| dc.contributor.advisor | Schwappach-Pignataro, Blanche Prof. Dr. | |
| dc.contributor.author | Menzel, Julia | |
| dc.date.accessioned | 2021-02-01T13:37:11Z | |
| dc.date.available | 2021-07-16T00:50:06Z | |
| dc.date.issued | 2021-02-01 | |
| dc.identifier.uri | http://hdl.handle.net/21.11130/00-1735-0000-0005-1561-6 | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-8419 | |
| dc.language.iso | eng | de |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/4.0/ | |
| dc.subject.ddc | 610 | |
| dc.title | Novel protein-protein interactions contribute to the regulation of cardiac excitation and Ca2+ handling | de |
| dc.type | doctoralThesis | de |
| dc.contributor.referee | Schwappach-Pignataro, Blanche Prof. Dr. | |
| dc.date.examination | 2020-07-17 | |
| dc.description.abstracteng | Cardiac function is defined by the process of excitation and contraction. During this process, the electrical excitation of the surface membrane is coupled to Ca2+-ion release at the sarcoplasmic reticulum, resulting in contraction. The small membrane-protein phospholamban (PLN) is an essential regulator of cardiac contraction by inhibiting the SR Ca2+-ATPase (SERCA), which modulates the reuptake of Ca2+-ions at the SR and ultimately results in cardiac relaxation. The inhibitory effect of PLN on SERCA is disrupted upon its phosphorylation by two different kinases. Beta-adrenergic stimulation activates protein kinase A (PKA), which phosphorylates PLN at S16, meanwhile increased intracellular Ca2+ levels stimulate Ca2+-calmodulin-dependent protein kinase II (CaMKII) phosphorylation of PLN at T17, causing a longer-lasting activation of SERCA.
This study focused on the identification and characterization of novel protein-protein interactions involved in the regulation of cardiac excitation and Ca2+ metabolism.
Pull-down approaches from rodent and human cardiac membranes, proximity labelling in live cardiac myocytes and immunoprecipitation experiments identified the phospho-adaptor protein 14-3-3 as a novel PLN interaction partner. Pull-down experiments with monomeric or pentameric PLN demonstrated that 14-3-3 preferentially binds phosphorylated PLN pentamers. The potential 14-3-3 binding motif in PLN was further dissected with recombinant binding assays and surface plasmon resonance measurements revealing that phosphorylated S16 or T17 transform PLN into a 14-3-3 target with different binding affinity sites. In line, molecular dynamic simulations uncovered different placement of PLN, phosphorylated at either S16 or T17, in the 14-3-3 binding groove resulting in different binding energies, favoring phosphorylated T17 PLN. Functionally, upon beta-adrenergic stimulation of cardiac myocytes PKA activity was stimulated and endogenous 14-3-3 was found to be enriched in these membrane fractions. Recombinant dephosphorylation assays showed that 14-3-3 bound to phosphorylated PLN protects PLN from rapid dephosphorylation. In line, increased SERCA activity was observed upon PKA stimulation in the presence of 14-3-3 in cardiomyocytes during patch clamp analysis. The observation that CaMKII-phosphorylated PLN at T17 results in a high affinity binding of 14-3-3 suggests that 14-3-3 may regulate the less dynamic pool of PLN and raises the question of whether 14-3-3 binding is involved in the regulation of PLN disease mutations (R14del, R9C), for which PKA phosphorylation is abolished. Recombinant binding assays confirmed that 14-3-3 could still interact with PLN R9C or R14del mutants, which are associated with dilated cardiomyopathy and heart failure.
Additionally, 14-3-3 together with the coat complex COPI are well characterized regulators of the potassium ion channel family TASK, responsible for phosphorylation dependent trafficking and cell surface expression. TASK-1 is strongly expressed in the atrium, involved in setting the resting membrane potential and associated with atrial fibrillation. Little is known about regulation and trafficking of these channels in the heart. A mass spectrometry approach and recombinant binding assays performed from cardiac membranes identified the PDZ domain containing proteins Dlg1 and Dlg4 as direct TASK-1 interactors. Further analysis of the PDZ binding site in TASK-1 confirmed the existence of a previously hypothesized PDZ-binding motif, which overlaps with the well characterized 14-3-3 binding motif located at the distal C-terminus of TASK-1. This finding suggests a phosphorylation dependent regulation of TASK-1 via 14-3-3 and PDZ domain containing proteins at the plasma membrane, yielding insight into trafficking of TASK-1 beyond the secretory pathway.
Taken together, in this study 14-3-3 was identified as a novel interaction partner of PLN. It was further demonstrated that 14-3-3 binding to PLN results in a stabilization of phosphorylated pentamers and prolonged SERCA activity, indicating a novel mechanism of PLN regulation dependent on cardiac kinase activity. In addition, the PDZ domain containing protein Dlg1 was identified as a direct interaction partner of TASK-1 in cardiac tissue. These findings expand our knowledge about the interactome and regulation of the cardiac protein PLN and the ion-channel TASK-1 underlining the importance of regulatory proteins such as 14-3-3 or Dlg1 in cellular processes like Ca2+ handling or protein trafficking. | de |
| dc.contributor.coReferee | Katschinski, Dörthe Prof. Dr. | |
| dc.contributor.thirdReferee | Lehnart, Stephan E. Prof. Dr. | |
| dc.contributor.thirdReferee | Meinecke, Michael Prof. Dr. | |
| dc.contributor.thirdReferee | Wienands, Jürgen Prof. Dr. | |
| dc.contributor.thirdReferee | Shattock, Michael J Prof. Dr. | |
| dc.subject.eng | Phospholamban | de |
| dc.subject.eng | 14-3-3 | de |
| dc.subject.eng | TASK-1 | de |
| dc.subject.eng | Cardiac excitation | de |
| dc.subject.eng | Protein-protein interactions | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-21.11130/00-1735-0000-0005-1561-6-4 | |
| dc.affiliation.institute | Medizinische Fakultät | |
| dc.subject.gokfull | Medizin (PPN619874732) | de |
| dc.description.embargoed | 2021-07-16 | |
| dc.identifier.ppn | 1746675349 | |