Funktionale Charakterisierung herzmuskelspezifischer Proteinvarianten

Dieding M. Funktionale Charakterisierung herzmuskelspezifischer Proteinvarianten. Bielefeld: Universität Bielefeld; 2016.Genetisch bedingte Kardiomyopathien sind schwere Erkrankungen des Herzmuskels. Sie führen oft zu Herzinsuffizienz, deren einzige kausale Therapie die Herztransplantation ist. Die genetische Prädisposition der Kardiomyopathie ist zudem die Hauptursache für den plötzlichen Herztod bei jungen Sportlern (D’Silva u. a. 2015). Ursächlich für die Erkrankung sind Mutationen in verschiedensten Genen. Die Mechanismen, die von der genetischen Veränderung zu einer Symptomatik führen, sind bisher nur teilweise verstanden. Häufig zeigt der Genotyp eine unvollständige Penetranz und bildet variable Phänotypen aus. Um die Mechanismen und damit die Ursache der Erkrankung zu verstehen, ist die interdisziplinäre Forschung von medizinischen, biochemischen und biophysikalischen Ansätzen erforderlich. Wird beispielsweise in Verbindung mit einer Erkrankung eine neue Genvariante entdeckt, helfen funktionale biophysikalische und biochemische Analysen die molekularen und zellularen Effekte der Genvariation aufzuklären und zu klassifizieren (Richards u. a. 2015). Die Klassifizierung findet Anwendung in der genetischen Beratung der betroffenen Familien. Mutationen, die im Zusammenhang mit Kardiomyopathien stehen, kodieren verschiedene Proteine in Herzmuskelzellen. Von diesen wurden in der vorliegenden Arbeit die Proteine Desmin und Desmoglein2 biophysikalisch charakterisiert. Das Intermediärfilament Desmin ist ein wichtiger Bestandteil des Cytoskeletts der Muskelzelle. Desminfilamente bilden ein flexibles Gerüst im Cytoplasma und geben der Zelle strukturellen und mechanischen Halt. Mutationen im Desmin-Gen (DES) verursachen eine große Bandbreite schwerer Skelett- und Herzmuskelerkrankungen. Das Cadherin Desmoglein gehört zu den Desmosomen, welche punktuell zwei Zellen mechanisch miteinander verknüpfen. Desmoglein2 ist eines von zwei Transmembranproteinen des kardialen Desmosoms, deren extrazellulare Domänen den Raum zwischen den Zellen überbrücken und mit gegenüberliegenden Domänen einen adhäsiven Kontakt herstellen. Somit erhalten Desmosomen die mechanische Integrität des Herzmuskels. Mutationen im Desmoglein2-Gen (DSG2) sind häufig Ursache der seltenen arrhythmogenen rechtsventrikulären Kardiomyopathie. Die Erforschung der Eigenschaften und Funktionen des Proteinwildtyps auf molekularer Ebene und der zellulare Kontext sind essenziell für das Verständnis der Pathomechanismen von Proteinvarianten. Vor diesem Hintergrund wurden die isolierten Proteine mittels Rasterkraftmikroskopie und -spektroskopie untersucht. Das Rasterkraftmikroskop (AFM) ist ein vielseitiges Instrument mit dem biologische Proben abgebildet und auf mechanische Eigenschaften untersucht werden können. Neben einem konventionellen AFM-Setup wurde ein aperturloses optisches Rasternahfeldmikroskop genutzt, welches - zusätzlich zur Topografie - Fluoreszenz detektieren kann. Die Eigenschaft von Desmin zu Filamenten zu assemblieren lag im Fokus bei diesem Protein beziehungsweise Proteinkomplex. Die in vitro assemblierten Desminfilamente wurden mit AFM und aperturlosem optischen Rasternahfeldmikroskop abgebildet. Dabei lag der Forschungsschwerpunkt bei dem Vergleich des Assemblierungsverhaltens von verschiedenen Varianten mit dem Wildtyp. Es wurde sowohl die homophile Assemblierung als auch die heterophile Assemblierung, also ein Gemisch aus Wildtyp und der jeweiligen Variante, untersucht. Einige der untersuchten Desminvarianten zeigten schwere Beeinträchtigung bei der Bildung zu nativen Filamenten. Unter dem Aspekt der Bindungseigenschaften wurde die adhäsive Region von Desmoglein2 untersucht. Mit dynamischer Kraftspektroskopie wurde die homophile Bindungskinetik von Desmoglein2-Proteinfragmenten betrachtet und darüber hinaus wurde die freie Enthalpie der Bindung abgeschätzt. Dabei zeigte der Wildtyp eine Bindungscharakteristik, die von homologen Cadherinen bekannt ist. Neben des Erkenntnisgewinns des nativen Charakters von Desmin und Desmoglein2 wurden diese Methoden herangezogen, um Kardiomyopathie assoziierte Varianten dem jeweiligen Wildtyp gegenüberzustellen und funktionale Unterschiede aufzudecken. Die Ergebnisse können dabei helfen die Auswirkung der Genvariation auf molekularer Ebene zu verstehen und sind ein wichtiges Puzzlestück für die Klassifizierung der Varianten

Arrhythmogenic cardiomyopathy related DSG2 mutations affect desmosomal cadherin binding kinetics

Dieding M, Debus JD, Kerkhoff R, et al. Arrhythmogenic cardiomyopathy related DSG2 mutations affect desmosomal cadherin binding kinetics. Scientific Reports. 2017;7(1): 13791.Cadherins are calcium dependent adhesion proteins that establish the intercellular mechanical contact by bridging the gap to adjacent cells. Desmoglein-2 (Dsg2) is a specific cadherin of the cell-cell contact in cardiac desmosomes. Mutations in the DSG2-gene are regarded to cause arrhythmogenic (right ventricular) cardiomyopathy (ARVC) which is a rare but severe heart muscle disease. The molecular pathomechanisms of the vast majority of DSG2 mutations, however, are unknown. Here, we investigated the homophilic binding of wildtype Dsg2 and two mutations which are associated with ARVC. Using single molecule force spectroscopy and applying Jarzynski's equality we determined the kinetics and thermodynamics of Dsg2 homophilic binding. Notably, the free energy landscape of Dsg2 dimerization exposes a high activation barrier which is in line with the proposed strand-swapping binding motif. Although the binding motif is not directly affected by the mutations the binding kinetics differ significantly from the wildtype. Furthermore, we applied a dispase based cell dissociation assay using HT1080 cell lines over expressing Dsg2 wildtype and mutants, respectively. Our molecular and cellular results consistently demonstrate that Dsg2 mutations can heavily affect homophilic Dsg2 interactions. Furthermore, the full thermodynamic and kinetic description of Dsg2 dimerization provides a consistent model of the so far discussed homophilic cadherin binding

Apertureless scanning near-field optical microscopy of sparsely labeled tobacco mosaic viruses and the intermediate filament desmin

Harder A, Dieding M, Walhorn V, et al. Apertureless scanning near-field optical microscopy of sparsely labeled tobacco mosaic viruses and the intermediate filament desmin. Beilstein Journal of Nanotechnology. 2013;4:510-516.Both fluorescence imaging and atomic force microscopy (AFM) are highly versatile and extensively used in applications ranging from nanotechnology to life sciences. In fluorescence microscopy luminescent dyes serve as position markers. Moreover, they can be used as active reporters of their local vicinity. The dipolar coupling of the tip with the incident light and the fluorophore give rise to a local field and fluorescence enhancement. AFM topographic imaging allows for resolutions down to the atomic scale. It can be operated in vacuum, under ambient conditions and in liquids. This makes it ideal for the investigation of a wide range of different samples. Furthermore an illuminated AFM cantilever tip apex exposes strongly confined non-propagating electromagnetic fields that can serve as a coupling agent for single dye molecules. Thus, combining both techniques by means of apertureless scanning near-field optical microscopy (aSNOM) enables concurrent high resolution topography and fluorescence imaging. Commonly, among the various (apertureless) SNOM approaches metallic or metallized probes are used. Here, we report on our custom-built aSNOM setup, which uses commercially available monolithic silicon AFM cantilevers. The field enhancement confined to the tip apex facilitates an optical resolution down to 20 nm. Furthermore, the use of standard mass-produced AFM cantilevers spares elaborate probe production or modification processes. We investigated tobacco mosaic viruses and the intermediate filament protein desmin. Both are mixed complexes of building blocks, which are fluorescently labeled to a low degree. The simultaneous recording of topography and fluorescence data allows for the exact localization of distinct building blocks within the superordinate structures

Analytical Tools in Minicircle Production

Rischmüller A, Viefhues M, Dieding M, et al. Analytical Tools in Minicircle Production. In: Schleef M, ed. Minicircle and Miniplasmid DNA Vectors: The Future of Nonviral and Viral gene Transfer. Weinheim: Wiley-Blackwell; 2013: 71-91

A novel desmin (DES) indel mutation causes severe atypical cardiomyopathy in combination with atrioventricular block and skeletal myopathy

Schirmer I, Dieding M, Klauke B, et al. A novel desmin (DES) indel mutation causes severe atypical cardiomyopathy in combination with atrioventricular block and skeletal myopathy. Molecular Genetics & Genomic Medicine. 2018;6(2):288-293.Background DES mutations cause different cardiac and skeletal myopathies. Most of them are missense mutations. Methods Using a next-generation sequencing cardiac 174 gene panel, we identified a novel heterozygous in-frame indel mutation (DES-c.493_520del28insGCGT, p.Q165_A174delinsAS) in a Caucasian patient with cardiomyopathy in combination with atrioventricular block and skeletal myopathy. This indel mutation is located in the coding region of the first exon. Family anamnesis revealed a history of sudden cardiac death. We performed cell transfection experiments and in vitro assembly experiments to prove the pathogenicity of this novel DES indel mutation. Results These experiments revealed a severe filament formation defect of mutant desmin supporting the pathogenicity. In addition, we labeled a skeletal muscle biopsy from the mutation carrier revealing cytoplasmic desmin positive protein aggregates. In summary, we identified and functionally characterized a pathogenic DES indel mutation causing cardiac and skeletal myopathy. Conclusion Our study has relevance for the clinical and genetic interpretation of further DES indel mutations causing cardiac or skeletal myopathies and might be helpful for risk stratification

Functional characterization of desmin mutant p.P419S

Brodehl A, Dieding M, Cakar H, et al. Functional characterization of desmin mutant p.P419S. European journal of human genetics : EJHG. 2013;21(6):589-590

Functional characterization of the novel DES mutation p.L136P associated with dilated cardiomyopathy reveals a dominant filament assembly defect

Brodehl A, Dieding M, Biere N, et al. Functional characterization of the novel DES mutation p.L136P associated with dilated cardiomyopathy reveals a dominant filament assembly defect. JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY. 2016;91:207-214.Background: Dilated cardiomyopathy (DCM) could be caused by mutations in more than 40 different genes. However, the pathogenic impact of specific mutations is in most cases unknown complicating the genetic counseling of affected families. Therefore, functional studies could contribute to distinguish pathogenic mutations and benign variants. Here, we present a novel heterozygous DES missense variant (c.407C > T; p.L136P) identified by next generation sequencing in a DCM patient. DES encodes the cardiac intermediate filament protein desmin, which has important functions in mechanical stabilization and linkage of the cell structures in cardiomyocytes. Methods and results: Cell transfection experiments and assembly assays of recombinant desmin in combination with atomic force microscopy were used to investigate the impact of this novel DES variant on filament formation. Desmin-p.L136P forms cytoplasmic aggregates indicating a severe intrinsic filament assembly defect of this mutant. Co-transfection experiments of wild-type and mutant desmin conjugated to different fluorescence proteins revealed a dominant affect of this mutant on filament assembly. These experiments were complemented by apertureless scanning near-field optical microscopy. Conclusion: In vitro analysis demonstrated that desmin-p.L136P is unable to form regular filaments and accumulate instead within the cytoplasm. Therefore, we classified DES-p.L136P as a likely pathogenic mutation. In conclusion, the functional characterization of DES-p.L136P might have relevance for the genetic counseling of affected families with similar DES mutations and could contribute to distinguish pathogenic mutations from benign rare variants. (C) 2016 Elsevier Ltd. All rights reserved

Rational design of a cytotoxic dinuclear Cu2 complex that binds by molecular recognition at two neighboring phosphates of the DNA backbone

Jany T, Moreth A, Gruschka C, et al. Rational design of a cytotoxic dinuclear Cu2 complex that binds by molecular recognition at two neighboring phosphates of the DNA backbone. Inorganic chemistry. 2015;54(6):2679-2690.The mechanism of the cytotoxic function of cisplatin and related anticancer drugs is based on their binding to the nucleobases of DNA. The development of new classes of anticancer drugs requires establishing other binding modes. Therefore, we performed a rational design for complexes that target two neighboring phosphates of the DNA backbone by molecular recognition resulting in a family of dinuclear complexes based on 2,7-disubstituted 1,8-naphthalenediol. This rigid backbone preorganizes the two metal ions for molecular recognition at the distance of two neighboring phosphates in DNA of 6-7 Å. Additionally, bulky chelating pendant arms in the 2,7-position impede nucleobase complexation by steric hindrance. We successfully synthesized the Cu(II)2 complex of the designed family of dinuclear complexes and studied its binding to dsDNA by independent ensemble and single-molecule methods like gel electrophoresis, precipitation, and titration experiments followed by UV-vis spectroscopy, atomic force microscopy (AFM), as well as optical tweezers (OT) and magnetic tweezers (MT) DNA stretching. The observed irreversible binding of our dinuclear Cu(II)2 complex to dsDNA leads to a blocking of DNA synthesis as studied by polymerase chain reactions and cytotoxicity for human cancer cells

Rational Design of a Cytotoxic Dinuclear Cu₂ Complex That Binds by Molecular Recognition at Two Neighboring Phosphates of the DNA Backbone

The mechanism of the cytotoxic function of cisplatin and related anticancer drugs is based on their binding to the nucleobases of DNA. The development of new classes of anticancer drugs requires establishing other binding modes. Therefore, we performed a rational design for complexes that target two neighboring phosphates of the DNA backbone by molecular recognition resulting in a family of dinuclear complexes based on 2,7-disubstituted 1,8-naphthalenediol. This rigid backbone preorganizes the two metal ions for molecular recognition at the distance of two neighboring phosphates in DNA of 6–7 Å. Additionally, bulky chelating pendant arms in the 2,7-position impede nucleobase complexation by steric hindrance. We successfully synthesized the Cu^II₂ complex of the designed family of dinuclear complexes and studied its binding to dsDNA by independent ensemble and single-molecule methods like gel electrophoresis, precipitation, and titration experiments followed by UV–vis spectroscopy, atomic force microscopy (AFM), as well as optical tweezers (OT) and magnetic tweezers (MT) DNA stretching. The observed irreversible binding of our dinuclear Cu^II₂ complex to dsDNA leads to a blocking of DNA synthesis as studied by polymerase chain reactions and cytotoxicity for human cancer cells