DNA double-strand break repair machinery in Penaeid crustaceans: A focus on the Non-Homologous End-Joining pathway.

DNA double-strand breaks (DSBs) are repaired through three major pathways: Non-Homologous End-Joining (NHEJ), Microhomology-Mediated End-Joining (MMEJ), and Homology-Directed Repair (HDR), each requiring a specific set of diverse proteins. Such pathways and their proteins have been studied in model organisms, including arthropods; however, DSB repair pathways are scarcely described in Crustacea, a taxon that includes the commercially valuable penaeid shrimps (Crustacea: Decapoda: Penaeidae). In this work, transcriptome and proteome databases of Penaeus vannamei and other Crustacea species were scrutinized for each protein of the NHEJ pathway. The structural and functional attributes of such proteins in penaeids were determined using bioinformatics. Additionally, the expression of the NHEJ-related Ku70, Ku80, DNA-PKcs, DNA ligase 4 (Lig4), and HDR- and MMEJ-related protein transcripts were assessed in P. vannamei gills, midgut gland, hemocytes, and muscle by RT-PCR. DSB repair protein transcripts were found expressed in the four assayed tissues, particularly in the gills and midgut gland. Among DSB repair proteins, all the analyzed transcripts of proteins related to the NHEJ pathway were present in gills. To the best of our knowledge, this is the first report on the expression of DSB repair proteins in Decapoda. Together, proteomic, transcriptomic, and expression data suggest the functionality of NHEJ, HDR, and MMEJ pathways in P. vannamei and other decapods. The information presented here contributes to understanding the response to DSB breaks in shrimps, describing possible outcomes in oxidative stress studies and also in the designing of gene editing strategies, which have not been developed in Penaeidae.

Proteolytic profile of larval developmental stages of Penaeus vannamei: An activity and mRNA expression approach.

In arthropods, the cleavage of specific proteins by peptidases has pivotal roles in multiple physiological processes including oogenesis, immunity, nutrition, and parasitic infection. These enzymes are also key players in the larval development, and well-described triggers of molting and metamorphosis. In this work the peptidase complement throughout the larvae development of Penaeus vannamei was quantified at the transcript and activity level using qPCR and fluorogenic substrates designed to be hydrolyzed by class-specific peptidases respectively, providing a detailed identification of the proteolytic repertoire in P. vannamei larvae. Significant changes in the peptidase activity profile were observed. During the lecithotrophic naupliar instars, the dominant peptidase activity and expression derive from cysteine peptidases, suggesting that enzymes of this class hydrolyze the protein components of yolk as the primary amino acid source. At the first feeding instar, zoea, dominant serine peptidase activity was found where trypsin activity is particularly high, supporting previous observations that during zoea the breakdown of food protein is primarily enzymatic. At decapodid stages the peptidase expression and activity is more diverse indicating that a multienzyme network achieves food digestion. Our results suggest that proteolytic enzymes fulfill specific functions during P. vannamei larval development.

Litopenaeus vannamei oxygen consumption and HSP gene expression at cyclic conditions of hyperthermia and hypoxia.

Although Litopenaeus vannamei is a widely studied species, the information on how the organisms respond to natural daily variations of environmental conditions such as temperature and dissolved oxygen, and how such conditions alter the physiological responses, is scarce. In the present work, the strategies used by shrimps to cope with temperature and dissolved oxygen fluctuations during 24 days were investigated through the evaluation of oxygen consumption and heat shock proteins (HSP) gene expression. During daily fluctuations, no change in oxygen consumption in the short-term, but a significant increase in the long-term during hyperthermia conditions was registered, whereas a significant decrease during hypoxia was observed during all the bioassay. On the other hand, HSP70 and HSP90 gene expression increased in gills under thermal stress but was down-regulated under hypoxia, in both the short- and the long-term. This study highlights that to counteract environmental variations of temperature and dissolved oxygen, the shrimps use molecular compensatory mechanisms (HSP gene expression) that are different to those used under constant hypoxic conditions, suggesting that hypoxia can compromise physiological cytoprotection.

Eco-Evolutionary Processes Generating Diversity Among Bottlenose Dolphin, Tursiops truncatus, Populations off Baja California, Mexico.

For highly mobile species that nevertheless show fine-scale patterns of population genetic structure, the relevant evolutionary mechanisms determining structure remain poorly understood. The bottlenose dolphin (Tursiops truncatus) is one such species, exhibiting complex patterns of genetic structure associated with local habitat dependence in various geographic regions. Here we studied bottlenose dolphin populations in the Gulf of California and Pacific Ocean off Baja California where habitat is highly structured to test associations between ecology, habitat dependence and genetic differentiation. We investigated population structure at a fine geographic scale using both stable isotope analysis (to assess feeding ecology) and molecular genetic markers (to assess population structure). Our results show that there are at least two factors affecting population structure for both genetics and feeding ecology (as indicated by stable isotope profiles). On the one hand there is a signal for the differentiation of individuals by ecotype, one foraging more offshore than the other. At the same time, there is differentiation between the Gulf of California and the west coast of Baja California, meaning that for example, nearshore ecotypes were both genetically and isotopically differentiated either side of the peninsula. We discuss these data in the context of similar studies showing fine-scale population structure for delphinid species in coastal waters, and consider possible evolutionary mechanisms.

American lobster Cathepsin D, an aspartic peptidase resistant to proteolysis and active in organic solvents, non-ionic detergents and salts.

Suitable peptidases for biotechnological applications are those active at low temperature, in organic solvents, detergents or proteolytic additives. American lobster cathepsin D1 (CD1) is an enzyme highly efficient at 5-50°C and at pH 2.5-5.5. We assessed the effect of common industrial additives on CD1 activity. CD1 was isolated from lobster gastric fluid by chromatography. The proteolytic activity was measured using a fluorogenic specific substrate and the conformation by intrinsic fluorescence. Non-ionic detergents Tween-20 and Triton X-100 stabilize the peptidase activity. Ethanol, methanol and isopropanol [5-15% (v/v)] increased the enzyme activity up to 80%. The enzyme is active until 2.5M urea and is resistant to proteolysis by papain and renin. In this work, a crustacean peptidase that remains active when exposed to different chemical and proteolytic additives is reported, evincing that crustaceans are a good model for discovery of novel stable peptidases for future pharmaceutical, cosmetic and alimentary applications.

Is digestive cathepsin D the rule in decapod crustaceans?

Cathepsin D is an aspartic endopetidase with typical characteristics of lysosomal enzymes. Cathepsin D activity has been reported in the gastric fluid of clawed lobsters where it acts as an extracellular digestive enzyme. Here we investigate whether cathepsin D is unique in clawed lobsters or, instead, common in decapod crustaceans. Eleven species of decapods belonging to six infraorders were tested for cathepsin D activity in the midgut gland, the muscle tissue, the gills, and when technically possible, in the gastric fluid. Cathepsin D activity was present in the midgut gland of all 11 species and in the gastric fluid from the seven species from which samples could be taken. All sampled species showed higher activities in the midgut glands than in non-digestive organs and the activity was highest in the clawed lobster. Cathepsin D mRNA was obtained from tissue samples of midgut gland, muscle, and gills. Analyses of deduced amino acid sequence confirmed molecular features of lysosomal cathepsin D and revealed high similarity between the enzymes from Astacidea and Caridea on one side, and the enzymes from Penaeoidea, Anomura, and Brachyura on the other side. Our results support the presence of cathepsin D activity in the midgut glands and in the gastric fluids of several decapod species suggesting an extracellular function of this lysosomal enzyme. We discuss whether cathepsin D may derive from the lysosomal-like vacuoles of the midgut gland B-cells and is released into the gastric lumen upon secretion by these cells.

Phenotypic, chemical and functional characterization of cyclic nucleotide phosphodiesterase 4 (PDE4) as a potential anthelmintic drug target.

BACKGROUND: Reliance on just one drug to treat the prevalent tropical disease, schistosomiasis, spurs the search for new drugs and drug targets. Inhibitors of human cyclic nucleotide phosphodiesterases (huPDEs), including PDE4, are under development as novel drugs to treat a range of chronic indications including asthma, chronic obstructive pulmonary disease and Alzheimer's disease. One class of huPDE4 inhibitors that has yielded marketed drugs is the benzoxaboroles (Anacor Pharmaceuticals). METHODOLOGY/PRINCIPAL FINDINGS: A phenotypic screen involving Schistosoma mansoni and 1,085 benzoxaboroles identified a subset of huPDE4 inhibitors that induced parasite hypermotility and degeneration. To uncover the putative schistosome PDE4 target, we characterized four PDE4 sequences (SmPDE4A-D) in the parasite's genome and transcriptome, and cloned and recombinantly expressed the catalytic domain of SmPDE4A. Among a set of benzoxaboroles and catechol inhibitors that differentially inhibit huPDE4, a relationship between the inhibition of SmPDE4A, and parasite hypermotility and degeneration, was measured. To validate SmPDE4A as the benzoxaborole molecular target, we first generated Caenorhabditis elegans lines that express a cDNA for smpde4a on a pde4(ce268) mutant (hypermotile) background: the smpde4a transgene restored mutant worm motility to that of the wild type. We then showed that benzoxaborole inhibitors of SmPDE4A that induce hypermotility in the schistosome also elicit a hypermotile response in the C. elegans lines that express the smpde4a transgene, thereby confirming SmPDE4A as the relevant target. CONCLUSIONS/SIGNIFICANCE: The orthogonal chemical, biological and genetic strategies employed identify SmPDE4A's contribution to parasite motility and degeneration, and its potential as a drug target. Transgenic C. elegans is highlighted as a potential screening tool to optimize small molecule chemistries to flatworm molecular drug targets.

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

Crustaceans are a diverse group, distributed in widely variable environmental conditions for which they show an equally extensive range of biochemical adaptations. Some digestive enzymes have been studied by purification/characterization approaches. However, global analysis is crucial to understand how digestive enzymes interplay. Here, we present the first proteomic analysis of the digestive fluid from a crustacean (Homarus americanus) and identify glycosidases and peptidases as the most abundant classes of hydrolytic enzymes. The digestion pathway of complex carbohydrates was predicted by comparing the lobster enzymes to similar enzymes from other crustaceans. A novel and unbiased substrate profiling approach was used to uncover the global proteolytic specificity of gastric juice and determine the contribution of cysteine and aspartic acid peptidases. These enzymes were separated by gel electrophoresis and their individual substrate specificities uncovered from the resulting gel bands. This new technique is called zymoMSP. Each cysteine peptidase cleaves a set of unique peptide bonds and the S2 pocket determines their substrate specificity. Finally, affinity chromatography was used to enrich for a digestive cathepsin D1 to compare its substrate specificity and cold-adapted enzymatic properties to mammalian enzymes. We conclude that the H. americanus digestive peptidases may have useful therapeutic applications, due to their cold-adaptation properties and ability to hydrolyze collagen.

Excretion/secretion products from Schistosoma mansoni adults, eggs and schistosomula have unique peptidase specificity profiles.

Schistosomiasis is one of a number of chronic helminth diseases of poverty that severely impact personal and societal well-being and productivity. Peptidases (proteases) are vital to successful parasitism, and can modulate host physiology and immunology. Interference of peptidase action by specific drugs or vaccines can be therapeutically beneficial. To date, research on peptidases in the schistosome parasite has focused on either the functional characterization of individual peptidases or their annotation as part of global genome or transcriptome studies. We were interested in functionally characterizing the complexity of peptidase activity operating at the host-parasite interface, therefore the excretory-secretory products of key developmental stages of Schistosoma mansoni that parasitize the human were examined. Using class specific peptidase inhibitors in combination with a multiplex substrate profiling assay, a number of unique activities derived from endo- and exo-peptidases were revealed in the excretory-secretory products of schistosomula (larval migratory worms), adults and eggs. The data highlight the complexity of the functional degradome for each developmental stage of this parasite and facilitate further enquiry to establish peptidase identity, physiological and immunological function, and utility as drug or vaccine candidates.

A chymotrypsin from the Digestive Tract of California Spiny Lobster, Panulirus interruptus: Purification and Biochemical Characterization.

A chymotrypsin was purified from the gastric juice of California spiny lobster (Panulirus interrutpus), using preparative electrophoresis and affinity chromatography on agarose-p-aminobenzamidine. The molecular mass was estimated by polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions to be 28 kDa. Chymotrypsin activity was totally inhibited by phenylmethylsulfonyl fluoride (PMSF) and chymostatin. Lobster chymotrypsin had optimal pH 7.0-8.0 and temperature of 55 °C. The enzyme is highly stable under a wide range of pH (retaining up to 80 % of activity after 1 h of incubation at pH 3.0, 5.0, and 12.0), showing higher stability at pH 8.0, and was inactivated after 20 min at 55 °C. Lobster chymotrypsin was able to hydrolyze protein substrates at as low as pH 3.0. These results are consistent with the findings of enzyme stability. Activity was assessed after incubation of enzyme with different organic solvents (in the range of 10-50 %); when tested in the presence of acetone, ethanol, propanol, and butanol, lobster chymotrypsin residual activity was >80 %; whereas in the presence of dimethyl sulfoxide (DMSO) and toluene, lobster chymotrypsin residual activity was <80 %. Deduced amino acid sequence, corroborated by mass spectrometry, was determined.

The QDREC web server: determining dose-response characteristics of complex macroparasites in phenotypic drug screens.

SUMMARY: Neglected tropical diseases (NTDs) caused by helminths constitute some of the most common infections of the world's poorest people. The etiological agents are complex and recalcitrant to standard techniques of molecular biology. Drug screening against helminths has often been phenotypic and typically involves manual description of drug effect and efficacy. A key challenge is to develop automated, quantitative approaches to drug screening against helminth diseases. The quantal dose-response calculator (QDREC) constitutes a significant step in this direction. It can be used to automatically determine quantitative dose-response characteristics and half-maximal effective concentration (EC50) values using image-based readouts from phenotypic screens, thereby allowing rigorous comparisons of the efficacies of drug compounds. QDREC has been developed and validated in the context of drug screening for schistosomiasis, one of the most important NTDs. However, it is equally applicable to general phenotypic screening involving helminths and other complex parasites. AVAILABILITY AND IMPLEMENTATION: QDREC is publically available at: http://haddock4.sfsu.edu/qdrec2/. Source code and datasets are at: http://tintin.sfsu.edu/projects/phenotypicAssays.html. CONTACT: rahul@sfsu.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Trypsin- and Chymotrypsin-like serine proteases in schistosoma mansoni-- 'the undiscovered country'.

BACKGROUND: Blood flukes (Schistosoma spp.) are parasites that can survive for years or decades in the vasculature of permissive mammalian hosts, including humans. Proteolytic enzymes (proteases) are crucial for successful parasitism, including aspects of invasion, maturation and reproduction. Most attention has focused on the 'cercarial elastase' serine proteases that facilitate skin invasion by infective schistosome larvae, and the cysteine and aspartic proteases that worms use to digest the blood meal. Apart from the cercarial elastases, information regarding other S. mansoni serine proteases (SmSPs) is limited. To address this, we investigated SmSPs using genomic, transcriptomic, phylogenetic and functional proteomic approaches. METHODOLOGY/PRINCIPAL FINDINGS: Genes encoding five distinct SmSPs, termed SmSP1 - SmSP5, some of which comprise disparate protein domains, were retrieved from the S. mansoni genome database and annotated. Reverse transcription quantitative PCR (RT- qPCR) in various schistosome developmental stages indicated complex expression patterns for SmSPs, including their constituent protein domains. SmSP2 stood apart as being massively expressed in schistosomula and adult stages. Phylogenetic analysis segregated SmSPs into diverse clusters of family S1 proteases. SmSP1 to SmSP4 are trypsin-like proteases, whereas SmSP5 is chymotrypsin-like. In agreement, trypsin-like activities were shown to predominate in eggs, schistosomula and adults using peptidyl fluorogenic substrates. SmSP5 is particularly novel in the phylogenetics of family S1 schistosome proteases, as it is part of a cluster of sequences that fill a gap between the highly divergent cercarial elastases and other family S1 proteases. CONCLUSIONS/SIGNIFICANCE: Our series of post-genomics analyses clarifies the complexity of schistosome family S1 serine proteases and highlights their interrelationships, including the cercarial elastases and, not least, the identification of a 'missing-link' protease cluster, represented by SmSP5. A framework is now in place to guide the characterization of individual proteases, their stage-specific expression and their contributions to parasitism, in particular, their possible modulation of host physiology.

Chemical and genetic validation of the statin drug target to treat the helminth disease, schistosomiasis.

The mevalonate pathway is essential in eukaryotes and responsible for a diversity of fundamental synthetic activities. 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is the rate-limiting enzyme in the pathway and is targeted by the ubiquitous statin drugs to treat hypercholesterolemia. Independent reports have indicated the cidal effects of statins against the flatworm parasite, S. mansoni, and the possibility that SmHMGR is a useful drug target to develop new statin-based anti-schistosome therapies. For six commercially available statins, we demonstrate concentration- and time-dependent killing of immature (somule) and adult S. mansoni in vitro at sub-micromolar and micromolar concentrations, respectively. Cidal activity trends with statin lipophilicity whereby simvastatin and pravastatin are the most and least active, respectively. Worm death is preventable by excess mevalonate, the product of HMGR. Statin activity against somules was quantified both manually and automatically using a new, machine learning-based automated algorithm with congruent results. In addition, to chemical targeting, RNA interference (RNAi) of HMGR also kills somules in vitro and, again, lethality is blocked by excess mevalonate. Further, RNAi of HMGR of somules in vitro subsequently limits parasite survival in a mouse model of infection by up to 80%. Parasite death, either via statins or specific RNAi of HMGR, is associated with activation of apoptotic caspase activity. Together, our genetic and chemical data confirm that S. mansoni HMGR is an essential gene and the relevant target of statin drugs. We discuss our findings in context of a potential drug development program and the desired product profile for a new schistosomiasis drug.