Wilson disease (WD) is a hepatoneurologic disorder caused by mutations in the copper-transporter ... more Wilson disease (WD) is a hepatoneurologic disorder caused by mutations in the copper-transporter ATP7B. Copper accumulation in the liver is a hallmark of WD. Current therapy is based on copper chelation, which decreases the manifestations of liver disease, but often worsens neurologic symptoms. We demonstrate that in Atp7b(-/-) mice, an animal model of WD, liver function can be significantly improved without copper chelation. Analysis of transcriptional and metabolic changes in samples from WD patients and Atp7b(-/-) mice identified disregulation of nuclear receptors (NR), especially the LXR/RXR heterodimer, as an important event in WD pathogenesis. Treating Atp7b(-/-) mice with the LXR agonist T0901317 ameliorated disease manifestations despite significant copper overload. Genetic markers of liver fibrosis and inflammatory cytokines were significantly decreased, lipid profiles normalized and liver function and histology was improved. In conclusion, the results demonstrate the major...
The slow afterhyperpolarization that follows an action potential is generated by the activation o... more The slow afterhyperpolarization that follows an action potential is generated by the activation of small-conductance calcium-activated potassium channels (SK channels). The slow afterhyperpolarization limits the firing frequency of repetitive action potentials (spike-frequency adaptation) and is essential for normal neurotransmission. SK channels are voltage-independent and activated by submicromolar concentrations of intracellular calcium. They are high-affinity calcium sensors that transduce fluctuations in intracellular calcium concentrations into changes in membrane potential. Here we study the mechanism of calcium gating and find that SK channels are not gated by calcium binding directly to the channel alpha-subunits. Instead, the functional SK channels are heteromeric complexes with calmodulin, which is constitutively associated with the alpha-subunits in a calcium-independent manner. Our data support a model in which calcium gating of SK channels is mediated by binding of cal...
The membrane-bound fraction of purified Na,K-ATPase was characterized following extensive proteol... more The membrane-bound fraction of purified Na,K-ATPase was characterized following extensive proteolytic digestion in the presence of various physiological ligands which stabilize different conformational states of the sodium pump. There are distinctive conformational changes of the protein which are revealed by amino-terminal amino acid sequence analysis of the digests following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The changes in cleavage patterns result from alterations in domain-domain interactions of the protein. We provide evidence in the alpha-subunit for (i) tight interaction between part of the cytoplasmic ATP binding domain and the membrane-bound portion of the protein; (ii) involvement of the cytoplasmic loop between M2 and M3 in structural rearrangements upon phosphorylation or ion binding; (iii) generation of the same digested products when either ouabain or potassium (rubidium) is present. Similarly, evidence is provided for conformational sensitivity...
The biologically and clinically important membrane transporters are challenging proteins to study... more The biologically and clinically important membrane transporters are challenging proteins to study because of their low level of expression, multidomain structure, and complex molecular dynamics that underlies their activity. ATP7B is a copper transporter that traffics between the intracellular compartments in response to copper elevation. The N-terminal domain of ATP7B (N-ATP7B) is involved in binding copper, but the role of this domain in trafficking is controversial. To clarify the role of N-ATP7B, we generated nanobodies that interact with ATP7B in vitro and in cells. In solution NMR studies, nanobodies revealed the spatial organization of N-ATP7B by detecting transient functionally relevant interactions between metal-binding domains 1-3. Modulation of these interactions by nanobodies in cells enhanced relocalization of the endogenous ATP7B toward the plasma membrane linking molecular and cellular dynamics of the transporter. Stimulation of ATP7B trafficking by nanobodies in the ...
Proceedings of the National Academy of Sciences, 1998
Wilson's disease (WND) is an inherited disorder of copper homeostasis characterized by ab... more Wilson's disease (WND) is an inherited disorder of copper homeostasis characterized by abnormal accumulation of copper in several tissues, particularly in the liver, brain, and kidney. The disease-associated gene encodes a copper-transporting P-type ATPase, the WND protein, the subcellular location of which could be regulated by copper. We demonstrate that the WND protein is present in cells in two forms, the 160-kDa and the 140-kDa products. The 160-kDa product was earlier shown to be targeted to trans-Golgi network. The 140-kDa product identified herein is located in mitochondria as evidenced by the immunofluorescent staining of HepG2 cells with specific mitochondria markers and polyclonal antibody directed against the C terminus of the WND molecule. The mitochondrial location for the 140-kDa WND product was confirmed by membrane fractionation and by analysis of purified human mitochondria. The antibody raised against a repetitive sequence in the N-terminal portion of the WND molecule detects an additional 16-kDa protein, suggesting that the 140-kDa product was formed after proteolytic cleavage of the full-length WND protein at the N terminus. Thus, the WND protein is a P-type ATPase with an unusual subcellular localization. The mitochondria targeting of the WND protein suggests its important role for copper-dependent processes taking place in this organelle.
Wilson disease (WD) is a hepatoneurologic disorder caused by mutations in the copper-transporter ... more Wilson disease (WD) is a hepatoneurologic disorder caused by mutations in the copper-transporter ATP7B. Copper accumulation in the liver is a hallmark of WD. Current therapy is based on copper chelation, which decreases the manifestations of liver disease, but often worsens neurologic symptoms. We demonstrate that in Atp7b(-/-) mice, an animal model of WD, liver function can be significantly improved without copper chelation. Analysis of transcriptional and metabolic changes in samples from WD patients and Atp7b(-/-) mice identified disregulation of nuclear receptors (NR), especially the LXR/RXR heterodimer, as an important event in WD pathogenesis. Treating Atp7b(-/-) mice with the LXR agonist T0901317 ameliorated disease manifestations despite significant copper overload. Genetic markers of liver fibrosis and inflammatory cytokines were significantly decreased, lipid profiles normalized and liver function and histology was improved. In conclusion, the results demonstrate the major...
The slow afterhyperpolarization that follows an action potential is generated by the activation o... more The slow afterhyperpolarization that follows an action potential is generated by the activation of small-conductance calcium-activated potassium channels (SK channels). The slow afterhyperpolarization limits the firing frequency of repetitive action potentials (spike-frequency adaptation) and is essential for normal neurotransmission. SK channels are voltage-independent and activated by submicromolar concentrations of intracellular calcium. They are high-affinity calcium sensors that transduce fluctuations in intracellular calcium concentrations into changes in membrane potential. Here we study the mechanism of calcium gating and find that SK channels are not gated by calcium binding directly to the channel alpha-subunits. Instead, the functional SK channels are heteromeric complexes with calmodulin, which is constitutively associated with the alpha-subunits in a calcium-independent manner. Our data support a model in which calcium gating of SK channels is mediated by binding of cal...
The membrane-bound fraction of purified Na,K-ATPase was characterized following extensive proteol... more The membrane-bound fraction of purified Na,K-ATPase was characterized following extensive proteolytic digestion in the presence of various physiological ligands which stabilize different conformational states of the sodium pump. There are distinctive conformational changes of the protein which are revealed by amino-terminal amino acid sequence analysis of the digests following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The changes in cleavage patterns result from alterations in domain-domain interactions of the protein. We provide evidence in the alpha-subunit for (i) tight interaction between part of the cytoplasmic ATP binding domain and the membrane-bound portion of the protein; (ii) involvement of the cytoplasmic loop between M2 and M3 in structural rearrangements upon phosphorylation or ion binding; (iii) generation of the same digested products when either ouabain or potassium (rubidium) is present. Similarly, evidence is provided for conformational sensitivity...
The biologically and clinically important membrane transporters are challenging proteins to study... more The biologically and clinically important membrane transporters are challenging proteins to study because of their low level of expression, multidomain structure, and complex molecular dynamics that underlies their activity. ATP7B is a copper transporter that traffics between the intracellular compartments in response to copper elevation. The N-terminal domain of ATP7B (N-ATP7B) is involved in binding copper, but the role of this domain in trafficking is controversial. To clarify the role of N-ATP7B, we generated nanobodies that interact with ATP7B in vitro and in cells. In solution NMR studies, nanobodies revealed the spatial organization of N-ATP7B by detecting transient functionally relevant interactions between metal-binding domains 1-3. Modulation of these interactions by nanobodies in cells enhanced relocalization of the endogenous ATP7B toward the plasma membrane linking molecular and cellular dynamics of the transporter. Stimulation of ATP7B trafficking by nanobodies in the ...
Proceedings of the National Academy of Sciences, 1998
Wilson's disease (WND) is an inherited disorder of copper homeostasis characterized by ab... more Wilson's disease (WND) is an inherited disorder of copper homeostasis characterized by abnormal accumulation of copper in several tissues, particularly in the liver, brain, and kidney. The disease-associated gene encodes a copper-transporting P-type ATPase, the WND protein, the subcellular location of which could be regulated by copper. We demonstrate that the WND protein is present in cells in two forms, the 160-kDa and the 140-kDa products. The 160-kDa product was earlier shown to be targeted to trans-Golgi network. The 140-kDa product identified herein is located in mitochondria as evidenced by the immunofluorescent staining of HepG2 cells with specific mitochondria markers and polyclonal antibody directed against the C terminus of the WND molecule. The mitochondrial location for the 140-kDa WND product was confirmed by membrane fractionation and by analysis of purified human mitochondria. The antibody raised against a repetitive sequence in the N-terminal portion of the WND molecule detects an additional 16-kDa protein, suggesting that the 140-kDa product was formed after proteolytic cleavage of the full-length WND protein at the N terminus. Thus, the WND protein is a P-type ATPase with an unusual subcellular localization. The mitochondria targeting of the WND protein suggests its important role for copper-dependent processes taking place in this organelle.
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Papers by S. Lutsenko