Abstract
TRIM25 is a member of the tripartite motif family of E3 ligases. This family of proteins regulate many cellular processes, including development, cell growth, differentiation, cancer, and innate immune response. TRIM25 is best characterized as an important factor for anti-viral innate immunity, but also functions in diverse RNA-dependent pathways. Previous TRIM25 studies have identified cellular substrates and touched on its E3 ligase activity, but provided little insight into the requirements for TRIM25 catalytic activation. By elucidating the mechanisms involved in TRIM25 anti-viral activation, we can provide a fundamental understanding of TRIM25-mediated processes. This work employs structural, biophysical, and biochemical techniques complemented with cell biology to analyze TRIM25’s tertiary and quaternary structures, as well as essential factors for TRIM25 mediated anti-viral activity.
The cellular PRRs RIG-I and ZAP independently modulate an effective anti-viral innate immune response through induction of IFN signaling and translational inhibition of viral proteins, respectively. The E3 ligase TRIM25 enhances both RIG-I and ZAP anti-viral activities through polyubiquitination. In order for ubiquitin synthesis to occur, TRIM25 must be catalytically activated by substrate-induced higher-order oligomerization.
The TRIM25 coiled-coil domain is the initial site of oligomerization. Structural studies reveal that the coiled-coil subunits are antiparallel in orientation and composed of heptad-hendecad-heptad repeats of hydrophobic residues. This pattern is maintained within other TRIMs as well. Many RING E3 ligases require dimerization for catalytic activity. However, the central antiparallel coiled-coil dictates that the catalytic RING domains remain sequestered at opposite ends of the dimer.
Further structural and biochemical studies revealed that the RING domain is a second site for TRIM25 oligomerization. Purified RING protein is monomeric in solution, as demonstrated with analytical ultracentrifugation. A structure of the RING domain in complex with a ubiquitin-conjugated E2 protein demonstrated that the interaction requires RING dimerization. Mutations designed to disrupt RING dimerization reduced TRIM25 E3 ligase activity and anti-viral activity in vitro. Furthermore, purified full-length TRIM25 forms a tetrameric species in solution and the introduction of either L69A or V72A result in only dimeric TRIM25.
Full-length TRIM25 co-purifies with nucleic acids. Biochemical studies revealed that RNA enhances TRIM25 E3 ligase activity. A mutational analysis identified a cooperative nucleic acid-binding mechanism within TRIM25. Lys and Arg residues within the coiled-coil, Linker 2 region, and SPRY domain coordinate nucleic acid binding as well as grant binding specificity to RNA over DNA. Moreover, a reduction in RNA binding affinity correlated with a reduction of TRIM25-mediated anti-viral activity.
Overall, this thesis provides a biochemical and structural basis for understanding the mechanisms of TRIM25 catalytic activation, and how this modulates an RNA-dependent cellular anti-viral response.
