Antibodies response induced by recombinant virus-like particles from Triatoma virus and chimeric antigens from Trypanosoma cruzi.

BACKGROUND: The infection caused by the protozoan Trypanosoma cruzi affects humans and is called as Chagas disease. Currently, the main measures available to reduce the incidence of this disease are drug treatment and vector control. Traditionally, the development of vaccines occurs mainly through the use of antigenic candidates of the etiologic agent in the form of a vaccine preparation. Virus-like particles (VLPs) are structures analogous to viral capsids composed essentially of structural proteins and are widely used in vaccination protocols because of their immunostimulatory properties. In this context, the objective of this study was to use strategies in a murine immunization model to characterize the immunostimulatory capacity of VLPs from Triatoma virus (TrV-VLPs), analysed in the presence or absence of the aluminium vaccine adjuvant. In parallel, to characterize the immunogenic behaviour of four T. cruzi chimeric recombinant proteins (mix-IBMP) associated with TrV-VLPs or aluminium vaccine adjuvant. METHOD: We immunized BALB/c mice once or twice, depending on the strategy, and collected serum samples at 15, 30 and 45 days after the immunization. Subsequently, serum samples from animals immunized with TrV-VLPs were used to determine total IgG, IgG1, IgG2a, IgG2b and IgG3 anti-TrV-VLPs by enzyme-linked immunosorbent assay (ELISA). RESULTS: Data obtained demonstrate the ability of TrV-VLPs to preferably induce IgG2b and IgG3 type antibodies in the absence of aluminium adjuvant. In fact, the use of aluminium did not interfere with the total IgG profile of anti-TrV-VLPs. Interestingly, mix-IBMP had a better profile of total IgG, IgG1 and IgG3 subclasses when mixed with TrV-VLPs. CONCLUSION: In conclusion, these results suggest the potential of TrV-VLPs as a vaccine adjuvant and the use of T. cruzi chimeric antigens as a rational strategy for the development of vaccines against the experimental model of Chagas disease.

Identifying Structural Determinants of Product Specificity in Leishmania major Farnesyl Diphosphate Synthase.

Farnesyl diphosphate synthase (FPPS) is an isoprenoid chain elongation enzyme that catalyzes the sequential condensation of dimethylallyl diphosphate (C5) with isopentenyl diphosphate (IPP; C5) and the resulting geranyl diphosphate (GPP; C10) with another molecule of IPP, eventually producing farnesyl diphosphate (FPP; C15), which is a precursor for the biosynthesis of a vast majority of isoprenoids. Previous studies of FPPS have highlighted the importance of the structure around the hydrophobic chain elongation path in determining product specificity. To investigate what structural features define the final chain length of the product in FPPS from Leishmania major, we designed and expressed six mutants of LmFPPS by replacing small amino acids around the binding pocket with bulky residues. Using enzymatic assays, binding kinetics, and crystallographic studies, we analyzed the effects of these mutations on the activity and product specificity of FPPS. Our results revealed that replacement of Thr-164 with tryptophan and phenylalanine completely abolished the activity of FPPS. Intriguingly, the T164Y substitution displayed dual product specificity and produced a mixture GPP and FPP as final products, with an activity for FPP synthesis that was lower than that of the wild-type enzyme. These data indicate that Thr-164 is a potential regulator of product specificity.

Bisphosphonate-Based Molecules as Potential New Antiparasitic Drugs.

Neglected tropical diseases such as Chagas disease and leishmaniasis affect millions of people around the world. Both diseases affect various parts of the globe and drugs traditionally used in therapy against these diseases have limitations, especially with regard to low efficacy and high toxicity. In this context, the class of bisphosphonate-based compounds has made significant advances regarding the chemical synthesis process as well as the pharmacological properties attributed to these compounds. Among this spectrum of pharmacological activity, bisphosphonate compounds with antiparasitic activity stand out, especially in the treatment of Chagas disease and leishmaniasis caused by Trypanosoma cruzi and Leishmania spp., respectively. Some bisphosphonate compounds can inhibit the mevalonate pathway, an essential metabolic pathway, by interfering with the synthesis of ergosterol, a sterol responsible for the growth and viability of these parasites. Therefore, this review aims to present the information about the importance of these compounds as antiparasitic agents and as potential new drugs to treat Chagas disease and leishmaniasis.

Getting the Most Out of Your Crystals: Data Collection at the New High-Flux, Microfocus MX Beamlines at NSLS-II.

Advances in synchrotron technology are changing the landscape of macromolecular crystallography. The two recently opened beamlines at NSLS-II-AMX and FMX-deliver high-flux microfocus beams that open new possibilities for crystallographic data collection. They are equipped with state-of-the-art experimental stations and automation to allow data collection on previously intractable crystals. Optimized data collection strategies allow users to tailor crystal positioning to optimally distribute the X-ray dose over its volume. Vector data collection allows the user to define a linear trajectory along a well diffracting volume of the crystal and perform rotational data collection while moving along the vector. This is particularly well suited to long, thin crystals. We describe vector data collection of three proteins-Akt1, PI3Kα, and CDP-Chase-to demonstrate its application and utility. For smaller crystals, we describe two methods for multicrystal data collection in a single loop, either manually selecting multiple centers (using H108A-PHM as an example), or "raster-collect", a more automated approach for a larger number of crystals (using CDP-Chase as an example).

Effects of copper occupancy on the conformational landscape of peptidylglycine α-hydroxylating monooxygenase.

The structures of metalloproteins that use redox-active metals for catalysis are usually exquisitely folded in a way that they are prearranged to accept their metal cofactors. Peptidylglycine α-hydroxylating monooxygenase (PHM) is a dicopper enzyme that catalyzes hydroxylation of the α-carbon of glycine-extended peptides for the formation of des-glycine amidated peptides. Here, we present the structures of apo-PHM and of mutants of one of the copper sites (H107A, H108A, and H172A) determined in the presence and absence of citrate. Together, these structures show that the absence of one copper changes the conformational landscape of PHM. In one of these structures, a large interdomain rearrangement brings residues from both copper sites to coordinate a single copper (closed conformation) indicating that full copper occupancy is necessary for locking the catalytically competent conformation (open). These data suggest that in addition to their required participation in catalysis, the redox-active metals play an important structural role.

Kinetic and structural analyses reveal residues in phosphoinositide 3-kinase α that are critical for catalysis and substrate recognition.

Phosphoinositide 3-kinases (PI3Ks) are ubiquitous lipid kinases that activate signaling cascades controlling cell survival, proliferation, protein synthesis, and vesicle trafficking. PI3Ks have dual kinase specificity: a lipid kinase activity that phosphorylates the 3'-hydroxyl of phosphoinositides and a protein-kinase activity that includes autophosphorylation. Despite the wealth of biochemical and structural information on PI3Kα, little is known about the identity and roles of individual active-site residues in catalysis. To close this gap, we explored the roles of residues of the catalytic domain and the regulatory subunit of human PI3Kα in lipid and protein phosphorylation. Using site-directed mutagenesis, kinetic assays, and quantitative mass spectrometry, we precisely mapped key residues involved in substrate recognition and catalysis by PI3Kα. Our results revealed that Lys-776, located in the P-loop of PI3Kα, is essential for the recognition of lipid and ATP substrates and also plays an important role in PI3Kα autophosphorylation. Replacement of the residues His-936 and His-917 in the activation and catalytic loops, respectively, with alanine dramatically changed PI3Kα kinetics. Although H936A inactivated the lipid kinase activity without affecting autophosphorylation, H917A abolished both the lipid and protein kinase activities of PI3Kα. On the basis of these kinetic and structural analyses, we propose possible mechanistic roles of these critical residues in PI3Kα catalysis.

Identification of allosteric binding sites for PI3Kα oncogenic mutant specific inhibitor design.

PIK3CA, the gene that encodes the catalytic subunit of phosphatidylinositol 3-kinase α (PI3Kα), is frequently mutated in breast and other types of cancer. A specific inhibitor that targets the mutant forms of PI3Kα could maximize treatment efficiency while minimizing side-effects. Herein we describe the identification of novel binding pockets that may provide an opportunity for the design of mutant selective inhibitors. Using a fragment-based approach, we screened a library of 352 fragments (MW<300Da) for binding to PI3Kα by X-ray crystallography. Five novel binding pockets were identified, each providing potential opportunities for inhibitor design. Of particular interest was a binding pocket near Glu542, which is located in one of the two most frequently mutated domains.

Plasmodium falciparum CTP:phosphocholine cytidylyltransferase possesses two functional catalytic domains and is inhibited by a CDP-choline analog selected from a virtual screening.

Phosphatidylcholine is the major lipid component of the malaria parasite membranes and is required for parasite multiplication in human erythrocytes. Plasmodium falciparum CTP:phosphocholine cytidylyltransferase (PfCCT) is the rate-limiting enzyme of the phosphatidylcholine biosynthesis pathway and thus considered as a potential antimalarial target. In contrast to its mammalian orthologs, PfCCT contains a duplicated catalytic domain. Here, we show that both domains are catalytically active with similar kinetic parameters. A virtual screening strategy allowed the identification of a drug-size molecule competitively inhibiting the enzyme. This compound also prevented phosphatidylcholine biosynthesis in parasites and exerted an antimalarial effect. This study constitutes the first step towards a rationalized design of future new antimalarial agents targeting PfCCT.

Biochemical characterization of Plasmodium falciparum CTP:phosphoethanolamine cytidylyltransferase shows that only one of the two cytidylyltransferase domains is active.

The intra-erythrocytic proliferation of the human malaria parasite Plasmodium falciparum requires massive synthesis of PE (phosphatidylethanolamine) that together with phosphatidylcholine constitute the bulk of the malaria membrane lipids. PE is mainly synthesized de novo by the CDP:ethanolamine-dependent Kennedy pathway. We previously showed that inhibition of PE biosynthesis led to parasite death. In the present study we characterized PfECT [P. falciparum CTP:phosphoethanolamine CT (cytidylyltransferase)], which we identified as the rate-limiting step of the PE metabolic pathway in the parasite. The cellular localization and expression of PfECT along the parasite life cycle were studied using polyclonal antibodies. Biochemical analyses showed that the enzyme activity follows Michaelis-Menten kinetics. PfECT is composed of two CT domains separated by a linker region. Activity assays on recombinant enzymes upon site-directed mutagenesis revealed that the N-terminal CT domain was the only catalytically active domain of PfECT. Concordantly, three-dimensional homology modelling of PfECT showed critical amino acid differences between the substrate-binding sites of the two CT domains. PfECT was predicted to fold as an intramolecular dimer suggesting that the inactive C-terminal domain is important for dimer stabilization. Given the absence of PE synthesis in red blood cells, PfECT represents a potential antimalarial target opening the way for a rational conception of bioactive compounds.