The Role of DmCatD, a Cathepsin D-Like Peptidase, and Acid Phosphatase in the Process of Follicular Atresia in Dipetalogaster maxima (Hemiptera: Reduviidae), a Vector of Chagas' Disease

In this work, we have investigated the involvement of DmCatD, a cathepsin D-like peptidase, and acid phosphatase in the process of follicular atresia of Dipetalogaster maxima, a hematophagous insect vector of Chagas' disease. For the studies, fat bodies, ovaries and hemolymph were sampled from anautogenous females at representative days of the reproductive cycle: pre-vitellogenesis, vitellogenesis as well as early and late atresia. Real time PCR (qPCR) and western blot assays showed that DmCatD was expressed in fat bodies and ovaries at all reproductive stages, being the expression of its active form significantly higher at the atretic stages. In hemolymph samples, only the immunoreactive band compatible with pro-DmCatD was observed by western blot. Acid phosphatase activity in ovarian tissues significantly increased during follicular atresia in comparison to pre-vitellogenesis and vitellogenesis. A further enzyme characterization with inhibitors showed that the high levels of acid phosphatase activity in atretic ovaries corresponded mainly to a tyrosine phosphatase. Immunofluorescence assays demonstrated that DmCatD and tyrosine phosphatase were associated with yolk bodies in vitellogenic follicles, while in atretic stages they displayed a different cellular distribution. DmCatD and tyrosine phosphatase partially co-localized with vitellin. Moreover, their interaction was supported by FRET analysis. In vitro assays using homogenates of atretic ovaries as the enzyme source and enzyme inhibitors demonstrated that DmCatD, together with a tyrosine phosphatase, were necessary to promote the degradation of vitellin. Taken together, the results strongly suggested that both acid hydrolases play a central role in early vitellin proteolysis during the process of follicular atresia.Fil: Leyria, Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Fruttero, Leonardo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Nazar, Magalí. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Canavoso, Lilian Etelvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentin

Proteomic analysis of the excretory-secretory products from larval stages of Ascaris suum reveals high abundance of glycosyl hydrolases

Background: Ascaris lumbricoides and Ascaris suum are socioeconomically important and widespread parasites of humans and pigs, respectively. The excretory-secretory (ES) molecules produced and presented at the parasite-host interface during the different phases of tissue invasion and migration are likely to play critical roles in the induction and development of protective immune and other host responses. Methodology/Principal Findings: The aim of this study was to identify the ES proteins of the different larval stages (L3-egg, L3-lung and L4) by LC-MS/MS. In total, 106 different proteins were identified, 20 in L3-egg, 45 in L3-lung stage and 58 in L4. Although most of the proteins identified were stage-specific, 15 were identified in the ES products of at least two stages. Two proteins, i.e. a 14-3-3-like protein and a serpin-like protein, were present in the ES products from the three different larval stages investigated. Interestingly, a comparison of ES products from L4 with those of L3-egg and L3-lung showed an abundance of metabolic enzymes, particularly glycosyl hydrolases. Further study indicated that most of these glycolytic enzymes were transcriptionally upregulated from L4 onwards, with a peak in the adult stage, particularly in intestinal tissue. This was also confirmed by enzymatic assays, showing the highest glycosidase activity in protein extracts from adult worms gut. Conclusions/Significance: The present proteomic analysis provides important information on the host-parasite interaction and the molecular of migratory stages of A. suum. In particularly, the high transcriptionally upregulated of glycosyl hydrolases from L4 onwards reveals indicate that the degradation of complex carbohydrates forms an essential part of the energy metabolism of this parasite once it establishes in the small intestine

Characterization of key triacylglycerol biosynthesis processes in rhodococci.

Oleaginous microorganisms have considerable potential for biofuel and commodity chemical production. Under nitrogen-limitation, Rhodococcus jostii RHA1 grown on benzoate, an analog of lignin depolymerization products, accumulated triacylglycerols (TAGs) to 55% of its dry weight during transition to stationary phase, with the predominant fatty acids being C16:0 and C17:0. Transcriptomic analyses of RHA1 grown under conditions of N-limitation and N-excess revealed 1,826 dysregulated genes. Genes whose transcripts were more abundant under N-limitation included those involved in ammonium assimilation, benzoate catabolism, fatty acid biosynthesis and the methylmalonyl-CoA pathway. Of the 16 atf genes potentially encoding diacylglycerol O-acyltransferases, atf8 transcripts were the most abundant during N-limitation (~50-fold more abundant than during N-excess). Consistent with Atf8 being a physiological determinant of TAG accumulation, a Δatf8 mutant accumulated 70% less TAG than wild-type RHA1 while atf8 overexpression increased TAG accumulation 20%. Genes encoding type-2 phosphatidic acid phosphatases were not significantly expressed. By contrast, three genes potentially encoding phosphatases of the haloacid dehalogenase superfamily and that cluster with, or are fused with other Kennedy pathway genes were dysregulated. Overall, these findings advance our understanding of TAG metabolism in mycolic acid-containing bacteria and provide a framework to engineer strains for increased TAG production

Proteomic analysis of Bifidobacterium longum subsp. infantis reveals the metabolic insight on consumption of prebiotics and host glycans.

Bifidobacterium longum subsp. infantis is a common member of the intestinal microbiota in breast-fed infants and capable of metabolizing human milk oligosaccharides (HMO). To investigate the bacterial response to different prebiotics, we analyzed both cell wall associated and whole cell proteins in B. infantis. Proteins were identified by LC-MS/MS followed by comparative proteomics to deduce the protein localization within the cell. Enzymes involved in the metabolism of lactose, glucose, galactooligosaccharides, fructooligosaccharides and HMO were constitutively expressed exhibiting less than two-fold change regardless of the sugar used. In contrast, enzymes in N-Acetylglucosamine and sucrose catabolism were induced by HMO and fructans, respectively. Galactose-metabolizing enzymes phosphoglucomutase, UDP-glucose 4-epimerase and UTP glucose-1-P uridylytransferase were expressed constitutively, while galactokinase and galactose-1-phosphate uridylyltransferase, increased their expression three fold when HMO and lactose were used as substrates for cell growth. Cell wall-associated proteomics also revealed ATP-dependent sugar transport systems associated with consumption of different prebiotics. In addition, the expression of 16 glycosyl hydrolases revealed the complete metabolic route for each substrate. Mucin, which possesses O-glycans that are structurally similar to HMO did not induced the expression of transport proteins, hydrolysis or sugar metabolic pathway indicating B. infantis do not utilize these glycoconjugates

Properties of epoxide hydrolase from the yeast Rhodotorula glutinis

Epoxide hydrolases are ubiquitous enzymes that can be found in nearly all living organisms. Some of the enzymes play an important role in detoxifying xenobiotic and metabolic compounds. Others are important in the growth of organisms like the juvenile hormone in some insects. The role of these enzymes in some organisms is still not fully understood.Epoxides are highly reactive valuable intermediates used by the pharmaceutical industry. Enantiopure epoxides are of high value in the production of pharmaceuticals like pain-killers or protease-inhibitors. There are a number of ways to produce enantiopure epoxides, but nowadays an environmentally friendly manner has a high preference. One such environmentally friendly method is the use of the epoxide hydrolases. These enzymes are able to enantioselectively hydrolyze one epoxide-enantiomer to its vicinal diol. By this so-called kinetic resolution, it is possible to obtain both the epoxide and diol enantiopure. Enantiopure diols are also of high value in the fine and pharmaceutical chemistry.The first goal of this project is achieved: the epoxide hydrolase from Rhodotorula glutinis has been isolated and purified. The second goal (optimization of the reaction conditions) has been performed but it is still favorable to further optimize them. Initial experiments of enzyme stability towards temperature and pH has been performed with crude enzyme extracts, not with the purified enzyme. With respect to the third goal (a suitable method for the isolation and separation of epoxide and diol), the use of a recently described membrane reactor is recommended. The performance of this reactor, however, has not been verified for the EH studied.The enzyme is partially characterized. The EH was found to be a membrane associated enzyme. Whether or not it is actually a membrane bound enzyme (and how many times it passes the membrane) is still unknown. The enzyme consists of two (most probably) identical subunits with a molecular mass of 45 kDa. Amino acid analysis revealed that the enzyme belongs to thea/b-hydrolase fold family, because the characteristic catalytic histidine motive (G H F) can be found in the amino acid sequence. The N-terminal sequence, however, could not be detected. The amount of purified enzyme was too low to establish its the three-dimensional structure.With partially purified enzyme sample, the specific activity and enantioselectivity could be enhanced when detergents were added. Non-ionic detergents had the largest positive effects, e.g. the specific activity for 1,2-epoxyhexane and styrene oxide was enhanced three and eight times, respectively. In the same way, the enantioselectivity for 1,2-epoxyhexane and styrene oxide could be enhanced over 10 and nearly 5 times, respectively. In addition, non-ionic detergents had an enzyme stabilizing effect. Anionic detergents had a very clear negative effect: enzyme activities were reduced to 20%.Another method investigated to influence the stability, the activity and the enantioselectivity consists of polymerizing the epoxide hydrolase in a network. The enantioselective conversion of (±)-1,2-epoxyoctane was reversed from a preference for ( R )-1,2-epoxyoctane to ( S )-1,2-epoxyoctane when the enzyme had been imprinted with ( S )-1,2-epoxyoctane prior to co-polymerization. This is the first time that the above mentioned method was successfully performed with a membrane-associated enzyme of thea/b-hydrolase fold family to which EH belongs. The half-life of the immobilized and imprinted biocatalyst was enhanced at least 7-fold. Most remarkable was that washing the immobilized EH with HCl, followed by washing it with buffer, resulted in about 50% of the residual activity, while native EH completely lost its activityThe effect of increasing epoxide amounts (up to 10 mmol per 10 mL of water, leading to phase separations) on both the activity and enantioselectivity has been studied, including the effect of detergents on such two-phase enzymatic conversions. It appeared that cell-free extracts without detergents gave the highest activity at 10 mmol epoxide per 10 mL of water added, without loss of enantioselectivity as compared to 1 mmol epoxide per 10 mL of water emulsions.It is recommended either to use whole cells (overexpressing EH or not) in a bioreactor to produce enantiopure epoxides and diols in large quantities or to use an immobilized-imprinted enzyme polymer for the conversion of smaller quantities of epoxides in an enantioselectivity of your choice. Further investigations to improve both methods (whole cells, the enzyme properties and the immobilization and imprinting procedures) are required to optimize this type of conversions for practical applications

Cloning, preparation and preliminary crystallographic studies of penicillin V acylase autoproteolytic processing mutants

The crystallization of three catalytically inactive mutants of penicillin Vacylase (PVA) from Bacillus sphaericus in precursor and processed forms is reported. The mutant proteins crystallize in different primitive monoclinic space groups that are distinct from the crystal forms for the native enzyme. Directed mutants and clone constructs were designed to study the post-translational autoproteolytic processing of PVA. The catalytically inactive mutants will provide threedimensional structures of precursor PVA forms, plus open a route to the study of enzyme-substrate complexes for this industrially important enzyme

The Toxoplasma gondii active serine hydrolase 4 regulates parasite division and intravacuolar parasite architecture

ABSTRACT Hydrolase are enzymes that regulate diverse biological processes, including posttranslational protein modifications. Recent work identified four active serine hydrolases (ASHs) in Toxoplasma gondii as candidate depalmitoylases. However, only TgPPT1 (ASH1) has been confirmed to remove palmitate from proteins. ASH4 (TgME49_264290) was reported to be refractory to genetic disruption. We demonstrate that recombinant ASH4 is an esterase that processes short acyl esters but not palmitoyl thioesters. Genetic disruption of ASH4 causes defects in cell division and premature scission of parasites from residual bodies. These defects lead to the presence of vacuoles with a disordered intravacuolar architecture, with parasites arranged in pairs around multiple residual bodies. Importantly, we found that the deletion of ASH4 correlates with a defect in radial dispersion from host cells after egress. This defect in dispersion of parasites is a general phenomenon that is observed for disordered vacuoles that occur at low frequency in wild-type parasites, suggesting a possible general link between intravacuolar organization and dispersion after egress. IMPORTANCE This work defines the function of an enzyme in the obligate intracellular parasite Toxoplasma gondii. We show that this previously uncharacterized enzyme is critical for aspects of cellular division by the parasite and that loss of this enzyme leads to parasites with cell division defects and which also are disorganized inside their vacuoles. This leads to defects in the ability of the parasite to disseminate from the site of an infection and may have a significant impact on the parasite's overall infectivity of a host organism