The Experts below are selected from a list of 279 Experts worldwide ranked by ideXlab platform
Elena E Pohl - One of the best experts on this subject based on the ideXlab platform.
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inhibition of mitochondrial ucp1 and ucp3 by Purine Nucleotides and phosphate
Biochimica et Biophysica Acta, 2018Co-Authors: Gabriel Macher, Melanie Koehler, Anne Rupprecht, Jurgen Kreiter, Peter Hinterdorfer, Elena E PohlAbstract:Abstract Mitochondrial membrane uncoupling protein 3 (UCP3) is not only expressed in skeletal muscle and heart, but also in brown adipose tissue (BAT) alongside UCP1, which facilitates a proton leak to support non-shivering thermogenesis. In contrast to UCP1, the transport function and molecular mechanism of UCP3 regulation are poorly investigated, although it is generally agreed upon that UCP3, analogous to UCP1, transports protons, is activated by free fatty acids (FFAs) and is inhibited by Purine Nucleotides (PNs). Because the presence of two similar uncoupling proteins in BAT is surprising, we hypothesized that UCP1 and UCP3 are differently regulated, which may lead to differences in their functions. By combining atomic force microscopy and electrophysiological measurements of recombinant proteins reconstituted in planar bilayer membranes, we compared the level of protein activity with the bond lifetimes between UCPs and PNs. Our data revealed that, in contrast to UCP1, UCP3 can be fully inhibited by all PNs and IC50 increases with a decrease in PN-phosphorylation. Experiments with mutant proteins demonstrated that the conserved arginines in the PN-binding pocket are involved in the inhibition of UCP1 and UCP3 to different extents. Fatty acids compete with all PNs bound to UCP1, but only with ATP bound to UCP3. We identified phosphate as a novel inhibitor of UCP3 and UCP1, which acts independently of PNs. The differences in molecular mechanisms of the inhibition between the highly homologous transporters UCP1 and UCP3 indicate that UCP3 has adapted to fulfill a different role and possibly another transport function in BAT.
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binding mechanism of Purine Nucleotides to mitochondrial uncoupling proteins explored by recognition force spectroscopy
Biophysical Journal, 2016Co-Authors: Melanie Koehler, Gabriel Macher, Anne Rupprecht, Elena E Pohl, Hermann J Gruber, Peter HinterdorferAbstract:Regulated transport of protons across the inner mitochondrial membrane is essential for physiological processes such as ATP synthesis and heat production. Besides the involvement in thermogenesis, uncoupling protein family members (UCP1-UCP3) were proposed to regulate reactive oxygen species (ROS) and cell metabolism [1]. Although it is accepted that UCP activity is inhibited by Purine Nucleotides, the exact molecular mechanism of inhibition and binding is still unclear. Previously, we hypothesized that PNs bind to UCP1 from cis- and trans-side, although only cis-binding led to protein inhibition [2]. In this work we aimed to elucidate the binding of various Nucleotides to UCP1-UCP3 on the single-molecule level. For this we reconstituted UCPs in bilayer lipid membranes at low density and probed the interaction forces between UCP and PNs coupled to AFM cantilevers using recognition force spectroscopy [3]. Our studies revealed that the life time of the UCP-PN bond tended to increase with the phosphorylation degree of the nucleotide. However comparison between UCP1, UCP2 and UCP3 revealed different strengths of binding. Moreover, mutations of three arginine residues (R276L, R83T, R182Q) known to be crucial for nucleotide binding were studied to reveal a detailed picture about the binding mechanism of the Nucleotides to the UCPs on a structural level.1) Rupprecht et al., PLOS One (2014) 9 (2):e88474.2) Zhu et al., JACS (2013) 135, 3640.3) Koehler et al., Biophysical Journal 106.2 (2014): 223a.
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combined single molecule recognition imaging and force spectroscopy to study the interactions between uncoupling proteins and Purine Nucleotides
Biophysical Journal, 2014Co-Authors: Melanie Kohler, Anne Rupprecht, Elena E Pohl, Hermann J Gruber, Gabriel Purstinger, Peter HinterdorferAbstract:We combined recognition imaging and force spectroscopy so as to study the interactions between receptors and ligands on the single molecule level. This method allowed the selection of a single receptor molecule reconstituted in a supported lipid membrane at low density, with the subsequent quantification of the receptor-ligand unbinding force. Based on atomic force microscopy (AFM) tapping mode a cantilever tip carrying a ligand molecule was oscillated across a membrane. Topography and recognition Images of reconstituted receptors were recorded simultaneously by analysing the downwards and upwards parts of the oscillation, respectively. Functional receptor molecules were selected from the recognition image with nanometer resolution before the AFM was switched to the force spectroscopy mode, using positional feedback control. The combined mode allowed for dynamic force probing on different pre-selected molecules, resulting in a higher throughput when compared with force mapping. We applied this method for a quantitative characterization of the interaction between uncoupling proteins (UCP) and Purine Nucleotides (PN). The UCPs are mitochondrial proton transporters, which are proposed to be involved in thermogenesis, reactive oxygen sites (ROS) regulation and metabolism. Recently we hypothesized that PNs bind to UCP1 from cis- and transsite, although only cis-binding led to protein inhibition [1]. So as to get better insight into the molecular mechanism of this interaction we characterized the oligomeric state of UCPs reconstituted in lipid membranes and probed their interaction force with PNs using dynamic force spectroscopy. In the latter mode, the dynamics of force loading was varied, which elucidated the recognition dynamics and yielded information about binding pocket, binding energy barriers, and chemical reaction rates. This study was supported by the FWF project FWFP25357000. [1] Zhu, et al. JACS 135 (2013) 3640.
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Purine Nucleotides similarly regulate uncoupling protein 3 and 1
Biophysical Journal, 2014Co-Authors: Gabriel Purstinger, Anne Rupprecht, Peter Hinterdorfer, Melanie Kohler, Elena E PohlAbstract:ATP generation is fueled by an electrical potential across the inner mitochondrial membrane (IMM), which can be decreased by an uncoupling protein (UCP) facilitated proton leak. Apart from UCP1, which supports non-shivering thermogenesis, the function of the other UCPs, such as UCP3, remains unknown. In contrast to UCP1, few results on UCP3 regulation from studies on isolated mitochondria or liposomes imply that ADP/GDP has a stronger inhibitory effect on UCP3 than ATP/GTP. In light of the fact that both, UCP1 and UCP3 were found in brown adipose tissue, we now test whether UCP3 and UCP1 are regulated differently. For this we compare the inhibition of UCP1 and UCP3 by PNs of varying phosphorylation and concentration, using a system of planar bilayers reconstituted with recombinant protein and FAs (1). In contrast to liposomes, this enables us to directly apply the membrane potential necessary for UCP3 function under physiological conditions. These results show that ATP and not ADP is the most potent UCP3 inhibitor and this is likewise similar to UCP1 and UCP2 (2, 3). UCP3 conductance is more strongly inhibited by the same PN concentrations than is UCP1 conductance, and adenosine Nucleotides are more effective inhibitors than guanosines. Our results demonstrate that inhibition of UCP3 is similar to that of UCP1. We anticipate these findings as a starting point to examine whether different factors (in comparison to UCP1) are required to activate UCP3.(1) Beck et al. (2006) Biochim Biophys Acta. 1757(5-6):474-9.(2) Beck et al. (2007) FASEB J. 21(4):1137-44.(3) Rupprech et al. (2010) Biophys J. 98(8):1503-11.
Peter Hinterdorfer - One of the best experts on this subject based on the ideXlab platform.
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inhibition of mitochondrial ucp1 and ucp3 by Purine Nucleotides and phosphate
Biochimica et Biophysica Acta, 2018Co-Authors: Gabriel Macher, Melanie Koehler, Anne Rupprecht, Jurgen Kreiter, Peter Hinterdorfer, Elena E PohlAbstract:Abstract Mitochondrial membrane uncoupling protein 3 (UCP3) is not only expressed in skeletal muscle and heart, but also in brown adipose tissue (BAT) alongside UCP1, which facilitates a proton leak to support non-shivering thermogenesis. In contrast to UCP1, the transport function and molecular mechanism of UCP3 regulation are poorly investigated, although it is generally agreed upon that UCP3, analogous to UCP1, transports protons, is activated by free fatty acids (FFAs) and is inhibited by Purine Nucleotides (PNs). Because the presence of two similar uncoupling proteins in BAT is surprising, we hypothesized that UCP1 and UCP3 are differently regulated, which may lead to differences in their functions. By combining atomic force microscopy and electrophysiological measurements of recombinant proteins reconstituted in planar bilayer membranes, we compared the level of protein activity with the bond lifetimes between UCPs and PNs. Our data revealed that, in contrast to UCP1, UCP3 can be fully inhibited by all PNs and IC50 increases with a decrease in PN-phosphorylation. Experiments with mutant proteins demonstrated that the conserved arginines in the PN-binding pocket are involved in the inhibition of UCP1 and UCP3 to different extents. Fatty acids compete with all PNs bound to UCP1, but only with ATP bound to UCP3. We identified phosphate as a novel inhibitor of UCP3 and UCP1, which acts independently of PNs. The differences in molecular mechanisms of the inhibition between the highly homologous transporters UCP1 and UCP3 indicate that UCP3 has adapted to fulfill a different role and possibly another transport function in BAT.
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binding mechanism of Purine Nucleotides to mitochondrial uncoupling proteins explored by recognition force spectroscopy
Biophysical Journal, 2016Co-Authors: Melanie Koehler, Gabriel Macher, Anne Rupprecht, Elena E Pohl, Hermann J Gruber, Peter HinterdorferAbstract:Regulated transport of protons across the inner mitochondrial membrane is essential for physiological processes such as ATP synthesis and heat production. Besides the involvement in thermogenesis, uncoupling protein family members (UCP1-UCP3) were proposed to regulate reactive oxygen species (ROS) and cell metabolism [1]. Although it is accepted that UCP activity is inhibited by Purine Nucleotides, the exact molecular mechanism of inhibition and binding is still unclear. Previously, we hypothesized that PNs bind to UCP1 from cis- and trans-side, although only cis-binding led to protein inhibition [2]. In this work we aimed to elucidate the binding of various Nucleotides to UCP1-UCP3 on the single-molecule level. For this we reconstituted UCPs in bilayer lipid membranes at low density and probed the interaction forces between UCP and PNs coupled to AFM cantilevers using recognition force spectroscopy [3]. Our studies revealed that the life time of the UCP-PN bond tended to increase with the phosphorylation degree of the nucleotide. However comparison between UCP1, UCP2 and UCP3 revealed different strengths of binding. Moreover, mutations of three arginine residues (R276L, R83T, R182Q) known to be crucial for nucleotide binding were studied to reveal a detailed picture about the binding mechanism of the Nucleotides to the UCPs on a structural level.1) Rupprecht et al., PLOS One (2014) 9 (2):e88474.2) Zhu et al., JACS (2013) 135, 3640.3) Koehler et al., Biophysical Journal 106.2 (2014): 223a.
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combined single molecule recognition imaging and force spectroscopy to study the interactions between uncoupling proteins and Purine Nucleotides
Biophysical Journal, 2014Co-Authors: Melanie Kohler, Anne Rupprecht, Elena E Pohl, Hermann J Gruber, Gabriel Purstinger, Peter HinterdorferAbstract:We combined recognition imaging and force spectroscopy so as to study the interactions between receptors and ligands on the single molecule level. This method allowed the selection of a single receptor molecule reconstituted in a supported lipid membrane at low density, with the subsequent quantification of the receptor-ligand unbinding force. Based on atomic force microscopy (AFM) tapping mode a cantilever tip carrying a ligand molecule was oscillated across a membrane. Topography and recognition Images of reconstituted receptors were recorded simultaneously by analysing the downwards and upwards parts of the oscillation, respectively. Functional receptor molecules were selected from the recognition image with nanometer resolution before the AFM was switched to the force spectroscopy mode, using positional feedback control. The combined mode allowed for dynamic force probing on different pre-selected molecules, resulting in a higher throughput when compared with force mapping. We applied this method for a quantitative characterization of the interaction between uncoupling proteins (UCP) and Purine Nucleotides (PN). The UCPs are mitochondrial proton transporters, which are proposed to be involved in thermogenesis, reactive oxygen sites (ROS) regulation and metabolism. Recently we hypothesized that PNs bind to UCP1 from cis- and transsite, although only cis-binding led to protein inhibition [1]. So as to get better insight into the molecular mechanism of this interaction we characterized the oligomeric state of UCPs reconstituted in lipid membranes and probed their interaction force with PNs using dynamic force spectroscopy. In the latter mode, the dynamics of force loading was varied, which elucidated the recognition dynamics and yielded information about binding pocket, binding energy barriers, and chemical reaction rates. This study was supported by the FWF project FWFP25357000. [1] Zhu, et al. JACS 135 (2013) 3640.
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Purine Nucleotides similarly regulate uncoupling protein 3 and 1
Biophysical Journal, 2014Co-Authors: Gabriel Purstinger, Anne Rupprecht, Peter Hinterdorfer, Melanie Kohler, Elena E PohlAbstract:ATP generation is fueled by an electrical potential across the inner mitochondrial membrane (IMM), which can be decreased by an uncoupling protein (UCP) facilitated proton leak. Apart from UCP1, which supports non-shivering thermogenesis, the function of the other UCPs, such as UCP3, remains unknown. In contrast to UCP1, few results on UCP3 regulation from studies on isolated mitochondria or liposomes imply that ADP/GDP has a stronger inhibitory effect on UCP3 than ATP/GTP. In light of the fact that both, UCP1 and UCP3 were found in brown adipose tissue, we now test whether UCP3 and UCP1 are regulated differently. For this we compare the inhibition of UCP1 and UCP3 by PNs of varying phosphorylation and concentration, using a system of planar bilayers reconstituted with recombinant protein and FAs (1). In contrast to liposomes, this enables us to directly apply the membrane potential necessary for UCP3 function under physiological conditions. These results show that ATP and not ADP is the most potent UCP3 inhibitor and this is likewise similar to UCP1 and UCP2 (2, 3). UCP3 conductance is more strongly inhibited by the same PN concentrations than is UCP1 conductance, and adenosine Nucleotides are more effective inhibitors than guanosines. Our results demonstrate that inhibition of UCP3 is similar to that of UCP1. We anticipate these findings as a starting point to examine whether different factors (in comparison to UCP1) are required to activate UCP3.(1) Beck et al. (2006) Biochim Biophys Acta. 1757(5-6):474-9.(2) Beck et al. (2007) FASEB J. 21(4):1137-44.(3) Rupprech et al. (2010) Biophys J. 98(8):1503-11.
Anne Rupprecht - One of the best experts on this subject based on the ideXlab platform.
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inhibition of mitochondrial ucp1 and ucp3 by Purine Nucleotides and phosphate
Biochimica et Biophysica Acta, 2018Co-Authors: Gabriel Macher, Melanie Koehler, Anne Rupprecht, Jurgen Kreiter, Peter Hinterdorfer, Elena E PohlAbstract:Abstract Mitochondrial membrane uncoupling protein 3 (UCP3) is not only expressed in skeletal muscle and heart, but also in brown adipose tissue (BAT) alongside UCP1, which facilitates a proton leak to support non-shivering thermogenesis. In contrast to UCP1, the transport function and molecular mechanism of UCP3 regulation are poorly investigated, although it is generally agreed upon that UCP3, analogous to UCP1, transports protons, is activated by free fatty acids (FFAs) and is inhibited by Purine Nucleotides (PNs). Because the presence of two similar uncoupling proteins in BAT is surprising, we hypothesized that UCP1 and UCP3 are differently regulated, which may lead to differences in their functions. By combining atomic force microscopy and electrophysiological measurements of recombinant proteins reconstituted in planar bilayer membranes, we compared the level of protein activity with the bond lifetimes between UCPs and PNs. Our data revealed that, in contrast to UCP1, UCP3 can be fully inhibited by all PNs and IC50 increases with a decrease in PN-phosphorylation. Experiments with mutant proteins demonstrated that the conserved arginines in the PN-binding pocket are involved in the inhibition of UCP1 and UCP3 to different extents. Fatty acids compete with all PNs bound to UCP1, but only with ATP bound to UCP3. We identified phosphate as a novel inhibitor of UCP3 and UCP1, which acts independently of PNs. The differences in molecular mechanisms of the inhibition between the highly homologous transporters UCP1 and UCP3 indicate that UCP3 has adapted to fulfill a different role and possibly another transport function in BAT.
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binding mechanism of Purine Nucleotides to mitochondrial uncoupling proteins explored by recognition force spectroscopy
Biophysical Journal, 2016Co-Authors: Melanie Koehler, Gabriel Macher, Anne Rupprecht, Elena E Pohl, Hermann J Gruber, Peter HinterdorferAbstract:Regulated transport of protons across the inner mitochondrial membrane is essential for physiological processes such as ATP synthesis and heat production. Besides the involvement in thermogenesis, uncoupling protein family members (UCP1-UCP3) were proposed to regulate reactive oxygen species (ROS) and cell metabolism [1]. Although it is accepted that UCP activity is inhibited by Purine Nucleotides, the exact molecular mechanism of inhibition and binding is still unclear. Previously, we hypothesized that PNs bind to UCP1 from cis- and trans-side, although only cis-binding led to protein inhibition [2]. In this work we aimed to elucidate the binding of various Nucleotides to UCP1-UCP3 on the single-molecule level. For this we reconstituted UCPs in bilayer lipid membranes at low density and probed the interaction forces between UCP and PNs coupled to AFM cantilevers using recognition force spectroscopy [3]. Our studies revealed that the life time of the UCP-PN bond tended to increase with the phosphorylation degree of the nucleotide. However comparison between UCP1, UCP2 and UCP3 revealed different strengths of binding. Moreover, mutations of three arginine residues (R276L, R83T, R182Q) known to be crucial for nucleotide binding were studied to reveal a detailed picture about the binding mechanism of the Nucleotides to the UCPs on a structural level.1) Rupprecht et al., PLOS One (2014) 9 (2):e88474.2) Zhu et al., JACS (2013) 135, 3640.3) Koehler et al., Biophysical Journal 106.2 (2014): 223a.
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combined single molecule recognition imaging and force spectroscopy to study the interactions between uncoupling proteins and Purine Nucleotides
Biophysical Journal, 2014Co-Authors: Melanie Kohler, Anne Rupprecht, Elena E Pohl, Hermann J Gruber, Gabriel Purstinger, Peter HinterdorferAbstract:We combined recognition imaging and force spectroscopy so as to study the interactions between receptors and ligands on the single molecule level. This method allowed the selection of a single receptor molecule reconstituted in a supported lipid membrane at low density, with the subsequent quantification of the receptor-ligand unbinding force. Based on atomic force microscopy (AFM) tapping mode a cantilever tip carrying a ligand molecule was oscillated across a membrane. Topography and recognition Images of reconstituted receptors were recorded simultaneously by analysing the downwards and upwards parts of the oscillation, respectively. Functional receptor molecules were selected from the recognition image with nanometer resolution before the AFM was switched to the force spectroscopy mode, using positional feedback control. The combined mode allowed for dynamic force probing on different pre-selected molecules, resulting in a higher throughput when compared with force mapping. We applied this method for a quantitative characterization of the interaction between uncoupling proteins (UCP) and Purine Nucleotides (PN). The UCPs are mitochondrial proton transporters, which are proposed to be involved in thermogenesis, reactive oxygen sites (ROS) regulation and metabolism. Recently we hypothesized that PNs bind to UCP1 from cis- and transsite, although only cis-binding led to protein inhibition [1]. So as to get better insight into the molecular mechanism of this interaction we characterized the oligomeric state of UCPs reconstituted in lipid membranes and probed their interaction force with PNs using dynamic force spectroscopy. In the latter mode, the dynamics of force loading was varied, which elucidated the recognition dynamics and yielded information about binding pocket, binding energy barriers, and chemical reaction rates. This study was supported by the FWF project FWFP25357000. [1] Zhu, et al. JACS 135 (2013) 3640.
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Purine Nucleotides similarly regulate uncoupling protein 3 and 1
Biophysical Journal, 2014Co-Authors: Gabriel Purstinger, Anne Rupprecht, Peter Hinterdorfer, Melanie Kohler, Elena E PohlAbstract:ATP generation is fueled by an electrical potential across the inner mitochondrial membrane (IMM), which can be decreased by an uncoupling protein (UCP) facilitated proton leak. Apart from UCP1, which supports non-shivering thermogenesis, the function of the other UCPs, such as UCP3, remains unknown. In contrast to UCP1, few results on UCP3 regulation from studies on isolated mitochondria or liposomes imply that ADP/GDP has a stronger inhibitory effect on UCP3 than ATP/GTP. In light of the fact that both, UCP1 and UCP3 were found in brown adipose tissue, we now test whether UCP3 and UCP1 are regulated differently. For this we compare the inhibition of UCP1 and UCP3 by PNs of varying phosphorylation and concentration, using a system of planar bilayers reconstituted with recombinant protein and FAs (1). In contrast to liposomes, this enables us to directly apply the membrane potential necessary for UCP3 function under physiological conditions. These results show that ATP and not ADP is the most potent UCP3 inhibitor and this is likewise similar to UCP1 and UCP2 (2, 3). UCP3 conductance is more strongly inhibited by the same PN concentrations than is UCP1 conductance, and adenosine Nucleotides are more effective inhibitors than guanosines. Our results demonstrate that inhibition of UCP3 is similar to that of UCP1. We anticipate these findings as a starting point to examine whether different factors (in comparison to UCP1) are required to activate UCP3.(1) Beck et al. (2006) Biochim Biophys Acta. 1757(5-6):474-9.(2) Beck et al. (2007) FASEB J. 21(4):1137-44.(3) Rupprech et al. (2010) Biophys J. 98(8):1503-11.
Gabriel Purstinger - One of the best experts on this subject based on the ideXlab platform.
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combined single molecule recognition imaging and force spectroscopy to study the interactions between uncoupling proteins and Purine Nucleotides
Biophysical Journal, 2014Co-Authors: Melanie Kohler, Anne Rupprecht, Elena E Pohl, Hermann J Gruber, Gabriel Purstinger, Peter HinterdorferAbstract:We combined recognition imaging and force spectroscopy so as to study the interactions between receptors and ligands on the single molecule level. This method allowed the selection of a single receptor molecule reconstituted in a supported lipid membrane at low density, with the subsequent quantification of the receptor-ligand unbinding force. Based on atomic force microscopy (AFM) tapping mode a cantilever tip carrying a ligand molecule was oscillated across a membrane. Topography and recognition Images of reconstituted receptors were recorded simultaneously by analysing the downwards and upwards parts of the oscillation, respectively. Functional receptor molecules were selected from the recognition image with nanometer resolution before the AFM was switched to the force spectroscopy mode, using positional feedback control. The combined mode allowed for dynamic force probing on different pre-selected molecules, resulting in a higher throughput when compared with force mapping. We applied this method for a quantitative characterization of the interaction between uncoupling proteins (UCP) and Purine Nucleotides (PN). The UCPs are mitochondrial proton transporters, which are proposed to be involved in thermogenesis, reactive oxygen sites (ROS) regulation and metabolism. Recently we hypothesized that PNs bind to UCP1 from cis- and transsite, although only cis-binding led to protein inhibition [1]. So as to get better insight into the molecular mechanism of this interaction we characterized the oligomeric state of UCPs reconstituted in lipid membranes and probed their interaction force with PNs using dynamic force spectroscopy. In the latter mode, the dynamics of force loading was varied, which elucidated the recognition dynamics and yielded information about binding pocket, binding energy barriers, and chemical reaction rates. This study was supported by the FWF project FWFP25357000. [1] Zhu, et al. JACS 135 (2013) 3640.
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Purine Nucleotides similarly regulate uncoupling protein 3 and 1
Biophysical Journal, 2014Co-Authors: Gabriel Purstinger, Anne Rupprecht, Peter Hinterdorfer, Melanie Kohler, Elena E PohlAbstract:ATP generation is fueled by an electrical potential across the inner mitochondrial membrane (IMM), which can be decreased by an uncoupling protein (UCP) facilitated proton leak. Apart from UCP1, which supports non-shivering thermogenesis, the function of the other UCPs, such as UCP3, remains unknown. In contrast to UCP1, few results on UCP3 regulation from studies on isolated mitochondria or liposomes imply that ADP/GDP has a stronger inhibitory effect on UCP3 than ATP/GTP. In light of the fact that both, UCP1 and UCP3 were found in brown adipose tissue, we now test whether UCP3 and UCP1 are regulated differently. For this we compare the inhibition of UCP1 and UCP3 by PNs of varying phosphorylation and concentration, using a system of planar bilayers reconstituted with recombinant protein and FAs (1). In contrast to liposomes, this enables us to directly apply the membrane potential necessary for UCP3 function under physiological conditions. These results show that ATP and not ADP is the most potent UCP3 inhibitor and this is likewise similar to UCP1 and UCP2 (2, 3). UCP3 conductance is more strongly inhibited by the same PN concentrations than is UCP1 conductance, and adenosine Nucleotides are more effective inhibitors than guanosines. Our results demonstrate that inhibition of UCP3 is similar to that of UCP1. We anticipate these findings as a starting point to examine whether different factors (in comparison to UCP1) are required to activate UCP3.(1) Beck et al. (2006) Biochim Biophys Acta. 1757(5-6):474-9.(2) Beck et al. (2007) FASEB J. 21(4):1137-44.(3) Rupprech et al. (2010) Biophys J. 98(8):1503-11.
Melanie Kohler - One of the best experts on this subject based on the ideXlab platform.
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combined single molecule recognition imaging and force spectroscopy to study the interactions between uncoupling proteins and Purine Nucleotides
Biophysical Journal, 2014Co-Authors: Melanie Kohler, Anne Rupprecht, Elena E Pohl, Hermann J Gruber, Gabriel Purstinger, Peter HinterdorferAbstract:We combined recognition imaging and force spectroscopy so as to study the interactions between receptors and ligands on the single molecule level. This method allowed the selection of a single receptor molecule reconstituted in a supported lipid membrane at low density, with the subsequent quantification of the receptor-ligand unbinding force. Based on atomic force microscopy (AFM) tapping mode a cantilever tip carrying a ligand molecule was oscillated across a membrane. Topography and recognition Images of reconstituted receptors were recorded simultaneously by analysing the downwards and upwards parts of the oscillation, respectively. Functional receptor molecules were selected from the recognition image with nanometer resolution before the AFM was switched to the force spectroscopy mode, using positional feedback control. The combined mode allowed for dynamic force probing on different pre-selected molecules, resulting in a higher throughput when compared with force mapping. We applied this method for a quantitative characterization of the interaction between uncoupling proteins (UCP) and Purine Nucleotides (PN). The UCPs are mitochondrial proton transporters, which are proposed to be involved in thermogenesis, reactive oxygen sites (ROS) regulation and metabolism. Recently we hypothesized that PNs bind to UCP1 from cis- and transsite, although only cis-binding led to protein inhibition [1]. So as to get better insight into the molecular mechanism of this interaction we characterized the oligomeric state of UCPs reconstituted in lipid membranes and probed their interaction force with PNs using dynamic force spectroscopy. In the latter mode, the dynamics of force loading was varied, which elucidated the recognition dynamics and yielded information about binding pocket, binding energy barriers, and chemical reaction rates. This study was supported by the FWF project FWFP25357000. [1] Zhu, et al. JACS 135 (2013) 3640.
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Purine Nucleotides similarly regulate uncoupling protein 3 and 1
Biophysical Journal, 2014Co-Authors: Gabriel Purstinger, Anne Rupprecht, Peter Hinterdorfer, Melanie Kohler, Elena E PohlAbstract:ATP generation is fueled by an electrical potential across the inner mitochondrial membrane (IMM), which can be decreased by an uncoupling protein (UCP) facilitated proton leak. Apart from UCP1, which supports non-shivering thermogenesis, the function of the other UCPs, such as UCP3, remains unknown. In contrast to UCP1, few results on UCP3 regulation from studies on isolated mitochondria or liposomes imply that ADP/GDP has a stronger inhibitory effect on UCP3 than ATP/GTP. In light of the fact that both, UCP1 and UCP3 were found in brown adipose tissue, we now test whether UCP3 and UCP1 are regulated differently. For this we compare the inhibition of UCP1 and UCP3 by PNs of varying phosphorylation and concentration, using a system of planar bilayers reconstituted with recombinant protein and FAs (1). In contrast to liposomes, this enables us to directly apply the membrane potential necessary for UCP3 function under physiological conditions. These results show that ATP and not ADP is the most potent UCP3 inhibitor and this is likewise similar to UCP1 and UCP2 (2, 3). UCP3 conductance is more strongly inhibited by the same PN concentrations than is UCP1 conductance, and adenosine Nucleotides are more effective inhibitors than guanosines. Our results demonstrate that inhibition of UCP3 is similar to that of UCP1. We anticipate these findings as a starting point to examine whether different factors (in comparison to UCP1) are required to activate UCP3.(1) Beck et al. (2006) Biochim Biophys Acta. 1757(5-6):474-9.(2) Beck et al. (2007) FASEB J. 21(4):1137-44.(3) Rupprech et al. (2010) Biophys J. 98(8):1503-11.
