Titel: Biophysical characterization of the pyrophosphatase NUDT9 and the NUDT9-homologous domain of the TRPM2 channel
Sprache: Englisch
Autor*in: Gattkowski, Ellen
Schlagwörter: ADPR; TRPM2; NUDT9; Calmodulin; biophysics; structural biology
Erscheinungsdatum: 2021-05
Tag der mündlichen Prüfung: 2021-08-20
The pyrophosphatase NUDT9 exhibits high ADPR specificity and hydrolyzes the nucleotide to AMP and ribose 5-phosphate in the presence of Mg2+ and thereby controls cellular ADPR levels. NUDT9 is widely expressed among various cell types and localized in mitochondria and the cytosol. 2′-deoxy-ADPR was identified as a novel NUDT9 substrate and the binding kinetics as well as the binding affinities for ADPR and 2′-deoxy-ADPR were determined to be comparable. Both nucleotides bind to an identical binding site as shown by TROSY-NMR experiments. Backbone resonance assignment identified residues so far unknown to be involved in the ligand binding. Interestingly, ligand binding not only thermally stabilized NUDT9 but also induced a more compact conformation of the complex. Based on these results, a prototype of a NUDT9 FRET sensor chemically labelled with Alexa Fluor was developed, which will visualize ADPR signaling pathways in cells in the future.

The TRPM2 channel is a non-selective cation channel in the plasma membrane permeable for Ca2+, Na+ and K+ ions. The C-terminal domain is homologous to NUDT9 and the channel is activated by cooperative binding of ADPR and Ca2+. Further, TRPM2 is regulated by Calmodulin and temperature. In addition to the established Calmodulin binding site in the N-terminus of TRPM2, two novel potential Calmodulin binding sites were identified. The first one is located C-terminal but in vicinity to the transmembrane section of the channel. Apo and Ca2+-Calmodulin bound to respective peptides but the exact binding mode could not be determined as it as it contained both endothermic and exothermic parts. The second novel Calmodulin binding site is located in the NUDT9H domain. The formation of a complex with Ca2+-Calmodulin thermally stabilized the isolated NUDT9H domain. Alanine substitution of the anchor residues Trp1355 and Ile1368 in the binding site abolished Calmodulin-dependent temperature sensitivity of TRPM2.
URL: https://ediss.sub.uni-hamburg.de/handle/ediss/9613
URN: urn:nbn:de:gbv:18-ediss-100552
Dokumenttyp: Dissertation
Betreuer*in: Tidow, Henning
Enthalten in den Sammlungen:Elektronische Dissertationen und Habilitationen

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20220504_phd_thesis_final_EGattkowski.pdfDissertation Ellen Gattkowski041f90e27f5f37979056b4384bb7acda4.27 MBAdobe PDFÖffnen/Anzeigen
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