Specific targeting to the Mycobacterium tuberculosis P-type ATPase Membrane Transporter, CtpF, of antituberculous compounds obtained by structure-based design.

Background: The resurgence of Mycobacterium tuberculosis (Mtb) strains that resist anti-tuberculosis (anti-TB) drugs used currently stresses the search for more effective low-toxicity drugs against new targets. Due to their role in ion homeostasis and virulence, Mtb plasma membrane P-type ATPases are interesting anti-TB targets, in particular, the Ca2+ transporting P2-type ATPase CtpF which is involved in oxidative stress response and persistence. Methods: In this study, the effect on the transcription level of the ctpF gene and other Mtb P2-type ATPases of two anti-Mtb hits was assessed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Both anti-Mtb hits ZINC14541509 and ZINC63908257 had been previously identified using pharmacophore-based virtual screening and MM-GBSA binding free energy. In addition, the bacterial activity of both compounds on Mycobacterium bovis was evaluated to see whether or not there is an effect on other mycobacteria of the Mtb complex. Results: qRT-PCR experiments showed that the ctpF transcription level was significantly higher in the presence of both compounds, especially ZINC14541509, strongly suggesting that CtpF may be a specific target of the selected compound. Conclusions: ZINC14541509 should be considered as an alternative for the structural-based design of novel anti-TB drugs.

Isolation of the Sarcoplasmic Reticulum Ca2+-ATPase from Rabbit Fast-Twitch Muscle.

The sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) is a membrane protein that is destabilized during purification in the absence of calcium ions. The disaccharide trehalose is a protein stabilizer that accumulates in the yeast cytoplasm when under stress. In the present work, SERCA was purified by including trehalose in the purification protocol. The purified SERCA showed high protein purity (~95%) and ATPase activity. ATP hydrolysis was dependent on the presence of Ca2+ and the enzyme kinetics showed a hyperbolic dependence on ATP (Km = 12.16 ± 2.25 µM ATP). FITC labeling showed the integrity of the ATP-binding site and the identity of the isolated enzyme as a P-type ATPase. Circular dichroism (CD) spectral changes at a wavelength of 225 nm were observed upon titration with ATP, indicating α-helical rearrangements in the nucleotide-binding domain (N-domain), which correlated with ATP affinity (Km). The presence of Ca2+ did not affect FITC labeling or the ATP-mediated structural changes at the N-domain. The use of trehalose in the SERCA purification protocol stabilized the enzyme. The isolated SERCA appears to be suitable for structural and ligand binding studies, e.g., for testing newly designed or natural inhibitors. The use of trehalose is recommended for the isolation of unstable enzymes.

The Functioning of Na+-ATPases from Protozoan Parasites: Are These Pumps Targets for Antiparasitic Drugs?

The ENA ATPases (from exitus natru: the exit of sodium) belonging to the P-type ATPases are structurally very similar to the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA); they exchange Na+ for H+ and, therefore, are also known as Na+-ATPases. ENA ATPases are required in alkaline milieu, as in the case for Aspergillus, where other transporters cannot mediate an uphill Na+ efflux. They are also important for salt tolerance, as described for Arabidopsis. During their life cycles, protozoan parasites might encounter a high pH environment, thus allowing consideration of ENA ATPases as possible targets for controlling certain severe parasitic diseases, such as Chagas' Disease. Phylogenetic analysis has now shown that, besides the types IIA, IIB, IIC, and IID P-type ATPases, there exists a 5th subgroup of ATPases classified as ATP4-type ATPases, found in Plasmodium falciparum and Toxoplasma gondii. In malaria, for example, some drugs targeting PfATP4 destroy Na+ homeostasis; these drugs, which include spiroindolones, are now in clinical trials. The ENA P-type (IID P-type ATPase) and ATP4-type ATPases have no structural homologue in mammalian cells, appearing only in fungi, plants, and protozoan parasites, e.g., Trypanosoma cruzi, Leishmania sp., Toxoplasma gondii, and Plasmodium falciparum. This exclusivity makes Na+-ATPase a potential candidate for the biologically-based design of new therapeutic interventions; for this reason, Na+-ATPases deserves more attention.

CtpB is a plasma membrane copper (I) transporting P-type ATPase of Mycobacterium tuberculosis.

BACKGROUND: The intracellular concentration of heavy-metal cations, such as copper, nickel, and zinc is pivotal for the mycobacterial response to the hostile environment inside macrophages. To date, copper transport mediated by P-type ATPases across the mycobacterial plasma membrane has not been sufficiently explored. RESULTS: In this work, the ATPase activity of the putative Mycobacterium tuberculosis P1B-type ATPase CtpB was associated with copper (I) transport from mycobacterial cells. Although CtpB heterologously expressed in M. smegmatis induced tolerance to toxic concentrations of Cu2+ and a metal preference for Cu+, the disruption of ctpB in M. tuberculosis cells did not promote impaired cell growth or heavy-metal accumulation in whole mutant cells in cultures under high doses of copper. In addition, the Cu+ ATPase activity of CtpB embedded in the plasma membrane showed features of high affinity/slow turnover ATPases, with enzymatic parameters KM 0.19 ± 0.04 µM and Vmax 2.29 ± 0.10 nmol/mg min. In contrast, the ctpB gene transcription was activated in cells under culture conditions that mimicked the hostile intraphagosomal environment, such as hypoxia, nitrosative and oxidative stress, but not under high doses of copper. CONCLUSIONS: The overall results suggest that M. tuberculosis CtpB is associated with Cu+ transport from mycobacterial cells possibly playing a role different from copper detoxification.

CtpB is a plasma membrane copper (I) transporting P-type ATPase of Mycobacterium tuberculosis

BACKGROUND: The intracellular concentration of heavy-metal cations, such as copper, nickel, and zinc is pivotal for the mycobacterial response to the hostile environment inside macrophages. To date, copper transport mediated by P-type ATPases across the mycobacterial plasma membrane has not been sufficiently explored. RESULTS: In this work, the ATPase activity of the putative Mycobacterium tuberculosis P1B-type ATPase CtpB was associated with copper (I) transport from mycobacterial cells. Although CtpB heterologously expressed in M. smegmatis induced tolerance to toxic concentrations of Cu2+ and a metal preference for Cu+, the disruption of ctpB in M. tuberculosis cells did not promote impaired cell growth or heavy-metal accumulation in whole mutant cells in cultures under high doses of copper. In addition, the Cu+ ATPase activity of CtpB embedded in the plasma mem-brane showed features of high affinity/slow turnover ATPases, with enzymatic parametersKM 0.19 ± 0.04 µM and Vmax 2.29 ± 0.10 nmol/mg min. In contrast, the ctpB gene transcription was activated in cells under culture conditions that mimicked the hostile intraphagosomal environment, such as hypoxia, nitrosative and oxidative stress, but not under high doses of copper. CONCLUSIONS: The overall results suggest that M. tuberculosis CtpB is associated with Cu+ transport from mycobacterial cells possibly playing a role different from copper detoxification.

Iron overload impact on P-ATPases.

Iron is a chemical element that is active in the fundamental physiological processes for human life, but its burden can be toxic to the body, mainly because of the stimulation of membrane lipid peroxidation. For this reason, the action of iron on many ATPases has been studied, especially on P-ATPases, such as the Na+,K+-ATPase and the Ca2+-ATPase. On the Fe2+-ATPase activity, the free iron acts as an activator, decreasing the intracellular Fe2+ and playing a protection role for the cell. On the Ca2+-ATPase activity, the iron overload decreases the enzyme activity, raising the cytoplasmic Ca2+ and decreasing the sarco/endoplasmic reticulum and the Golgi apparatus Ca2+ concentrations, which could promote an enzyme oxidation, nitration, and fragmentation. However, the iron overload effect on the Na+,K+-ATPase may change according to the tissue expressions. On the renal cells, as well as on the brain and the heart, iron promotes an enzyme inactivation, whereas its effect on the erythrocytes seems to be the opposite, directly stimulating the ATPase activity, or stimulating it by signaling pathways involving ROS and PKC. Modulations in the ATPase activity may impair the ionic transportation, which is essential for cell viability maintenance, inducing irreversible damage to the cell homeostasis. Here, we will discuss about the iron overload effect on the P-ATPases, such as the Na+,K+-ATPase, the Ca2+-ATPase, and the Fe2+-ATPase.

Spermine modulates fungal morphogenesis and activates plasma membrane H+-ATPase during yeast to hyphae transition.

Polyamines play a regulatory role in eukaryotic cell growth and morphogenesis. Despite many molecular advances, the underlying mechanism of action remains unclear. Here, we investigate a mechanism by which spermine affects the morphogenesis of a dimorphic fungal model of emerging relevance in plant interactions, Yarrowia lipolytica, through the recruitment of a phytohormone-like pathway involving activation of the plasma membrane P-type H+-ATPase. Morphological transition was followed microscopically, and the H+-ATPase activity was analyzed in isolated membrane vesicles. Proton flux and acidification were directly probed at living cell surfaces by a non-invasive selective ion electrode technique. Spermine and indol-3-acetic acid (IAA) induced the yeast-hypha transition, influencing the colony architecture. Spermine induced H+-ATPase activity and H+ efflux in living cells correlating with yeast-hypha dynamics. Pharmacological inhibition of spermine and IAA pathways prevented the physio-morphological responses, and indicated that spermine could act upstream of the IAA pathway. This study provides the first compelling evidence on the fungal morphogenesis and colony development as modulated by a spermine-induced acid growth mechanism analogous to that previously postulated for the multicellular growth regulation of plants.

The Parkinson-associated human P5B-ATPase ATP13A2 protects against the iron-induced cytotoxicity.

P-type ion pumps are membrane transporters that have been classified into five subfamilies termed P1-P5. The ion transported by the P5-ATPases is not known. Five genes named ATP13A1-ATP13A5 that belong to the P5-ATPase group are present in humans. Loss-of-function mutations in the ATP13A2 gene (PARK9, OMIM 610513) underlay a form of Parkinson's disease (PD) known as the Kufor-Rakeb syndrome (KRS), which belongs to the group of syndromes of neurodegeneration with brain iron accumulation (NBIA). Here we report that the cytotoxicity induced by iron exposure was two-fold reduced in CHO cells stably expressing the ATP13A2 recombinant protein (ATP13A2). Moreover, the iron content in ATP13A2 cells was lower than control cells stably expressing an inactive mutant of ATP13A2. ATP13A2 expression caused an enlargement of lysosomes and late endosomes. ATP13A2 cells exhibited a reduced iron-induced lysosome membrane permeabilization (LMP). These results suggest that ATP13A2 overexpression improves the lysosome membrane integrity and protects against the iron-induced cell damage.

Cross-talk between reactive oxygen species and polyamines in regulation of ion transport across the plasma membrane: implications for plant adaptive responses.

Many stresses are associated with increased accumulation of reactive oxygen species (ROS) and polyamines (PAs). PAs act as ROS scavengers, but export of putrescine and/or PAs to the apoplast and their catabolization by amine oxidases gives rise to H2O2 and other ROS, including hydroxyl radicals ((•)OH). PA catabolization-based signalling in apoplast is implemented in plant development and programmed cell death and in plant responses to a variety of biotic and abiotic stresses. Central to ROS signalling is the induction of Ca(2+) influx across the plasma membrane. Different ion conductances may be activated, depending on ROS, plant species, and tissue. Both H2O2 and (•)OH can activate hyperpolarization-activated Ca(2+)-permeable channels. (•)OH is also able to activate both outward K(+) current and weakly voltage-dependent conductance (ROSIC), with a variable cation-to-anion selectivity and sensitive to a variety of cation and anion channel blockers. Unexpectedly, PAs potentiated (•)OH-induced K(+) efflux in vivo, as well as ROSIC in isolated protoplasts. This synergistic effect is restricted to the mature root zone and is more pronounced in salt-sensitive cultivars compared with salt-tolerant ones. ROS and PAs suppress the activity of some constitutively expressed K(+) and non-selective cation channels. In addition, both (•)OH and PAs activate plasma membrane Ca(2+)-ATPase and affect H(+) pumping. Overall, (•)OH and PAs may provoke a substantial remodelling of cation and anion conductance at the plasma membrane and affect Ca(2+) signalling.

Toluene permeabilization differentially affects F- and P-type ATPase activities present in the plasma membrane of Streptococcus mutans

Streptococcus mutans membrane-bound P- and F-type ATPases are responsible for H+ extrusion from the cytoplasm thus keeping intracellular pH appropriate for cell metabolism. Toluene-permeabilized bacterial cells have long been used to study total membrane-bound ATPase activity, and to compare the properties of ATPase in situ with those in membrane-rich fractions. The aim of the present research was to determine if toluene permeabilization can significantly modify the activity of membrane-bound ATPase of both F-type and P-type. ATPase activity was assayed discontinuously by measuring phosphate release from ATP as substrate. Treatment of S. mutans membrane fractions with toluene reduced total ATPase activity by approximately 80 percent and did not allow differentiation between F- and P-type ATPase activities by use of the standard inhibitors vanadate (3 µM) and oligomycin (4 µg/mL). Transmission electron microscopy shows that, after S. mutans cells permeabilization with toluene, bacterial cell wall and plasma membrane are severely injured, causing cytoplasmic leakage. As a consequence, loss of cell viability and disruption of H+ extrusion were observed. These data suggest that treatment of S. mutans with toluene is an efficient method for cell disruption, but care should be taken in the interpretation of ATPase activity when toluene-permeabilized cells are used, because results may not reflect the real P- and F-type ATPase activities present in intact cell membranes. The mild conditions used for the preparation of membrane fractions may be more suitable to study specific ATPase activity in the presence of biological agents, since this method preserves ATPase selectivity for standard inhibitors.

Probing the SERCA1a sarcoplasmic reticulum Ca2+-ATPase phosphorylation-site mutant D351E with inorganic phosphate

The expression of sarcoplasmic reticulum SERCA1a Ca2+-ATPase wild-type and D351E mutants was optimized in yeast under the control of a galactose promoter. Fully active wild-type enzyme was recovered in yeast microsomal membrane fractions in sufficient amounts to permit a rapid and practical assay of ATP hydrolysis and phosphoenzyme formation from ATP or Pi. Mutant and wild-type Ca2+-ATPase were assayed for phosphorylation by Pi under conditions that are known to facilitate this reaction in the wild-type enzyme, including pH 6.0 or 7.0 at 25°C in the presence of dimethylsulfoxide. Although glutamyl (E) and aspartyl (D) residue side chains differ by only one methylene group, no phosphoenzyme could be detected in the D351E mutant, even upon the addition of 40 percent dimethylsulfoxide and 1 mM 32Pi in the presence of 10 mM EGTA and 5 mM MgCl2. These results show that in the D351E mutant, increasing hydrophobicity of the site with inorganic solvent was not a sufficient factor for the required abstraction of water in the reaction of E351 with Pi to form a glutamylphosphate (P-E351) phosphoenzyme moiety. Mutation D351E may disrupt the proposed alignment of the reactive water molecule with the aspartylphosphate (P-D351) moiety in the phosphorylation site, which may be an essential alignment both in the forward reaction (hydrolysis of aspartylphosphate) and in the reverse reaction (abstraction of water upon formation of an aspartylphosphate intermediate).

Geobacillus stearothermophilus LV cadA gene mediates resistance to cadmium, lead and zinc in zntA mutants of Salmonella entérica serovar Typhimurium

Salmonella entérica serovar Typhimurium cells expressing the cadA gene of Geobacillus stearothermophilus LV exhibit a hypersensitive phenotype to cadmium chloride. Deletion of the ORF STM3576 from the Salmonella genome resulted in cadmium, lead and zinc sensitivity, confirming that this ORF is a homologue of the zntA gene. The observed sensitivity was reverted upon expression of the G. stearothermophilus LV cadA gene. These results indicate that the cadA gene product is involved in Cd, Pb and Zn resistance as a classical P-type ATPase and strongly suggest that the observed hypersensitive phenotype to these metals can be related to the function of the host ·zntA gene product.