Adenosine triphosphate (ATP) is the molecular currency of intracellular energy transfer in living organisms. The enzyme ATP synthase is primarily responsible for ATP production in eukaryotes. In archaea and some bacteria, ATP is synthesized by V-ATPase that is related to ATP synthase both in structure and function. Both of these enzymes are reversible rotary motors capable of catalyzing ATP synthesis or hydrolysis. The rotation of the central rotor, which is powered by the flow of proton (or sometimes sodium ion) down the electrochemical gradient through the membrane-bound Fo/Vo region, leads to the chemical synthesis of ATP in F1/V1 region. The F1/V1 region, on the other hand, can catalyze ATP hydrolysis, which in turn leads to proton (or sodium) pumping across the membrane through rotation of the central rotor in the opposite direction. This thesis describes structure determination of both the intact F-type and V-type enzymes using single particle electron cryomicroscopy (cryo-EM), with the aim of better understanding their overall architecture, subunit organization and the mechanism of proton translocation. Our cryo-EM structural analysis on the F-type ATP synthase from Saccharomyces cerevisiae uncovered the arrangement of subunits a, b, c, and the two dimer-specific subunits e and g within the membrane-bound region of Fo. A model of oligomerization of the ATP synthase involving two distinct dimerization interfaces was proposed.The rotor-stator interaction within the membrane-bound region of both enzymes is responsible for proton translocation. Our cryo-EM structures of the V-ATPase from Thermus thermophilus reveal that the interaction between the rotary ring (rotor) and the I-subunit (stator) is surprisingly small, with only two subunits from the ring making contact with the I-subunit near the middle of the membrane. Furthermore, the spatial arrangement of transmembrane helices resolved in subunit I can form two passageways that could provide proton access through the membrane-bound region and is consistent with a two-channel model of proton translocation.

TGF-β negatively affects the maintenance and expansion of hematopoietic stem cells ex vivo and its inhibition has been widely studied as a treatment for numerous hematopoietic disorders and cancers. Current inhibitory strategies (small molecule ATP competitors and neutralizing antibodies) are compared to a novel cell-permeable peptide-based inhibitor of TGF-β RI. Multiple levels of assay from biochemical to functional are utilized with the aim of applying the most successful inhibitor to hematopoietic stem cell culture. The neutralizing antibody proved ineffective in the short-term biochemical assay but was extremely effective at neutralizing TGF-β signalling in proliferation and hematopoietic colony-forming cell assays with no evidence of toxicity. The small molecule inhibitors (SD-208 and Pyrazole TGF-β RI inhibitor) were equally effective at micromolar levels in all forms of assay, with SD-208 being slightly more potent. The novel peptide inhibitor proved ineffective in all assays, which is likely a result of its rapid degradation in live cells.

Background: Modulation of ischemia-dependent pathways alters electrophysiological evolution of ventricular fibrillation(VF). Hypothesis: 1)There is regional disease-related expression of KATP-channels in human cardiomyopathic hearts. 2)KATP-channel blockade promotes spontaneous VF termination by attenuating spatiotemporal dispersion of refractoriness(ΔERP). Methods and Results: Electric mapping of control(n=6) and treatment(n=9) (10 μmol/L glibenclamide) isolated human cardiomyopathic hearts was performed. Spontaneous defibrillation and KATP-subunit gene expression were studied. Spontaneous VF termination occurred in 1/6 control and 7/8 treated hearts (P=0.026). After 180 seconds of ischemia, LV transmural dispersion in VF cycle length was observed(p=0.001), which was attenuated by glibenclamide. There was greater gene expression of all KATP-subunit on the endocardium compared with the epicardium(P<0.02). In ischemic rat heart model, ΔERP was verified with pacing protocols (36±5ms vs 4.9±4ms, p=0.019). Conclusions: KATP channel subunit gene expression is heterogeneously altered in the cardiomyopathic human heart. Blockade of KATP channels promotes spontaneous defibrillation by attenuating ischemia-dependent ΔERP during VF.

Background: Modulation of ischemia-dependent pathways alters electrophysiological evolution of ventricular fibrillation(VF). Hypothesis: 1)There is regional disease-related expression of KATP-channels in human cardiomyopathic hearts. 2)KATP-channel blockade promotes spontaneous VF termination by attenuating spatiotemporal dispersion of refractoriness(ΔERP). Methods and Results: Electric mapping of control(n=6) and treatment(n=9) (10 μmol/L glibenclamide) isolated human cardiomyopathic hearts was performed. Spontaneous defibrillation and KATP-subunit gene expression were studied. Spontaneous VF termination occurred in 1/6 control and 7/8 treated hearts (P=0.026). After 180 seconds of ischemia, LV transmural dispersion in VF cycle length was observed(p=0.001), which was attenuated by glibenclamide. There was greater gene expression of all KATP-subunit on the endocardium compared with the epicardium(P<0.02). In ischemic rat heart model, ΔERP was verified with pacing protocols (36±5ms vs 4.9±4ms, p=0.019). Conclusions: KATP channel subunit gene expression is heterogeneously altered in the cardiomyopathic human heart. Blockade of KATP channels promotes spontaneous defibrillation by attenuating ischemia-dependent ΔERP during VF.

The research was designed to investigate the effects of AIT-082, a derivative of the purine hypoxanthine containing a para-amino benzoic acid moiety, on neural cells. AIT-082 has been shown to possess a number of neurotrophic and neuroprotective properties and to enhance memory. Furthermore, AIT-082 is undergoing clinical trials as a potential treatment for Alzheimers disease.<p>The first part of the study investigated the ability of AIT-082 to influence cellular ATP levels in cortical neurons. Decreased energy metabolism is a key point in Yings (Ying, 1996a) theory of the development of Alzheimers disease. Previous work with AIT-082 had shown that it could protect hippocampal neurons from cellular damage caused by sublethal doses of glutamate. Specifically, AIT-082 prevented neurite degeneration. Also, AIT-082 was shown to increase mitochondrial membrane potential, especially at the distal tips of the neurites, in hippocampal neurons. I hypothesized that AIT-082 was protecting the neurons by increasing the ability of the mitochondria to generate ATP and thereby increasing the amount of ATP available to the cell. ATP was collected and measured from cortical neuron cultures that were exposed to glutamate, AIT-082, glutamate and AIT-082. The ATP levels were compared to the ATP levels from cortical neuron cultures that were exposed to vehicle for glutamate and AIT-082. The results did not significantly increase ATP levels in cortical neurons following glutamate exposure. <p>The next set of experiments involved investigations into the ability of AIT-082 to influence phosphorylation events in neural cells. AIT-082 shares some neurotrophic and neuroprotective properties with a group of drugs called the immunophilin ligands. The neuroprotective properties of the immunophilin ligands are mainly due to their ability to influence protein phosphorylation by inhibiting the activity of calcineurin a protein phosphatase. The first set of experiments used western blot techniques to measure serine peptide and threonine peptide phosphorylation levels in proteins from whole brain homogenates that were incubated with vehicle, AIT-082, and GMP. Both AIT-082 and GMP caused an increase in the level of serine peptide phosphorylation compared to vehicle but only the increase caused by GMP treatment proved to be significant. Further, threonine phosphorylation levels were significantly increased by GMP but not AIT-082. Phosphorylation levels of short peptide sequences containing either a phosphorylated serine or threonine residue were also measured in neuronal and astrocytic cultures. The neuronal cultures were exposed to 4 h of hypoxia to mimic the conditions of reduced energy availability observed in Alzheimers disease brains. Astrocyte cultures were exposed to 4 h of hypoxia/ischemia for the same reason. Both cell types were allowed to recover for 0, 1, 4, 12 and 24 hours with or without AIT-082 following the insult. AIT-082 treatment did not significantly affect phosphorylation levels of proteins harvested from either neuron or astrocyte cultures at any time period. I conclude therefore, that AIT-082 is not able to influence phosphorylation of the short amino acid sequences containing phosphorylated serine or threonine residues that could be detected by the primary antibodies used in my experiments.

The research was designed to investigate the effects of AIT-082, a derivative of the purine hypoxanthine containing a para-amino benzoic acid moiety, on neural cells. AIT-082 has been shown to possess a number of neurotrophic and neuroprotective properties and to enhance memory. Furthermore, AIT-082 is undergoing clinical trials as a potential treatment for Alzheimers disease.<p>The first part of the study investigated the ability of AIT-082 to influence cellular ATP levels in cortical neurons. Decreased energy metabolism is a key point in Yings (Ying, 1996a) theory of the development of Alzheimers disease. Previous work with AIT-082 had shown that it could protect hippocampal neurons from cellular damage caused by sublethal doses of glutamate. Specifically, AIT-082 prevented neurite degeneration. Also, AIT-082 was shown to increase mitochondrial membrane potential, especially at the distal tips of the neurites, in hippocampal neurons. I hypothesized that AIT-082 was protecting the neurons by increasing the ability of the mitochondria to generate ATP and thereby increasing the amount of ATP available to the cell. ATP was collected and measured from cortical neuron cultures that were exposed to glutamate, AIT-082, glutamate and AIT-082. The ATP levels were compared to the ATP levels from cortical neuron cultures that were exposed to vehicle for glutamate and AIT-082. The results did not significantly increase ATP levels in cortical neurons following glutamate exposure. <p>The next set of experiments involved investigations into the ability of AIT-082 to influence phosphorylation events in neural cells. AIT-082 shares some neurotrophic and neuroprotective properties with a group of drugs called the immunophilin ligands. The neuroprotective properties of the immunophilin ligands are mainly due to their ability to influence protein phosphorylation by inhibiting the activity of calcineurin a protein phosphatase. The first set of experiments used western blot techniques to measure serine peptide and threonine peptide phosphorylation levels in proteins from whole brain homogenates that were incubated with vehicle, AIT-082, and GMP. Both AIT-082 and GMP caused an increase in the level of serine peptide phosphorylation compared to vehicle but only the increase caused by GMP treatment proved to be significant. Further, threonine phosphorylation levels were significantly increased by GMP but not AIT-082. Phosphorylation levels of short peptide sequences containing either a phosphorylated serine or threonine residue were also measured in neuronal and astrocytic cultures. The neuronal cultures were exposed to 4 h of hypoxia to mimic the conditions of reduced energy availability observed in Alzheimers disease brains. Astrocyte cultures were exposed to 4 h of hypoxia/ischemia for the same reason. Both cell types were allowed to recover for 0, 1, 4, 12 and 24 hours with or without AIT-082 following the insult. AIT-082 treatment did not significantly affect phosphorylation levels of proteins harvested from either neuron or astrocyte cultures at any time period. I conclude therefore, that AIT-082 is not able to influence phosphorylation of the short amino acid sequences containing phosphorylated serine or threonine residues that could be detected by the primary antibodies used in my experiments.

Adenosine triphosphate (ATP) is the molecular currency of intracellular energy transfer in living organisms. The enzyme ATP synthase is primarily responsible for ATP production in eukaryotes. In archaea and some bacteria, ATP is synthesized by V-ATPase that is related to ATP synthase both in structure and function. Both of these enzymes are reversible rotary motors capable of catalyzing ATP synthesis or hydrolysis. The rotation of the central rotor, which is powered by the flow of proton (or sometimes sodium ion) down the electrochemical gradient through the membrane-bound Fo/Vo region, leads to the chemical synthesis of ATP in F1/V1 region. The F1/V1 region, on the other hand, can catalyze ATP hydrolysis, which in turn leads to proton (or sodium) pumping across the membrane through rotation of the central rotor in the opposite direction. This thesis describes structure determination of both the intact F-type and V-type enzymes using single particle electron cryomicroscopy (cryo-EM), with the aim of better understanding their overall architecture, subunit organization and the mechanism of proton translocation. Our cryo-EM structural analysis on the F-type ATP synthase from Saccharomyces cerevisiae uncovered the arrangement of subunits a, b, c, and the two dimer-specific subunits e and g within the membrane-bound region of Fo. A model of oligomerization of the ATP synthase involving two distinct dimerization interfaces was proposed.The rotor-stator interaction within the membrane-bound region of both enzymes is responsible for proton translocation. Our cryo-EM structures of the V-ATPase from Thermus thermophilus reveal that the interaction between the rotary ring (rotor) and the I-subunit (stator) is surprisingly small, with only two subunits from the ring making contact with the I-subunit near the middle of the membrane. Furthermore, the spatial arrangement of transmembrane helices resolved in subunit I can form two passageways that could provide proton access through the membrane-bound region and is consistent with a two-channel model of proton translocation.

TGF-β negatively affects the maintenance and expansion of hematopoietic stem cells ex vivo and its inhibition has been widely studied as a treatment for numerous hematopoietic disorders and cancers. Current inhibitory strategies (small molecule ATP competitors and neutralizing antibodies) are compared to a novel cell-permeable peptide-based inhibitor of TGF-β RI. Multiple levels of assay from biochemical to functional are utilized with the aim of applying the most successful inhibitor to hematopoietic stem cell culture. The neutralizing antibody proved ineffective in the short-term biochemical assay but was extremely effective at neutralizing TGF-β signalling in proliferation and hematopoietic colony-forming cell assays with no evidence of toxicity. The small molecule inhibitors (SD-208 and Pyrazole TGF-β RI inhibitor) were equally effective at micromolar levels in all forms of assay, with SD-208 being slightly more potent. The novel peptide inhibitor proved ineffective in all assays, which is likely a result of its rapid degradation in live cells.

ABCA1 is a key plasma membrane protein required for the efflux of cellular cholesterol to extracellular acceptors, particularly to apoA-I. This process is essential to maintain cholesterol homeostasis in the body. The detailed molecular mechanisms, however, are still insufficiently understood. Also, the molecular identity of ABCA1, i.e. channel, pump or flippase, remains unknown. In this study we analyzed the extracellular ATP levels in the medium of ABCA1-expressing BHK cells and RAW macrophages and compared them to the medium of relevant non-expressing cells. We found that the extracellular ATP concentrations are significantly elevated when cells express ABCA1. Importantly, a dysfunctional ABCA1 mutant (A937V), when expressed similarly as WT-ABCA1, is unable to raise extracellular ATP concentration. This suggests a causal relationship between functional ABCA1 and elevated extracellular ATP. To explore the physiological role of elevated extracellular ATP, we analyzed ABCA1-mediated cholesterol efflux under the conditions where extracellular ATP levels were modulated. We found that increasing extracellular ATP within the physiological range, i.e. < μM, promotes cholesterol efflux to apoA-I. On the other hand, removing extracellular ATP, either by adding apyrase to the medium or by expressing a plasma membrane bound ecto-nucleotidase CD39, abolishes cholesterol efflux to apoA-I. Based on these results we conclude that, through direct or indirect mechanisms, ABCA1 functions to raise ATP levels in the medium. This elevated extracellular ATP is required for ABCA1-mediated cholesterol efflux to apoA-I.

L' ATP es una molecula clau en el metabolisme cel.lular, actuant com a donador d'energia lliure i acoplant reaccions endergoniques i exergoniques. L'ATP es sintetitzat a la mitocondria en un proces anomenat fosforil.lacio oxidativa despres d'una serie de reaccions a la cadena de citocroms que es troba en la membrana interna de l'organel.la. La font d'energia necessaria per aquesta sintesi s'obte en les cel.lules animals dels nutrients de l'ingestio, i de la llum solar en les plantes. Existeix una via alternativa de sintesi d'ATP extramitocondrial, la glicolisi, que s'inicia amb la fosforilacio de la glucosa per l'enzim hexokinasa.Interaccio llum-materiaL'energia electromagnetica l'ona de la qual oscil.la en una longitud d'ona de nanometres es anomenada llum. En aquestes frequencies, l'energia promou excitacio electronica de certs atoms i molecules. Existeix una interaccio diferent de la llum amb molecules, produida per el camp electromagnetic que per definicio la llum provoca en qualsevol medi. El camp electric resultant desplaca els electrons dels enllacos quimics produint una polaritzacio del medi sense que existeixi absorcio de l'energia. Aquest es un mecanisme interactiu que existeix sempre, i es l'unic que es dona en molecules que son transparents (no absorbeixen) per a una determinada frequencia de la llum, com es el cas de l'ATP per l'energia visible i infravermella propera. Experiments, resultats i discussioQuan l'ATP es excitat amb fotons ultraviolats, es produiex una fluorescencia en longituts d'ona visibles. L'io magnesi s'ha utilitzat per estudiar com la llum visible i infravermella propera produiex un desplacament de carregues electriques a la molecula d'ATP. La construccio d'un interferometre de Michelson ha servit per observar l'interaccio no absortiva de la llum i l'ATP. La mesura directa de l'index de refraccio d'una solucio d'ATP dona informacio sobre les caracteristiques electriques del medi. L'observacio de que aquest index canvia despres d'irradiar la solucio amb longituds d'ona visibles i infravermelles properes, confirmen que la llum provoca canvis electrics significatius en l'ATP. En aquest treball tambe s'ha estudiat el comportament bioquimic de l'ATP irradiat quant forma part de dues reaccions quimiques diferents: la de la luciferina-luciferasa i la de la hexoquinasa. En tots dos casos, l'us d'aquest ATP irradiat ha produit una alteracio dels parametres cinetics estudiats, V0 i k en la reaccio de la luciferina-luciferasa, i km i vmax en la reaccio de l'hexoquinasa.ConclusionsAquesta interaccio no absortiva de la llum amb l'ATP es la primera descrita per a una biomolecula. El mecanisme aporta noves dades per explicar els efectes observats en el metabolisme cel.lular despres de l'irradiacio d'organismes, teixits i cultius cel.lulars amb llum visible i infravermella propera. / ATP is a key molecule in cellular metabolism. In this thesis, I examined the effects of visible (635 and 655 nm) and near-infrared (810 and 830 nm) light on ATP in solution. I also examined were the biochemical behavior of light-exposed ATP in the luciferine-luciferase reaction and hexokinase reaction, the initial step in glycolysis that begins extra mithocondrial ATP synthesis. Irradiated groups in the luciferine-luciferase reaction showed an improvement in the kinetic parameters V0 and k, and more ATP molecules reacted with the enzyme when they were excited by light. When irradiated ATP was added to the hexokinase reaction, the experimental groups showed significant differences in the Michaelis-Menten kinetic parameters (km for ATP and vmax) and the rate of product synthesis was greater. Changes in both reactions were wavelength and dose dependant. When ATP was excited with UV photons, it fluoresced. This fluorescence decreased when Mg2+ was added, probably because the ion binds the phosphates, which are the part of the molecule responsible for light emission. Irradiating the ATP-Mg2+ solution with 655 nm and 830 nm light increased the fluorescence resulting from a displacement of charges in the phosphor-oxygen bond that repels Mg2+. The refraction of light in an ATP solution was observed by the Michelson interferometer and by directly measuring the refractive index. The refractive index changed after red and near-infrared light interaction due to a change in the electrical permittivity of the medium. Since ATP in water is transparent to visible and near-infrared light, and is therefore not a chromophore for those wavelengths, I conclude that the observed light interaction with ATP is not due to photon absorption but to the electromagnetic disturbance produced by the light, which leads to a polarization of the dielectric molecule that is ATP. This interaction of visible and near-infrared electromagnetic energy with ATP offers new perspectives for explaining light interaction at subcellular level.