13 research outputs found
Investigation of the mechanism of activation of the NO-sensitive guanylate cyclase and its subcellular localization
Der NO / cGMP-Signalweg spielt eine zentrale Rolle bei der Regulation des Blutdrucks und der Relaxation der glatten Muskulatur und bei der Thrombozytenaggregation. Das zentrale Enzym dieses Signalwegs ist die Stickstoffmonoxid-sensitive Guanylat-Cyclase (NOsGC).
Gegenstand der Arbeit war die Untersuchung der dynamischen Konformationsänderungen der NOsGC bei NO-Stimulation unter Verwendung der endogenen Tryptophane als FRET-Donatoren und dem Substratanalogon 2´-Mant-3´-dGTP als FRET-Akzeptor. Die Ergebnisse weisen darauf hin, dass das NO-Aktivierungssignal von der HNOX-Domäne über zwei unterschiedliche Domänen auf die katalytische Domäne übertragen wird. Die Bewegung von Helices aus diesen beiden Domänen in Richtung der katalytischen Domäne führen dazu, dass sich diese schließt und in ihre aktive Konformation übergeht.
Das Polaritätsprotein Scribble wurde im Rahmen dieser Arbeit als möglicher Interaktionspartner der α2-Untereinheit identifiziert. Untersuchungen der Lokalisation der α2 / β1 Isoform an Zell-Zell-Kontakten ergaben, dass nur das Heterodimer aus α2- und β1-Untereinheit die besondere Lokalisation zeigt, während die einzelne α2 Untereinheit zytosolisch verteilt ist.
Zur Untersuchung der intrazelluläre und lokalisationsbezogene Aktivität der NOsGC wurden auf Basis des cGMP-Fluoreszenzindikators FlincG und den beiden Isoformen der NOsGC zwei monomolekulare Fluoreszenzsensoren charakterisiert. Der Einsatz der Fluoreszenzsensoren ergab, dass die besondere Lokalisation der α2 / β1-Isoform an Zell-Zell-Kontakten dem Abfangen von NO an der Membran dienen könnte und dass der NOsGC-Aktivator Cinaciguat einen schnellen Anstieg der intrazellulären NOsGC-Aktivität verursacht.
Zusammenfassend konnte in dieser Arbeit das Verständnis der Signaltransduktion der NO-Aktivierung innerhalb der NOsGC erweitert werden. Zusätzlich konnten Einblicke in den Wirkmechanismus neuer Arzneistoffgruppen gewonnen werden, deren Zielstruktur die NOsGC ist.The NO / cGMP pathway plays a crucial role in the regulation of blood pressure and smooth muscle relaxation, platelet aggregation and in the central and peripheral neurotransmission. The key enzyme of this pathway is the nitric oxide-sensitive guanylate cyclase (NOsGC).
In this study the dynamic conformational changes to NOsGC upon NO stimulation were examined through the use of the endogenous tryptophan residues of NOsGC as FRET donors and the substrate analogue 2'-Mant-3'- dGTP as a FRET acceptor. The results indicate that the activation signal from the amino terminal heme and NO binding domain (HNOX) is transmitted via two different domains to the carboxy terminal catalytic domain. After NO stimulation the movement of two helices from this domains leads to a closure of the catalytic domain and induces its active conformation.
The regulator of cell polarity, scribble, was identified as an interaction partner of α2 / β1 isoform. Investigation of the localization of this isoform at cell-cell contacts revealed that only the heterodimer of α2 and β1 subunit is located at cell-cell contacts.
To investigate the intracellular localization and activity of NOsGC two monomolecular fluorescent sensors based on the cGMP fluorescence indicator FlincG and the two NOsGC isoforms (α1 / β1 and α2 / β1) were developed and established. The use of the fluorescence sensors led to the observation that the special localization of the α2 / β1 isoform at cell-cell contacts serves to intercept NO on the membrane and reveal that the NOsGC activator cinaciguat causes a rapid increase in intracellular NOsGC activity.
In summary, this study furthers the understanding of NO-signal transduction within the intact full-length NOsGC. In addition, experiments give insight into the activation mechanism of new NOsGC activator and stimulator drugs
The Amino-Terminus of Nitric Oxide Sensitive Guanylyl Cyclase α1 Does Not Affect Dimerization but Influences Subcellular Localization
BACKGROUND: Nitric oxide sensitive guanylyl cyclase (NOsGC) is a heterodimeric enzyme formed by an α- and a β₁-subunit. A splice variant (C-α₁) of the α₁-subunit, lacking at least the first 236 amino acids has been described by Sharina et al. 2008 and has been shown to be expressed in differentiating human embryonic cells. Wagner et al. 2005 have shown that the amino acids 61-128 of the α₁-subunit are mandatory for quantitative heterodimerization implying that the C-α₁-splice variant should lose its capacity to dimerize quantitatively. METHODOLOGY/PRINCIPAL FINDINGS: In the current study we demonstrate preserved quantitative dimerization of the C-α₁-splice by co-purification with the β₁-subunit. In addition we used fluorescence resonance energy transfer (FRET) based on fluorescence lifetime imaging (FLIM) using fusion proteins of the β₁-subunit and the α₁-subunit or the C-α₁ variant with ECFP or EYFP. Analysis of the respective combinations in HEK-293 cells showed that the fluorescence lifetime was significantly shorter (≈0.3 ns) for α₁/β₁ and C-α₁/β₁ than the negative control. In addition we show that lack of the amino-terminus in the α₁ splice variant directs it to a more oxidized subcellular compartment. CONCLUSIONS/SIGNIFICANCE: We conclude that the amino-terminus of the α₁-subunit is dispensable for dimerization in-vivo and ex-vivo, but influences the subcellular trafficking
Heme Oxygenase Isoforms Differ in Their Subcellular Trafficking during Hypoxia and Are Differentially Modulated by Cytochrome P450 Reductase
Heme oxygenase (HO) degrades heme in concert with NADPH cytochrome P450 reductase (CPR) which donates electrons to the reaction. Earlier studies reveal the importance of the hydrophobic carboxy-terminus of HO-1 for anchorage to the endoplasmic reticulum (ER) which facilitates the interaction with CPR. In addition, HO-1 has been shown to undergo regulated intramembrane proteolysis of the carboxy-terminus during hypoxia and subsequent translocation to the nucleus. Translocated nuclear HO-1 was demonstrated to alter binding of transcription factors and to alter gene expression. Little is known about the homologous membrane anchor of the HO-2 isoform. The current work is the first systematic analysis in a eukaryotic system that demonstrates the crucial role of the membrane anchor of HO-2 for localization at the endoplasmic reticulum, oligomerization and interaction with CPR. We show that although the carboxy-terminal deletion mutant of HO-2 is found in the nucleus, translocation of HO-2 to the nucleus does not occur under conditions of hypoxia. Thus, we demonstrate that proteolytic regulation and nuclear translocation under hypoxic conditions is specific for HO-1. In addition we show for the first time that CPR prevents this translocation and promotes oligomerization of HO-1. Based on these findings, CPR may modulate gene expression via the amount of nuclear HO-1. This is of particular relevance as CPR is a highly polymorphic gene and deficiency syndromes of CPR have been described in humans
FRET efficiencies of HO-CPR co-transfections in HEK293 cells.
<p>FRET efficiencies (%) with SEM were calculated after measuring the lifetimes of the samples in HEK 293 cells. CFP-HO-1 single transfection served as donor control. The mean calculation was carried out after three independent measurements.</p
Comparison of enzyme activity of the carboxy-terminally deleted HO isoforms.
<p>Measurements were made in the presence (black columns) and absence (grey columns) of exogenous CPR. (A) Activity measured in the cytosolic fractions from Sf9 cells expressing the HO mutants. (B) Measurement in homogenates from infected Sf9 cells. (C) Enzyme activity assay after purification using the <i>Strep</i>-tag/Streptavidin system. Data are shown as means ± SEM (n = 3, *p<0.05).</p
Comparison of enzyme activity of full-length HO isoforms.
<p>Measurements were made in the presence (black columns) and absence (grey columns) of exogenous CPR. (A) HO activity measured in cytosolic fractions from Sf9 cells expressing the indicated HO variants. (B) Same experiments made in homogenates from Sf9 cells. (C) Enzyme activity assay after purification using the <i>Strep</i>-tag/Streptavidin system. Data are shown as means ± SEM (n = 3, *p<0.05).</p
Confocal laser scanning analysis of GFP-tagged HO isoforms in HEK293 under hypoxia.
<p>The left panels show the cells at time 0 h and the right panels after incubation with 1% oxygen for 42 h. (A) GFP-HO-1; (B) GFP-HO1ΔC266; (C) GFP-HO-2; (D) GFP-HO-2ΔC289.</p
Influence of CPR on the hypoxic translocation of HO in HEK293 cells using confocal microscopy.
<p>HEK293 cells were transfected with YFP-HO-1 (A-row 1) and YFP-HO2 (B-row 1) or co-transfected with YFP-HO1 and CPR-CFP (A-row 2) or YFP-HO2 and CPR-CFP (B-row 2). Cells were analyzed under normoxic conditions using the CFP-channel (column 1) and YFP-channel (column 2) or after hypoxia in the CFP-channel (column 3) or YFP-channel (column 4). The red arrows in panel A point to typical cells where the translocation of HO-1 (row 1) and its prevention in the presence of CPR (row 2) are visible. The white bars correspond to 10 µm.</p