Investigation of the Dimerization of the Nitric Oxide-Sensitive Guanylyl Cyclase

Die Stickstoffmonoxid-sensitive Guanylyl-Cyclase ist ein wichtiges Ziel für Arzneimittel zur Behandlung von Herzkreislauferkrankungen. Das Enzym besteht aus einer α1- und einer β1 Untereinheit. Im Rahmen der vorliegenden Arbeit konnten wir eine neue besonders effiziente Anreinigung dieses Heterodimers etablieren. Mit Hilfe dieser neuen Methode konnten wir nachweisen, dass eine humane Spleißvariante der α1 Untereinheit mit der β1 Untereinheit ein katalytisch aktives Heterodimer bildet. Darüber hinaus zeigte sich in der konfokalen Fluoreszenzmikroskopie, dass die aminoterminal um 258 Aminosäuren verkürzte α1-Variante eine spezifische Lokalisation am endoplasmatischen Retikulum aufweist. Dieser Befund deckt sich mit Ergebnissen zur subzellulären Verteilung der Spleißvariante bei Differenzierungsprozessen in embryonalen Stammzellen. In einem zweiten Teil der Arbeit wurden die Bedingungen der Dimerisierung und Expression der Stickstoffmonoxid-sensitiven Guanylyl-Cyclase näher untersucht. Dabei konnten wir zeigen, dass eine weitergehende aminoterminale Deletion der α1 Untereinheit bei Koexpression mit der β1 Untereinheit zu einem Verlust der Aktivierbarkeit durch NO führt. Die Bindung von Häm und die Dimerisierung blieben hierbei erhalten. Dagegen führte die analoge aminoterminale Deletion der β1 Untereinheit nach Koexpression mit der α1 Untereinheit zu einem hämfreien Enzymkomplex. In zellfreien Expressionsystemen konnten wir die Untereinheiten des Enzyms erfolgreich exprimieren. Es kam allerdings nicht zur Bildung von funktionell aktiven Heterodimeren. Die Ergebnisse deuten auf eine wichtige Rolle von zellulären Bestandteilen für die Bildung eines intakten heterodimeren Enzyms hin.The nitric oxide-sensitive guanylyl cyclase is an important target for drugs to treat cardiovascular diseases. The enzyme consists of an α1-and β1 subunit. In the present study we were able to establish a new efficient purification method of this heterodimer. Using this new protocol we were able to demonstrate that a splice variant of human α1 subunit is able to build a catalytically active heterodimer with β1 subunit. Moreover, we could show that this truncated α1-variant has a specific localization at the endoplasmic reticulum. This finding is consistent with results of the subcellular distribution of the splice variant in differentiation processes in embryonic stem cells. In a second part, the terms of the dimerization and expression of nitric oxide-sensitive guanylyl cyclase were examined. We have shown that a more extensive amino-terminal deletion of the α1 subunit leads to a loss of activation by NO. The binding of heme and dimerization were obtained here. In contrast, the analogous amino-terminal deletion of the β1 subunit coexpression led to a heme free enzyme complex. In cell-free expression systems we were able to express the subunits of the enzyme successfully. However, the formation of functionally active heterodimers could not be shown. The results suggest an important role of cellular components for the formation of an intact heterodimeric enzyme

Investigating the zoonotic origin of the West African Ebola epidemic

The severe Ebola virus disease epidemic occurring in West Africa stems from a single zoonotic transmission event to a 2‐year‐old boy in Meliandou, Guinea. We investigated the zoonotic origins of the epidemic using wildlife surveys, interviews, and molecular analyses of bat and environmental samples. We found no evidence for a concurrent outbreak in larger wildlife. Exposure to fruit bats is common in the region, but the index case may have been infected by playing in a hollow tree housing a colony of insectivorous free‐tailed bats (Mops condylurus). Bats in this family have previously been discussed as potential sources for Ebola virus outbreaks, and experimental data have shown that this species can survive experimental infection. These analyses expand the range of possible Ebola virus sources to include insectivorous bats and reiterate the importance of broader sampling efforts for understanding Ebola virus ecology

Investigating the zoonotic origin of the West African Ebola epidemic

The severe Ebola virus disease epidemic occurring in West Africa stems from a single zoonotic transmission event to a 2‐year‐old boy in Meliandou, Guinea. We investigated the zoonotic origins of the epidemic using wildlife surveys, interviews, and molecular analyses of bat and environmental samples. We found no evidence for a concurrent outbreak in larger wildlife. Exposure to fruit bats is common in the region, but the index case may have been infected by playing in a hollow tree housing a colony of insectivorous free‐tailed bats (Mops condylurus). Bats in this family have previously been discussed as potential sources for Ebola virus outbreaks, and experimental data have shown that this species can survive experimental infection. These analyses expand the range of possible Ebola virus sources to include insectivorous bats and reiterate the importance of broader sampling efforts for understanding Ebola virus ecology

Data from: Investigating the zoonotic origin of the West African Ebola epidemic

The severe Ebola virus disease epidemic occurring in West Africa stems from a single zoonotic transmission event to a 2-year-old boy in Meliandou, Guinea. We investigated the zoonotic origins of the epidemic using wildlife surveys, interviews, and molecular analyses of bat and environmental samples. We found no evidence for a concurrent outbreak in larger wildlife. Exposure to fruit bats is common in the region, but the index case may have been infected by playing in a hollow tree housing a colony of insectivorous free-tailed bats (Mops condylurus). Bats in this family have previously been discussed as potential sources for Ebola virus outbreaks, and experimental data have shown that this species can survive experimental infection. These analyses expand the range of possible Ebola virus sources to include insectivorous bats and reiterate the importance of broader sampling efforts for understanding Ebola virus ecology

Differential transcriptional responses to Ebola and Marburg virus infection in bat and human cells

The unprecedented outbreak of Ebola in West Africa resulted in over 28,000 cases and 11,000 deaths, underlining the need for a better understanding of the biology of this highly pathogenic virus to develop specific counter strategies. Two filoviruses, the Ebola and Marburg viruses, result in a severe and often fatal infection in humans. However, bats are natural hosts and survive filovirus infections without obvious symptoms. The molecular basis of this striking difference in the response to filovirus infections is not well understood. We report a systematic overview of differentially expressed genes, activity motifs and pathways in human and bat cells infected with the Ebola and Marburg viruses, and we demonstrate that the replication of filoviruses is more rapid in human cells than in bat cells. We also found that the most strongly regulated genes upon filovirus infection are chemokine ligands and transcription factors. We observed a strong induction of the JAK/STAT pathway, of several genes encoding inhibitors of MAP kinases (DUSP genes) and of PPP1R15A, which is involved in ER stress-induced cell death. We used comparative transcriptomics to provide a data resource that can be used to identify cellular responses that might allow bats to survive filovirus infections

Partial mitochondrial cytochrome b and 16S sequences from bats captured in southeastern Guinea, 2014

Comprises all Sanger sequences generated for this study. Individuals were only assigned to the genus-level in the field. BLAST results were combined with this field assignment and further biological information (species distribution) to perform the final assignment. Sequence names are built as follows: sample #_final assignment

Partial mitochondrial 16S sequences from soil samples from Meliandou, Guinea, 2014

This ZIP file comprises demultiplexed FASTQ files corresponding to 30 PCR products sequenced on a MiSeq. A tab-delimited list is provided as the ReadMe

Differential transcriptional responses to Ebola and Marburg virus infection in bat and human cells

The unprecedented outbreak of Ebola in West Africa resulted in over 28,000 cases and 11,000 deaths, underlining the need for a better understanding of the biology of this highly pathogenic virus to develop specific counter strategies. Two filoviruses, the Ebola and Marburg viruses, result in a severe and often fatal infection in humans. However, bats are natural hosts and survive filovirus infections without obvious symptoms. The molecular basis of this striking difference in the response to filovirus infections is not well understood. We report a systematic overview of differentially expressed genes, activity motifs and pathways in human and bat cells infected with the Ebola and Marburg viruses, and we demonstrate that the replication of filoviruses is more rapid in human cells than in bat cells. We also found that the most strongly regulated genes upon filovirus infection are chemokine ligands and transcription factors. We observed a strong induction of the JAK/STAT pathway, of several genes encoding inhibitors of MAP kinases (DUSP genes) and of PPP1R15A, which is involved in ER stress-induced cell death. We used comparative transcriptomics to provide a data resource that can be used to identify cellular responses that might allow bats to survive filovirus infections