Analysis of SPP interaction of a double fluorescence-tagged HO-1 in HEK293 cells.

<p><b>(A)</b> Confocal laser scanning analysis of HO variants under normoxic or hypoxic conditions (1% O₂, 48 h) and in co-transfection with wild type HA-tagged SPP or the inactive mutant. Figure shows representative data from five pictures per sample out of three independent experiments. Bar represents 20 μm. <b>(B)</b> Co-immunoprecipitations with HA-antibody, detected with GFP-antibody. Western blots of cell extracts used for co-immunoprecipitation were incubated with GFP-antibody to detect HO variants and were incubated with HA-antibody to detect SPP as homodimer at around 95 kDa. Representative blots of one out of three independent experiments are shown. WT: wild type, mut: inactive mutant, right: ladder [kDa].</p

Analysis of SPP binding and cutting of HO variants, CPR and BVR in HEK293 cells by co-immunoprecipitation.

<p>Western blots of co-immunoprecipitations (Co-IPs) of fluorescent protein fused HO variants <b>(A and B)</b> CPR and BVR <b>(C)</b> with HA-antibody, detected with GFP-antibody. <b>(D)</b> Western blots of co-immunoprecipitations of fluorescent protein fused HO variants with HA- or IgG control-antibody, detected with GFP-antibody. Western blots of cell extracts used for co-immunoprecipitation were incubated with GFP-antibody to detect HO variants, CPR and BVR and were incubated with HA-antibody to detect SPP as homodimer at around 95 kDa as described by Nyborg et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188344#pone.0188344.ref034" target="_blank">34</a>]. Representative blots of one out of three independent experiments are shown. WT: wild type, mut: inactive mutant, right: ladder [kDa].</p

Translocation rates of HO mutants and chimeras before and after incubation with hypoxia or in co-transfection with HA-SPP.

<p>Translocation rates of HO mutants and chimeras before and after incubation with hypoxia or in co-transfection with HA-SPP.</p

Confocal laser scanning analysis of GFP-tagged HO chimeras.

<p>HO variants were imaged under normoxic or hypoxic conditions (1% O₂, 48 h) and co-transfected with wild type HA-tagged SPP or the inactive mutant in HEK293 cells. <b>(A)</b> Representative CLSM pictures. Bar represents 20 μm. WT1: Wild type HO-1. WT2: Wild type HO-2. C1: HO-1-HO-2 chimera. C2.1-C2.3: HO-2-HO-1 chimeras. Models: Red: HO-1 part. Blue: HO-2 part. Green square: PEST domain. Yellow ellipse: NSS. <b>(B)</b> Statistical analysis. Data from five pictures per sample out of three independent experiments was counted and statistically analyzed. Bars: means. Error bars: SEM. * significant less translocation compared to HO-1 (p < 0.05).</p

Analysis of SPP binding of HO-1 deletion variants by co-immunoprecipitation.

<p><b>(A)</b> HO-1 deletion variants were co-immunoprecipitated with endogenous SPP and detected with anti-GFP-antibody under normoxia. <b>(B)</b> HO-1 deletion variants in co-transfection with SPP mut were co-immunoprecipitated with HA-antibody and detected with anti-GFP-antibody. Western blots of cell extracts used for co-immunoprecipitation were incubated with GFP-antibody to detect HO-1 variants and incubated with HA-antibody or SPP-antibody to detect SPP as homodimer. Representative blots of one out of three independent experiments are shown. right: ladder [kDa]. <b>(C)</b> Models of the HO-1 deletion variants: WT1: Wild type HO-1. D1: HO-1-ΔC266. D2.1: HO-1-ΔN181. D2.2: HO-1-ΔN262. Red: HO-1 part. Green square: PEST domain. Yellow ellipse: NSS.</p

Binding analysis of SPP PAL mutants to wild type HO-1 and wild type HO-2 by co-immunoprecipitation.

<p><b>(A)</b> HO-1. <b>(B)</b> HO-2. Both show Western blots of co-immunoprecipitations of PAL mutants of SPP and HO-1 or HO-2 with HA-antibody, detected with GFP-antibody. Western blots of cell extracts used for co-immunoprecipitation are incubated with GFP-antibody to detect HO-1 or HO-2 and HA-antibody to detect SPP as homodimer. Representative blots of one out of three independent experiments are shown. right: ladder [kDa].</p

Alignment of human wild type HO-1 and HO-2.

<p>Human wild type HO-1 and HO-2 were aligned using Clustal W 2.0 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188344#pone.0188344.ref035" target="_blank">35</a>]. Wildtype HOs, regions where chimeras shift between HO-1 and HO-2, mutations and beginning or ending of deletion mutants are highlighted. A leucine-rich region or nuclear shuttle sequence (NSS) is shown in yellow, a degradation signal sequence (PEST-domain) in green and the anchor, a lipophilic membrane domain, for HO-1 in red and for HO-2 in blue. HO-1’s anchor contains the SPP cleavage site SF275/276, marked as described in Hsu et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188344#pone.0188344.ref017" target="_blank">17</a>]. Identical amino acids are marked with *, strong and weak conservation is marked with: and. . WT1: Wild type HO-1. WT2: Wild type HO-2. M1: HO-1 H25A. C2.3: HO-2-HO-1-NSS-PEST-anchor. D2.1: HO-1-ΔN181. C2.2: HO-2-HO-1-PEST-anchor. D2.2: HO-1-ΔN262. C1: HO-1-HO-2-anchor. C2.1: HO-2-HO-1-anchor. D1: HO-1-ΔC266. M2: HO-1 SF275/276AL.</p

Insights into the mechanism of isoenzyme-specific signal peptide peptidase-mediated translocation of heme oxygenase

<div><p>It has recently been shown that signal peptide peptidase (SPP) can catalyze the intramembrane cleavage of heme oxygenase-1 (HO-1) that leads to translocation of HO-1 into the cytosol and nucleus. While there is consensus that translocated HO-1 promotes tumor progression and drug resistance, the physiological signals leading to SPP-mediated intramembrane cleavage of HO-1 and the specificity of the process remain unclear. In this study, we used co-immunoprecipitation and confocal laser scanning microscopy to investigate the translocation mechanism of HO-1 and its regulation by SPP. We show that HO-1 and the closely related HO-2 isoenzyme bind to SPP under normoxic conditions. Under hypoxic conditions SPP mediates intramembrane cleavage of HO-1, but not HO-2. In experiments with an inactive HO-1 mutant (H25A) we show that translocation is independent of the catalytic activity of HO-1. Studies with HO-1 / HO-2 chimeras indicate that the membrane anchor, the PEST-domain and the nuclear shuttle sequence of HO-1 are necessary for full cleavage and subsequent translocation under hypoxic conditions. In the presence of co-expressed exogenous SPP, the anchor and the PEST-domain are sufficient for translocation. Taken together, we identified the domains involved in HO-1 translocation and showed that SPP-mediated cleavage is isoform-specific and independent of HO-activity. A closer understanding of the translocation mechanism of HO-1 is of particular importance because nuclear HO-1 seems to lead to tumor progression and drug resistance.</p></div

Analysis of endogenous SPP binding and cutting of HO variants, CPR and BVR in HEK293 cells by co-immunoprecipitation under normoxia and after 48 h of hypoxia (1% O₂).

<p>Western blots of co-immunoprecipitations of HO variants, CPR and BVR with SPP-antibody or IgG control-antibody-were detected with GFP-antibody. Western blots of cell extracts used for co-immunoprecipitation were incubated with GFP-antibody to detect HO variants, CPR and BVR and were incubated with SPP-antibody to detect SPP as homodimer. Representative blots of one out of three independent experiments are shown. right: ladder [kDa].</p

Confocal laser scanning analysis of GFP-tagged HO mutants.

<p>HO variants were imaged under normoxic or hypoxic conditions (1% O₂, 48 h) and co-transfected with wild type HA-tagged SPP or the inactive mutant in HEK293 cells. <b>(A)</b> Representative CLSM pictures. Bar represents 20 µm. <b>(B)</b> Statistical analysis. Data from five pictures per sample out of three independent experiments was counted and statistically analyzed. Bars: means. Error bars: SEM. * significantly less translocation compared to HO-1 (p < 0.05).</p