MKRN1 Induces Degradation of West Nile Virus Capsid Protein by Functioning as an E3 Ligase

Abstract

West Nile virus capsid protein (WNVCp) displays pathogenic toxicity via the apoptotic pathway. However, a cellular mechanism protective against this toxic effect has not been observed so far. Here, we identified Makorin ring finger protein 1 (MKRN1) as a novel E3 ubiquitin ligase for WNVCp. The cytotoxic effects of WNVCp as well as its expression levels were inhibited in U2OS cells that stably expressed MKRN1. Immunoprecipitation analyses revealed an interaction between MKRN1 and WNVCp. Domain analysis indicated that the C terminus of MKRN1 and the N terminus of WNVCp were required for the interaction. MKRN1 could induce WNVCp ubiquitination and degradation in a proteasome-dependent manner. Interestingly, the WNVCp mutant with amino acids 1 to 105 deleted WNVCp was degraded by MKRN1, whereas the mutant with amino acids 1 to 90 deleted was not. When three lysine sites at positions 101, 103, and 104 of WNVCp were replaced with alanine, MKRN1-mediated ubiquitination and degradation of the mutant were significantly inhibited, suggesting that these sites are required for the ubiquitination. Finally, U2OS cell lines stably expressing MKRN1 were resistant to cytotoxic effects of WNV. In contrast, cells depleted of MKRN1 were more susceptible to WNVCp cytotoxicity. Confirming this, overexpression of MKRN1 significantly reduced, but depletion of MKRN1 increased, WNV proliferation in 293T cells. Taken together, our results suggest that MKRN1 can protect cells from WNV by inducing WNVCp degradation.

Uncoupling cis -Acting RNA Elements from Coding Sequences Revealed a Requirement of the N-Terminal Region of Dengue Virus Capsid Protein in Virus Particle Formation

“…It has been reported previously that the C protein of flaviviruses is involved in the induction of apoptosis and the breakdown of cellular tight junctions (26,37,44). A growing number of host proteins that interact with flavivirus C proteins have been reported (4,5,18,28). For instance, by use of twohybrid systems, several cellular proteins that interact with WNV C have been identified (29,42).…”

LEP-2/Makorin regulates LIN-28 stability to promote the juvenile-to-adult transition in Caenorhabditis elegans

“…Like other Mkrns, LEP-2 has a RING domain that is typical of many E3 ligases (Bohne et al, 2010;Gray et al, 2000). Indeed, Mkrn1 in mammals functions as an E3 ligase, targeting several proteins and even itself, although has not yet been identified as one of its targets (Kim et al, 2005;Salvatico et al, 2010;Lee et al, 2009Lee et al, , 2012Ko et al, 2012;Shimada et al, 2009;Ko et al, 2010;Kim et al, 2014;Cassar et al, 2015). In other contexts, Mkrns are also RNA-binding proteins, are localized to ribonucleoprotein granules, and have been found to sequester some mRNAs or promote their translation (Cano et al, 2010;Cassar et al, 2015;Gajdos et al, 2010;Kwon et al, 2013;Miroci et al, 2012;Cheung et al, 2010;Yang et al, 2008).…”

Ubiquitination and degradation of the FADD adaptor protein regulate death receptor-mediated apoptosis and necroptosis

“…As MKRN1 is an E3 ubiquitin ligase 24,26,27 , we next asked whether MKRN1 E3 ligase activity affected the stability of proteins involved in DISC complexes. When MKRN1 was knocked-down by siRNA or short-hairpin RNA (shRNA), MKRN1 protein was barely detected and MKRN1 messenger RNA levels were reduced up to 70-80% of control mRNAs (Fig.…”

“…4e). We next investigated the role of the E3 ligase activity of MKRN1 on FADD ubiquitination using the enzymatically inactive H307E MKRN1 mutant 24,26,27 . The H307E mutant failed to induce FADD ubiquitination despite its ability to bind to FADD ( Fig.…”

“…MKRN1, a protein highly conserved from invertebrates to vertebrates, has a ubiquitous expression pattern in various tissues 25 and functions as an E3 ubiquitin ligase for various substrates 24,26,27 . Previous reports showed that MKRN1 was able to suppress p53 function, and regulate cell growth and apoptosis.…”