Frankfurt (Main), Univ., Diss., 2001. / Computerdatei im Fernzugriff.

Frankfurt (Main), Univ., Diss., 2001. / Computerdatei im Fernzugriff.

Frankfurt (Main), Universiẗat, Diss., 2001.

No description available.

Phospholamban (PLN) reguliert in der Herzmuskelzelle die Aktivität der Kalzium-ATPase SERCA2a und damit maßgeblich die Kinetik des myozytären Kalzium-Kreislaufs. PLN liegt im Herz in Form von Monomeren und Pentameren vor, wobei angenommen wird, dass nur die Monomere die Aktivität der SERCA2a durch direkte Interaktion hemmen. Die Funktion der Pentamere ist noch immer unklar. In der vorliegenden Arbeit sollte untersucht werden, ob PLN-Pentamere für die PKA-abhängige Phosphorylierung des PLN und damit für die Regulation der PLN-Aktivität von Bedeutung sein können. Mit Hilfe transfizierter HEK293AD-Zellen und verschiedener PLN-Mutanten wurde gezeigt, dass sowohl PLN-Monomere als auch -Pentamere durch die PKA phosphoryliert werden, wobei die Phosphorylierung der Monomere in Anwesenheit von Pentameren geringer ist und verzögert abläuft. Ohne Pentamer war die Phosphorylierung der Monomere dagegen bereits basal und nach moderater PKA-Stimulation stärker. Ursache dafür schien eine höhere Affinität der PKA für PLN-Pentamere als für Monomere zu sein. Darüber hinaus konnte gezeigt werden, dass nicht nur PLN-Monomere sondern auch das PLN-Pentamer mit der SERCA2a interagieren und das Oligomer im Gegensatz zum PLN-Monomer nach PLN-Phosphorylierung zu einem kleinen Anteil an die SERCA2a gebunden bleibt. Auch spiegelten sich die unterschiedlichen Phosphorylierungsmuster von PLN-Pentamer und Monomer in den SERCA2a-Aktivitäten wieder. Messungen der SERCA2a-Aktivität in Mäuseherzen mit (Wildtyp und TgPLN) und ohne (TgAFA-PLN) PLN-Pentamere zeigten, dass Wildtyp-PLN und TgPLN die SERCA2a stärker inhibieren als TgAFA-PLN, was auf die stärkere basale Phosphorylierung des TgAFA-PLN zurückzuführen war. Nach PKA-Stimulation war der Anstieg der Enzymaktivität in Anwesenheit von TgPLN fast dreimal höher als in TgAFA-PLN. Analog zeigte TgPLN eine deutlichere Steigerung der Phosphorylierung der PLN-Monomere als TgAFA-PLN. Zusammenfassend konnte gezeigt werden, dass PLN-Pentamere durch Hemmung der Monomer-Phosphorylierung deren Aktivität erhöhen mit der Folge einer verstärkten Inhibition der SERCA2a. Da die inhibitorische Wirkung durch PKA-Stimulation vollständig aufgehoben werden kann, erhöhen die Pentamere die Regulationsmöglichkeiten der SERCA2a-Aktivität. / Phospholamban (PLN) regulates the activity of the calcium ATPase SERCA2a and thus the kinetics of myocyte calcium cycling. In the heart, PLN occurs in monomeric and pentameric form, however, only monomers are thought to inhibit the activity of SERCA2a by direct interaction. The function of the pentamer is still unclear. The aim of the present work was to investigate whether PLN pentamers may play a role for PKA dependent PLN phosphorylation and thus for regulating PLN activity. Using transfected HEK293AD cells and various PLN mutants, it was shown that both PLN monomers and pentamers get phosphorylated by PKA. Intriguingly, phosphorylation of monomers was delayed in the presence of pentamers but increased in the absence of pentamers, both under basal conditions and moderate PKA stimulation. The underlying reason for this observation turned out to be a higher affinity of PKA for PLN pentamers compared to monomers. Furthermore, not only PLN monomers but also PLN pentamers interacted with SERCA2a. Unlike monomers, a small proportion of PLN oligomers was still bound to SERCA2a following PLN phosphorylation. Further, SERCA2a activity reflected the different phosphorylation patterns of monomers and pentamers. Measurements of SERCA2a activity in mouse hearts with (Wildtyp-PLN; TgPLN) and without PLN pentamers (TgAFA-PLN) showed that wild-type PLN and TgPLN strongly inhibit SERCA2a due to stronger phosphorylation of TgAFA-PLN. After PKA stimulation, the increase of SERCA2a enzyme activity was almost three times higher in TgPLN than in TgAFA-PLN. Likewise, the increase of monomer phosphorylation was more pronounced in TgPLN than in TgAFA-PLN. Taken together, it was shown that PLN pentamers increase the activity of PLN monomers by attenuating monomer phosphorylation leading to increased inhibition of SERCA2a. Since this inhibition can be completely abolished by PKA stimulation, we conclude that PLN pentamers augment the regulatory range of SERCA2a.

No description available.

No description available.

Phospholamban (PLN) is an integral membrane protein that regulates the sarco(endo)plasmic Ca2+-ATPase (SERCA2a) within the cardiac sarcoplasmic reticulum (CSR). SERCA2a regulates intracellular Ca2+- handling and thus plays a critical role in initiating cardiac contraction and relaxation. It is believed that dysregulation of SERCA2a is a contributing factor in human heart failure patients. Even though there have been substantial advancements in understanding heart failure pharmacological therapies, patient prognosis remains poor. Nitroxyl (HNO), a new candidate heart failure drug therapy, has been shown to enhance overall cardiovascular function in both healthy and failing hearts, at least in part, by increasing Ca²⁺ re-uptake into the CSR. Previous research has shown that activation of SERCA2a by HNO is PLN-dependent; however, the mechanism of action of HNO remains unknown. We propose that HNO, a thiol oxidant, modifies one or more of the three PLN cysteine residues (C36, C41, C46) affecting the regulatory potency of PLN toward SERCA2a. To test this hypothesis, a series of PLN mutants were constructed containing single, double and triple cysteine substitutions. Using the baculovirus expression insect cell system, each PLN cysteine mutant was expressed alone and co-expressed with SERCA2a in insect cells and isolated in cellular endoplasmic reticulum (ER) microsomes. Samples were treated with Angeli's salt (an HNO donor) to determine the role of each PLN cysteine residue in the mechanism of SERCA2a activation by nitroxyl. Using a standard phosphate activity assay and SDS-PAGE/immunoblot techniques, we determined that the PLN cysteine residues at positions 41 and 46 are important in HNO activation of SERCA2a. Both SERCA2a + 41C PLN and SERCA2a + 46C PLN microsomal samples showed a ΔK0.5 of ~0.33 μM and evidence of reversible HNO induced disulfide bond formation. These studies provide important new insight into the mechanism of action of HNO on cardiac SR and thereby help evaluate the drug as a candidate therapy for congestive heart failure. / Ph. D.

The Ca-ATPase is an integral membrane enzyme which translocates two calcium ions from the cytoplasm of the cell to the sarcoplasmic reticulum lumen utilizing ATP breakdown as its energy source, in order to promote muscle relaxation. The focus of this research is the cardiac isoform of the Ca-ATPase which undergoes allosteric regulation by the phosphoprotein phospholamban (PLN). The Ca-ATPase is thought to be a target for nitrative stress and is affected by several chronic diseases of the heart. In the heart, age-based nitration of the Ca-ATPase inhibits Ca²⁺ transport activity but the physical mechanism by which nitration inhibits Ca-ATPase activity is not understood. Conversely, nitroxyl (HNO), a new candidate for drug therapy for congestive heart failure (CHF), improves overall cardiovascular function by increasing Ca-ATPase activity in the heart. However, the physical mechanism for this activation is unknown. Therefore, we have used enzyme kinetics, fluorescence spectroscopy, and EPR spectroscopy studies to determine the effects of ONOO⁻ and HNO on the Ca-ATPase and the physical regulation of the Ca-ATPase by PLN. Treatment of Ca-ATPase with a nitrating agent, ONOO⁻, inhibited Ca-ATPase activity, and the [ONOO⁻]-dependent inhibition of the Ca-ATPase was more effective in the presence of PLN. ONOO⁻ did not affect the [Ca²]-dependence of Ca-ATPase activity either in the presence or absence of PLN. ONOO⁻ had no effect on Ca-ATPase rotational mobility or oligomeric interactions, as affected by PLN, but ONOO⁻ decreased the amplitude of the Ca²⁺-dependent E2 to E1•Ca2 conformational change, both in the absence and presence of PLN. Treatment with HNO had no affect on the [Ca²⁺]-dependence of Ca-ATPase activity in the absence of PLN; however in the presence of PLN, the [Ca²⁺]-dependent activity was shifted to lower Ca²⁺ levels and corresponded to the uncoupling of PLN from the Ca-ATPase. HNO decreased Ca-ATPase rotational mobility and increased the Ca-ATPase Ca²⁺-dependent conformational transition, consistent with uncoupling PLN from the Ca-ATPase. Taken together, these results suggest that ONOO⁻ inactivates a fraction of enzyme units to lower overall enzyme activity, whereas HNO uncouples PLN from the Ca-ATPase with increases in Ca-ATPase conformational flexibility and Ca-ATPase activity. / Ph. D.

No description available.