Exploring protein myristoylation in Toxoplasma gondii.

Toxoplasma gondii is an important human and veterinary pathogen and the causative agent of toxoplasmosis, a potentially severe disease especially in immunocompromised or congenitally infected humans. Current therapeutic compounds are not well-tolerated, present increasing resistance, limited efficacy and require long periods of treatment. On this context, searching for new therapeutic targets is crucial to drug discovery. In this sense, recent works suggest that N-myristoyltransferase (NMT), the enzyme responsible for protein myristoylation that is essential in some parasites, could be the target of new anti-parasitic compounds. However, up to date there is no information on NMT and the extent of this modification in T. gondii. In this work, we decided to explore T. gondii genome in search of elements related with the N-myristoylation process. By a bioinformatics approach it was possible to identify a putative T. gondii NMT (TgNMT). This enzyme that is homologous to other parasitic NMTs, presents activity in vitro, is expressed in both intra- and extracellular parasites and interacts with predicted TgNMT substrates. Additionally, NMT activity seems to be important for the lytic cycle of Toxoplasma gondii. In parallel, an in silico myristoylome predicts 157 proteins to be affected by this modification. Myristoylated proteins would be affecting several metabolic functions with some of them being critical for the life cycle of this parasite. Together, these data indicate that TgNMT could be an interesting target of intervention for the treatment of toxoplasmosis.

Autocatalytic maturation of the Tat-dependent halophilic subtilase Nep produced by the archaeon Natrialba magadii.

Halolysins are subtilisin-like extracellular proteases produced by haloarchaea that possess unique protein domains and are salt dependent for structural integrity and functionality. In contrast to bacterial subtilases, the maturation mechanism of halolysins has not been addressed. The halolysin Nep is secreted by the alkaliphilic haloarchaeon Natrialba magadii, and the recombinant active enzyme has been synthesized in Haloferax volcanii. Nep contains an N-terminal signal peptide with the typical Tat consensus motif (GRRSVL), an N-terminal propeptide, the protease domain, and a C-terminal domain. In this study, we used Nep as a model protease to examine the secretion and maturation of halolysins by using genetic and biochemical approaches. Mutant variants of Nep were constructed by site-directed mutagenesis and expressed in H. volcanii, which were then analyzed by protease activity and Western blotting. The Tat dependence of Nep secretion was demonstrated in Nep RR/KK variants containing double lysine (KK) in place of the twin arginines (RR), in which Nep remained cell associated and the extracellular activity was undetectable. High-molecular-mass Nep polypeptides without protease activity were detected as cell associated and extracellularly in the Nep S/A variant, in which the catalytic serine 352 had been changed by alanine, indicating that Nep protease activity was needed for precursor processing and activation. Nep NSN 1-2 containing a modification in two potential cleavage sites for signal peptidase I (ASA) was not efficiently processed and activated. This study examined for the first time the secretion and maturation of a Tat-dependent halophilic subtilase.

An efficient protocol for the synthesis of quinoxaline derivatives at room temperature using recyclable alumina-supported heteropolyoxometalates.

We report a suitable quinoxaline synthesis using molybdophosphovanadates supported on commercial alumina cylinders as catalysts. These catalysts were prepared by incipient wetness impregnation. The catalytic test was performed under different reaction conditions in order to know the performance of the synthesized catalysts. The method shows high yields of quinoxaline derivatives under heterogeneous conditions. Quinoxaline formation was obtained using benzyl, o-phenylenediamine, and toluene as reaction solvent at room temperature. The CuH(2)PMo(11)VO(40) supported on alumina showed higher activity in the tested reaction. Finally, various quinoxalines were prepared under mild conditions and with excellent yields.

Structure of the class XI myosin globular tail reveals evolutionary hallmarks for cargo recognition in plants.

The plant-specific class XI myosins (MyoXIs) play key roles at the molecular, cellular and tissue levels, engaging diverse adaptor proteins to transport cargoes along actin filaments. To recognize their cargoes, MyoXIs have a C-terminal globular tail domain (GTD) that is evolutionarily related to those of class V myosins (MyoVs) from animals and fungi. Despite recent advances in understanding the functional roles played by MyoXI in plants, the structure of its GTD, and therefore the molecular determinants for cargo selectivity and recognition, remain elusive. In this study, the first crystal structure of a MyoXI GTD, that of MyoXI-K from Arabidopsis thaliana, was elucidated at 2.35 Šresolution using a low-identity and fragment-based phasing approach in ARCIMBOLDO_SHREDDER. The results reveal that both the composition and the length of the α5-α6 loop are distinctive features of MyoXI-K, providing evidence for a structural stabilizing role for this loop, which is otherwise carried out by a molecular zipper in MyoV GTDs. The crystal structure also shows that most of the characterized cargo-binding sites in MyoVs are not conserved in plant MyoXIs, pointing to plant-specific cargo-recognition mechanisms. Notably, the main elements involved in the self-regulation mechanism of MyoVs are conserved in plant MyoXIs, indicating this to be an ancient ancestral trait.

A suitable preparation of N-sulfonyl-1,2,3,4-tetrahydroisoquinolines and their ring homologs with a reusable Preyssler heteropolyacid as catalyst.

The preparation of N-sulfonyl-1,2,3,4-tetrahydroisoquinolines, N-sulfonyl-2,3,4,5-tetrahydro-1H-2-benzazepines and N-sulfonyl-1,2,3,4,5,6-hexahydrobenzazocine was catalyzed by a Preyssler heteropolyacid, H(14)[NaP(5)W(30)O(110)], (PA), supported on silica (PASiO(2)40) with excellent yields by means of the Pictet-Spengler reaction of N-aralkylsulfonamides with s-trioxane. The reactions proceed with 0.5 mol% of silica-supported catalyst in toluene at 70°C. The catalyst can be recycled without appreciable loss of the catalytic activity.

Emerging Therapeutic Targets Against Toxoplasma gondii: Update on DNA Repair Response Inhibitors and Genotoxic Drugs.

Toxoplasma gondii is the causative agent of toxoplasmosis in animals and humans. This infection is transmitted to humans through oocysts released in the feces of the felines into the environment or by ingestion of undercooked meat. This implies that toxoplasmosis is a zoonotic disease and T. gondii is a foodborne pathogen. In addition, chronic toxoplasmosis in goats and sheep is the cause of recurrent abortions with economic losses in the sector. It is also a health problem in pets such as cats and dogs. Although there are therapies against this infection in its acute stage, they are not able to permanently eliminate the parasite and sometimes they are not well tolerated. To develop better, safer drugs, we need to elucidate key aspects of the biology of T. gondii. In this review, we will discuss the importance of the homologous recombination repair (HRR) pathway in the parasite's lytic cycle and how components of these processes can be potential molecular targets for new drug development programs. In that sense, the effect of different DNA damage agents or HHR inhibitors on the growth and replication of T. gondii will be described. Multitarget drugs that were either associated with other targets or were part of general screenings are included in the list, providing a thorough revision of the drugs that can be tested in other scenarios.

Evaluation of ATM Kinase Inhibitor KU-55933 as Potential Anti-Toxoplasma gondii Agent.

Toxoplasma gondii is an apicomplexan protozoan parasite with a complex life cycle composed of multiple stages that infect mammals and birds. Tachyzoites rapidly replicate within host cells to produce acute infection during which the parasite disseminates to tissues and organs. Highly replicative cells are subject to Double Strand Breaks (DSBs) by replication fork collapse and ATM, a member of the phosphatidylinositol 3-kinase (PI3K) family, is a key factor that initiates DNA repair and activates cell cycle checkpoints. Here we demonstrate that the treatment of intracellular tachyzoites with the PI3K inhibitor caffeine or ATM kinase-inhibitor KU-55933 affects parasite replication rate in a dose-dependent manner. KU-55933 affects intracellular tachyzoite growth and induces G1-phase arrest. Addition of KU-55933 to extracellular tachyzoites also leads to a significant reduction of tachyzoite replication upon infection of host cells. ATM kinase phosphorylates H2A.X (γH2AX) to promote DSB damage repair. The level of γH2AX increases in tachyzoites treated with camptothecin (CPT), a drug that generates fork collapse, but this increase was not observed when co-administered with KU-55933. These findings support that KU-55933 is affecting the Toxoplasma ATM-like kinase (TgATM). The combination of KU-55933 and other DNA damaging agents such as methyl methane sulfonate (MMS) and CPT produce a synergic effect, suggesting that TgATM kinase inhibition sensitizes the parasite to damaged DNA. By contrast, hydroxyurea (HU) did not further inhibit tachyzoite replication when combined with KU-55933.

Functional roles of C-terminal extension (CTE) of salt-dependent peptidase activity of the Natrialba magadii extracellular protease (NEP).

Nep (Natrialba magadii extracellular protease) is a halolysin-like peptidase secreted by the haloalkaliphilic archaeon Natrialba magadii. Many extracellular proteases have been characterized from archaea to bacteria as adapted to hypersaline environments retaining function and stability until 4.0M NaCl. As observed in other secreted halolysins, this stability can be related to the presence of a C-terminal extension (CTE) sequence. In the present work, we compared the biochemical properties of recombinant Nep protease with the truncated form at the 134 amino acids CTE (Nep∆CTE), that was more active in 4M NaCl than the non-truncated wild type enzyme. Comparable to the wild type, Nep∆CTE protease is irreversibly inactivated at low salt solutions. The substrate specificity of the truncated Nep∆CTE was similar to that of wild type form as demonstrated by a combinatorial library of FRET substrates. The enzyme stability, the effect of different salts and the thermodynamics assays using different lengths of substrates demonstrated similarities between the two forms. Altogether, these data provide further information on the stability and structural determinants of halolysins under different salinities, especially concerning the enzymatic behavior.

Effects of the linker region on the structure and function of modular GH5 cellulases.

The association of glycosyl hydrolases with catalytically inactive modules is a successful evolutionary strategy that is commonly used by biomass-degrading microorganisms to digest plant cell walls. The presence of accessory domains in these enzymes is associated with properties such as higher catalytic efficiency, extension of the catalytic interface and targeting of the enzyme to the proper substrate. However, the importance of the linker region in the synergistic action of the catalytic and accessory domains remains poorly understood. Thus, this study examined how the inter-domain region affects the structure and function of modular GH5 endoglucanases, by using cellulase 5A from Bacillus subtilis (BsCel5A) as a model. BsCel5A variants featuring linkers with different stiffnesses or sizes were designed and extensively characterized, revealing that changes in flexibility or rigidity in this region differentially affect kinetic behavior. Regarding the linker length, we found that precise inter-domain spacing is required to enable efficient hydrolysis because excessively long or short linkers were equally detrimental to catalysis. Together, these findings identify molecular and structural features that may contribute to the rational design of chimeric and multimodular glycosyl hydrolases.

Structure and function of a novel cellulase 5 from sugarcane soil metagenome.

Cellulases play a key role in enzymatic routes for degradation of plant cell-wall polysaccharides into simple and economically-relevant sugars. However, their low performance on complex substrates and reduced stability under industrial conditions remain the main obstacle for the large-scale production of cellulose-derived products and biofuels. Thus, in this study a novel cellulase with unusual catalytic properties from sugarcane soil metagenome (CelE1) was isolated and characterized. The polypeptide deduced from the celE1 gene encodes a unique glycoside hydrolase domain belonging to GH5 family. The recombinant enzyme was active on both carboxymethyl cellulose and ß-glucan with an endo-acting mode according to capillary electrophoretic analysis of cleavage products. CelE1 showed optimum hydrolytic activity at pH 7.0 and 50 °C with remarkable activity at alkaline conditions that is attractive for industrial applications in which conventional acidic cellulases are not suitable. Moreover, its three-dimensional structure was determined at 1.8 Å resolution that allowed the identification of an insertion of eight residues in the ß8-α8 loop of the catalytic domain of CelE1, which is not conserved in its psychrophilic orthologs. This 8-residue-long segment is a prominent and distinguishing feature of thermotolerant cellulases 5 suggesting that it might be involved with thermal stability. Based on its unconventional characteristics, CelE1 could be potentially employed in biotechnological processes that require thermotolerant and alkaline cellulases.

Correlation between catalysis and tertiary structure arrangement in an archaeal halophilic subtilase.

Nep (Natrialba magadii extracellular protease) is a halolysin-like peptidase secreted by the haloalkaliphilic archaeon N. magadii that exhibits optimal activity and stability in salt-saturated solutions. In this work, the effect of salt on the function and structure of Nep was investigated. In absence of salt, Nep became unfolded and aggregated, leading to the loss of activity. The enzyme did not recover its structural and functional properties even after restoring the ideal conditions for catalysis. At salt concentrations higher than 1 M (NaCl), Nep behaved as monomers in solution and its enzymatic activity displayed a nonlinear concave-up dependence with salt concentration resulting in a 20-fold activation at 4 M NaCl. Although transition from a high to a low-saline environment (3-1 M NaCl) did not affect its secondary structure contents, it diminished the enzyme stability and provoked large structural rearrangements, changing from an elongated shape at 3 M NaCl to a compact conformational state at 1 M NaCl. The thermodynamic analysis of peptide hydrolysis by Nep suggests a significant enzyme reorganization depending on the environmental salinity, which supports in solution SAXS and DLS studies. Moreover, solvent kinetic isotopic effect (SKIE) data indicates the general acid-base mechanism as the rate-limiting step for Nep catalysis, like classical serine-peptidases. All these data correlate the Nep conformational states with the enzymatic behavior providing a further understanding on the stability and structural determinants for the functioning of halolysins under different salinities.

Sustainable synthesis of flavonoid derivatives, QSAR study and insecticidal activity against the fall armyworm, Spodoptera frugiperda (Lep.: Noctuidae).

A simple, clean, solvent-free preparation of flavones by the use of a silica-supported Preyssler heteropolyacid as reusable catalyst is described. High selectivity and very good yields (87-94%) were obtained in short reaction times (7-13 min). Bioassays for insecticidal activity against Spodoptera frugiperda were carried out with a set of flavones. Bioassays showed that some of the flavones had moderate insecticidal activity. Quantitative structure-activity relationships were established on the available data with the purpose of predicting the insecticidal activity of a number of structurally related flavones. A relationship between the molecular structure and biological activity is proposed.

QSAR analysis on Spodoptera litura antifeedant activities for flavone derivatives.

We establish useful models that relate experimentally measured biological activities of compounds to their molecular structure. The pED(50) feeding inhibition on Spodoptera litura species exhibited by aurones, chromones, 3-coumarones and flavones is analyzed in this work through the hypothesis encompassed in the Quantitative Structure-Activity Relationships (QSAR) Theory. This constitutes a first necessary computationally based step during the design of more bio-friendly repellents that could lead to insights for improving the insecticidal activities of the investigated compounds. After optimizing the molecular structure of each furane and pyrane benzoderivative with the semiempirical molecular orbitals method PM3, more than a thousand of constitutional, topological, geometrical and electronic descriptors are calculated and multiparametric linear regression models are established on the antifeedant potencies. The feature selection method employed in this study is the Replacement Method, which has proven to be successful in previous analyzes. We establish the QSAR both for the complete molecular set of compounds and also for each chemical class, so that acceptably describing the variation of the inhibitory activities from the knowledge of their structure and thus achieving useful predictive results. The main interest of developing trustful QSAR models is that these enable the prediction of compounds having no experimentally measured activities for any reason. Therefore, the structure-activity relationships are further employed for investigating the antifeedant activity on previously synthesized 2-,7-substituted benzopyranes, which do not pose any measured values on the biological expression. One of them, 2-(alpha-naphtyl)-4H-1-benzopyran-4-one, results in a promising structure to be experimentally analyzed as it has predicted pED(50)=1.162.

Gene cloning and heterologous synthesis of a haloalkaliphilic extracellular protease of Natrialba magadii (Nep).

The gene encoding the protease Nep secreted by the haloalkaliphilic archaeon Natrialba magadii was cloned and sequenced. Upstream of the nep gene, a region related to haloarchaeal TATA-box and BRE-like consensus sequences was identified. The nep-encoded polypeptide had a molecular mass of 56.4 kDa, a pI of 3.77 and included a 121-amino acid propeptide not present in the mature Nep. A Tat motif (GRRSVL) was also identified at residues 10-15 suggesting it is a substrate of the Tat pathway. The primary sequence of Nep was closely related to serine proteases of the subtilisin family from archaea and bacteria (50-85% similarity). The nep gene was expressed in Escherichia coli and Haloferax volcanii resulting in production of active Nep protease. In contrast to the recombinant E. coli strains in which Nep activity was only detected in cell lysate, high levels of Nep protein and activity were detected in the culture medium of stationary phase recombinant Hfx. volcanii strains. The Hfx. volcanii synthesized protease was active in high salt, high pH and high DMSO. This study provides the first molecular characterization of a halolysin-like protease from alkaliphilic haloarchaea and is the first description of a recombinant system that facilitates high-level secretion of a haloarchaeal protease.

Effect of organic solvents on the activity and stability of an extracellular protease secreted by the haloalkaliphilic archaeon Natrialba magadii.

The effect of various organic solvents on the activity and stability of an extracellular protease produced by the haloalkaliphilic archaeon Natrialba magadii was tested. This protease was active and stable in aqueous-organic solvent mixtures containing 1.5 M NaCl and glycerol, dimethylsulfoxide (DMSO), N,N-dimethyl formamide, propylenglycol, and dioxane. Among the solvents tested, DMSO, propylenglycol, and glycerol were effective in preserving enzyme stability in suboptimal NaCl concentrations. The stabilizing effect of DMSO on this haloalkaliphilic protease was more efficient at pH 8 than at pH 10, suggesting that DMSO may not substitute for salt to allow halophilic proteins to withstand the effect of high pH values. These results show that Nab. magadii extracellular protease is a solvent tolerant enzyme and suggest a potential application of this haloalkaliphilic protease in aqueous-organic solvent biocatalysis.