Binding of cytohesin 2 to the plasma membrane and its relationship with the EGFR

Eukaryotic cells are compartmentalized by lipid membranes to achieve the spatial separation of biological processes and signaling pathways. Controlled trafficking of proteins between these compartments as well as the recruitment of proteins to the membranes themselves are crucial for trouble-free function of the cell. Previous research has revealed several possibilities for interaction between proteins and membranes. Phosphoinositides can specifically interact with certain protein domains, anionic lipids attract positively charged protein domains by electrostatic force and lipids can even be attached to proteins post-translationally to enable integration into the membrane. This work focuses on the guanine nucleotide exchange factor ARNO, a multidomain protein that activates small GTPases like Arf6 and therefore is directly involved in the vesicle trafficking machinery of the cell. In its autoinhibited form, ARNO is localized in the cytoplasm, whereas recruitment to the plasma membrane is a prerequisite for its activation of Arf. So far, research has been centered on the interaction of the PH-domain and the PBR-domain of ARNO with artificial membrane systems. To expand these findings and account for the enormous complexity of the inner leaflet of the cellular plasma membrane, in this study, membrane sheets are employed. The ability of a variety of protein constructs consisting of different ARNO domains to bind to these sheets is analyzed. It seems that the different domains of ARNO aid to the interaction with the membrane in a cooperative manner. While the PH-domain is absolutely required for association with the membrane, it is not sufficient for sequestration of ARNO in the membrane. Moreover, its interaction with the phosphoinositides could be altered by the concentration of calcium in the binding buffer. Hypothetically, this might be due to the formation of PIP-bridges permitted by the crosslinking of individual PIP molecules by the Ca2+ ions. This results in a loss of accessibility of the PIPs for binding by the ARNO PH-domain. In this work, binding studies of the other ARNO domains conclusively show that the PBR-domain, the Sec7-domain as well as the coiled-coil domain participate in plasma membrane binding. Moreover, dimerization of ARNO also improves its binding ability, most probably by an increase of the local avidity. Once bound to the plasma membrane, ARNO proteins form clusters. Brightfield as well as STED microscopy reveals that these overlap with clusters of the endogenous EGFR to a non-random degree. Having observed this colocalization, a possible biological relationship between ARNO and the EGFR is assessed. Overexpression of ARNO in HeLa cells results in a tendency towards increased activation of the EGFR after stimulation with EGF. Upon activation, the EGFR can be translocated to the nucleus by retrograde endosomal trafficking. Its JM-domain of the EGFR has been proposed as a possible binding partner for ARNO in previous studies and contains the nuclear localization signal of the EGFR. This work explores in how far overexpression or inhibition of ARNO influences the nuclear translocation of the EGFR. However, this does not seem to be the case

Deguelin Attenuates Reperfusion Injury and Improves Outcome after Orthotopic Lung Transplantation in the Rat

The main goal of adequate organ preservation is to avoid further cellular metabolism during the phase of ischemia. However, modern preservation solutions do rarely achieve this target. In donor organs hypoxia and ischemia induce a broad spectrum of pathologic molecular mechanisms favoring primary graft dysfunction (PGD) after transplantation. Increased hypoxia-induced transcriptional activity leads to increased vascular permeability which in turn is the soil of a reperfusion edema and the enhancement of a pro-inflammatory response in the graft after reperfusion. We hypothesize that inhibition of the respiration chain in mitochondria and thus inhibition of the hypoxia induced mechanisms might reduce reperfusion edema and consecutively improve survival in vivo. In this study we demonstrate that the rotenoid Deguelin reduces the expression of hypoxia induced target genes, and especially VEGF-A, dose-dependently in hypoxic human lung derived cells. Furthermore, Deguelin significantly suppresses the mRNA expression of the HIF target genes VEGF-A, the pro-inflammatory CXCR4 and ICAM-1 in ischemic lungs vs. control lungs. After lung transplantation, the VEGF-A induced reperfusion-edema is significantly lower in Deguelin-treated animals than in controls. Deguelin-treated rats exhibit a significantly increased survival-rate after transplantation. Additionally, a downregulation of the pro-inflammatory molecules ICAM-1 and CXCR4 and an increase in the recruitment of immunomodulatory monocytes (CD163+ and CD68+) to the transplanted organ involving the IL4 pathway was observed. Therefore, we conclude that ischemic periods preceding reperfusion are mainly responsible for the increased vascular permeability via upregulation of VEGF. Together with this, the resulting endothelial dysfunction also enhances inflammation and consequently lung dysfunction. Deguelin significantly decreases a VEGF-A induced reperfusion edema, induces the recruitment of immunomodulatory monocytes and thus improves organ function and survival after lung transplantation by interfering with hypoxia induced signaling

Specificity of Deguelin.

<p>Western blot analysis of total AKT (tAKT), phospho AKT (pAKT) and ACTB of transplanted lungs derived from animals that were either treated with Deguelin or without (control). However a trend to lower levels of pAKT (n.s.; P = 0.2403) may be seen, no significant differences are observed between the two groups, underlining HIF-1 downregulating potency of Deguelin.</p

Deguelin effectively inhibits pro-edema and pro-inflammatory genes during hypoxia <i>in vivo</i>.

<p>Lungs from animals pretreated with or without Deguelin are explanted. After 1 hour incubation at 37°C, simulating warm ischemia, the ischemic lungs are processed for further analysis. (A) Scheme representing the ischemia experiment. (B) Gene expression of VEGF-A, CXCR4 and ICAM-1 in ischemic lungs treated with or without Deguelin. Beta actin served as negative control. Groups are compared to native Lungs (sham). Sham  =  native lungs without ischemia, w.i.  =  ischemic lungs without treatment, w.i.D  =  ischemic lungs with Deguelin treatment. Measurements were performed in triplicate. Columns and error bars represent means ± SEM. * indicates significance level vs. sham, † indicates significance level vs. w.i.; *, P<0.05; ***, P<0.0005; ††, P<0.005; †††, P<0.0005; one-way ANOVA and unpaired t test.</p

VEGF-A activity correlates with edema formation.

<p>The transplantation experiments compromise two groups, one control (perfadex only) and one Deguelin treated group. Animals receiving Deguelin (donor and recipient) are pretreated 3 days prior transplantation and treatment is kept upright in the recipients until the end of the experiment after 48 hours after transplantation. (A) Graphs and representative western blot images representing tissue VEGF-A mRNA and protein levels after transplantation and reperfusion. Animals that received Deguelin are compared vs. controls. (B) Right graph representing the wet-to-dry ratio evaluating the extent of tissue edema. Left graph representing the mean survival expressed in hours of both groups (controls vs. animals that received Deguelin). (C) Corresponding micrographs (10× magnification, H&E) show microstructural changes that occur after reperfusion. Calibration bar represents 100 µm. Columns and error bars represent means ± SEM. * indicates significance level vs. control. ***, P<0.0005; one-way ANOVA and unpaired t test.</p

Therapeutic schedule.

<p>Scheme representing the therapy (gavages) and transplantation schedule. The lightning marks the day of transplantation. Experiment ends 48 hours after transplantation.</p

Layout of the first experimental stage.

<p>Layout of the first experimental stage.</p

Layout of the second experimental stage.

<p>Layout of the second experimental stage.</p

Deguelin prevents from ischemia induced edema and loss of lung microstructure.

<p>From the explantation experiment, micrographs are analyzed to detect structural edema as sign for organ damage. (A) Representative H&E micrographs (purple) from native lungs (sham), ischemic lungs without treatment (w.i.) and ischemic lungs with deguelin treatment (w.i.D). The blue graphs represent planimetric evaluation of the H&E stains to evaluate the area/field occupied by tissue as measurement for edema. Magnifications used: 10× and 40×. Bar in the 10× magnified micrographs represent 100 µm and in the 40× magnified micrographs represent 50 µm. From each H&E stain representing always one animal, 3 different areas were photographed and evaluated. (B) Graph representing the evaluation of the planimetric measurements. Columns and error bars represent means ± SEM. * indicates significance level vs. sham, † indicates significance level vs. w.i.; ***, P<0.0005; †††, P<0.0001; one-way ANOVA and unpaired t test.</p

Anti-inflammatory macrophages are recruited to Deguelin treated lungs.

<p>Figure representing micrographs of cellular invasion into transplanted lungs at the end of the reperfusion phase. The left micrographs represent control animals and the right micrographs represent animals that received Deguelin treatment. From top to bottom, micrographs represent DAB immunostainings from RM-4 (pan-macrophage), ICAM-1, CXCR4, CD68 and CD163. Magnification was set at 40× and 100× for pictures in picture. CXCR4 micrographs are represented in 20× magnification. Calibration bar represents 50 µm (40×). The graphs represent the statistical evaluation of each cell type. The upper graph represents a total macrophage count (RM-4+ cells), followed by ICAM-1+ cell count and CXCR4staining. The last two graphs represent CD68+ cell count, and finally a CD 163+ cell count. Arrowheads mark positively stained cells. Evaluation for CXCR4 is performed by calculating the ratio between positively and negatively stained amount of cells to avoid bias from alveoli that contain no cells. Arrowheads mark positively stained cells. Columns and error bars represent means ± SEM. * indicates significance level vs. control. ***, P<0.0001; one-way ANOVA and unpaired t test.</p