Robust ATP Research Articles

A genomic database furnishes minimal functional glycyl-tRNA synthetases homologous to other, designed class II urzymes.

The hypothesis that conserved core catalytic sites could represent ancestral aminoacyl-tRNA synthetases (AARS) drove the design of functional TrpRS, LeuRS, and HisRS 'urzymes'. We describe here new urzymes detected in the genomic record of the arctic fox, Vulpes lagopus. They are homologous to the α-subunit of bacterial heterotetrameric Class II glycyl-tRNA synthetase (GlyRS-B) enzymes. AlphaFold2 predicted that the N-terminal 81 amino acids would adopt a 3D structure nearly identical to our designed HisRS urzyme (HisCA1). We expressed and purified that N-terminal segment and the spliced open reading frame GlyCA1-2. Both exhibit robust single-turnover burst sizes and ATP consumption rates higher than those previously published for HisCA urzymes and comparable to those for LeuAC and TrpAC. GlyCA is more than twice as active in glycine activation by adenosine triphosphate as the full-length GlyRS-B α2 dimer. Michaelis-Menten rate constants for all three substrates reveal significant coupling between Exon2 and both substrates. GlyCA activation favors Class II amino acids that complement those favored by HisCA and LeuAC. Structural features help explain these results. These minimalist GlyRS catalysts are thus homologous to previously described urzymes. Their properties reinforce the notion that urzymes may have the requisite catalytic activities to implement a reduced, ancestral genetic coding alphabet.

UAV air combat autonomous trajectory planning method based on robust adversarial reinforcement learning

The poor robustness of the air combat autonomous trajectory planning strategy (ATP) trained through vanilla reinforcement learning (RL) methods is attributed to its dependence on the source environment. When this ATP is transformed from the source to the target environment, it may fail to lead to an effective dogfight victory due to disturbances, which can potentially threaten the flight safety of the unmanned combat aerial vehicle (UCAV). In order to enhance the robustness of ATP, we propose a robust RL-based method for generating an ATP strategy. This method can effectively consider uncertainties in the state and potential model misspecifications. First, a jointly trained adversary is designed to apply disturbances in the environment, forming a two-player zero-sum game with the ATP agent. To solve this game problem and learn a robust ATP, the robust adversarial RL method (RARL) is applied. Additionally, in order to prevent non-convergence of strategies resulting from the introduction of the RARL algorithm, a curriculum learning method is proposed that emulates the way human pilots learn, gradually progressing from simpler to more challenging courses. Finally, a quantitative method for evaluating the robustness of the ATP is proposed, taking into consideration the differences in ATP performance between the source and target combat environments. The results of robustness estimations demonstrate that the RARL algorithm, which is proposed herein, effectively enhances the ATP's robustness.

Extracellular histone proteins activate P2XR7 channel current.

Extracellular histone proteins are elevated in circulation after injury or activation of the innate immune response. In resistance-size arteries, extracellular histone proteins increased endothelial cell (EC) Ca2+ influx and propidium iodide (PI) labeling, but paradoxically decreased vasodilation. These observations could be explained by the activation of an EC resident non-selective cation channel. We tested the hypothesis that the ionotropic purinergic receptor 7 (P2XR7), a non-selective cation channel associated with cationic dye uptake, is activated by histone proteins. We expressed mouse P2XR7 (C57BL/6J variant 451L) in heterologous cells and measured inward cation current using two-electrode voltage clamp (TEVC). Cells expressing mouse P2XR7 had robust ATP- and histone-evoked inward cation currents. ATP- and histone-evoked currents reversed approximately at the same potential. Current decay with agonist removal was slower for histone-evoked than ATP- or BzATP-evoked currents. As with ATP-evoked P2XR7 currents, histone-evoked currents were inhibited by non-selective P2XR7 antagonists (Suramin, PPADS, and TNP-ATP). Selective P2XR7 antagonists, AZ10606120, A438079, GW791343, and AZ11645373, inhibited ATP-evoked P2XR7 currents but did not inhibit histone-evoked P2XR7 currents. As previously reported with ATP-evoked currents, histone-evoked P2XR7 currents were also increased in conditions of low extracellular Ca2+. These data demonstrate that P2XR7 is necessary and sufficient for histone-evoked inward cation currents in a heterologous expression system. These results provide insight into a new allosteric mechanism of P2XR7 activation by histone proteins.

Extracellular histone proteins are novel activators of P2XR7 channel current

Extracellular histone proteins have been identified as important damage associated molecules that contribute to vascular dysfunction. Histones are elevated in circulation after traumatic injury and activation of the innate immune response. In prior work, we demonstrated that extracellular histone proteins induced endothelial cell (EC) Ca 2+ influx, increased EC propidium iodide labeling, and decreased vasodilatory responses. These paradoxical observations could be explained by activation of a non-selective cation channel. The ionotropic purinergic receptor 7 (P2XR7) stands out as a candidate as it has been shown to form a high-conductance, poorly selective, cation channel capable of conducting large cationic dyes. Herein, we test the hypothesis that P2XR7 channels are activated by histone proteins. We expressed mouse P2XR7 (C57BL/6J variant 451L) in heterologous cells ( Xenopus oocytes) and measured inward cation current using two-electrode voltage clamp (TEVC). Cells expressing mouse P2XR7 had robust ATP- and histone-evoked inward cation currents. ATP- and histone-evoked currents reversed approximately at the same potential. Current decay with agonist removal was much slower for histone-evoked than ATP- or BzATP-evoked currents. As with ATP-evoked P2XR7 currents, histone-evoked currents were inhibited by non-selective P2XR7 antagonists (Suramin, PPADS, and TNP-ATP). GW791343, a selective P2XR7 antagonist with a known binding site distinct from the ATP binding site, inhibited ATP-evoked currents but did not inhibit histone-evoked currents. As with ATP-evoked currents, histone-evoked P2XR7 currents were also increased in conditions of low extracellular Ca 2+ . These data demonstrate that P2XR7 is necessary and sufficient for histone-evoked inward cation currents. These results suggest a new allosteric mechanism of P2XR7 activation by histone proteins that may guide the development of more effective P2XR7 pharmacology and therapeutic strategies for a wide array of pathologies that involve P2XR7 function. DMC is supported by NIH R00HL133451 and R01HL155180 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Fall in the ATP levels in the red blood cells in ApoE-LDLR double-deficient mice model prior to atherosclerosis development

Abstract Background It was previously reported that red blood cells (RBCs) regulate blood flow via RBC-derived ATP [1]. Nevertheless, to the best of our knowledge, no study was performed to characterize possible alterations in RBC ATP levels in ApoE/LDLR−/− mice [2] which constitute a reliable model of human atherosclerosis, displaying distinct erythropathy [3]. Interestingly, young ApoE/LDLR−/− mice display higher exercise capacity and higher O2 carrying capacity of RBCs, as compared to their age-matched control [3,4]. However, it is not known whether increased exerise capacity in ApoE/LDLR−/− mice is linked to altered ATP release from RBCs. It was previously reported that prostacyclin analogs, known atheroprotective agents, which preserve vascular endothelium functions in various diseases [5,6], induce ATP release from human RBCs [1,7]. Purpose To characterize intra- and extra-cellular ATP levels in RBCs isolated from ApoE/LDLR−/− mice in comparison to control mice. Methods All experiments were conducted according to the Guidelines for Animal Care and Treatment of the EU and to the Local Ethical Committee on Animal Testing at our University. For experiments, 8- and 24-week-old C57BL/6 control mice (N=4–6 and N=5–8, respectively) and ApoE/LDLR−/− mice (N=4–7 and N=4–5, respectively) were used. The complete blood count, RBC morphology, biochemistry of blood plasma, RBC deformability, and RBC phosphatidylserine exposure were assessed. The intra- and extra-cellular ATP levels and ATP release from RBCs due to administration of iloprost (100 nM, 1 μM, 10 μm) were studied. Results Intracellular ATP level in RBCs isolated from 8-week-old ApoE/LDLR−/− mice was considerably lower as compared to their age-matched control (7.72±0.77 and 21.23±3.40 pmoles/1x106 RBCs, respectively). In 24-week-old mice, intracellular ATP in RBCs was low not only in ApoE/LDLR−/− mice but also in control mice (8.70±1.30 and 6.27±0.96 pmoles/1x106 RBCs, respectively). Basal extracellular ATP released from RBCs over 30 min incubation was 400 times lower than corresponding intracellular level, and mirrored intracellular ATP levels in all studied groups. Iloprost (100 nM–10 μM) did not produce robust ATP release in any of the studied groups, with only some effects when the highest concentration was used (10 μM). Conclusion(s) Hypercholesterolemia-dependent changes in young ApoE/LDLR−/− mice prior to atherosclerotic plaque development may induce a severe fall in intracellular ATP levels in the RBCs that might be linked to a possible diversion of glycolysis to 2,3-DPG to increase oxygen delivery, and might contribute to the alterations in RBC-dependent regulation of blood flow in ApoE/LDLR−/−. Further studies are required to mechanistically explain these findings. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): 1) National Science Centre, Poland (UMO 2016/23/B/ST4/00795) 2) the Innovation Incubator 4.0 project funded by the Ministry of Science and Higher Education, Poland (POIR 2014-2020)

Interactions of human mesenchymal stromal cells with peripheral blood mononuclear cells in a Mitogenic proliferation assay

BackgroundImmunomodulation by mesenchymal stromal cells (MSCs) is a potentially important therapeutic modality. MSCs suppress peripheral blood mononuclear cell (PBMC) proliferation in vitro, suggesting a mechanism for suppressing inflammatory responses in vivo. This study details the interactions of PBMCs and MSCs. MethodsPooled human PBMCs and MSCs were co-cultured at different MSC:PBMC ratios and harvested from 0 to 120 h, with and without phytohaemagglutin A (PHA) stimulation. Proliferation of adherent MSCs and non-adherent PBMCs was assessed by quantitation of ATP levels. PBMC surface marker expression was analyzed by flow cytometry. Indoleamine 2,3-dioxygenase (IDO) activity was determined by kynurenine assay and IDO mRNA by RT-PCR. Cytokine release was measured by ELISA. Immunofluorescent microscopy detected MSC, PBMC, monocyte (CD14+) and apoptotic events. ResultsPBMC proliferation in response to PHA gave a robust ATP signal by 72 h, which was suppressed by co-culture with densely plated MSCs. Very low level MSC seeding densities relative to PBMC number reproducibly stimulated PBMC proliferation. The CD4+/CD3+ population significantly decreased over time while the CD8+/CD3+ population significantly increased. No change in CD4+/CD8+ ratio is seen with high density MSC co-culture; very low density MSCs augment the changes seen in PHA stimulated PBMCs alone. IDO activity in MSCs co-cultured with PBMCs correlated with PBMC suppression. MSCs increased the secretion of IL-10 and IL-6 from stimulated co-cultures and decreased TNF-α secretion. In stimulated co-culture, low density MSCs decreased in number; fluorescence immunomicroscopy detected association of PBMC with MSC and phosphatidyl serine externalization in both cell populations. ConclusionsA bidirectional interaction between MSCs and PBMCs occurs during co-culture. High numbers of MSCs inhibit PHA-stimulated PBMC proliferation and the PBMC response to stimulation; low numbers of MSCs augment these responses. Low density MSCs are susceptible to attrition, apparently by PBMC-induced apoptosis. These results may have direct application when considering therapeutic dosing of patients; low MSC doses may have unintended detrimental consequences.

The Mineralization Regulator ANKH Mediates Cellular Efflux of ATP, Not Pyrophosphate.

The plasma membrane protein ankylosis homologue (ANKH, mouse ortholog: Ank) prevents pathological mineralization of joints by controlling extracellular levels of the mineralization inhibitor pyrophosphate (PPi). It was long thought that ANKH acts by transporting PPi into the joints. We recently showed that when overproduced in HEK293 cells, ANKH mediates release of large amounts of nucleoside triphosphates (NTPs), predominantly ATP, into the culture medium. ATP is converted extracellularly into PPi and AMP by the ectoenzyme ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). We could not rule out, however, that cells also release PPi directly via ANKH. We now addressed the question of whether PPi leaves cells via ANKH using HEK293 cells that completely lack ENPP1. Introduction of ANKH in these ENPP1-deficient HEK293 cells resulted in robust cellular ATP release without the concomitant increase in extracellular PPi found in ENPP1-proficient cells. Ank activity was previously shown to be responsible for about 75% of the PPi found in mouse bones. However, bones of Enpp1-/- mice contained <2.5% of the PPi found in bones of wild-type mice, showing that Enpp1 activity is also a prerequisite for Ank-dependent PPi incorporation into the mineralized bone matrix in vivo. Hence, ATP release precedes ENPP1-mediated PPi formation. We find that ANKH also provides about 25% of plasma PPi, whereas we have previously shown that 60% to 70% of plasma PPi is derived from the NTPs extruded by the ABC transporter, ABCC6. Both transporters that keep plasma PPi at sufficient levels to prevent pathological calcification therefore do so by extruding NTPs rather than PPi itself. © 2022 American Society for Bone and Mineral Research (ASBMR).

Kinetic coupling of the respiratory chain with ATP synthase, but not proton gradients, drives ATP production in cristae membranes

Mitochondria have a characteristic ultrastructure with invaginations of the inner membrane called cristae that contain the protein complexes of the oxidative phosphorylation system. How this particular morphology of the respiratory membrane impacts energy conversion is currently unknown. One proposed role of cristae formation is to facilitate the establishment of local proton gradients to fuel ATP synthesis. Here, we determined the local pH values at defined sublocations within mitochondria of respiring yeast cells by fusing a pH-sensitive GFP to proteins residing in different mitochondrial subcompartments. Only a small proton gradient was detected over the inner membrane in wild type or cristae-lacking cells. Conversely, the obtained pH values did barely permit ATP synthesis in a reconstituted system containing purified yeast F1F0 ATP synthase, although, thermodynamically, a sufficiently high driving force was applied. At higher driving forces, where robust ATP synthesis was observed, a P-side pH value of 6 increased the ATP synthesis rate 3-fold compared to pH 7. In contrast, when ATP synthase was coreconstituted with an active proton-translocating cytochrome oxidase, ATP synthesis readily occurred at the measured, physiological pH values. Our study thus reveals that the morphology of the inner membrane does not influence the subcompartmental pH values and is not necessary for robust oxidative phosphorylation in mitochondria. Instead, it is likely that the dense packing of the oxidative phosphorylation complexes in the cristae membranes assists kinetic coupling between proton pumping and ATP synthesis.

Analysis of Platelet Alpha and Dense Granule Release Using Small Volumes of Blood

A significant limitation of the gold-standard platelet function test, light transmission aggregometry (LTA), is the relatively large volume of blood required, as well as need for a platelet count in the resulting platelet-rich plasma of not much less than 100,000/μL, thus potentially precluding this test in pediatric and thrombocytopenic patients. Flow cytometric assessment of platelet activation in diluted whole blood avoids these limitations, typically employing anti-CD62P (P-Selectin) binding reflecting alpha granule release and PAC-1 binding reflecting conversion of GPIIb/IIIa to its active conformation. We have recently reported the use of tandem mass spectrometry to assess platelet dense granule release of deuterated serotonin in comparably diluted whole blood samples (https://doi.org/10.1093/ajcp/aqz094). In the present study we examine the possibility of adapting and optimizing the luciferin-luciferase (L-L) approach often used together with LTA for the assay of platelet dense granule ATP release from 10-fold diluted whole blood in parallel with flow cytometric analysis. We optimized the L-L reaction with commercially available reagents and used a conventional lumi-aggregometer to measure real-time secretion of platelet dense granules following agonist stimulation in 10-fold diluted whole blood obtained from healthy adult volunteers, with or without specimen stirring at 1000 RPM. Direct comparison of optimized reagents to a commercial L-L reagent kit showed an improved lower limit of detection for exogenously added ATP by at least one order of magnitude. Initial studies were performed under non-stirring conditions. Dose-dependency of agonist was observed, with representative results for released ATP of 243.1 ± 29.7 pmol/107 platelets in response to 40 μM TRAP (thrombin receptor-activating peptide) and 18.9 ± 2.4 pmol/107 platelets in response to 1 nM convulxin (GPVI receptor activator) (mean ± SEM, N = 15). In contrast, virtually no ATP secretion was observed in response to 10 μM ADP or to 10 μg/mL collagen. Despite the quite low platelet count (as low as 11,000/μL to date) following the 10-fold dilution of whole blood, introducing 1000 RPM stirring to these samples now permitted robust ATP release in response to collagen as low as 2 μg/mL. There continued to be little or no ATP release, however, in response to 10 μM ADP even with the addition of stirring. Flow cytometry was used to interrogate agonist-stimulated alpha granule release and GPIIb/IIIa conformation change. Following incubations with agonist and fluorescently-labeled antibodies, the 10-fold diluted whole blood was further diluted an additional 10-fold, and gating based on light scatter and binding of anti-CD61 antibody used to identify individual platelets. Under non-stirring conditions, TRAP, ADP, and convulxin all produced strong P-selectin exposure, as well as conversion of GPIIb/IIIa to its active conformation. Analysis of samples that had undergone 1000 RPM stirring during incubation with agonist showed quite similar results in response to TRAP. While still strongly positive, the responses to ADP in the stirred specimens showed decreases typically of at least 50%. Without stirring, platelet responses to 2-10 μg/mL collagen were virtually absent, whereas in the stirred specimens significantly positive anti-CD62P and PAC-1 binding were now observed. The combined flow cytometric and L-L studies required &lt;1 mL blood. The results demonstrate the dramatic differences observed in the release of platelet alpha and dense granules, depending upon conditions and assay systems employed. We have now shown that with agonist addition to the same 10-fold diluted whole blood samples employed in flow cytometric platelet analysis, dense granule release may also be assessed by modifications of the L-L methodology already employed for lumi-aggregation studies in many laboratories. We have additionally shown that by employing stirring of the specimen during incubation with agonist, collagen itself may be employed as platelet stimulus, rather than having to rely upon convulxin. The difference in requirements for alpha versus dense granule release, however, remains exemplified by ADP: While a potent stimulus of alpha granule release, ADP employed in the 10-fold diluted whole blood setting appeared incapable of producing significant dense granule release either in the absence or presence of sample stirring. Disclosures Wool: Diagnostica Stago: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract B199: Potent induction of immunogenic cell death by PT-112

Abstract Purpose: To characterize the ability of PT-112 to induce immunogenic cell death (ICD) in vitro and in vivo. Background: PT-112 is a novel chemical entity consisting of a platinum core conjugated to a pyrophosphate and diaminocyclohexane groups. In vitro studies demonstrated that PT-112 has both cytostatic and cytotoxic effects on human cancer cells, two effects that are seen in the absence of robust binding to nuclear DNA. Accordingly, PT-112 potency is largely unaffected by functional DNA repair pathways, suggesting that PT-112 operates with mechanisms that differ from conventional DNA-damaging chemotherapies. PT-112 is currently under phase I/II clinical development in patients with solid tumors and hematologic malignancies, both as monotherapy and in combination with a PD-L1 inhibitor. Interim reports have established encouraging tolerability and signals of single-agent anticancer activity. Previous work with human colorectal cancer HCT-116 cells demonstrated that PT-112 causes the release of ICD-relevant damage-associated molecular patterns (DAMPs). ICD is a particularly relevant form of cell death for cancer therapy as it can drive tumor-targeting immune responses of clinical relevance. Here, we tested the ability of PT-112 to induce bona fide ICD in murine breast carcinoma TSA cells by in vitro DAMP release studies and in vivo gold-standard vaccination experiments. Methods: TSA cells were exposed to PT-112, cisplatin (negative ICD control) or mitoxantrone (MTX, positive ICD control) at different concentrations for 24 and 48 hours, followed by cytofluorometric assessment of cell number, viability, cell cycle distribution and exposure of the membrane-assocaited DAMP calreticulin (CRT). In addition, culture supernatants were assayed for the release of two other DAMPs, namely ATP and HMGB1, by luminometric tests and ELISA, respectively. BALB/c mice were vaccinated with TSA cells succumbing to optimal PT-112 (or MTX and cisplatin, as positive and negative controls, respectively) concentrations or mock vaccinated (with PBS). After 14 days this was followed by a rechallenge with living TSA cells and scoring of tumor-free survival (TFS) and tumor growth. Results: In vitro, PT-112 caused a robust cytostatic and cytotoxic effect on TSA cells that was accompanied by robust CRT exposure, ATP secretion and HMGB1 release. In line with such an ICD-compatible profile, 10/10 mice vaccinated with PT-112-treated TSA cells were tumor-free after the 1st rechallenge, and also rejected a subsequent challenge performed 1 month later, suggesting the establishment of long-term immunologic anticancer memory. As expected, cisplatin-treated TSA cells failed to mediate a similar effect (TFS at D7 after rechallenge: 10%), as did the mock vaccination arm (TFS at D7: 20%). MTX-treated cells conferred partial protection (TFS at D7: 40%), and the developing tumors had significantly delayed kinetics as compared to tumors evolving in mock-vaccinated mice. Conclusions: In the TSA model system of ICD vaccination, PT-112 is superior to MTX at inducing ICD and the associated establishment of immunologic anticancer memory in vivo. These data provide a robust rationale to combining PT-112 with checkpoint inhibitors in the clinic, to achieve maximal therapeutic effects potentially coupled with long-term protection from metastatic reactivation. Citation Format: Takahiro Yamazaki, Tyler David Ames, Lorenzo Galluzzi. Potent induction of immunogenic cell death by PT-112 [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B199.

ORP4L is essential for T-cell acute lymphoblastic leukemia cell survival.

Metabolic pathways are reprogrammed in cancer to support cell survival. Here, we report that T-cell acute lymphoblastic leukemia (T-ALL) cells are characterized by increased oxidative phosphorylation and robust ATP production. We demonstrate that ORP4L is expressed in T-ALL but not normal T-cells and its abundance is proportional to cellular ATP. ORP4L acts as an adaptor/scaffold assembling CD3ɛ, Gαq/11 and PLCβ3 into a complex that activates PLCβ3. PLCβ3 catalyzes IP3 production in T-ALL as opposed to PLCγ1 in normal T-cells. Up-regulation of ORP4L thus results in a switch in the enzyme responsible for IP3-induced endoplasmic reticulum Ca2+ release and oxidative phosphorylation. ORP4L knockdown results in suboptimal bioenergetics, cell death and abrogation of T-ALL engraftment in vivo. In summary, we uncovered a signalling pathway operating specifically in T-ALL cells in which ORP4L mediates G protein-coupled ligand-induced PLCβ3 activation, resulting in an increase of mitochondrial respiration for cell survival. Targeting ORP4L might represent a promising approach for T-ALL treatment.

Conformational Changes of the Multidrug Transporter P-Glycoprotein in Solution and Lipid Discs

P-glycoprotein (Pgp) exports hundreds of chemically unrelated, hydrophobic compounds out of cells. Since Pgp can greatly affect bioavailability, pharmacokinetics and efficacy of therapeutic drugs, there is great interest in understanding the mechanism by which drugs are extruded. Pgp is a prototype ABC transporter with two nucleotide binding domains (NBDs) that bind and hydrolyze ATP, leading to extensive conformational changes across the transmembrane domains that result in substrate translocation across the cell membrane. According to current alternating access models, binding of ATP promotes NBDs dimerization (outward-facing conformation), and ATP-hydrolysis leads to their dissociation (inward-facing conformation). Despite decades of biochemical/biophysical studies, the mechanism by which the NBDs control these conformational changes is still controversial. We have used luminescence resonance energy transfer (LRET) to measure distances between the two NBDs during the ATP hydrolysis cycle. Pgp was labeled with the LRET probes, reconstituted in a lipid bilayer nanodisc, and the distance between the NBDs was measured at 37°C in nucleotide-free, ATP-bound, MgATP hydrolysis conditions. Pgp in nanodiscs showed increased basal ATPase activity and robust verapamil-stimulated ATP hydrolysis. Our LRET results suggest that the NBDs associate and dissociate in response to ATP binding and hydrolysis. Like the bacterial homolog MsbA, Pgp showed two main conformations (dimeric NBDs and dissociated NBDs) that coexist in equilibrium. However, the proportion of molecules in each conformation and the ATP-hydrolsysis driven changes are distinctly different suggesting that Pgp predominantly resides in the dissociated NBD state. The results are consistent with a recent snapshots analysis at ambient temperature by negative stain electron microscopy. The impact on substrate translocation mechanisms in Pgp vs. MsbA will be discussed. The LRET studies open new possibilities of studying conformational changes at physiological temperatures under native-like conditions.

Subunit asymmetry and roles of conformational switching in the hexameric AAA+ ring of ClpX.

The hexameric AAA+ ring of Escherichia. coli ClpX, an ATP-dependent protein unfolding and translocation machine, functions with the ClpP peptidase to degrade target substrates. For efficient function, ClpX subunits must switch between nucleotide-loadable (L) and nucleotide-unloadable (U) conformations, but the roles of switching are uncertain. Moreover, it is controversial whether working AAA+ ring enzymes assume symmetric or asymmetric conformations. Here, we show that a covalent ClpX ring with one subunit locked in the U conformation catalyzes robust ATP-hydrolysis, with each unlocked subunit able to bind and hydrolyze ATP, albeit with highly asymmetric position-specific affinities. Preventing U⇔L interconversion in one subunit alters the cooperativity of ATP hydrolysis and reduces the efficiency of substrate binding, unfolding, and degradation, showing that conformational switching enhances multiple aspects of wild-type ClpX function. These results support an asymmetric and probabilistic model of AAA+ ring activity.

Evolutionarily Conserved Roles of the Dicer Helicase Domain in Regulating RNA Interference Processing

The enzyme Dicer generates 21-25 nucleotide RNAs that target specific mRNAs for silencing during RNA interference and related pathways. Although their active sites and RNA binding regions are functionally conserved, the helicase domains have distinct activities in the context of different Dicer enzymes. To examine the evolutionary origins of Dicer helicase functions, we investigated two related Dicer enzymes from the thermophilic fungus Sporotrichum thermophile. RNA cleavage assays showed that S. thermophile Dicer-1 (StDicer-1) can process hairpin precursor microRNAs, whereas StDicer-2 can only cleave linear double-stranded RNAs. Furthermore, only StDicer-2 possesses robust ATP hydrolytic activity in the presence of double-stranded RNA. Deletion of the StDicer-2 helicase domain increases both StDicer-2 cleavage activity and affinity for hairpin RNA. Notably, both StDicer-1 and StDicer-2 could complement the distantly related yeast Schizosaccharomyces pombe lacking its endogenous Dicer gene but only in their full-length forms, underscoring the importance of the helicase domain. These results suggest an in vivo regulatory function for the helicase domain that may be conserved from fungi to humans.

De novo Fatty Acid Biosynthesis Contributes Significantly to Establishment of a Bioenergetically Favorable Environment for Vaccinia Virus Infection

The poxvirus life cycle, although physically autonomous from the host nucleus, is nevertheless dependent upon cellular functions. A requirement for de novo fatty acid biosynthesis was implied by our previous demonstration that cerulenin, a fatty acid synthase inhibitor, impaired vaccinia virus production. Here we show that additional inhibitors of this pathway, TOFA and C75, reduce viral yield significantly, with partial rescue provided by exogenous palmitate, the pathway's end-product. Palmitate's major role during infection is not for phospholipid synthesis or protein palmitoylation. Instead, the mitochondrial import and β-oxidation of palmitate are essential, as shown by the impact of etomoxir and trimetazidine, which target these two processes respectively. Moreover, the impact of these inhibitors is exacerbated in the absence of exogenous glucose, which is otherwise dispensable for infection. In contrast to glucose, glutamine is essential for productive viral infection, providing intermediates that sustain the TCA cycle (anaplerosis). Cumulatively, these data suggest that productive infection requires the mitochondrial β-oxidation of palmitate which drives the TCA cycle and energy production. Additionally, infection causes a significant rise in the cellular oxygen consumption rate (ATP synthesis) that is ablated by etomoxir. The biochemical progression of the vaccinia life cycle is not impaired in the presence of TOFA, C75, or etomoxir, although the levels of viral DNA and proteins synthesized are somewhat diminished. However, by reversibly arresting infections at the onset of morphogenesis, and then monitoring virus production after release of the block, we determined that virion assembly is highly sensitive to TOFA and C75. Electron microscopic analysis of cells released into C75 revealed fragmented aggregates of viroplasm which failed to be enclosed by developing virion membranes. Taken together, these data indicate that vaccinia infection, and in particular virion assembly, relies on the synthesis and mitochondrial import of fatty acids, where their β-oxidation drives robust ATP production.

A Conserved Mechanism of RNA Substrate Recognition and Cleavage by Fungal Dicers

Dicer is a central enzyme for processing small RNAs in RNA interference. It is able to cleave both hairpin and long double-stranded RNA precursors into microRNAs and small interfering RNAs respectively. While humans have one Dicer that is able to process both substrates, many organisms have multiple copies. Understanding how organisms with two Dicers chose specific RNAs for cleavage could give insight into how the human protein functions. We have turned to the thermophilic fungus Sporotrichum thermophile as a model system to purify stable Dicers for biochemical dissection. While the function of these two Dicers was thought to be partially redundant, our results show that they cleave different RNA substrates, similar to what is observed in the Drosophila melanogaster system. S. thermophile Dicer-1 dices hairpin precursor microRNAs faster than long double-stranded RNAs while S. thermophile Dicer-2 can only cleave perfect RNA duplexes accurately with the requirement for ATP. Removal of the helicase domain from Dicer-2 relieves the requirement of ATP for cleavage and allows the enzyme to process hairpin substrates. In addition, only Dicer-2 shows robust ATP hydrolysis in the presence of dsRNA and partial stimulation in the presence of single-stranded RNA or an RNA/DNA hybrid. The conserved function of these two Dicers may indicate a common mechanism used by eukaryotes to process small RNAs.

Roles of the N domain of the AAA+ Lon protease in substrate recognition, allosteric regulation and chaperone activity

Degron binding regulates the activities of the AAA+ Lon protease in addition to targeting proteins for degradation. The sul20 degron from the cell-division inhibitor SulA is shown here to bind to the N domain of Escherichia coli Lon, and the recognition site is identified by cross-linking and scanning for mutations that prevent sul20-peptide binding. These N-domain mutations limit the rates of proteolysis of model sul20-tagged substrates and ATP hydrolysis by an allosteric mechanism. Lon inactivation of SulA in vivo requires binding to the N domain and robust ATP hydrolysis but does not require degradation or translocation into the proteolytic chamber. Lon-mediated relief of proteotoxic stress and protein aggregation in vivo can also occur without degradation but is not dependent on robust ATP hydrolysis. In combination, these results demonstrate that Lon can function as a protease or a chaperone and reveal that some of its ATP-dependent biological activities do not require translocation.

Mutations on the N-Terminal Edge of the DELSEED Loop in either the α or β Subunit of the Mitochondrial F 1 -ATPase Enhance ATP Hydrolysis in the Absence of the Central γ Rotor

F(1)-ATPase is a rotary molecular machine with a subunit stoichiometry of α(3)β(3)γ(1)δ(1)ε(1). It has a robust ATP-hydrolyzing activity due to effective cooperativity between the three catalytic sites. It is believed that the central γ rotor dictates the sequential conformational changes to the catalytic sites in the α(3)β(3) core to achieve cooperativity. However, recent studies of the thermophilic Bacillus PS3 F(1)-ATPase have suggested that the α(3)β(3) core can intrinsically undergo unidirectional cooperative catalysis (T. Uchihashi et al., Science 333:755-758, 2011). The mechanism of this γ-independent ATP-hydrolyzing mode is unclear. Here, a unique genetic screen allowed us to identify specific mutations in the α and β subunits that stimulate ATP hydrolysis by the mitochondrial F(1)-ATPase in the absence of γ. We found that the F446I mutation in the α subunit and G419D mutation in the β subunit suppress cell death by the loss of mitochondrial DNA (ρ(o)) in a Kluyveromyces lactis mutant lacking γ. In organello ATPase assays showed that the mutant but not the wild-type γ-less F(1) complexes retained 21.7 to 44.6% of the native F(1)-ATPase activity. The γ-less F(1) subcomplex was assembled but was structurally and functionally labile in vitro. Phe446 in the α subunit and Gly419 in the β subunit are located on the N-terminal edge of the DELSEED loops in both subunits. Mutations in these two sites likely enhance the transmission of catalytically required conformational changes to an adjacent α or β subunit, thereby allowing robust ATP hydrolysis and cell survival under ρ(o) conditions. This work may help our understanding of the structural elements required for ATP hydrolysis by the α(3)β(3) subcomplex.

Function without form: an ongoing search for maxi-anion channel proteins. Focus on “Maxi-anion channel and pannexin 1 hemichannel constitute separate pathways for swelling-induced ATP release in murine L929 fibrosarcoma cells”

<i>Function without form: an ongoing search for maxi-anion channel proteins</i>. Focus on “Maxi-anion channel and pannexin 1 hemichannel constitute separate pathways for swelling-induced ATP release in murine L929 fibrosarcoma cells”

Activity of Clinically Relevant Antimalarial Drugs on Plasmodium falciparum Mature Gametocytes in an ATP Bioluminescence “Transmission Blocking” Assay

BackgroundCurrent anti-malarial drugs have been selected on the basis of their activity against the symptom-causing asexual blood stage of the parasite. Which of these drugs also target gametocytes, in the sexual stage responsible for disease transmission, remains unknown. Blocking transmission is one of the main strategies in the eradication agenda and requires the identification of new molecules that are active against gametocytes. However, to date, the main limitation for measuring the effect of molecules against mature gametocytes on a large scale is the lack of a standardized and reliable method. Here we provide an efficient method to produce and purify mature gametocytes in vitro. Based on this new procedure, we developed a robust, affordable, and sensitive ATP bioluminescence-based assay. We then assessed the activity of 17 gold-standard anti-malarial drugs on Plasmodium late stage gametocytes.Methods and FindingsDifficulties in producing large amounts of gametocytes have limited progress in the development of malaria transmission blocking assays. We improved the method established by Ifediba and Vanderberg to obtain viable, mature gametocytes en masse, whatever the strain used. We designed an assay to determine the activity of antimalarial drugs based on the intracellular ATP content of purified stage IV–V gametocytes after 48 h of drug exposure in 96/384-well microplates. Measurements of drug activity on asexual stages and cytotoxicity on HepG2 cells were also obtained to estimate the specificity of the active drugs.ConclusionsThe work described here represents another significant step towards determination of the activity of new molecules on mature gametocytes of any strain with an automated assay suitable for medium/high-throughput screening. Considering that the biology of the forms involved in the sexual and asexual stages is very different, a screen of our 2 million-compound library may allow us to discover novel anti-malarial drugs to target gametocyte-specific metabolic pathways.