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Ring finger protein 34 (RNF34) interacts with and promotes ?-aminobutyric acid type-A receptor degradation via ubiquitination of the ?2 subunit.

ABSTRACT: We have found that the large intracellular loop of the ?2 GABAA receptor (R) subunit (?2IL) interacts with RNF34 (an E3 ubiquitin ligase), as shown by yeast two-hybrid and in vitro pulldown assays. In brain extracts, RNF34 co-immunoprecipitates with assembled GABAARs. In co-transfected HEK293 cells, RNF34 reduces the expression of the ?2 GABAAR subunit by increasing the ratio of ubiquitinated/nonubiquitinated ?2. Mutating several lysines of the ?2IL into arginines makes the ?2 subunit resistant to RNF34-induced degradation. RNF34 also reduces the expression of the ?2 subunit when ?1 and ?3 subunits are co-assembled with ?2. This effect is partially reversed by leupeptin or MG132, indicating that both the lysosomal and proteasomal degradation pathways are involved. Immunofluorescence of cultured hippocampal neurons shows that RNF34 forms clusters and that a subset of these clusters is associated with GABAergic synapses. This association is also observed in the intact rat brain by electron microscopy immunocytochemistry. RNF34 is not expressed until the 2nd postnatal week of rat brain development, being highly expressed in some interneurons. Overexpression of RNF34 in hippocampal neurons decreases the density of ?2 GABAAR clusters and the number of GABAergic contacts that these neurons receive. Knocking down endogenous RNF34 with shRNA leads to increased ?2 GABAAR cluster density and GABAergic innervation. The results indicate that RNF34 regulates postsynaptic ?2-GABAAR clustering and GABAergic synaptic innervation by interacting with and ubiquitinating the ?2-GABAAR subunit promoting GABAAR degradation.

SUBMITTER: Jin H

PROVIDER: S-EPMC4200290 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Ring finger protein 34 (RNF34) interacts with and promotes γ-aminobutyric acid type-A receptor degradation via ubiquitination of the γ2 subunit.

Jin Hongbing H Chiou Tzu-Ting TT Serwanski David R DR Miralles Celia P CP Pinal Noelia N De Blas Angel L AL

The Journal of biological chemistry 20140905 42

We have found that the large intracellular loop of the γ2 GABAA receptor (R) subunit (γ2IL) interacts with RNF34 (an E3 ubiquitin ligase), as shown by yeast two-hybrid and in vitro pulldown assays. In brain extracts, RNF34 co-immunoprecipitates with assembled GABAARs. In co-transfected HEK293 cells, RNF34 reduces the expression of the γ2 GABAAR subunit by increasing the ratio of ubiquitinated/nonubiquitinated γ2. Mutating several lysines of the γ2IL into arginines makes the γ2 subunit resistant ...[more]

PMID: 25193658

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Project description:Ubiquitination, as one of the most prevalent posttranslational modifications of proteins, enables a tight control of host immune responses. Many viruses hijack the host ubiquitin system to regulate host antiviral responses for their survival. Here, we found that the fish pathogen nervous necrosis virus (NNV) recruited Lateolabrax japonicus E3 ubiquitin ligase ring finger protein 34 (LjRNF34) to inhibit the RIG-I-like receptor (RLR)-mediated interferon (IFN) response via ubiquitinating Lateolabrax japonicus TANK-binding kinase 1 (LjTBK1) and interferon regulatory factor 3 (LjIRF3). Ectopic expression of LjRNF34 greatly enhanced NNV replication and prevented IFN production, while deficiency of LjRNF34 led to the opposite effect. Furthermore, LjRNF34 targeted LjTBK1 and LjIRF3 via its RING domain. Of note, the interactions between LjRNF34 and LjTBK1 or LjIRF3 were conserved in different cellular models derived from fish. Mechanically, LjRNF34 promoted K27- and K48-linked ubiquitination and degradation of LjTBK1 and LjIRF3, which in turn diminished LjTBK1-induced translocation of LjIRF3 from the cytoplasm to the nucleus. Ultimately, NNV capsid protein (CP) was found to bind with LjRNF34, CP induced LjTBK1 and LjIRF3 degradation, and IFN suppression depended on LjRNF34. Our finding demonstrates a novel mechanism by which NNV CP evaded host innate immunity via LjRNF34 and provides a potential drug target for the control of NNV infection. IMPORTANCE Ubiquitination plays an essential role in the regulation of innate immune responses to pathogens. NNV, a type of RNA virus, is the causal agent of a highly destructive disease in a variety of marine and freshwater fish. A previous study reported NNV could hijack the ubiquitin system to manipulate the host's immune responses; however, how NNV utilizes ubiquitination to facilitate its own replication is not well understood. Here, we identified a novel distinct role of E3 ubiquitin ligase LjRNF34 as an IFN antagonist to promote NNV infection. NNV capsid protein utilized LjRNF34 to target LjTBK1 and LjIRF3 for K27- and K48-linked ubiquitination and degradation. Importantly, the interactions between LjRNF34 and CP, LjTBK1, or LjIRF3 are conserved in different cellular models derived from fish, suggesting it is a general immune evasion strategy exploited by NNV to target the IFN response via RNF34.

Dataset Information

Ring finger protein 34 (RNF34) interacts with and promotes ?-aminobutyric acid type-A receptor degradation via ubiquitination of the ?2 subunit.

Publications

Ring finger protein 34 (RNF34) interacts with and promotes γ-aminobutyric acid type-A receptor degradation via ubiquitination of the γ2 subunit.

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