Congratulations Sergio
On 2nd September, Sergio D'Anna defended his MSc thesis in Biology-Biotechnology. Sergio’s project was entitled "Characterizing ABCC6 substrate specificity – A long-sought key to prevent vascular calcification”.
MSc student Sergio D'Anna and supervisor Hussam Nour-Eldin after the examination
Abstract: The top two leading death causes worldwide are ischemic heart disease and strokes, whose incidence is significantly higher in patients suffering from severe vascular calcification (VC). VC is a process that leads to calcium phosphate salts deposition in blood vessels. Many therapies are currently being tested to halt calcium deposition. However, prolonged use would probably jeopardize bone health as calcium phosphate salts are its main constituents. Therefore, it is vital to highlight differences in VC and physiological bone formation to target blood vessels specifically.
The key to new treatments could lie in understanding the underlying causes of VC-promoting diseases, especially the monogenic ones, as their onset is more straightforward than the multifactorial ones. For example, pseudoxanthoma elasticum (PXE) is a Mendelian disorder caused by inactivating mutations in the ABCC6 transporter.
ABCC6 is known to physiologically inhibit VC by mediating the release of cellular ATP, which is rapidly converted into the mineralization inhibitor pyrophosphate. However, vesicular transport assays failed to demonstrate ABCC6-mediated ATP export. Therefore, it is suspected that the transporter might translocate an unknown substrate somehow responsible for subsequent ATP efflux. Since the disease causes ectopic calcification in blood vessels without altering bone health, elucidating ABCC6's role on PXE could lead to developing new VC treatments with little side-effects.
This thesis aimed to characterize ABCC6 substrate specificity using a novel method initially developed to screen plant transporters with thousands of metabolites. In addition, ABCC6 specificity towards ATP was also tested to verify the previous vesicular studies.
Through heterologous expression in Xenopus oocytes, we first demonstrated ABCC6 functionality through an export assay with leukotriene C4 (LTC4), one of its known substrates. Then, through a luciferase-based ATP detection assay, we could not detect ABCC6-mediated ATP export, which confirms that ATP is likely not a direct ABCC6 substrate. Finally, the novel screening assay was conducted after a long optimization process, generating a massive data set. The limited time available for this thesis permitted routine/standard data analyses, which did not identify any new ABCC6 substrates. However, the optimization process set the first essential steps in tuning the novel method to screen mammalian transporters. Future in-depth data analyses and method optimization could lead not only to elucidating ABCC6 substrate specificity but to a new paradigm in identifying the substrate of any other human transporter of interest.
Sergio was supervised by Associate Professor Hussam Nour-Eldin and co-supervised by Henrik Dimre, University of Southern Denmark.