During my outgoing phase, I was working at Dr. Ju Chen´s lab at UCSD. His world-leading group is specializing in murine models for cardiovascular research, where I obtained here high-end training in mouse genetics and cardiovascular phenotyping. Using advanced CRISPR/Cas9 technology, I have made significant progress in developing the N2A region deficient mouse line. During my outgoing phase, I worked closely with Dr. Mayans on structural characterization of N2A-CARP interaction. We have developed well-behaved constructs of CARP and N2A region amenable for structural studies. We have demonstrated that the monomer of the N2A region displaces the homodimer of CARP to form 1:1 complex. In contrast to previous reports, we have shown that the N2A region is not intrinsically unfolded entropic spring but adopts exceptionally thermostable helical fold. We have solved the high-resolution crystal structure of adjacent I81 domain and revealed unique loop extensions contributing to complex formation. The results of this study have been published.
During my return phase in Europe, I have received office and lab space at ZI, where I further studied N2A-CARP interaction and mastered mouse phenotyping methods using advanced small animal imaging facility available in Mannheim. Besides, at the ZI, I am using state-of-the-art animal house facility and molecular biology equipped labs, suitable for recombinant protein expression and small molecule screens.
ZI helped to set up a small research group at Vilnius University with additional funding from Lithuanian Research Council and Leducq Foundation-funded 13CVD research network. I am currently supervising Ph.D. student Ieva Rinkūnaitė at Vilnius University. Further phenotypic characterization of mouse models and testing of N2A-CARP interaction modulators have to be performed in ZI and Vilnius University.
In collaboration with Olga Mayans, I have performed deuterium/hydrogen exchange experiments using mass spectroscopy, to further characterize the 3D structure of single proteins and the binding surface of the N2A-CARP complex. These results helped to elucidate the N2A-CARP binding surface at the atomic level, which served for targeted development interaction inhibitors. Moreover, we discovered that the N2A-CARP complex interacts with an unexpected ligand that is essential for cardiac function.
In summary, my outgoing phase research period successfully contributed to research in Dr. Chen´s lab on the molecular mechanisms that couple mechanical cardiomyocyte strain and cardiomyocyte loss during the heart failure involving the mechanosensing titin filament. During my secondment to UCSD, I have acquired new technical qualifications for the generation of genetic mouse models for cardiovascular research and murine cardiovascular phenotyping. I brought these newly obtained competencies back to the EU, where I implemented them while working at ZI and setting up my research group at Vilnius university. The results of my work have been published in 9 scientific publications, presented in numerous scientific conferences, and two more manuscripts are currently in preparation. In the framework of promoting public awareness of science, the results of this project were communicated to the general audience as short communications, interviews, and podcasts.
Competences acquired during this fellowship were a key component allowing me to become a mentor for a Ph.D. student and initiate a transgenic core facility at Vilnius University.
