Current projects
Mechanisms of Oxygen-sensing
Project manager: Prof. Dr. D.M. Katschinski
staff: S. Krull
Although in bacteria the mechanisms of oxygen sensing are well described, the oxygen sensing mechanisms in mammals are not totally understood. The PAS protein FixL is triggering oxygen-mediated gene epression in nitrogen fixing rhizobiae. Recently, we described a mammalian protein, which is a homologue to FixL, named the protein PASKIN and developed a PASKIN knock out mouse. We are investigating the organ-specific expression as well as PASKIN binding partners, which should help to understand the development of oxygen sensing mechanisms from an evolutionary point of view. Funding: DFG Ka 1269/7-1
Age-dependent expression and function of the cellular oxygen sensing prolylhydroxylase 1-3 in the heart
Project manager: Prof. Dr. D.M. Katschinski
Staff: Dr. S. Vogel
HIF-1α is important for the adaptation of cells towards a decreased partial oxygen pressure. We have described a decrease of HIF-1α expression but increased expression of the HIFa destructing PHD3 with advanced age in murine and human heart samples. Compared to the other two PHDs, for PHD3 highest levels were found in human and murine heart tissue. To identify the importance of HIF-1α and PHD3 for the age-dependent heart phenotype, a cDNA library was screened with PHD3 as bait. The importance of three PHD3 interacting transcription factors/transcriptional co-activators (activating transcription factor 4, friend of GATA-2 and megakaryoblastic leukemia factor 1) will be further investigated. Since we also identified the folding helper enzyme (FKBP38) as PHD interacting partner, we are investigating, if this protein-protein interaction can be of use to modulate PHD activity. Funding: DFG Ka 1269/8-1
Translational regulation of HIF-1a via polyadenylation of the 3`UTR
Project manager: Dr. S. Hägele
Staff: M. von Ahlen
HIF-1α, the master regulator of the oxygen-regulated gene expression is mainly regulated via protein stability. In the 3`untranslated region (UTR) of the HIF-1α gene several conserved polyadenylation consensus sequences can be found. We identified the cytoplasmic polyadenylation binding protein (CPEB) as interaction partner of the 3`UTR of HIF-1α as well as the HIF-1α protein. We are currently investigating, if the translation rate of HIF-1a is regulated by CPEB and if via protein-protein interaction HIF-1α can influence its own translation efficiency. Funding: junior research funding program of the Georg August University Göttingen
Identification of new PHD targets
Project manager: Dr. J. Köditz
Staff: Dipl. Biol. M. Wottawa
The activating transcription factor-4 (ATF-4) is translationally induced under anoxic conditions, mediates part of the unfolded protein response following ER stress, and is a critical regulator of cell fate. Here, we identified the zipper II domain of ATF-4 to interact with the oxygen sensor prolyl-4-hydroxylase domain 3 (PHD3). The PHD inhibitors DMOG, CoCl2 and hypoxia, or proteasomal inhibition, all induced ATF-4 protein levels. Hypoxic induction of ATF-4 was due to increased protein stability but independent of the ubiquitin ligase pVHL. A novel oxygen-dependent degradation (ODD) domain was identified adjacent to the zipper II domain. Mutations of five proline residues within this ODD domain or siRNA-mediated downregulation of PHD3, but not of PHD2, was sufficient to stabilize ATF-4 under normoxic conditions. These data demonstrate that PHD-dependent oxygen-sensing recruits both the HIF and ATF-4 systems and hence not only confers adaptive responses but also cell fate decisions. Further studies are needed to understand the oxygen-dependent degradation pathway of ATF-4.
Unfogging the function of Fog-2 – a putative link between tissue hypoxia and coronary artery development
Project manager: Dr. M. Kleinschmidt
Staff: S. Krull
In a yeast two hybrid screen we identified the transcription factor Fog-2 as a protein interactor of the cellular oxygen sensor PHD3. In addition, Fog-2 has been found to be expressed in an oxygen-dependent manner. Fog-2 is responsible for coronary artery development during embryogenesis. To this end, we will analyze the mechanisms responsible for the oxygen- dependent expression of Fog-2. Using in vivo studies we plan to determine, if Fog-2 is also upregulated in adult tissue as a consequence of hypoxia.
Identification of PHD mediated signal transduction pathways
Project manager: Dr. S. Vogel
Staff: S. Le-Hu
For the identification of PHD-specific signal transduction pathways we are currently establishing stable and inducible PHD2 and 3 shRNA cell clones. These will be analyzed for their response towards hypoxia and hypoxia-/reoxygenation and will open the possibility to analyze the redundancy of the PHDs. Furthermore the cell clones will be analyzed by Affymetrix Chip assays.