The ubiquitin ligase F-box/G-domain protein 1 promotes the degradation of the disease-linked protein torsinA through the ubiquitin–proteasome pathway and macroautophagy

Abstract

DYT1 dystonia is a dominantly inherited, disabling neurological disorder with low penetrance that is caused by the deletion of a glutamic acid (ΔE) in the protein torsinA. We previously showed that torsinA(wt) is degraded through macroautophagy while torsinA(ΔE) is targeted to the ubiquitin proteasome pathway (UPP). The different catabolism of torsinA(wt) and (ΔE) potentially modulates torsinA(wt):torsinA(ΔE) stoichiometry. Therefore, gaining a mechanistic understanding on how the protein quality control machinery clears torsinA(ΔE) in neurons may uncover important regulatory steps in disease pathogenesis. Here, we asked whether FBG1, a ubiquitin ligase known to degrade neuronal glycoproteins, is implicated in the degradation of torsinA(ΔE) by the UPP. In a first set of studies completed in cultured cells, we show that FBG1 interacts with and influences the steady-state levels of torsinA(wt) and (ΔE). Interestingly, FBG1 achieves this effect promoting the degradation of torsinA not only through the UPP, but also by macroautophagy. To determine the potential clinical significance of these findings, we asked if eliminating expression of Fbg1 triggers a motor phenotype in torsinA(ΔE) knock in mice, a model of non-manifesting DYT1 mutation carriers. We detected differences in spontaneous locomotion between aged torsinA(ΔE) knock in-Fbg1 knock out and control mice. Furthermore, neuronal levels of torsinA were unaltered in Fbg1 null mice, indicating that redundant systems likely compensate in vivo for the absence of this ubiquitin ligase. In summary, our studies support a non-essential role for FBG1 on the degradation of torsinA and uncover a novel link of FBG1 to the autophagy pathway.

The BiP Molecular Chaperone Plays Multiple Roles during the Biogenesis of TorsinA, an AAA+ ATPase Associated with the Neurological Disease Early-onset Torsion Dystonia

“…3 and 6). In addition, we obtained preliminary evidence that the proteins are ubiquitinated (data not shown), which is in agreement with a recent study showing that the E3 ubiquitin ligase, FBG1, is involved in the degradation of torsinA and torsinA⌬E (137). We also found that Kar2/BiP plays a pro-degradative role when the stability of torsinA and torsinA⌬E is compromised (Figs.…”

FBG1 Is the Final Arbitrator of A1AT-Z Degradation

“…We asked if the ability of FBG1 to decrease A1AT-Z levels depended on its ubiquitin ligase function. To demonstrate whether the ubiquitin ligase activity of FBG1 is required to degrade A1AT-Z, we co-transfected A1AT-Z with pCMV, FLAG-tagged full-length FBG1 (FBG1), or a FLAG-tagged N-terminal deletion of FBG1 (FBG1ΔN), which deletes the SKP1 binding site, necessary for ubiquitination [ 25 ]. After 48 hours, cells were harvested and separated on SDS-PAGE stain-free gels ( Fig 6A ).…”

“…Furthermore, inhibition of the proteasome in this Atg5-deficient cells failed to increase A1AT-Z levels further indicating that the ALS pathway was the primary means of degradation of A1AT-Z [ 16 ]. Since past research has shown that FBG1 can function through both the UPP and ALS to clear misfolded proteins [ 25 ], we investigated whether FBG1 could degrade A1AT-Z through autophagy.…”

“…Mission shRNA (SHCLNV-NM_012168, Sigma) and control vector were obtained as lentivirus particles and transduced as previously described [ 25 ]. Briefly, 5μg of lentivirus particle were added to HepG2 cells with 8μg/ml hexadimethrine bromide and media was replaced after 18 hours [ 25 ]. Two days after transduction HepG2 cells were split and seeded at 25% confluence.…”

Transcriptome profiles in brains of mice heterozygous for a DYT1 dystonia-associated mutation in the endogenous Tor1a gene

“…The third point is the nature of the pathways and individual genes that are affected in Tor1a +/ΔE mice. Several of these had been implicated previously, for example, the eukaryotic translation initiation (Beauvais et al, 2018;Beauvais et al, 2019;Rittiner et al, 2016), protein ubiquitination pathway (Gordon et al, 2012;Nery et al, 2011) and cell cycle (Ledoux et al, 2013). Others were of particular interest, such as the mitochondrial function, intracellular trafficking and axonal guidance: they are involved in regulating neuronal functions including excitability.…”