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Das Schwermetall Kupfer ist von essentieller Bedeutung für zahlreiche zelluläre Funktionen. Aufgrund seines Redoxpotentials muss die Kupferhomöostase im Organismus eng reguliert werden. Die Schlüsselrolle spielt dabei das Kupfertransportprotein ATP7B (Wilson ATPase) in der Leber. Ein Funktionsverlust dieses Proteins wird in der autosomal-rezessiv vererbten Erkrankung Morbus Wilson deutlich. Der Kupfertransportdefekt der Hepatozyten führt zu einer Kupferüberladung in der Leber mit nachfolgender Schädigung. Ferner kommt es zu einer Kupferakkumulation im Zentralnervensystem mit neurologischen Störungen. Das bei der Erkrankung betroffene Gen ATP7B wurde 1993 kloniert. Bis heute sind über 500 krankheitsverursachende Genmutationen entdeckt worden. Eine Schlüsselfunktion dieser Enzymgruppe ist die katalytische Phosphorylierung. Die Auswirkungen von Mutationen auf die Funktion des Proteins sind jedoch nur unzureichend verstanden. ATP7B kann mit Hilfe des Baculovirusexpressionssystems hergestellt und anschließend proteinbiochemischen Untersuchungen unterzogen werden. In dieser experimentellen Arbeit wurde untersucht, ob Punktmutationen diesen Phosphorylierungsmechanismus von ATP7B beeinflussen. Dafür wurden, neben dem Wildtyp-Protein, 25 patientenspezifische und eine noch nicht beim Menschen beobachtete Mutation der Phosphorylierungsstelle D1027A als Negativkontrolle generiert und die katalytische Aktivität in einem Phosphorylierungsassay untersucht. In der vorliegenden Arbeit wurde gezeigt, dass bestimmte Punktmutationen zum Funktionsverlust von ATP7B führen. Als weiterer Mechanismus der Mutationswirkung wurde, neben der Inaktivierung von ATP7B, die Hyperphosphorylierung entdeckt. Die biochemische Charakterisierung dieser Mutationen führt zu einem tieferen Verständnis in der Pathophysiologie des Morbus Wilson und ebnet den Weg für detaillierte Untersuchungen der Genotyp-Phänotyp-Korrelation sowie für innovative Diagnostik- und Therapiestrategien.

Vilsono liga (VL) ir HFE-hemochromatozė – monogeninės, pagal Mendelio dėsnius paveldimos retos ligos. Šių ligų priežastis yra ATP7B arba HFE genų mutacijos, sukeliančios gyvybei pavojingas lėtines ligas. Šio darbo metu buvo tirti ligonių, sergančių metabolinėmis kepenų ligomis – VL ir HFE–hemochromatoze genų mutacijos, jų dažnis, ligų fenotipinės charakteristikos bei HFE geno dažniausių mutavusių alelių dažnis Lietuvos savanorių kraujo donorų populiacijoje. Buvo nustatyta, kad Rytų ir Centrinės Europos šalių populiacijoms būdinga c.3207C>A (p.His1069Gln) mutacija ATP7B gene taip pat dažniausia ir Lietuvoje. Patvirtinta, kad VL jautriausias metodas - molekulinis genetinis diagnostikos metodas. Kitiems klinikiniams laboratoriniams tyrimo metodams buvo būdingas mažesnis jautrumas. Ištyrus Lietuvos savanorius kraujo donorus nustatyta, kad HFE–hemochromatozės geno c.845G>A (p.Cys282Tyr) ir c.187C>G (p.His63Asp) mutacijų dažnis yra artimiausias Lenkijai ir kitoms Rytų ir Centrinės Europos šalims. HFE-hemochromatozės išsivystymo rizika yra 1,3 proc. Lietuvos savanoriams kraujo donorams, kurių genotipas c.[845G>A]; [845G>A] (0,1 proc. tiriamųjų) arba genotipas c.[845G>A];[187C>G], (1,2 proc. tiriamųjų). Tiriant HFE geno mutacijų paplitimą tarp skirtinguose Lietuvos etnokultūriniuose regionuose gyvenančių savanorių kraujo donorų nustatyta, kad c.845G>A mutacijos dažnis statistiškai patikimai buvo dažnesnis Žemaitijoje. / Wilson’s disease (WD) and HFE-hemochromatosis are monogenic rare diseases inherited following Mendel’s laws. These diseases are caused by ATP7B or HFE gene mutations, which cause life-threatening chronic diseases. This study analyzed gene mutations in patients with metabolic liver diseases – WD and HFE–hemochromatosis, the frequency of such mutations, the phenotypic characteristics of these diseases, and the frequency of the most common mutations in the alleles of the HFE gene in the population of Lithuanian volunteer blood donors. The study showed that the ATP7B gene mutation c.3207C>A (p.His1069Gln), which is characteristic of the populations of Central and Eastern Europe, was also most common in Lithuania. The study confirmed that molecular genetic diagnostics was the most sensitive technique in detecting WD. Other clinical laboratory diagnostic techniques demonstrated lower sensitivity. The examination of Lithuanian volunteer blood donors showed that the frequency of HFE–hemochromatosis mutations c.845G>A (p.Cys282Tyr) and c.187C>G (p.His63Asp) was closest to that in Poland and other Eastern and Central European countries. The risk of developing HFE-hemochromatosis among Lithuanian volunteer blood donors with genotype c.[845G>A]; [845G>A] (0.1% of the subjects) or c.[845G>A];[187C>G] (1.2% of the subjects) was 1.3%. The analysis of the prevalence of HFE gene mutations among Lithuanian volunteer blood donors from different ethno-cultural regions of Lithuania showed that... [to full text]

Wilson disease (WD) is an autosomal recessive disorder characterized by deposition of copper in the body, mainly in the liver and brain. WD patients present with hepatic, neurological, and psychiatric problems. The diagnosis of WD is very challenging, and is performed by taking into account both clinical and biochemical parameters. The treatment of WD exists, which aims at initial chelation therapy followed by maintenance therapy. WD is caused by mutations in the ATP7B gene. Till date, more than 600 mutations in ATP7B have already been described from many countries, including India. However, there are a very few large cohort studies which are reported from Indian population. In this study, we have attempted to perform mutation analysis of ATP7B in a large cohort of WD families from Bangalore, south India, and further look into the molecular consequences of the novel mutations identified in the present study.

Das Schwermetall Kupfer ist von essentieller Bedeutung für zahlreiche zelluläre Funktionen. Aufgrund seines Redoxpotentials muss die Kupferhomöostase im Organismus eng reguliert werden. Die Schlüsselrolle spielt dabei das Kupfertransportprotein ATP7B (Wilson ATPase) in der Leber. Ein Funktionsverlust dieses Proteins wird in der autosomal-rezessiv vererbten Erkrankung Morbus Wilson deutlich. Der Kupfertransportdefekt der Hepatozyten führt zu einer Kupferüberladung in der Leber mit nachfolgender Schädigung. Ferner kommt es zu einer Kupferakkumulation im Zentralnervensystem mit neurologischen Störungen. Das bei der Erkrankung betroffene Gen ATP7B wurde 1993 kloniert. Bis heute sind über 500 krankheitsverursachende Genmutationen entdeckt worden. Eine Schlüsselfunktion dieser Enzymgruppe ist die katalytische Phosphorylierung. Die Auswirkungen von Mutationen auf die Funktion des Proteins sind jedoch nur unzureichend verstanden. ATP7B kann mit Hilfe des Baculovirusexpressionssystems hergestellt und anschließend proteinbiochemischen Untersuchungen unterzogen werden. In dieser experimentellen Arbeit wurde untersucht, ob Punktmutationen diesen Phosphorylierungsmechanismus von ATP7B beeinflussen. Dafür wurden, neben dem Wildtyp-Protein, 25 patientenspezifische und eine noch nicht beim Menschen beobachtete Mutation der Phosphorylierungsstelle D1027A als Negativkontrolle generiert und die katalytische Aktivität in einem Phosphorylierungsassay untersucht. In der vorliegenden Arbeit wurde gezeigt, dass bestimmte Punktmutationen zum Funktionsverlust von ATP7B führen. Als weiterer Mechanismus der Mutationswirkung wurde, neben der Inaktivierung von ATP7B, die Hyperphosphorylierung entdeckt. Die biochemische Charakterisierung dieser Mutationen führt zu einem tieferen Verständnis in der Pathophysiologie des Morbus Wilson und ebnet den Weg für detaillierte Untersuchungen der Genotyp-Phänotyp-Korrelation sowie für innovative Diagnostik- und Therapiestrategien.

Many processes in living systems occur through transient interactions among proteins. Those interactions are often weak and are driven by small changes in free energy. Due to the short-living nature of these interactions, our knowledge about driving forces, dynamics and structures of these types of protein-protein heterocomplexes are though limited. This is especially important for cellular copper (Cu) trafficking: Copper ions are essential for all eukaryotes and most bacteria. As a cofactor in many enzymes, copper is especially vital in respiration or detoxification. Since the same features that make copper useful also make it toxic, it needs to be controlled tightly. Additionally, in the reducing environment of the cytosol, Cu is present as insoluble Cu(I). To circumvent both toxicity and solubility issues, a system has evolved where copper is comforted by certain copper binding proteins, so-called Cu-chaperones. They transiently interact with each other to distribute the Cu atoms in a cell. In humans, one of them is Atox1. It binds copper with a binding site containing two thiol residues and transfers it to other binding sites, mostly those of a copper pump, ATP7B (also known as Wilsons disease protein). My work was aimed at understanding copper-mediated protein-protein interactions on a molecular and mechanistic level. Which amino acids interact with the metal? Which forces drive the transfer from one protein to the other? Using biophysical and biochemical methods such as chromatography and calorimetry on wild type and point-mutated proteins in vitro, we found that the copper is transferred via a dynamic intermediate complex that keeps the system flexible while shielding the copper against other interactions. Although similar transfer interactions can be observed in other organisms, and many conclusions in the copper field are drawn from bacterial and yeast analogs, we believe that it is important to investigate human proteins, too. Not only is their regulation different, but also only in humans we find the diseases linked to the proteins: Copper level regulation diseases are to be named first, but atypical copper levels have also been linked to tumors and amyloid dispositions. In summary, my observations and conclusions are of basic research character and can be of importance for both general copper and human medicinal research.

The intention of this analysis was to identify patients with treated Wilson disease (WD) and residual neurological symptoms in order to determine whether or not they were undergoing any treatment in addition to the common decoppering medication. Moreover, the effects of any symptomatic medication were analyzed. Two samples of WD patients were investigated either by a mailed questionnaire survey (n = 135) or by a retrospective analysis (n = 75). A considerable proportion of patients still suffered from neurological symptoms (n = 106, 50.5%), of whom a relatively small proportion was treated symptomatically (n = 33, 31.1%). The documented effects varied substantially, with anticholinergics and botulinum toxin (against dystonia) and primidone (against tremor) apparently being the most promising compounds. Further studies are required to analyze the symptomatic treatment of WD patients with residual neurological symptoms in more detail. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Copper is an integral component of key metabolic enzymes. Numerous physiological processes depend on a fine balance between the biosynthetic incorporation of copper into proteins and the export of excess copper from the cell. The homeostatic control of copper requires the activity of the copper transporting ATPases (Cu-ATPases). In Wilson disease the disruption in the function of the Cu-ATPase ATP7B results in the accumulation of excess copper and a marked deficiency of copper-dependent enzymes. In this work, the structure of ATP7B has been modeled by homology using the Ca-ATPase X-ray structure, enabling a mechanism of copper transport by ATP7B to be proposed. The fourth transmembrane helix (TM4) of Ca-ATPase contains conserved residues critical to cation binding and is predicted to correspond to TM6 of the ATP7B homology model, containing the highly conserved CXXCPC motif. The interaction with Cu(I) and the importance of the 3 cysteines in TM6 of ATP7B has been shown using model peptides. ATP7B has a large cytoplasmic N-terminus comprised of six copper-binding domains (WCBD1-6), each capable of binding one Cu(I). Protein-protein interactions between WCBDs and the copper chaperone Atox1 has been shown, contrary to previous reports, to occur even in the absence of copper. 15N relaxation measurements on the apo and Cu(I)-bound WCBD4-6 show that there is minimal change in the dynamic properties and the relative orientation of the domains in the two states. The domain 4-5 linker remains flexible, and domain 5-6 is not a rigid dimer, with flexibility between the domains. Copper transfer to and between WCBD1-6 likely occurs via protein interactions facilitated by the flexibility of the domains with respect to each other. The flexible linkers connecting the domains are important in giving the domains motional freedom to interact with Atox1, to transfer copper to other domains, and finally to transfer copper to the transmembrane site for transport across the membrane.

Copper is an integral component of key metabolic enzymes. Numerous physiological processes depend on a fine balance between the biosynthetic incorporation of copper into proteins and the export of excess copper from the cell. The homeostatic control of copper requires the activity of the copper transporting ATPases (Cu-ATPases). In Wilson disease the disruption in the function of the Cu-ATPase ATP7B results in the accumulation of excess copper and a marked deficiency of copper-dependent enzymes. In this work, the structure of ATP7B has been modeled by homology using the Ca-ATPase X-ray structure, enabling a mechanism of copper transport by ATP7B to be proposed. The fourth transmembrane helix (TM4) of Ca-ATPase contains conserved residues critical to cation binding and is predicted to correspond to TM6 of the ATP7B homology model, containing the highly conserved CXXCPC motif. The interaction with Cu(I) and the importance of the 3 cysteines in TM6 of ATP7B has been shown using model peptides. ATP7B has a large cytoplasmic N-terminus comprised of six copper-binding domains (WCBD1-6), each capable of binding one Cu(I). Protein-protein interactions between WCBDs and the copper chaperone Atox1 has been shown, contrary to previous reports, to occur even in the absence of copper. 15N relaxation measurements on the apo and Cu(I)-bound WCBD4-6 show that there is minimal change in the dynamic properties and the relative orientation of the domains in the two states. The domain 4-5 linker remains flexible, and domain 5-6 is not a rigid dimer, with flexibility between the domains. Copper transfer to and between WCBD1-6 likely occurs via protein interactions facilitated by the flexibility of the domains with respect to each other. The flexible linkers connecting the domains are important in giving the domains motional freedom to interact with Atox1, to transfer copper to other domains, and finally to transfer copper to the transmembrane site for transport across the membrane.

The intention of this analysis was to identify patients with treated Wilson disease (WD) and residual neurological symptoms in order to determine whether or not they were undergoing any treatment in addition to the common decoppering medication. Moreover, the effects of any symptomatic medication were analyzed. Two samples of WD patients were investigated either by a mailed questionnaire survey (n = 135) or by a retrospective analysis (n = 75). A considerable proportion of patients still suffered from neurological symptoms (n = 106, 50.5%), of whom a relatively small proportion was treated symptomatically (n = 33, 31.1%). The documented effects varied substantially, with anticholinergics and botulinum toxin (against dystonia) and primidone (against tremor) apparently being the most promising compounds. Further studies are required to analyze the symptomatic treatment of WD patients with residual neurological symptoms in more detail. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.