated × protein; bag [bax]), anti-apoptosis-related genes (tumor protein 53 [p53] and b-cell lymphoma-2 [bcl-2]), and the apoptotic Caenorhabditis elegans (C. elegans) death gene-3 like caspases (cysteine-aspartic proteases-3 [caspase-3], cysteine-aspartic proteases-8 [caspase-8], cysteine-aspartic proteases-9 [caspase-9], and cysteine-aspartic proteases-10 [caspase-10]) in liver, while suppressed the gene expression of the inflammatory factors [interleukin-1β (il-1β), interleukin-8 (il-8), and tumor necrosis factor, tnf ] in head kidney. At the same time, dietary inclusion of CAP elevated the protein expression of bcl-2, autophagy microtubule-associated protein light chain 3A/B (LC3A/B-I), and LC3A/B-II by inhibiting the phosphorylation of the mammalian target of rapamycin (mTOR; P < 0.05). Moreover, the apoptosis rate of the D3 and D4 groups was significantly increased (P < 0.05). Taken together, these results indicated that the optimal level of CAP-replacing fish meal should be <50% that has no negative effect on the growth performance and liver health of juvenile largemouth bass. In addition, excessive CAP inclusion may damage liver health by activating autophagy and apoptosis signaling pathways.

Understanding the Impact of Drought in Coffea Genotypes: Transcriptomic Analysis Supports a Common High Resilience to Moderate Water Deficit but a Genotype Dependent Sensitivity to Severe Water Deficit

Water scarcity is the most significant factor limiting coffee production, although some cultivars can still have important drought tolerance. This study analyzed leaf transcriptomes of two coffee cultivars with contrasting physiological responses, Coffea canephora cv. CL153 and Coffea. arabica cv. Icatu, subjected to moderate (MWD) or severe water deficits (SWD). We found that MWD had a low impact compared with SWD, where 10% of all genes in Icatu and 17% in CL153 reacted to drought, being mainly down-regulated upon stress. Drought triggered a genotype-specific response involving the up-regulation of reticuline oxidase genes in CL153 and heat shock proteins in Icatu. Responsiveness to drought also included desiccation protectant genes, but primarily, aspartic proteases, especially in CL153. A total of 83 Transcription Factors were found engaged in response to drought, mainly up-regulated, especially under SWD. Together with the enrollment of 49 phosphatases and 272 protein kinases, results suggest the involvement of ABA-signaling processes in drought acclimation. The integration of these findings with complementing physiological and biochemical studies reveals that both genotypes are more resilient to moderate drought than previously thought and suggests the existence of post-transcriptional mechanisms modulating the response to drought.

Structures of plasmepsin X from P. falciparum reveal a novel inactivation mechanism of the zymogen and molecular basis for binding of inhibitors in mature enzyme

Plasmodium falciparum plasmepsin X (PfPMX), involved in the invasion and egress of this deadliest malarial parasite, is essential for its survival and hence considered as an important drug target. We report the first crystal structure of PfPMX zymogen containing a novel fold of its prosegment. A unique twisted loop from the prosegment and arginine 244 from the mature enzyme are involved in zymogen inactivation; such mechanism, not previously reported, might be common for apicomplexan proteases similar to PfPMX. The maturation of PfPMX zymogen occurs through cleavage of its prosegment at multiple sites. Our data provide thorough insights into the mode of binding of a substrate and a potent inhibitor 49c to PfPMX. We present molecular details of inactivation, maturation, and inhibition of PfPMX that should aid in the development of potent inhibitors against pepsin-like aspartic proteases from apicomplexan parasites.

The Duality of Caspases in Cancer, as Told through the Fly

Caspases, a family of cysteine-aspartic proteases, have an established role as critical components in the activation and initiation of apoptosis. Alongside this a variety of non-apoptotic caspase functions in proliferation, differentiation, cellular plasticity and cell migration have been reported. The activity level and context are important factors in determining caspase function. As a consequence of their critical role in apoptosis and beyond, caspases are uniquely situated to have pathological roles, including in cancer. Altered caspase function is a common trait in a variety of cancers, with apoptotic evasion defined as a “hallmark of cancer”. However, the role that caspases play in cancer is much more complex, acting both to prevent and to promote tumourigenesis. This review focuses on the major findings in Drosophila on the dual role of caspases in tumourigenesis. This has major implications for cancer treatments, including chemotherapy and radiotherapy, with the activation of apoptosis being the end goal. However, such treatments may inadvertently have adverse effects on promoting tumour progression and acerbating the cancer. A comprehensive understanding of the dual role of caspases will aid in the development of successful cancer therapeutic approaches.

Barley Nepenthesin-Like Aspartic Protease HvNEP-1 Degrades Fusarium Phytase, Impairs Toxin Production, and Suppresses the Fungal Growth

Nepenthesins are categorized under the subfamily of the nepenthesin-like plant aspartic proteases (PAPs) that form a distinct group of atypical PAPs. This study describes the effect of nepenthesin 1 (HvNEP-1) protease from barley (Hordeum vulgare L.) on fungal histidine acid phosphatase (HAP) phytase activity. Signal peptide lacking HvNEP-1 was expressed in Pichia pastoris and biochemically characterized. Recombinant HvNEP-1 (rHvNEP-1) strongly inhibited the activity of Aspergillus and Fusarium phytases, which are enzymes that release inorganic phosphorous from phytic acid. Moreover, rHvNEP-1 suppressed in vitro fungal growth and strongly reduced the production of mycotoxin, 15-acetyldeoxynivalenol (15-ADON), from Fusarium graminearum. The quantitative PCR analysis of trichothecene biosynthesis genes (TRI) confirmed that rHvNEP-1 strongly repressed the expression of TRI4, TRI5, TRI6, and TRI12 in F. graminearum. The co-incubation of rHvNEP-1 with recombinant F. graminearum (rFgPHY1) and Fusarium culmorum (FcPHY1) phytases induced substantial degradation of both Fusarium phytases, indicating that HvNEP-1-mediated proteolysis of the fungal phytases contributes to the HvNEP-1-based suppression of Fusarium.

Tobacco BY2 cells expressing recombinant cardosin B as an alternative for production of active milk clotting enzymes

Cynara cardunculus L. or cardoon is a plant that is used as a source of milk clotting enzymes during traditional cheese manufacturing. This clotting activity is due to aspartic proteases (APs) found in the cardoon flower, named cyprosins and cardosins. APs from cardoon flowers display a great degree of heterogeneity, resulting in variable milk clotting activities and directly influencing the final product. Producing these APs using alternative platforms such as bacteria or yeast has proven challenging, which is hampering their implementation on an industrial scale. We have developed tobacco BY2 cell lines as an alternative plant-based platform for the production of cardosin B. These cultures successfully produced active cardosin B and a purification pipeline was developed to obtain isolated cardosin B. The enzyme displayed proteolytic activity towards milk caseins and milk clotting activity under standard cheese manufacturing conditions. We also identified an unprocessed form of cardosin B and further investigated its activation process. The use of protease-specific inhibitors suggested a possible role for a cysteine protease in cardosin B processing. Mass spectrometry analysis identified three cysteine proteases containing a granulin-domain as candidates for cardosin B processing. These findings suggest an interaction between these two groups of proteases and contribute to an understanding of the mechanisms behind the regulation and processing of plant APs. This work also paves the way for the use of tobacco BY2 cells as an alternative production system for active cardosins and represents an important advancement towards the industrial production of cardoon APs.

Absence of the CXCR4 antagonist EPI-X4 from pharmaceutical human serum albumin preparations

Background Endogenous Peptide Inhibitor of CXCR4 (EPI-X4) is a natural antagonist of the CXC chemokine receptor 4 (CXCR4). EPI-X4 is a 16-mer peptide that is released from human serum albumin (HSA) by acidic aspartic proteases such as Cathepsin D and E. Since human serum albumin (HSA) is an important medicinal substance we asked whether different pharmaceutical HSA products contain EPI-X4 which could have been generated during manufacturing and whether HSA can serve as a substrate for cathepsins despite of the presence of stabilizers like caprylate. Methods Eight pharmaceutical HSA preparations representing all currently used fractionation technologies were analyzed. The previously described specific EPI-X4 ELISA was used for quantification; in vitro EPI-X4 generation by acidification in the presence or absence of cathepsins was followed by quantification with ELISA. Results None of the pharmaceutical HSA preparations tested contained EPI-X4. Acidification of HSA did not generate EPI-X4. Addition of cathepsins D and E to acidified HSA yielded high concentrations of EPI-X4 in all HSA preparations, indistinguishable between individual products. Conclusion Medicinal HSA preparations per se do not contain EPI-X4, but will replenish its precursor which can be cleaved to EPI-X4 in vivo, environmental conditions permitting.

Absence of the CXCR4 Antagonist EPI-X4 from Pharmaceutical Human Serum Albumin Preparations

Background: Endogenous Peptide Inhibitor of CXCR4 (EPI-X4) is a natural antagonist of the CXC chemokine receptor 4 (CXCR4). EPI-X4 is a 16-mer peptide that is released from human serum albumin (HSA) by acidic aspartic proteases such as Cathepsin D and E. Since human serum albumin (HSA) is an important medicinal substance we asked whether different pharmaceutical HSA products contain EPI-X4 which could have been generated during manufacturing and whether HSA can serve as a substrate for cathepsins despite of the presence of stabilizers like caprylate. Methods: Eight pharmaceutical HSA preparations representing all currently used fractionation technologies were analyzed. The previously described specific EPI-X4 ELISA was used for quantification; in vitro EPI-X4 generation by acidification in the presence or absence of cathepsins was followed by quantification with ELISA. Results: None of the pharmaceutical HSA preparations tested contained EPI-X4. Acidification of HSA did not generate EPI-X4. Addition of cathepsins D and E to acidified HSA yielded high concentrations of EPI-X4 in all HSA preparations, indistinguishable between individual products. Conclusion: Medicinal HSA preparations per se do not contain EPI-X4, but will replenish its precursor which can be cleaved to EPI-X4 in vivo, environmental conditions permitting.