Palmitoylation of MIR2 is required for its function
Kaposi’s sarcoma-associated herpesvirus (KSHV) produces two RING finger E3 ubiquitin ligases, MIR1 and MIR2, which facilitate the ubiquitination and degradation of cellular proteins critical for an effective antiviral immune response. While MIR1 and MIR2 share 30% sequence identity, their substrate preferences differ significantly. MIR1 primarily targets major histocompatibility complex class I (MHC-I) for downregulation, whereas MIR2 can downregulate a broad array of cell surface proteins. Many of these substrates are found in lipid raft microdomains, specialized regions of the plasma membrane crucial for various signaling pathways. Palmitoylation, a posttranslational modification, enhances the recruitment of transmembrane proteins to these lipid rafts.
In this study, we explored the role of palmitoylation in MIR function. Our results demonstrate that palmitoylation is essential for MIR2’s ability to downregulate MHC-I and platelet endothelial cell adhesion molecule 1 (PECAM-1), but not for other substrates. The drug 2-bromohexadecanoic acid (2-Br), a palmitoylation inhibitor, obstructs this downregulation. Biochemical analyses reveal that MIR2 is directly palmitoylated at cysteine 146. Mutating this cysteine to phenylalanine prevents MIR2 from undergoing palmitoylation, thereby inhibiting its ability to downregulate MHC-I and PECAM-1, while leaving other substrates like B7.2 and intercellular adhesion molecule 1 (ICAM-1) unaffected. Unpalmitoylated MIR2 fails to interact with MHC-I, thereby impeding its ability to ubiquitinate and remove MHC-I from the cell surface. Additionally, we observed that MIR2 is palmitoylated in vivo during lytic infection. This suggests that palmitoylation may regulate MIR2’s function and localization during viral infection by enabling it to effectively interact with and downregulate multiple substrates involved in the host immune response.