Portrait of author

Ana Paula Cordeiro:
Structural and Functional studies of FICD. A new unfolded protein response target

Date: 22-06-2018    Supervisor: Lars Ellgaard

The FIC proteins are present in more than 3000 species that are distributed in all kingdoms of life. FIC proteins are widespread in bacteria, but there is a single representative in animals. It is known that the main ER-resident chaperone BiP is regulated by FICD AMPylation and de-AMPylation, which switches BiP to an inactive or active state according to unfolded protein load in the cell. This inactivation is accomplished by a defective J protein-driven ATP hydrolysis following BiP AMPylation that impairs stable engagement of substrates by BiP. In addition, FICD features an auto-AMPylation activity that it has not been well explored and its biological relevance remains unknown. In some FIC proteins, auto-modification of the protein relieves auto-inhibition and, therefore, is a prerequisite for target AMPylation. Still, there are many open questions about the regulation of the endogenous FICD activity to be addressed. Here we investigated further the interaction between FICD and BiP in addition to FICD auto-AMPylation. FICD binding to BiP was characterized by pull-down assays and the affinity of this interaction was quantified by MST measurements. Auto-AMPylation assays were performed for distinct FICD mutants to evaluate if changes in this self-modification affects target modification. Further, our findings showed that FICD auto-AMPylation can be detected as a gel-shift by SDS-PAGE analysis, and that oligomerization disruption of FICD can release auto-inhibition. Overall, our findings are a starting point to the importance of auto-AMPylation for FICD regulation in order to exert its function as an UPR target in the ER. However, further assays are still required to assess the exact mechanism in which auto-AMPylation impacts the target AMPylation.