Recombinant multiepitope proteins expressed in Escherichia coli cells and their potential for immunodiagnosis.

Recombinant multiepitope proteins (RMPs) are a promising alternative for application in diagnostic tests and, given their wide application in the most diverse diseases, this review article aims to survey the use of these antigens for diagnosis, as well as discuss the main points surrounding these antigens. RMPs usually consisting of linear, immunodominant, and phylogenetically conserved epitopes, has been applied in the experimental diagnosis of various human and animal diseases, such as leishmaniasis, brucellosis, cysticercosis, Chagas disease, hepatitis, leptospirosis, leprosy, filariasis, schistosomiasis, dengue, and COVID-19. The synthetic genes for these epitopes are joined to code a single RMP, either with spacers or fused, with different biochemical properties. The epitopes' high density within the RMPs contributes to a high degree of sensitivity and specificity. The RMPs can also sidestep the need for multiple peptide synthesis or multiple recombinant proteins, reducing costs and enhancing the standardization conditions for immunoassays. Methods such as bioinformatics and circular dichroism have been widely applied in the development of new RMPs, helping to guide their construction and better understand their structure. Several RMPs have been expressed, mainly using the Escherichia coli expression system, highlighting the importance of these cells in the biotechnological field. In fact, technological advances in this area, offering a wide range of different strains to be used, make these cells the most widely used expression platform. RMPs have been experimentally used to diagnose a broad range of illnesses in the laboratory, suggesting they could also be useful for accurate diagnoses commercially. On this point, the RMP method offers a tempting substitute for the production of promising antigens used to assemble commercial diagnostic kits.

New Approaches to the Prevention of Visceral Leishmaniasis: A Review of Recent Patents of Potential Candidates for a Chimeric Protein Vaccine.

The development of prophylactic vaccines is important in preventing and controlling diseases such as visceral leishmaniasis (VL), in addition to being an economic measure for public health. Despite the efforts to develop a vaccine against human VL caused by Leishmania infantum, none is available, and the focus has shifted to developing vaccines against canine visceral leishmaniasis (CVL). Currently, commercially available vaccines are targeted at CVL but are not effective. Different strategies have been applied in developing and improving vaccines, such as using chimeric proteins to expand vaccine coverage. The search for patents can be a way of tracking vaccines that have the potential to be marketed. In this context, the present work presents a summary of immunological aspects relevant to VL vaccine development with a focus on the composition of chimeric protein vaccines for CVL deposited in patent banks as an important approach for biotechnological development. The resulting data could facilitate the screening and selection of antigens to compose vaccine candidates with high performance against VL.

Glycosylation and charge distribution orchestrates the conformational ensembles of a biotechnologically promissory phytase in different pHs - a computational study.

Phytases [myo-inositol(1,2,3,4,5,6) hexakisphosphate phosphohydrolases] are phytate-specific phosphatases not present in monogastric animals. Nevertheless, they are an essential supplement to feeding such animals and for human special diets. It is crucial, hence, the biotechnological use of phytases with intrinsic stability and activity at the acid pHs from gastric environments. Here we use Metadynamics (METADY) simulations to probe the conformational space of the Aspergillus nidulans phytase and the differential effects of pH and glycosylation in this same space. The results suggest that strategic combinations of pH and glycosylation affect the stability of native-like conformations and alternate these structures from a metastable to a stable profile. Furthermore, the protein segments previously reported as more thermosensitive in phytases from this family present a pivotal role in the conformational changes at different conditions, especially H2, H5-7, L8, L10, L12, and L17. Also, the glycosylations and the pH-dependent charge balance modulate the mobility and interactions at these same regions, with consequences for the surface solvation and active site exposition. Finally, although the glycosylations have stabilized the native structure and improved the substrate docking at all the studied pHs, the data suggest a higher phytate receptivity at catalytic poses for the unglycosylated structure at pH 6.5 and the glycosylated one at pH 4.5. This behavior agrees with the exact change in optimum pH reported for this enzyme, expressed on low or high glycosylating systems. We hope the results and insights presented here will be helpful in future approaches for rational engineering of technologically promising phytases and intelligent planning of their heterologous expression systems and conditions for use.Communicated by Ramaswamy H. Sarma.

Probing the mechanism of flavin action in the oxidative decarboxylation catalyzed by salicylate hydroxylase.

Salicylate hydroxylase (NahG) is a FAD-dependent monooxygenase in which the reduced flavin activates O2 coupled to the oxidative decarboxylation of salicylate to catechol or uncoupled from substrate oxidation to afford H2O2. This chapter presents different methodologies in equilibrium studies, steady-state kinetics, and identification of reaction products, which were important to understand the SEAr mechanism of catalysis in NahG, the role of the different FAD parts for ligand binding, the extent of uncoupled reaction, and the catalysis of salicylate's oxidative decarboxylation. These features are likely familiar to many other FAD-dependent monooxygenases and offer a potential asset for developing new tools and strategies in catalysis.

The C-terminal mutation beyond the catalytic site of brown spider phospholipase D significantly impacts its biological activities.

Loxosceles spider envenomation results in dermonecrosis, principally due to phospholipases D (PLDs) present in the venom. These enzymes have a strongly conserved sequence, 273ATXXDNPW280, in the C-terminal region (SMD-tail) that make contact with ß-sheets of the TIM barrel, in which the amino acids Asp277 and Trp280 establish the energetically strongest contacts. The SMD-tail is conserved in PLDs from different species but absent in the non-toxic PLD ancestral glycerophosphodiester phosphodiesterases (GDPDs). This work aims to understand the role of the C-terminal region in the structural stability and/or function of phospholipases D. Through site-directed mutagenesis of the rLiD1 protein (recombinant Loxosceles intermedia dermonecrotic protein 1), we produced two mutants: rLiD1D277A and rLiD1W280A (both with sphingomyelinase activity), in which Asp277 and Trp280 were replaced by alanine. rLiD1D277A showed similar sphingomyelinase activity but at least 2 times more dermonecrotic activity than rLiD1 (wild-type protein). Conversely, while the rLiD1W280A displayed a slight increase in sphingomyelinase activity, its biological activity was similar or lower compared to rLiD1, potentially due to its decreased thermostability and formation of amyloid aggregates. In conclusion, these new findings provide evidence that SMD-tail mutants impact the structure and function of these proteins and point out that residues outside the active site can even increase the function of these enzymes.

Comparative structure of similarities and differences between tree and cognitive model of Cognitive Behavioral Theory / Estrutura comparativa de semelhanças e diferenças entre árvore e modelo cognitivo da Teoria Cognitivo-Comportamental

To contribute to studies in Psychology through a systematic and methodological analysis of the comparison between a tree and the cognitive model of the cognitive behavioral theory, establishing similarities and differences between the comparative domains. Method It is a qualitative, exploratory, documentary research, based on the Conceptual Metaphor, the Cognitive-Behavioral Theory, and on the Methodology of Teaching with Analogies. It starts from the hypothesis of the existence of a conceptual metaphor in which "the cognitive model is structured like a tree", transposing characteristics between both. It verifies, classifies, analyzes the comparison by means of analogical reasoning and the filling of comparative charts between vehicle and target. Results The results point to the complexity and potential of comparison if treated systematically and methodologically. Conclusion The research results corroborate the hypothesis that originated it.

Este artigo buscou analisar as percepções de pais e professores a respeito da trajetória escolar de crianças adotadas. Para isto, entrevistamos professores do Ensino Fundamental (3º ao 7º ano) e pais adotivos, investigando como compreendem a existência de possíveis sucessos e dificuldades escolares nas crianças adotadas, e se há relação entre adoção e atuação escolar. Método A pesquisa é qualitativa, exploratória, documental, fundamentada na Metáfora Conceptual, na Teoria Cognitivo-Comportamental e na Metodologia de Ensino com Analogias e parte da hipótese da existência de uma metáfora conceptual em que "o modelo cognitivo se estrutura como uma árvore", transpondo características entre ambos. Verifica-se, classifica-se e analisa-se a comparação por meio de raciocínio analógico e do preenchimento de quadros comparativos entre veículo e alvo. Resultados Os resultados apontam a complexidade e o potencial da comparação, caso seja tratada sistemática e metodologicamente. Conclusão A hipótese de pesquisa é corroborada.

The role of remote flavin adenine dinucleotide pieces in the oxidative decarboxylation catalyzed by salicylate hydroxylase.

Salicylate hydroxylase (NahG) has a single redox site in which FAD is reduced by NADH, the O2 is activated by the reduced flavin, and salicylate undergoes an oxidative decarboxylation by a C(4a)-hydroperoxyflavin intermediate to give catechol. We report experimental results that show the contribution of individual pieces of the FAD cofactor to the observed enzymatic activity for turnover of the whole cofactor. A comparison of the kinetic parameters and products for the NahG-catalyzed reactions of FMN and riboflavin cofactor fragments reveal that the adenosine monophosphate (AMP) and ribitol phosphate pieces of FAD act to anchor the flavin to the enzyme and to direct the partitioning of the C(4a)-hydroperoxyflavin reaction intermediate towards hydroxylation of salicylate. The addition of AMP or ribitol phosphate pieces to solutions of the truncated flavins results in a partial restoration of the enzymatic activity lost upon truncation of FAD, and the pieces direct the reaction of the C(4a)-hydroperoxyflavin intermediate towards hydroxylation of salicylate.

Rhipicephalus microplus: An overview of vaccine antigens against the cattle tick.

Rhipicephalus microplus, popularly known as the cattle tick, is the most important tick of livestock as it is responsible for significant economic losses. The use of chemical acaricides is still the most widely used control method despite its known disadvantages. Vaccination would be a safe alternative for the control of R. microplus and holds advantages over the use of chemical acaricides as it is environmental-friendly and leaves no residues in meat or milk. Two vaccines based on the Bm86 protein were commercialized, TickGARD® and Gavac®, with varying reported efficacies in different countries. The use of other vaccines, such as Tick Vac®, Go-Tick®, and Bovimune Ixovac® have been restricted to some countries. Several other proteins have been analyzed as possible antigens for more effective vaccines against R. microplus, including peptidases, serine proteinase inhibitors, glutathione S-transferases, metalloproteases, and ribosomal proteins, with efficacies ranging from 14% to 96%. Nonetheless, more research is needed to develop safe and efficient tick vaccines, such as the evaluation of the efficacy of antigens against other tick species to verify cross-reactivity and inclusion of additional antigens to promote the blocking of the infection and spreading of tick-borne diseases. This review summarizes the discoveries of candidate antigens for R. microplus tick vaccines as well as the methods used to test their efficacy.

Reenacting the Birth of a Function: Functional Divergence of HIUases and Transthyretins as Inferred by Evolutionary and Biophysical Studies.

Transthyretin was discovered in the 1940s, named after its ability to bind thyroid hormones and retinol. In the genomic era, transthyretins were found to be part of a larger family with homologs of no obvious function, then called transthyretin-related proteins. Thus, it was proposed that the transthyretin gene could be the result of gene duplication of an ancestral of this newly identified homolog, later found out to be an enzyme involved in uric acid degradation, then named HIUase (5-hydroxy-isourate hydrolase). Here, we sought to re-enact the evolutionary history of this protein family by reconstructing, from a phylogeny inferred from 123 vertebrate sequences, three ancestors corresponding to key moments in their evolution-before duplication; the common transthyretin ancestor after gene duplication and the common ancestor of Eutheria transthyretins. Experimental and computational characterization showed the reconstructed ancestor before duplication was unable to bind thyroxine and likely presented the modern HIUase reaction mechanism, while the substitutions after duplication prevented that activity and were enough to provide stable thyroxine binding, as confirmed by calorimetry and x-ray diffraction. The Eutheria transthyretin ancestor was less prone to characterization, but limited data suggested thyroxine binding as expected. Sequence/structure analysis suggests an early ability to bind the Retinol Binding Protein. We solved the X-ray structures from the two first ancestors, the first at 1.46 resolution, the second at 1.55 resolution with well-defined electron density for thyroxine, providing a useful tool for the understanding of structural adaptation from enzyme to hormone distributor.

Proteus: An algorithm for proposing stabilizing mutation pairs based on interactions observed in known protein 3D structures.

BACKGROUND: Protein engineering has many applications for industry, such as the development of new drugs, vaccines, treatment therapies, food, and biofuel production. A common way to engineer a protein is to perform mutations in functionally essential residues to optimize their function. However, the discovery of beneficial mutations for proteins is a complex task, with a time-consuming and high cost for experimental validation. Hence, computational approaches have been used to propose new insights for experiments narrowing the search space and reducing the costs. RESULTS: In this study, we developed Proteus (an acronym for Protein Engineering Supporter), a new algorithm for proposing mutation pairs in a target 3D structure. These suggestions are based on contacts observed in other known structures from Protein Data Bank (PDB). Proteus' basic assumption is that if a non-interacting pair of amino acid residues in the target structure is exchanged to an interacting pair, this could enhance protein stability. This trade is only allowed if the main-chain conformation of the residues involved in the contact is conserved. Furthermore, no steric impediment is expected between the proposed mutations and the surrounding protein atoms. To evaluate Proteus, we performed two case studies with proteins of industrial interests. In the first case study, we evaluated if the mutations suggested by Proteus for four protein structures enhance the number of inter-residue contacts. Our results suggest that most mutations proposed by Proteus increase the number of interactions into the protein. In the second case study, we used Proteus to suggest mutations for a lysozyme protein. Then, we compared Proteus' outcomes to mutations with available experimental evidence reported in the ProTherm database. Four mutations, in which our results agree with the experimental data, were found. This could be initial evidence that changes in the side-chain of some residues do not cause disturbances that harm protein structure stability. CONCLUSION: We believe that Proteus could be used combined with other methods to give new insights into the rational development of engineered proteins. Proteus user-friendly web-based tool is available at < http://proteus.dcc.ufmg.br >.

Phoneutria toxin PnTx3-5 inhibits TRPV1 channel with antinociceptive action in an orofacial pain model.

Capsaicin, an agonist of TRPV1, evokes intracellular [Ca2+] transients and glutamate release from perfused trigeminal ganglion. The spider toxin PnTx3-5, native or recombinant is more potent than the selective TRPV1 blocker SB-366791 with IC50 of 47 ±â€¯0.18 nM, 45 ±â€¯1.18 nM and 390 ±â€¯5.1 nM in the same experimental conditions. PnTx3-5 is thus more potent than the selective TRPV1 blocker SB-366791. PnTx3-5 (40 nM) and SB-366791 (3 µM) also inhibited the capsaicin-induced increase in intracellular Ca2+ in HEK293 cells transfected with TRPV1 by 75 ±â€¯16% and 84 ±â€¯3.2%, respectively. In HEK293 cells transfected with TRPA1, cinnamaldehyde (30 µM) generated an increase in intracellular Ca2+ that was blocked by the TRPA1 antagonist HC-030031 (10 µM, 89% inhibition), but not by PnTx3-5 (40 nM), indicating selectivity of the toxin for TRPV1. In whole-cell patch-clamp experiments on HEK293 cells transfected with TRPV1, capsaicin (10 µM) generated inward currents that were blocked by SB-366791 and by both native and recombinant PnTx3-5 by 47 ±â€¯1.4%; 54 ±â€¯7.8% and 56 ±â€¯9.0%, respectively. Intradermal injection of capsaicin into the rat left vibrissa induced nociceptive behavior that was blocked by pre-injection with either SB-366791 (3 nmol/site i.d., 83.3 ±â€¯7.2% inhibition) or PnTx3-5 (100 fmol/site, 89 ±â€¯8.4% inhibition). We conclude that both native and recombinant PnTx3-5 are potent TRPV1 receptor antagonists with antinociceptive action on pain behavior evoked by capsaicin.

Benzimidazole inhibitors of the major cysteine protease of Trypanosoma brucei.

Aim: Limitations in available therapies for trypanosomiases indicate the need for improved medicines. Cysteine proteases cruzain and rhodesain are validated targets for treatment of Chagas disease and human African trypanosomiasis. Previous studies reported a benzimidazole series as potent cruzain inhibitors. Results & methodology: Considering the high similarity between these proteases, we evaluated 40 benzimidazoles against rhodesain. We describe their structure-activity relationships (SAR), revealing trends similar to those observed for cruzain and features that lead to enzyme selectivity. This series comprises noncovalent competitive inhibitors (best Ki = 0.21 µM against rhodesain) and micromolar activity against Trypanosoma brucei brucei. A cheminformatics analysis confirms scaffold novelty, and the inhibitors described have favorable predicted physicochemical properties. Conclusion: Our results support this series as a starting point for new human African trypanosomiasis medicines.

Structural and immunological characterization of a new nucleotidyltransferase-like antigen from Paracoccidioides brasiliensis.

Pb27 antigen is an interesting alternative to immunological diagnosis of Paracoccidioidomycosis (PCM) and has demonstrated to be protective in experimental PCM. Its tertiary structure and possible function remained unknown till now. To study Pb27 at the atomic level, the recombinant protein was expressed in Escherichia coli BL21(DE3), purified, and its three-dimensional structure was solved by X-ray crystallography. Based on this structure, we performed a residue correlation analysis and in silico ligand search assays to address a possible biological function to Pb27. We identified Pb27 as a member of the extensive nucleotidyltransferase superfamily. The protein has an αßαßαß topology with two domains (N- and C-terminal domains) and adopts a monomeric form as its biological unit in solution. Structural comparisons with similar members of the superfamily clearly indicate Pb27 C-terminal domain is singular and may play an important role in its biological function. Bioinformatics analysis suggested that Pb27 might bind to ATP and CTP. This suggestion is corroborated by the fact that a magnesium cation is coordinated by two aspartic acid residues present at the active site (between N- and C-terminal domains), as evidenced by X-ray diffraction data. Besides, NMR assays (1H-15N HSQC spectra) confirmed the binding of CTP to Pb27, demonstrating for the first time an interaction between a nucleotide and this protein. Moreover, we evaluated the reactivity of sera from patients with Paracoccidioides brasiliensis infection against the recombinant form of Pb27 and showed that it was recognized by sera from infected and treated patients. Predicted B and T cell epitopes were synthesized and further evaluated against sera of PCM patients, providing information of the most reactive peptides in Pb27 primary structure which interact with specific Pb27 antibodies.

Evaluation of three recombinant proteins for the development of ELISA and immunochromatographic tests for visceral leishmaniasis serodiagnosis.

BACKGROUND: Visceral Leishmaniasis (VL) is an infectious disease that is a significant cause of death among infants aged under 1 year and the elderly in Brazil. Serodiagnosis is a mainstay of VL elimination programs; however, it has significant limitations due to low accuracy. OBJECTIVE: This study aimed to evaluate three recombinant Leishmania infantum proteins (rFc, rC9, and rA2) selected from previous proteomics and genomics analyses to develop enzyme-linked immunosorbent assay (ELISA) and immunochromatographic tests (ICT) for the serodiagnosis of human VL (HVL) and canine VL (CVL). METHODS: A total of 186 human (70 L. infantum-infected symptomatic, 20 other disease-infected, and 96 healthy) and 185 canine (82 L. infantum-infected symptomatic, 27 L. infantum-infected asymptomatic, and 76 healthy) sera samples were used for antibody detection. FINDINGS: Of the three proteins, rA2 (91.5% sensitivity and 87% specificity) and rC9 (95.7% sensitivity and 87.5% specificity) displayed the best performance in ELISA-HVL and ELISA-CVL, respectively. ICT-rA2 also displayed the best performance for HVL diagnosis (92.3% sensitivity and 88.0% specificity) and had high concordance with immunofluorescence antibody tests (IFAT), ELISA-rK39, IT-LEISH®, and ELISAEXT. ICT-rFc, ICT-rC9, and ICT-rA2 had sensitivities of 88.6%, 86.5%, and 87.0%, respectively, with specificity values of 84.0%, 92.0%, and 100%, respectively for CVL diagnosis. MAIN CONCLUSIONS: The three antigens selected by us are promising candidates for VL diagnosis regardless of the test format, although the antigen combinations and test parameters may warrant further optimisation.

Catalytic mechanism for the conversion of salicylate into catechol by the flavin-dependent monooxygenase salicylate hydroxylase.

Salicylate hydroxylase (NahG) is a flavin-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of salicylate into catechol in the naphthalene degradation pathway in Pseudomonas putida G7. We explored the mechanism of action of this enzyme in detail using a combination of structural and biophysical methods. NahG shares many structural and mechanistic features with other versatile flavin-dependent monooxygenases, with potential biocatalytic applications. The crystal structure at 2.0 Šresolution for the apo form of NahG adds a new snapshot preceding the FAD binding in flavin-dependent monooxygenases. The kcat/Km for the salicylate reaction catalyzed by the holo form is >105 M-1 s-1 at pH 8.5 and 25 °C. Hammett plots for Km and kcat using substituted salicylates indicate change in rate-limiting step. Electron-donating groups favor the hydroxylation of salicylate by a peroxyflavin to yield a Wheland-like intermediate, whereas the decarboxylation of this intermediate is faster for electron-withdrawing groups. The mechanism is supported by structural data and kinetic studies at different pHs. The salicylate carboxyl group lies near a hydrophobic region that aids decarboxylation. A conserved histidine residue is proposed to assist the reaction by general base/general acid catalysis.

Evaluation of three recombinant proteins for the development of ELISA and immunochromatographic tests for visceral leishmaniasis serodiagnosis

BACKGROUND Visceral Leishmaniasis (VL) is an infectious disease that is a significant cause of death among infants aged under 1 year and the elderly in Brazil. Serodiagnosis is a mainstay of VL elimination programs; however, it has significant limitations due to low accuracy. OBJECTIVE This study aimed to evaluate three recombinant Leishmania infantum proteins (rFc, rC9, and rA2) selected from previous proteomics and genomics analyses to develop enzyme-linked immunosorbent assay (ELISA) and immunochromatographic tests (ICT) for the serodiagnosis of human VL (HVL) and canine VL (CVL). METHODS A total of 186 human (70 L. infantum-infected symptomatic, 20 other disease-infected, and 96 healthy) and 185 canine (82 L. infantum-infected symptomatic, 27 L. infantum-infected asymptomatic, and 76 healthy) sera samples were used for antibody detection. FINDINGS Of the three proteins, rA2 (91.5% sensitivity and 87% specificity) and rC9 (95.7% sensitivity and 87.5% specificity) displayed the best performance in ELISA-HVL and ELISA-CVL, respectively. ICT-rA2 also displayed the best performance for HVL diagnosis (92.3% sensitivity and 88.0% specificity) and had high concordance with immunofluorescence antibody tests (IFAT), ELISA-rK39, IT-LEISH®, and ELISAEXT. ICT-rFc, ICT-rC9, and ICT-rA2 had sensitivities of 88.6%, 86.5%, and 87.0%, respectively, with specificity values of 84.0%, 92.0%, and 100%, respectively for CVL diagnosis. MAIN CONCLUSIONS The three antigens selected by us are promising candidates for VL diagnosis regardless of the test format, although the antigen combinations and test parameters may warrant further optimisation.

The in vivo and in vitro roles of Trypanosoma cruzi Rad51 in the repair of DNA double strand breaks and oxidative lesions.

In Trypanosoma cruzi, the etiologic agent of Chagas disease, Rad51 (TcRad51) is a central enzyme for homologous recombination. Here we describe the different roles of TcRad51 in DNA repair. Epimastigotes of T. cruzi overexpressing TcRAD51 presented abundant TcRad51-labeled foci before gamma irradiation treatment, and a faster growth recovery when compared to single-knockout epimastigotes for RAD51. Overexpression of RAD51 also promoted increased resistance against hydrogen peroxide treatment, while the single-knockout epimastigotes for RAD51 exhibited increased sensitivity to this oxidant agent, which indicates a role for this gene in the repair of DNA oxidative lesions. In contrast, TcRad51 was not involved in the repair of crosslink lesions promoted by UV light and cisplatin treatment. Also, RAD51 single-knockout epimastigotes showed a similar growth rate to that exhibited by wild-type ones after treatment with hydroxyurea, but an increased sensitivity to methyl methane sulfonate. Besides its role in epimastigotes, TcRad51 is also important during mammalian infection, as shown by increased detection of T. cruzi cells overexpressing RAD51, and decreased detection of single-knockout cells for RAD51, in both fibroblasts and macrophages infected with amastigotes. Besides that, RAD51-overexpressing parasites infecting mice also presented increased infectivity and higher resistance against benznidazole. We thus show that TcRad51 is involved in the repair of DNA double strands breaks and oxidative lesions in two different T. cruzi developmental stages, possibly playing an important role in the infectivity of this parasite.

Biochemical analysis and identification of linear B-cell epitopes from recombinant Sm21.7 antigen from Schistosoma mansoni.

Schistosoma mansoni tegument is a dynamic host-interactive layer that is an essential source of parasite antigens and a relevant field for schistosome vaccine research. Sm21.7 is a cytoskeleton antigen found in S. mansoni tegument that engenders protection in experimental challenge infection. Because of its crucial role in the parasite tegument and its promising protective capability, Sm21.7 is an exciting target for the development of therapeutic strategies. The present study describes Sm21.7 structural and biophysical features using circular dichroism spectroscopy and identifies linear B-cell epitopes of Sm21.7 using in-silico methods and immunoassay. The Sm21.7 gene was cloned into the pETDEST42 vector, and the recombinant protein was overexpressed in Escherichia coli DE3. The soluble protein was purified by affinity chromatography followed by ion-exchange chromatography. Purified recombinant Sm21.7 was analyzed by circular dichroism spectroscopy which demonstrated that the rSm21.7 structure was comprised of approximately 38% α-helices and its conformation remains stable at temperatures of up to 60 °C. Prediction of rSm21.7 B-cell epitopes was based on amino acid physicochemical properties. Sixteen peptides corresponding to predicted epitopes were synthesized and immunoreactivity assessed by spot peptide array using pooled rSm21.7-immunized mice sera or patients' sera with different clinical forms of S. mansoni infection. Immunoassays revealed that sera from rSm21.7-immunized mice reacted predominantly with peptides located in the dynein-light chain domain (DLC) at the C-terminal region of rSm21.7. Comparative analysis of the antibody response of acute, intestinal and hepatosplenic patients' sera to the Sm21.7 peptides showed that a differential recognition pattern of Sm21.7-derived peptides by intestinal patients' sera might contribute to down-regulate the immune response in chronic intestinal patients. Together, the results may help the development of S. mansoni vaccine strategies based on the rSm21.7 antigen.

Effect of ionizing radiation exposure on Trypanosoma cruzi ubiquitin-proteasome system.

In recent years, proteasome involvement in the damage response induced by ionizing radiation (IR) became evident. However, whether proteasome plays a direct or indirect role in IR-induced damage response still unclear. Trypanosoma cruzi is a human parasite capable of remarkable high tolerance to IR, suggesting a highly efficient damage response system. Here, we investigate the role of T. cruzi proteasome in the damage response induced by IR. We exposed epimastigotes to high doses of gamma ray and we analyzed the expression and subcellular localization of several components of the ubiquitin-proteasome system. We show that proteasome inhibition increases IR-induced cell growth arrest and proteasome-mediated proteolysis is altered after parasite exposure. We observed nuclear accumulation of 19S and 20S proteasome subunits in response to IR treatments. Intriguingly, the dynamic of 19S particle nuclear accumulation was more similar to the dynamic observed for Rad51 nuclear translocation than the observed for 20S. In the other hand, 20S increase and nuclear translocation could be related with an increase of its regulator PA26 and high levels of proteasome-mediated proteolysis in vitro. The intersection between the opposed peaks of 19S and 20S protein levels was marked by nuclear accumulation of both 20S and 19S together with Ubiquitin, suggesting a role of ubiquitin-proteasome system in the nuclear protein turnover at the time. Our results revealed the importance of proteasome-mediated proteolysis in T. cruzi IR-induced damage response suggesting that proteasome is also involved in T. cruzi IR tolerance. Moreover, our data support the possible direct/signaling role of 19S in DNA damage repair. Based on these results, we speculate that spatial and temporal differences between the 19S particle and 20S proteasome controls proteasome multiple roles in IR damage response.

A combined approach for enhancing the stability of recombinant cis-dihydrodiol naphthalene dehydrogenase from Pseudomonas putida G7 allowed for the structural and kinetic characterization of the enzyme.

The second enzyme of the naphthalene degradation pathway in Pseudomonas putida G7 is NahB, a dehydrogenase that converts cis-1,2-dihydroxy-1,2-dihydronaphthalene to 1,2-dihydroxynaphthalene. We report the cloning, optimization of expression, purification, kinetic studies and preliminary structural characterization of the recombinant NahB. The nahB gene was cloned into a T7 expression vector and the enzyme was overexpressed in Escherichia coli Rosetta (DE3) as an N-terminal hexa-histidine-tagged protein (6xHis-NahB). Using methods of enhancing protein stability in solution, we tested different expression, cell lysis, and purification protocols with and without ligand supplementation. The protein stability was evaluated by dynamic light scattering and circular dichroism spectroscopy assays. Best-derived protocols (expression at 18 °C, cell lysis with homogenizer, and three purification steps) were used to produce 20 mg of homogeneous 6xHis-NahB per liter of culture. The secondary and quaternary structures of 6xHis-NahB were assessed by circular dichroism and size-exclusion chromatography experiments, respectively. The enzyme was NAD+-dependent and active at pH 7.0 and 9.4 for the oxidation of the substrate. The Michaelis-Menten parameters determined at pH 7.0 and 25 °C for the substrate and cofactor, presented respective Km values of 6 and 350 µM, and a kcat value of 8.3 s-1. Furthermore, we identified conditions for the crystallization of 6xHis-NahB. X-ray diffraction data were collected from a single 6xHis-NahB crystal which diffracted to 2.21 Å. The crystal belongs to space group I222, with unit-cell parameters a = 63.62, b = 69.50, and c = 117.47 Å. The tertiary structure of 6xHis-NahB was determined using the molecular replacement method. Further structural refinement is currently underway.