CHARACTERIZATION OF THE MECHANISM OF 6-PHOSPHOGLUCONATE DEHYDROGENASE FROM TRYPANOSOMA BRUCEI AND ITS INTERACTION WITH INHIBITORS BY ISOTHERMAL TITRATION CALORIMETRY

6-Phosphogluconate dehydrogenase (6PGDH) converts 6PG to ribulose 5-phosphate and concomitantly provides NADPH, inside the pentose phosphate pathway. Its presence has been shown essential for growth of bloodstream form Trypanosoma brucei, a parasite responsible for African trypanosomiasis and it may be considered a validated drug target in this protozoan. Drugs are the principal means of intervention but new drugs are urgently needed for human African trypanosomiasis. Strong inhibitors have been found against 6PGDH, which show some selectivity versus the parasite enzyme compared the mammalian one. T. brucei 6PGDH shows only a 33% amino acid identity with the mammalian 6PGDH even if their structures have a similar overall fold and many residues nearest neighbours to the substrate are conserved. Regarding 6PGDH mechanism two residues, one acting as an acid and the other as a base, are postulated to assist all the three catalytic steps. These residues (E192 and K185 in the T. brucei) have been identified on the basis of crystallographic evidence and site-directed mutagenesis. Much remains to understand yet, for instance on which way at the end of the reaction the protonation state of the two catalytic groups changes into the opposite to that at the beginning of the reaction and on the homotropic cooperativity of the enzyme (homodimeric) and the induced enzyme changes at the substrate binding. To elucidate this and the inhibition mechanism by some lead compounds to develop as potential drugs, we have exploited isothermal titration calorimetry (ITC). ITC measurements were performed in a VP-ITC microcalorimeter and the data were fitted by nonlinear least-squares fitting using OriginTM software. The number of H+ exchanged has been measured after binding studies in different buffers. Binding was studied sometimes by fluorometry. Recombinant T. brucei 6PGDH has been purified from an overexpressing E. coli strain. Kinetics of the enzymes was studied spectrophotometrically, also in function of pH. Preparation of site-directed mutants of the enzyme has been a complementary technique to obtain information on the enzyme. cysteine reactivity has been assayed with the Ellmann reactant DTNB. Binding of the substrate and its analogues is entropy driven, while binding of coenzymes is enthalpy driven. Oxidized coenzyme and its analogue display a half-site reactivity in the ternary complex with the substrate or the inhibitors while reduced coenzyme displays full-site reactivity. Binding of 6PG and 5-phospho-ribonate (5PR) poorly affects the dissociation constant of the coenzymes, while binding of 4-phospho-erythronate (4PE), which is the most selective among the studied inhibitors for the parasite enzyme compared to the mammalian sheep liver one, decreases the dissociation constant of the coenzymes by two orders of magnitude. In a similar way the Kd of 4PE greatly decreases in the presence of the coenzymes. Results suggest that while 5PR acts as substrate analogue, 4PE mimics the transition-state of the dehydrogenation. The stronger affinity of 4PE is interpreted on the basis of the enzyme mechanism, suggesting that the inhibitor forces the catalytic K185 in the protonated state. pH curves of the mutants K185R and E192Q clearly show that other residues are involved in mechanism. It has been shown that a change from an “open” to a “closed” conformation is rate limiting. The conformational changes induced by substrate binding appear to be related with the release of about one H+/enzyme dimer, and with a drastic reduction of cysteine reactivity. The residues H188 and C372 are not directly involved in the substrate binding but are very close to the active site. The mutants H188L and C372S show a linear relationship between the residual activity and the number of H+ released and also with the cysteine reactivity. These data suggest that H188 and C372 are involved in the transition from the “open” to the “closed” form. Further studies have been performed to better characterize the mechanism of 6PGDH. Kinetic isotope effect studies on the reverse reaction, the reductive carboxylation of Ru5P to 6PG indicate that the presence of 6PG changes the rate limiting step of the reaction. In the absence of 6PG the rate limiting step is clearly identified in a conformational change of the enzyme-Ru5P complex. In the presence of 6PG this step become fast, and the rate limiting step can be identified in an isomerization of the enzyme-6PG complex according to the previously published data. These results add new support to the alternative-site model for the mechanism of 6PGDH

Thermodynamic characterization of substrate and inhibitor binding to Trypanosoma brucei 6-phosphogluconate dehydrogenase

6-Phosphogluconate dehydrogenase is a potential target for new drugs against African trypanosomiasis. Phosphorylated aldonic acids are strong inhibitors of 6-phosphogluconate dehydrogenase, and 4-phospho-d-erythronate (4PE) and 4-phospho-d-erythronohydroxamate are two of the strongest inhibitors of the Trypanosoma brucei enzyme. Binding of the substrate 6-phospho-d-gluconate (6PG), the inhibitors 5-phospho-d-ribonate (5PR) and 4PE, and the coenzymes NADP, NADPH and NADP analogue 3-amino-pyridine adenine dinucleotide phosphate to 6-phospho-d-gluconate dehydrogenase from T. brucei was studied using isothermal titration calorimetry. Binding of the substrate (K(d) = 5 microm) and its analogues (K(d) =1.3 microm and K(d) = 2.8 microm for 5PR and 4PE, respectively) is entropy driven, whereas binding of the coenzymes is enthalpy driven. Oxidized coenzyme and its analogue, but not reduced coenzyme, display a half-site reactivity in the ternary complex with the substrate or inhibitors. Binding of 6PG and 5PR poorly affects the dissociation constant of the coenzymes, whereas binding of 4PE decreases the dissociation constant of the coenzymes by two orders of magnitude. In a similar manner, the K(d) value of 4PE decreases by two orders of magnitude in the presence of the coenzymes. The results suggest that 5PR acts as a substrate analogue, whereas 4PE mimics the transition state of dehydrogenation. The stronger affinity of 4PE is interpreted on the basis of the mechanism of the enzyme, suggesting that the inhibitor forces the catalytic lysine 185 into the protonated state

HIV-1 Tat Promotes Integrin-Mediated HIV Transmission to Dendritic Cells by Binding Env Spikes and Competes Neutralization by Anti-HIV Antibodies

none44Use of Env in HIV vaccine development has been disappointing. Here we show that, in the presence of a biologically active Tat subunit vaccine, a trimeric Env protein prevents in monkeys virus spread from the portal of entry to regional lymph nodes. This appears to be due to specific interactions between Tat and Env spikes that form a novel virus entry complex favoring R5 or X4 virus entry and productive infection of dendritic cells (DCs) via an integrin-mediated pathway. These Tat effects do not require Tat-transactivation activity and are blocked by anti-integrin antibodies (Abs). Productive DC infection promoted by Tat is associated with a highly efficient virus transmission to T cells. In the Tat/Env complex the cysteine-rich region of Tat engages the Env V3 loop, whereas the Tat RGD sequence remains free and directs the virus to integrins present on DCs. V2 loop deletion, which unshields the CCR5 binding region of Env, increases Tat/Env complex stability. Of note, binding of Tat to Env abolishes neutralization of Env entry or infection of DCs by anti-HIV sera lacking anti-Tat Abs, which are seldom present in natural infection. This is reversed, and neutralization further enhanced, by HIV sera containing anti-Tat Abs such as those from asymptomatic or Tat-vaccinated patients, or by sera from the Tat/Env vaccinated monkeys. Thus, both anti-Tat and anti-Env Abs are required for efficient HIV neutralization. These data suggest that the Tat/Env interaction increases HIV acquisition and spreading, as a mechanism evolved by the virus to escape anti-Env neutralizing Abs. This may explain the low effectiveness of Env-based vaccines, which are also unlikely to elicit Abs against new Env epitopes exposed by the Tat/Env interaction. As Tat also binds Envs from different clades, new vaccine strategies should exploit the Tat/Env interaction for both preventative and therapeutic interventions. © 2012 Monini et al.openMonini P.; Cafaro A.; Srivastava I.; Moretti S.; Sharma V.; Andreini C.; Chiozzini C.; Ferrantelli F.; Pavone Cossut M.; Tripiciano A.; Nappi F.; Longo O; Bellino S.; Picconi O.; Fanales-Belasio E.;Borsetti A.; Toschi E.; Schiavoni I.; Bacigalupo I.; Kan E.; Sernicola L.; Maggiorella M.; Montin K.;Porcu M.; Leone P.; Leone P; Collacchi B.; Palladino C.; Ridolfi B.; Falchi M.; Macchia I.; UlmerJ.B.; Butto` S.; Sgadari C.; Magnani M.; Federico M.; Titti F.; Banci L.; Dallocchio F.; Rappuoli R.; Ensoli F.; Barnett S.W.; Garaci E.; Ensoli B.Monini, P.; Cafaro, A.; Srivastava, I.; Moretti, S.; Sharma, V.; Andreini, C.; Chiozzini, C.; Ferrantelli, F.; Pavone Cossut, M.; Tripiciano, A.; Nappi, F.; Longo, O; Bellino, S.; Picconi, O.; Fanales Belasio, E.; Borsetti, A.; Toschi, E.; Schiavoni, I.; Bacigalupo, I.; Kan, E.; Sernicola, L.; Maggiorella, M.; Montin, Katy; Porcu, M.; Leone, P.; Leone, P; Collacchi, B.; Palladino, C.; Ridolfi, B.; Falchi, M.; Macchia, I.; U. l. m. e. r. J., B.; Butto`, S.; Sgadari, C.; Magnani, M.; Federico, M.; Titti, F.; Banci, L.; Dallocchio, Franco Pasquale Filippo; Rappuoli, R.; Ensoli, F.; Barnett, S. W.; Garaci, E.; Ensoli, Barbar

Tat/Env complex and ternary Tat/Env/αvβ3 complex by modeling-docking calculations.

<p>(<b>A</b>) ribbon representation of the Tat/Env complex showing that the Env CD4 binding site and the RGD domain of Tat are both exposed. Color code: ΔV1-2 Env: blue; Tat: red; Tat-RGD: yellow. (<b>B</b>) Surface representation of the ternary Tat/Env/αvβ3 complex. Color code: ΔV1-2 Env: green; Tat: purple; αvβ3 integrin: grey. See experimental procedures for details.</p

Virological outcome in Tat/Env-vaccinated or control monkeys after intrarectal challenge with the SHIV_SF162P4cy (70 MID₅₀).

<p>Box plots of (A) Viral RNA, (B) proviral DNA in blood at 2, 3, 4 and 5 weeks after challenge, respectively; and (C) proviral DNA at week 4 after challenge in rectal tissue (RT) and inguinal lymph nodes (LN). Statistical analysis was performed by the one-sided Wilcoxon rank sum test. Red: monkeys vaccinated with Tat/Env (n = 6); blue: control animals (n = 6).</p

Rate and affinity constants for Tat/Env binding determined by surface plasmon resonance.

<p>k_on, association rate constant; k_off, dissociation rate constant; T_1/2, complex dissociation half life; K_d, binding dissociation constant. No binding was observed with monomeric wt Env.</p

Neutralization of Tat/Env complex entry or infection in MDDCs by sera from Tat-vaccinated HIV-infected individuals.

<p>(A) Percentage of MDDCs internalizing Env in the presence of sera from trial subjects (n = 7) at baseline (week 0, anti-Tat Ab negative, in blue) and after Tat vaccination (week 48, anti-Tat Ab positive, in red) in the absence (empty bars) or in the presence (filled bars) of Tat. The codes of the sera are indicated at the bottom of the bars. (B) Geometric mean (GM) of the ratio (week 48 vs baseline), with 95% confidence interval (CI) of the percentage of MDDCs internalizing Env in the presence or in the absence of Tat in trial subjects (n = 7). Statistical analysis was performed by the two-tailed Student’s <i>t</i>-test. (C) MDDC infection performed in duplicate for 48 h with pSF162LUC in the presence of coated cys₂₂ Tat (0.01 µM) or BSA (control) with sera from a representative trial subject before (baseline, anti-Tat Ab negative) and after Tat vaccination (anti-Tat positive). Sera prior to or after vaccination contained the same anti-Env Ab titers. Data are expressed as RLU.</p

Enhancement by soluble Tat of HIV-1 infection in MDDCs.

<p>Peak infection fold increase reached 3–8 days after infection with the R5 pSF162LUC in MDDCs from 10 different donors. The virus was pre-incubated with 1 µM cys₂₂ Tat or with PBS-0.1% BSA (control buffer). Fold increase were calculated as the ratio between the RLU from MDDCs infected with virus pre-incubated with cys₂₂ Tat and RLU from MDDCs infected with virus pre-incubated with control buffer (baseline RLU: 298.95) (both values had been previously subtracted of the uninfected MDDCs RLU background).</p

Tat-mediated entry of VLPs expressing R5 or X4 Env into MDDCs.

<p>(<b>A</b>) and (<b>B</b>): entry in immature MDDCs of null-VLPs (control) or (A) VLPs expressing R5-Env (HIV<b>-</b>1 BaL) (VLP-R5Env) or (B) VLPs expressing X4-Env (HIV<b>-</b>1 HXBc2) (VLP-X4Env), in the absence or presence of increasing concentrations of cys₂₂ Tat after 4 h of incubation, evaluated by flow cytometry. Results are shown as either dot-plot (A) or histograms (B). SSC: side scatter. Numbers in the insets represent the percentage of GFP positive cells. (<b>C</b>) Confocal microscopy analysis of VLP-R5Env entry into MDDCs in the presence of cys₂₂ Tat (60 nM) after 4 h of incubation (0.2 µm optical sections form the apical to the basal cell side).</p