Vaccinia-Related Kinase 2 Mediates Accumulation of Polyglutamine Aggregates via Negative Regulation of the Chaperonin TRiC

Abstract

e Misfolding of proteins containing abnormal expansions of polyglutamine (polyQ) repeats is associated with cytotoxicity in several neurodegenerative disorders, including Huntington's disease. Recently, the eukaryotic chaperonin TRiC hetero-oligomeric complex has been shown to play an important role in protecting cells against the accumulation of misfolded polyQ protein aggregates. It is essential to elucidate how TRiC function is regulated to better understand the pathological mechanism of polyQ aggregation. Here, we propose that vaccinia-related kinase 2 (VRK2) is a critical enzyme that negatively regulates TRiC. In mammalian cells, overexpression of wild-type VRK2 decreased endogenous TRiC protein levels by promoting TRiC ubiquitination, but a VRK2 kinase-dead mutant did not. Interestingly, VRK2-mediated downregulation of TRiC increased aggregate formation of a polyQ-expanded huntingtin fragment. This effect was ameliorated by rescue of TRiC protein levels. Notably, small interference RNA-mediated knockdown of VRK2 enhanced TRiC protein stability and decreased polyQ aggregation. The VRK2-mediated reduction of TRiC protein levels was subsequent to the recruitment of COP1 E3 ligase. Among the members of the COP1 E3 ligase complex, VRK2 interacted with RBX1 and increased E3 ligase activity on TRiC in vitro. Taken together, these results demonstrate that VRK2 is crucial to regulate the ubiquitination-proteosomal degradation of TRiC, which controls folding of polyglutamine proteins involved in Huntington's disease.

Vaccinia-Related Kinase 2 Controls the Stability of the Eukaryotic Chaperonin TRiC/CCT by Inhibiting the Deubiquitinating Enzyme USP25

“…Protein extraction and preparation, glutathione S-transferase (GST) pulldown, and immunoblotting were performed as previously described (14).…”

“…To further examine the physiological role of USP25 on TRiC protein stability, we evaluated polyQ aggregation using the polyQ-expanded Htt fragment fused to GFP when USP25 protein levels were modulated. Growing evidences reveal that functional TRiC inhibits polyQ aggregation at early stages and alleviates soluble or insoluble toxic speciesmediated harmful effects (10,14). Notably, overexpression of either a specific TRiC subunit or recombinant CCT1 significantly suppresses polyQ aggregation and neuronal cell death (23,24).…”

“…Because increased CCT4 ubiquitination is dependent on VRK2 enzymatic activity, we sought to identify substrate proteins that function in the ubiquitination of chaperonin (14). To delineate the molecular mechanisms of TRiC protein degradation, we attempted to identify a novel binding partner of TRiC.…”

“…In our previous study, VRK2 increased TRiC protein degradation by promoting ubiquitination, and TRiC ubiquitination was dependent on VRK2 enzymatic activity (14). However, the molecular mechanism of VRK2 and its putative substrates have remained largely unexplored.…”

“…TRiC protein levels are elevated by inhibition of the degradation pathway. Although the novel vaccinia-related kinase 2 (VRK2) is involved in TRiC turnover, the molecular mechanisms of TRiC protein degradation remain unclear (14). It is, therefore, necessary to examine VRK2 function in TRiC protein degradation to provide better therapeutic insights into TRiC-assisted alleviation of polyQ toxicity.…”