Studies on the differences of the heme oxygenase isoenzymes and their relationship to NO-sensitive guanylyl cyclase

Die Arbeit befasst sich mit den Unterschieden der Hämoxygenase-Isoenzyme (HO-1 und HO-2) bezüglich der Stabilität der carboxy-terminalen Membrananker und der Interaktion mit der NADPH-abhängigen Cytochrom-P450-Oxidoreduktase (CPR). Die erhöhte Stabilität der HO-2 konnte auf einen höheren Oligomerisierungsgrad zurückgeführt werden. Dies könnte ursächlich für das unterschiedliche Translokationsverhalten in den Zellkern nach Inkubation unter Hypoxie sein. Ein Beleg hierfür ist, dass die Anwesenheit der CPR zu einer Stabilisierung der HO-1 führte und die Translokation der HO-1 unter Hypoxie ausblieb. Weiterhin wurden die HO-2-spezifischen Hämbindemotive (CPF-Motive) untersucht, die allerdings keinen Einfluss auf die Stabilität, die Enzymaktivität und auf das Ausbleiben der nukleären Translokation der HO-2 haben. In der a1-Untereinheit der Stickstoffmonoxid-sensitiven Guanylyl-Cyclase (NOsGC) konnte ebenfalls ein CPF-Motiv identifiziert werden. Untersuchungen von Wildtyp und einer CPF-Mutante zeigten keine Unterschiede bei der Enzymaktivität und des Redoxstatus in der Zelle. Weiterführende Experimente könnten zur Aufklärung der Rolle des amino-terminalen Bereichs der a1-Untereinheit und des CPF-Motivs bei der NOsGC-Aktivierung beitragen. Koexpressionsversuche von HO und NOsGC im eukaryotischen Expressionssystem zeigten, dass die Anwesenheit von HO-1 und HO-2 die Aktivierung der NOsGC durch NO reduziert. Untersuchungen mit dem NOsGC-Aktivator Cinaciguat in An- und Abwesenheit des NOsGC-Inhibitors ODQ lassen darauf schließen, dass die HO-Koexpression zu einer verminderten Hämhaltigkeit der NOsGC führt, woraus auch der verringerte NOsGC-Gehalt resultieren kann. Die Koexpression der humanen Biliverdinreduktase schien durch die vermehrte Bildung des antioxidativen Bilirubins einen protektiven Einfluss auf die Hämgruppe zu besitzen. Die Ergebnisse zeigen, dass im rekombinanten Expressionssystem die HO-Formen und das hämabbauende System die NOsGC beeinflussen.This study deals with the differences between the heme oxygenase isoenzymes (HO-1 and HO-2) and focuses upon their different stability with respect to the carboxy-terminal membrane anchor and the interaction with the NADPH-cytochrome P450 oxidoreductase (CPR). The increased stability of HO-2 is most likely attributed to a higher degree of oligomerization. It is suggested that this was the reason for different nuclear translocation of HO-1 and HO-2 under hypoxic conditions. Further evidence for this is that the presence of CPR led to a stabilization of HO-1 that did not translocate. Another focus was the investigation of the heme-binding motifs (CPF-motifs) of HO-2, which are specific for this isoenzyme. The CPF-motifs have no influence on the stability, enzyme activity and nuclear translocation of HO-2. Through amino acid sequence analysis, a CPF-motif could also be identified in the a1-subunit of nitric oxide-sensitive guanylyl cyclase (NOsGC). Studies showed no differences between the wild type and an enzyme carrying a CPF-mutation, with respect to the enzyme activity and the redox status of the cell. Further experiments should be performed, since the role of a1-subunit´s amino-terminal region in activation of NOsGC was not sufficiently clarified. At the end HO and NOsGC were co-expressed in a eukaryotic expression system. It could be demonstrated that the presence of HO isoenzymes reduces NOsGC activation by NO. Studies, with NOsGC activator cinaciguat in the presence or absence of NOsGC inhibitor ODQ, suggest the co-expression of HO isoenzymes leads to a reduced heme content in NOsGC. The reduced NOsGC expression in the presence of HO is probably also a result of reduced heme levels. However, the co-expression of human biliverdin reductase (hBVR) showed a protective effect on the heme group through increased formation of the antioxidant bilirubin. The results show that in a recombinant expression system, HO forms and the heme degrading system influence NOsGC

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

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

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

FRET measurements of HO isoforms and CPR in homogenate (black column) or cytosol (grey column).

<p>YFP-GAFA-CFP served as positive control for FRET-interactions. CPR was tagged with CFP and HO-variants with YFP. Data are shown as means ± SEM (n = 3, p<0.05).</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

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