Cytoglobin, conserved but still mysterious. A structural and functional characterisation
24 November 2017
UAntwerp, Campus Drie Eiken, Building R, Auditorium R1 - Universiteitsplein 1 - 2610 Wilrijk (Antwerp) (route: UAntwerpen, Campus Drie Eiken
4:00 PM - 6:00 PM
Sylvia Dewilde, Wim Vanden Berghe
PhD defence Stijn Vermeylen - Department of Biomedical Sciences
Cytoglobin (Cygb) is besides hemoglobin and myoglobin, a member of the globin superfamily. Although different studies have questioned the role of Cygb, none have really been able to completely solve the function of this conserved hexa-coordinated globin. Several hypotheses have been proposed, such as a role as ROS-scavenger and a role in different diseases, like fibrosis and cancer. The conserved amino acid sequence among different Cygb orthologues points to the importance of this protein. Therefore the first main objective of the research project was to structurally and functionally characterise different Cygb orthologues that were recombinantly expressed in E. coli. The obtained results were compared to human Cygb (CYGB).
We demonstrated that the Antarctic fish Cygbs were structurally adapted to the extreme living conditions: i) by forming multimers, up to pentamers, in vitro; and ii) by having a lower affinity for the internal ligand, compared to zebrafish and human Cygb at room temperature. As they showed similar external ligand binding constants at lower temperature, we hypothesise that by forming multimers and by structurally changing the heme pocket structure, the globins are specifically adapted to function under these cold temperatures and as such protect the Antarctic fish.
The Cygbs paralogues of Danio rerio (Cygb-1 and Cygb-2) were also structurally characterised. Both Cygbs, with distinct expression patterns and probably functions, did not show large structural differences with CYGB. The most striking, yet preliminary, observation was made by EPR experiments, where we could suggest a more flexible heme pocket structure of D. rerio Cygb-1 compared to Cygb-2. This structural difference, together with the difference in ligand binding properties and localisation, suggests a more oxygen carrier function for Cygb-1 and a more protective role for Cygb-2. However more research is needed to uncover the exact functions of these paralogues.
The second main objective focused more on the ROS-scavenging and tumor suppressor function of Cygb. We optimised different experimental parameters for Cold Atmospheric Plasma (CAP) treatment of different melanoma cell lines. It has already been demonstrated that CAP treatment will change the oxidative balance by increasing the concentration of RONS. Defence mechanisms, such as anti-oxidants and DNA repair mechanisms will be activated to reduce the oxidative damage, which will be recovered faster in healthy cells. This pilot study has indicated that different experimental parameters are important and will influence the outcome of the in vitro experiments. The optimised parameters are now used in future research where the specific role of Cygb in the cellular response to CAP is investigated.