Functional analysis of point mutants of cardiac actin involved in dilative and hypertrophic cardiomyopathies
Zusammenfassung der Projektergebnisse
In this project we purified alpha cardiac actin variants, which have been made responsible for the generation of cardiomyopathies: wild type, one causing HCM: A295S and two causing DCM: R312K/H and E361G. These actin variants were expressed by the insect cell Sf9/baculovirus system and purified to homogeneity by affinity adsorption to gelsolin G4-6. Unfortunately, it was not possible to isolate the actins after N-terminal His-tagging. We purified cardiac actin form either bovine or pig heart acetone powder and compared the conventionally prepared with recombinant wt cardiac actin and did not find differences in the biochemical assays. The biochemical analyses of the isolated cardiac actin variants indicated that the HCM related A295S mutant possessed a decreased ability to polymerize and to stimulate the myosin-S1 ATPase. Similarly, both DCM related mutants possessed a reduced ability to polymerize and after decoration with tropomyosin and troponin complex both exhibited reduced calcium-ion sensitivity. In addition, the cooperativity of myosin-S1 binding to thin filaments containing the R312H mutant was clearly reduced probably due to the fact that the mutated residue is located with the tropomyosin-binding region. After infection of neonatal cardiomyocytes with adenovirus particles conferring these actins their incorporation into nascent thin filaments was observed. Wt and R312H actins appeared to be incorporated at their natural sites, the Z-lines, whereas the E361G mutant was preferentially incorporated at the thin filament minus ends, i.e. close to the middle of the sarcomeres. This behaviour might be due to the location of the mutated residue within the α-actinin binding region and suggest a cause for the reduced sarcomere mechanostability typical of DCM affected hearts. In addition, we investigated the effects of mutations of the TnI subunit of troponin complex that were recently identified in two newborns suffering of restrictive cardiomyopathy (RCM), which in most cases has a poor prognosis unless heart transplantation can be preformed. Both mutated TnIs resulted in an increased calcium-ion sensitivity as typically observed in RCM hearts. Structurally they led to thin filament bending and breakage when analysed with reconstituted thin filaments, however, these alterations were at least partially reversed by addition of the actin-binding domain of MyBP-C. Furthermore, we showed that FHOD1 (a member of the formin family that usually promote actin filament formation) is specifically located within the intercalated discs (ICDs) that represent membrane specialisations involved in connecting cardiac muscle cells. Into the ICDs terminate the last half sarcomeres of the cardiac muscle myofibrils and FHOD1 might well have an important role in conferring sarcomere stability. It will be interesting to analyse the interactions of FHOD1 with cardiac actin and its different mutants. As an extension to the cardiovascular system we participated in a study about the degradation of neutrophil released extracellular traps (NETs). Under pathological inflammatory conditions massive release of NETs by neutrophil leucocytes can lead to vascular occlusion. In order to overcome a dangerous obstruction of blood circulation the degradation of NETs must occur to prevent tissue damage. Using mice deficient in the two endonucleases present in blood plasma (DNase I and DNase 1-like3) it was shown that the presence or administration of either of these endonucleases leads to NET degradation and survival of the animals. Since NET formation has also been shown to occur in coronary vessels during infarction the therapeutic use of endonuclease administration might be considered.
Projektbezogene Publikationen (Auswahl)
- (2013): FHOD1 is a combined actin filament capping and bundling factor that selectively associates with actin arcs and stress fibers. J. Cell Sci. 126, 1891-1901
Schönichen, A. Mannherz, H.G., Behrmann, E., Mazur, A.J., Kühn, S., Fackler, O.T., Raunser, S., Dehmelt, L., and Geyer, M.
(Siehe online unter https://doi.org/10.1242/jcs.126706) - (2014): Molecular mechanisms of disease-related human β-actin mutations p.R183W and p.E364K. FEBS J. 2014 Dec;281(23):5279-91
Hundt N, Preller M, Swolski O, Ang AM, Mannherz HG, Manstein DJ, Müller M
(Siehe online unter https://doi.org/10.1111/febs.13068) - (2014): Thymosin ß4 inhibits ADF/cofilin stimulated F-actin cycling and HeLa cell migration: Reversal by active Arp2/3 complex. Cytoskeleton (Hoboken) 71: 95-107
Al Haj, A., Mazur, A.J., App, C., B., Theiss, C., Silvan, U., Schoenenberger, C.-A., Jockusch, B.M., Hannappel, E., Weeds, A.G., and Mannherz, H.G.
(Siehe online unter https://doi.org/10.1002/cm.21128) - (2015). Distinct actin oligomers modulate differently the activity of actin nucleators. FEBS J. 282(19): 3824-40
Qu Z, Silvan U, Jockusch BM, Aebi U, Schoenenberger CA, Mannherz HG
(Siehe online unter https://doi.org/10.1111/febs.13381) - (2015): Detection of molecules and cells using nuclear magnetic resonance with magnetic nanoparticles. Journal of Magnetism and Magnetic Materials 380, 271-275
Rümenapp, C., Gleich, B., Mannherz, H.G. and Haase, A.
(Siehe online unter https://doi.org/10.1016/j.jmmm.2014.09.009) - (2015): Distribution of Formins in Cardiac Muscle: FHOD1 is a component of intercalated discs and costameres. Eur. J. Cell Biol. 94, 101-113
Al Haj, A., Mazur, A.J., Radaszkiewicz, K., Radaszkiewicz, T., Makowiecka, A., Stopschinski, B.E., Schönichen, A., Geyer, M., Mannherz, H.G.
(Siehe online unter https://doi.org/10.1016/j.ejcb.2014.11.003) - (2015): Impaired DNase1-mediated degradation of neutrophil extracellular traps is associated with acute thrombotic microangiopathies. J Thromb Haemost. 13, 732-742
Jiménez-Alcázar M, Napirei M, Panda R, Köhler EC, Kremer-Hovinga JA, Mannherz HG, Peine S, Renné T, Lämmle B, Fuchs TA
(Siehe online unter https://doi.org/10.1111/jth.12796) - (2015): VEGF Signaling Regulates Cofilin and the Arp2/3-complex within the Axonal Growth Cone. Curr Neurovasc Res. 12(3):293-307
Dumpich M, Mannherz HG, Theiss C
(Siehe online unter https://doi.org/10.2174/1567202612666150603141144) - (2016): Contributions of the lower dimer to supramolecular actin patterning revealed by TIRF microscopy. J Struct Biol. 195(2): 159-166
Silvan U, Hyotyla J, Mannherz HG, Ringler P, Müller SA, Aebi U, Maier T, Schoenenberger CA
(Siehe online unter https://doi.org/10.1016/j.jsb.2016.05.008) - (2016): Gelsolin interacts with LamR, hnRNP U, nestin, Arp3 and β-tubulin in human melanoma cells as revealed by immunoprecipitation and mass spectrometry. Eur J Cell Biol. 95(1): 26-41
Mazur AJ, Radaszkiewicz T, Makowiecka A, Malicka-Błaszkiewicz M, Mannherz HG, Nowak D
(Siehe online unter https://doi.org/10.1016/j.ejcb.2015.11.001) - (2016): Reelin and cofilin cooperate during the migration of cortical neurons: a quantitative morphological analysis. Development 143(6): 1029-40
Chai X, Zhao S, Fan L, Zhang W, Lu X, Shao H, Wang S, Song L, Failla AV, Zobiak B, Mannherz HG, Frotscher M
(Siehe online unter https://doi.org/10.1242/dev.134163) - (2017): Actin ADP-ribosylation at Threonine148 by Photorhabdus luminescens toxin TccC3 induces aggregation of intracellular F-actin. Cell. Microbiol. 19 (1) e12636
Lang AE, Qu Z, Schwan C, Silvan U, Unger A, Schoenenberger CA, Aktories K, Mannherz HG
(Siehe online unter https://doi.org/10.1111/cmi.12636) - (2017): Actin: Structure, Function, Dynamics, and Interactions with Bacterial Toxins. Curr Top Microbiol Immunol. (Ed. H.G. Mannherz) 399:1-34
Kühn S, Mannherz HG
(Siehe online unter https://doi.org/10.1007/82_2016_45) - (2017): Host DNases prevent vascular occlusion by neutrophil extracellular traps. Science. 358(6367): 1202-1206
Jiménez-Alcázar M, Rangaswamy C, Panda R, Bitterling J, Simsek YJ, Long AT, Bilyy R, Krenn V, Renné C, Renné T, Kluge S, Panzer U, Mizuta R, Mannherz HG, Kitamura D, Herrmann M, Napirei M, Fuchs TA
(Siehe online unter https://doi.org/10.1126/science.aam8897) - (2017): Photorhabdus luminescens Toxins TccC3 and TccC5 Affect the Interaction of Actin with Actin- Binding Proteins Essential for Treadmilling. Curr Top Microbiol Immunol. (Ed. H.G. Mannherz) 399:53-67
Lang AE, Kühn S, Mannherz HG
(Siehe online unter https://doi.org/10.1007/82_2016_43) - (2018): Interaction of isolated cross-linked short actin oligomers with the skeletal muscle myosin motor domain. FEBS J. 285(9):1715-1729
Qu Z, Fujita-Becker S, Ballweber E, Ince S, Herrmann C, Schröder RR, Mannherz HG
(Siehe online unter https://doi.org/10.1111/febs.14442) - (2018): V EGF Triggers the Activation of Cofilin and the Arp2/3 Complex within the Growth Cone. Int. J. Mol. Sci. 19(2). pii: E384
Schlau M, Terheyden-Keighley D, Theis V, Mannherz HG, Theiss C
(Siehe online unter https://doi.org/10.3390/ijms19020384)
