Biophysical Studies of Rab GTPase Membrane Binding

Rab proteins are the largest subfamily of the Ras superfamily of small GTPases, with more than 60 known members, that are involved in a multitude of different processes regulating membrane traffic. Rab proteins cycle between the cytosol and association with membranes, whereby each Rab exhibits a characteristic and specific subcellular localisation. It remains obscure how Rab proteins, in spite of high sequence and structure identity, distinguish between different membranes in the cell with such specificity. Membrane biophysical properties, such as stored curvature elastic stress and bending rigidity, are increasingly found to be determinants for protein recruitment and activity, and other Ras related proteins have recently been shown to exhibit sensitivity towards lipid species and elastic membrane properties. In this study Rab membrane binding is for the first time correlated to membrane bending rigidity, suggesting that biophysical properties of lipid membranes may play a role in the regulation of Rab targeting. Furthermore, all Rab proteins tested were observed to bind membranes in the absence of other protein factors, questioning the function of protein targeting factors for the Rab membrane recruitment process. Another aspect of Rab membrane interaction is Rab extraction from membranes by GDI. A large scale in vitro screening of 17 Rab proteins revealed a broad range of extractability from membranes with GDI. No correlation was found between extractability and the C-terminal prenylation motif, and no difference in extractability was observed in direct comparison of the extraction potential with GDIα and β. However, Rab proteins that exhibited low extractability from membranes are involved in secretory processes, suggesting a functional correlation to extractability. Furthermore, Rab40c as the first mammalian Rab protein to date was shown to be palmitoylated

The role of guanine nucleotide exchange factors (GEFs) in EGF-receptor signalling : Screening for a small molecule inhibitor of the Rin1-mediated Rab5 activation

The small GTPase Rab5 is a key regulator of early endosomal trafficking. It functions as a molecular switch that can be activated by guanine nucleotide exchange factors (GEFs) and inactivated via hydrolysis of GTP with the help of catalytic GTPase activating proteins (GAPs). GEFs mediate the exchange of GTPase-bound GDP for GTP. One of at least nine Rab5 GEFs, that have been identified until today, is Rin1. However, the differences in the exact roles of these GEFs are not completely understood. Rin1 is a multi-domain protein that has multiple signalling functions alongside the Rab5 activation. It is for example also involved in ABL kinase signalling, where it increases ABL activation. Rab5 over-activation has been reported to be involved in the genesis and progression of many different types of cancer and it could often be traced back to Rin1 over-expression. On the other hand Rin1 has been found to have a tumour repressive effect, mediated by its ABL kinase signalling function. This study aimed on the identification of a small molecule inhibitor of the Rin1-mediated Rab5 activation. Such a small molecule inhibitor could be used as a tool to unravel parts of the complex signalling networks around Rab5. Ideally the inhibitor should be specific for Rin1 over other GEFs and moreover not influence its ABL kinase signalling function. A high-throughput in vitro screening of more than 20 000 small molecules has been performed. The screening assay monitored the Rin1-catalyzed nucleotide exchange on Rab5a by the use of the fluorescently labelled GTP analogue Bodipy-TR-GTP. Amongst other primary hits the compound CG3 05 A02 was identified. During specificity and aggregation studies it turned out to be the most promising candidate. Characterization of CG3 05 A02 revealed that its inhibitory effect in the Bodipy-TR-GTP nucleotide exchange assay is specific for Rin1 and Rabex-5 on Rab5a over several other GEF/GTPase pairs. The IC50 values were in both cases found to be in the low micromolar range. Rin1 and Rabex-5 share the homologous catalytic Vps9 domain and the compound might also be able to inhibit other Vps9 domain-containing GEFs. The compound did not induce aggregation of Rin1 or Rab5a and had no unspecific off-target effects in an unrelated insulin receptor auto-phosphorylation assay. It moreover did not influence the interaction between Rin1 and ABL1, indicating no intramolecular domain-unspecific inhibition. Unfortunately a control experiment with radioactively labelled GTP showed no inhibitory effect of the comound. The inhibition therefore depends on the use of Bodipy-TR-GTP but unlikely originates from CG3 05 A02 binding to the GTP analogue. In this case it would have inhibited the Bodipy-TR-GTP nucleotide exchange assay when GEFs/GTPases other than Rin1/Rabex-5 and Rab5 were used. This label-dependence classifies the compound unsuitable for cellular or in vivo application. The actual target of CG3 05 A02 could not be finally addressed but Bodipy-TR-GTP binding to Rab5a in absence of a GEF was not influenced by the compound. This argues against GTP-competitive binding as the mechanism of inhibition. The compound potentially targets either directly the Vps9 domains of Rin1 and Rabex-5, areas adjacent to these, or the complex between the GEFs and Rab5. A mechanism based on steric hindrance involving the compound and the Bodipy-TR moiety of the GTP analogue was proposed as the mode of action. Identification of a binding site for CG3 05 A02 in ongoing crystallization approaches could provide a starting point for in silico screenings that can result in a second generation of inhibitors of the Rin1-mediated Rab5 activation.Die Rolle der Guaninnukleotid-Austauschfaktoren (GEFs) im EGF-Rezeptor Signalweg : Die Suche nach einem niedermolekularen Inhibitor der Rin1-vermittelten Rab5 Aktivierung Rab5 ist eine kleine GTPase, die maßgeblich an der Regulierung des Membrantransportes zu- und -an frühen Endosomen beteiligt ist. GTPasen sind molekulare Schalter, die durch Guaninnukleotid Austauschfaktoren (GEFs) aktiviert und durch die Hydrolyse von GTP mit Hilfe katalytischer GTPase aktivierender Proteinen (GAPs) inaktiviert werden. GEFs vermitteln den Austausch von GTPase-gebundenem GDP gegen freies GTP. Einer von mindestens neun bisher beschriebenen Rab5 GEFs ist Rin1. Die genauen Unterschiede in Funktion und Wirkungsweise der verschiedenen Rab5 GEFs sind derzeit noch nicht im Gesamten entschlüsselt. Rin1 ist ein Multidomänenprotein mit verschiedenen Funktionen in der intrazellulären Signaltransduktion. Neben der Aktivierung von Rab5 spielt Rin1 ebenfalls eine Rolle in ABL Kinasen Signalwegen. Hier verstärkt es die Aktivierung der ABL Kinasen. Rab5 Überaktivierung wurde mit verschiedenen Krebsarten in Zusammenhang gebracht. In den meisten Fällen kann diese Überaktivierung auf Rin1 Überexpression zurückgeführt werden. Rin1 verfügt jedoch auch über eine krebsunterdrückende Funktion, die im Zusammenhang mit seiner Interaktion mit ABL Kinasen steht. Diese Arbeit zielt auf die Identifikation eines niedermolekularen Inhibitors der Rin1-vermittelten Rab5 Aktivierung ab. Der Inhibitor könnte helfen, die komplexen Signalkaskaden, an denen Rab5 beteiligt ist, aufzuschlüsseln. Idealerweise wäre er spezifisch für Rin1 gegenüber anderen GEFs. Außerdem sollte er die Interaktion zwischen Rin1 und den ABL Kinasen nicht beeinflussen. Hierfür wurde ein in vitro Hochdurchsatzscreening mit über 20 000 potentiellen niedermolekularen Inhibitoren durchgeführt. Das Screeningexperiment überwachte den Rin1-vermittelten Austausch von GDP gegen das Fluoreszenzmarkierte GTP Analogon Bodipy-TR-GTP an Rab5. So wurde unter Anderem der niedermolekulare Inhibitor CG3 05 A02 identifiziert. Während Spezifitäts- und Aggregationsstudien stellte sich dieser als der vielversprechendste Kandidat heraus. Seine Charakterisierung ergab Spezifität für Rin1 und Rabex-5 an der GTPase Rab5a im Bodipy-TR-GTP Nukleotidaustauschexperiment. Verschiedene andere GEF/GTPase Paare wurden nicht beeinflusst, obwohl deren Austauschmechanismus dem von Rin1 und Rabex-5 an Rab5 ähnelt. Die IC50 Werte, die in den Austauschexperimenten mit Rin1 und Rabex-5 ermittelt wurden, liegen beide im niedrigen micromolaren Bereich. Beide GEFs verfügen über eine homologe katalytische Vps9 Domäne. Es kann derzeit nicht ausgeschlossen werden, dass CG3 05 A02 auch weitere Vps9 Domänen GEFs inhibiert. Der Inhibitor führte keine Aggregation von Rin1 oder Rab5 herbei und hatte auch keinen unspezifischen Effekt in einem unverwandten Insulinrezeptor Autophosphorylierungsexperiment. Außerdem beeinflusste er die Interaktion zwischen Rin1 und ABL1 nicht, was gegen eine intramolekulare, domänenunspezifische Inhibition sprach. Bedauerlicherweise zeigte ein weiteres Kontrollexperiment keine Inhibition des Rin1-vermittelten Nukleotidaustausches, wenn radioaktiv markiertes GTP verwendet wurde. Dieses Ergebnis sprach für eine Bodipy-TR-GTP-Abhängigkeit der Inhibition. Diese stammt wahrscheinlich nicht von einer Interaktion zwischen dem Inhibitor und dem GTP Analog her, denn in diesem Fall wäre die Inhibition in allen Bodipy-TR-GTP Nukleotidaustauschexperimenten aufgetreten, auch wenn andere GEFs und GTPasen getestet wurden. Aufgrund dieser Markierungsabhängigkeit ist der Inhibitor für die zelluläre und die in vivo Anwendung ungeeignet. An welches Protein CG3 05 A02 bindet konnte nicht final aufgeklärt werden. Das Beladen von Rab5a mit Bodipy-TR-GTP in Abwesenheit eines GEFs wurde von CG3 05 A02 nicht beeinflusst, was gegen einen GTP-kompetitiven Mechanismus der Inhibition spricht. Der Inhibitor bindet entweder an die Vps9 Domänen von Rin1 und Rabex-5, in der Nähe von diesen, oder an den Komplex zwischen den GEFs und Rab5. Aufgrund der Datenlage wurde ein auf sterischer Hinderung basierender Inhibitionsmechanismus vermutet, bei dem der Bodipy-TR-Rest und der an den Proteinkomplex gebundene Inhibitor sich behindern, sodass das Nukleotid nicht in die Bindungstasche gelangen kann. Das deutlich kleinere, radioaktiv markierte GTP ist von dieser Hinderung nicht betroffen. Kristallisation der Proteine im Komplex mit CG3 05 A02 könnte zur Identifizierung einer Bindestelle führen, die als Ausgangspunkt für zukünftige in silico Screenings dienen könnte. Aus einem derartigen Screening könnte dann eine zweite Generation von Inhibitoren der Rin1-vermittelten Rab5 Aktivierung hervorgehen

Insights into the intracellular localization, protein associations and artemisinin resistance properties of Plasmodium falciparum K13

The emergence of artemisinin (ART) resistance in Plasmodium falciparum intra-erythrocytic parasites has led to increasing treatment failure rates with first-line ART-based combination therapies in Southeast Asia. Decreased parasite susceptibility is caused by K13 mutations, which are associated clinically with delayed parasite clearance in patients and in vitro with an enhanced ability of ring-stage parasites to survive brief exposure to the active ART metabolite dihydroartemisinin. Herein, we describe a panel of K13-specific monoclonal antibodies and gene-edited parasite lines co-expressing epitope-tagged versions of K13 in trans. By applying an analytical quantitative imaging pipeline, we localize K13 to the parasite endoplasmic reticulum, Rab-positive vesicles, and sites adjacent to cytostomes. These latter structures form at the parasite plasma membrane and traffic hemoglobin to the digestive vacuole wherein artemisinin-activating heme moieties are released. We also provide evidence of K13 partially localizing near the parasite mitochondria upon treatment with dihydroartemisinin. Immunoprecipitation data generated with K13-specific monoclonal antibodies identify multiple putative K13-associated proteins, including endoplasmic reticulum-resident molecules, mitochondrial proteins, and Rab GTPases, in both K13 mutant and wild-type isogenic lines. We also find that mutant K13-mediated resistance is reversed upon co-expression of wild-type or mutant K13. These data help define the biological properties of K13 and its role in mediating P. falciparum resistance to ART treatment

Novel Regulatory Mechanisms and Functions of MUC4 in Pancreatic Cancer

Mucins are high molecular weight glycoproteins and have critical functions in protecting epithelial cells from a myriad of cellular stress. However, mucins are expressed aberrantly under cancer conditions that allow tumors to progress and metastasize. Among many mucins, Mucin 4 (MUC4) serves as one of the top-differentially expressed proteins in pancreatic cancer (PC), however, the precise mechanism responsible for its aberrant expression is still not clear. The evolving view of cancer as an energetic and growing ecosystem underlines an intricate interplay between cancer and its microenvironment. In spite of being recognized as one of the most critical oncogenic proteins in PC, MUC4 regulation in terms of micro-environmental stress has not been determined. In my dissertation research, I have investigated the role of PC microenvironment in the regulation of MUC4. From my studies, I have demonstrated that MUC4 stability is significantly reduced due to hypoxia-mediated induction of reactive oxygen species (ROS), which promotes autophagy by inhibiting pAkt/mTORC1 pathway. Hypoxia-mediated degradation of MUC4 provides necessary metabolites to ensure the survival of highly stressed PC cells. The longstanding model of cancer development involves that presence of cytokines can trigger chronic inflammation and impact tumor development, including PC. In addition to cytokines, bile acids (BA) facilitated chronic inflammation has shown to induce intestinal metaplasia, but their role in PC is still elusive. Elevated levels of BA (p In addition to the regulation, I have pinpointed the novel functional roles of MUC4 in determining the fate of receptor tyrosine kinases (RTKs) in PC. Multiple studies have associated MUC4 overexpression with increased stability of RTKs for sustained proliferation; however, no studies have so far highlighted the implicated mechanism. I have demonstrated that the presence of MUC4 leads to increased internalization and recycling of EGFR and HER2 to the plasma membrane compared to MUC4 silenced PC cells. Mechanistically, the impact of MUC4 on RTKs trafficking is associated with its ability to regulate the activity of RAB5A, which is known to catalyze the rate-limiting step in receptor internalization. Lastly, I have detected the presence of MUC4 in pancreatic cancer associated stellate cells (PaSC). This was an unexpected finding given that MUC4 is normally expressed in the epithelial cells. These results indicate towards the involvement of MUC4 expression in determining the activation status of PaSC and provide us an additional strong rationale to therapeutically target MUC4. Altogether, in my dissertation research, I have elucidated the novel regulatory mechanisms and functions of MUC4 in PC condition

Role of Calsyntenin-1 in Hepatitis C Virus Pathogenesis

Approximately 150 million people worldwide have chronic HCV infection that can lead to hepatic fibrosis, liver failure and cancer. In a proteomic screen of the “secretome” from HCV infected hepatic cells we observed increased (43 fold) secretion of calsyntenin-1, compared to uninfected controls. Calsyntenin-1 is an adapter molecule that links microtubule associated molecular motor kinesin 1 to vesicular cargo in neurons. We showed that calsyntenin-1 is involved in early stages of the viral replication cycle, namely uptake into early endosomes while its loss altered early endosomal distribution, velocities and function. It was also found to be required for the establishment of new HCV replication complexes, their transport and distribution within cell. Furthermore, siRNA-mediated knockdown of calsyntenin-1 showed its association with intracellular trafficking of multivesicular bodies and recycling endosomes by affecting their distribution within HCV infected cells and also reduced exosome infectivity as determined by Deltavision microscopy and infectivity assay (TCID50) analysis. Our results suggest that calsyntenin-1 in involved in multiple stages of HCV replication cycle as an important host factor. The uptake of viral particles into early endosomes, trafficking of replication complexes and egress of virus containing exosomes involve kinesin-1 based transport dependent on membrane adaptor qualities of calsyntenin-1