For this summer, I am working in the Horner lab, a lab in the the molecular genetics and microbiology department of the Duke school of medicine. The Horner lab is widely investigating RNA virus-host interactions with the immune system, specifically focusing on positive sense RNA viruses such as Zika, Dengue, Sendai, and Hepatitis C viruses interacting with the innate immune system. My mentor Dr. Gutierrez, a postdoc in the lab, is currently investigating ufmylation, a post translational modification, and its potential substrates in the innate immune system pathway. My specific project is attempting to characterize the protein-protein binding site of RIG-I and 14-3-3e, two proteins at the start of the innate immune system.
We set to determine whether the LIR motifs in the 2-CARD domain of the RIG-I protein mediate binding between RIG-I and 14-3-3e. The decision to investigate this particular motif came about by the lab’s investigation into previous studies of these proteins. It was previously found that 14-3-3e often binds in a phosphorylation binding pocket. However, it is known that 14-3-3e also binds in other non-canonical methods not involving phosphorylation (Pennington et al, 2018). RIG-I is one of the proteins known to interact without phosphorylation and thus must use a different binding method (Snider et al, 2021). It is currently being explored whether 14-3-3e may also bind at the site of another post translational modification, ufmylation, as this modification has been shown to be present on 14-3-3e by the Horner lab.
It was previously found that RIG-I binds to 14-3-3e through the 2-CARD domain of the RIG-I protein (Liu et al, 2012). It is not known what part within this domain binds to 14-3-3e. RIG-I contains two LIR motifs in the 2-CARD domain. The lab suspected that RIG-I may bind through one or both of these LC3 Interacting Regions, also known as LIR motifs, because these regions have been shown to mediate binding at post translational modification sites for other protein-protein interactions.
With this background, I was given the project to determine if RIG-I binds to 14-3-3e through the LIR motifs in the 2-CARD domain of RIG-I. To test this, we will be utilizing a RIG-I KO cell line and inserting plasmids encoding for RIG-I with mutated LIR motifs, disabling the function of these motifs. These lines will be tested to ensure expression of the RIG-I protein with a western blot, to determine function of the innate immune pathway with the mutated protein through a luciferase assay for IFNb (a major product of the pathway), and for binding of RIG-I to 14-3-3e through co-immunoprecipitation. If a LIR motif is not integral to the binding of 14-3-3e and RIG-I, IFNb should still be produced and the two proteins should precipitate together while RIG-I is expressed with a mutant on that motif. If a LIR motif is integral to the binding of 14-3-3e and RIG-I, IFNb should not be produced and the two proteins should not precipitate together while RIG-I is expressed with a mutant on that motif.
Upon completion of this experiment, this project has two likely paths forward. If the LIR motifs are not likely to be necessary for RIG-I and 14-3-3e binding, more potential suspected binding sites on RIG-I for this interaction may be investigated. If the LIR motifs are likely to be necessary for RIG-I and 14-3-3e binding, the binding site for this interaction on 14-3-3e may be investigated.
Pennington, K., Chan, T., Torres, M. et al. The dynamic and stress-adaptive signaling hub of 14-3-3: emerging mechanisms of regulation and context-dependent protein–protein interactions. Oncogene 37, 5587–5604 (2018). https://doi.org/10.1038/s41388-018-0348-3
Snider, D. L., Park, M., Murphy, K. A., Beachboard, D. C., & Horner, S. M. (2021). Signaling from the RNA sensor rig-I is regulated by ufmylation. PNAS. https://doi.org/10.1101/2021.10.26.465929
Liu, H. M., Loo, Y.-M., Horner, S. M., Zornetzer, G. A., Katze, M. G., & Gale, M. (2012). The mitochondrial targeting chaperone 14-3-3ε regulates a rig-I translocon that mediates membrane association and innate antiviral immunity. Cell Host & Microbe, 11(5), 528–537. https://doi.org/10.1016/j.chom.2012.04.006
