Defective Proteasome Delivery of Polyubiquitinated Proteins by Ubiquilin-2 Proteins Containing ALS Mutations

Abstract

Ubiquilin proteins facilitate delivery of ubiquitinated proteins to the proteasome for degradation. Interest in the proteins has been heightened by the discovery that gene mutations in UBQLN2 cause dominant inheritance of amyotrophic lateral sclerosis (ALS). However, the mechanisms by which the mutations cause ALS are not known. Here we report on the underlying defect of ubiquilin-2 proteins containing ALS-linked mutations in affecting proteasome-mediated degradation. We found that overexpression of ubiquilin-2 proteins containing any one of five different ALS mutations slow degradation of Myc, a prototypic proteasome substrate. Examination of coprecipitating proteins indicated that the mutant proteins are generally capable of binding polyubiquitinated proteins, but defective in binding the proteasome. GST-pulldown studies revealed that many of the mutants bind weaker to the S5a subunit of the proteasome, compared with wild type (WT) ubiquilin-2 protein. The results suggest the mutant proteins are unable to deliver their captured cargo to the proteasome for degradation, which presumably leads to toxicity. Quantification of cell death is consistent with this idea. Measurement of protein turnover further indicated the mutant proteins have longer half-lives than WT ubiquilin-2. Our studies provide novel insight into the mechanism by which ALS-linked mutations in UBQLN2 interfere with protein degradation.

Motor neuron disease, TDP-43 pathology, and memory deficits in mice expressing ALS–FTD-linked UBQLN2 mutations

“…We speculate that this susceptibility is because they are vulnerable to disturbances in proteostasis, based on the defective properties of mutant UBQLN2 proteins and by extrapolation from other studies. First, our results, like those of others studies, clearly show the UBQLN2 mutants disrupt proteostasis, leading to accumulation of misfolded proteins (13,(28)(29)(30)(31). Second, accumulating evidence suggests disturbance in proteostasis, particularly induction of the UPR, as central in disease pathogenesis caused by ALS mutations in SOD1 and C9ORF72 (45)(46)(47)(48)(49).…”

“…Interestingly, overexpression of WT UBQLN2 was recently shown to induce neurodegenerative phenotypes and pathology in the brain similar to the expression of mutant P497H UBQLN2 protein (40). The capacity of WT hUBQLN2 to induce toxicity when highly overexpressed is consistent with an increase in cell death seen upon overexpression of the protein in cell cultures, although like our animal studies, there too, toxicity of the WT protein was less potent than ALS-FTD mutant UBQLN2 proteins (30,41). The toxicity caused by overexpression of WT UBQLN2 is not entirely surprising because overexpression of WT SOD1, TDP43, and FUS, all of which are encoded by genes in which mutations cause ALS, generate disease phenotypes (42)(43)(44).…”

“…Previous studies have shown that mtUBQLN2 proteins interfere with proteasomal degradation by retaining their ability to bind polyubiquitinated proteins, but inability to deliver the bound proteins to the proteasome for degradation from a failure to dock with the proteasome (30,32). Studies also suggest the mtUBQLN2 proteins interfere with autophagy (32).…”

“…Indeed, overexpression of the mutant UBQLN2 (mtUBQLN2) proteins in cells and animals support both possibilities. For example, overexpression of ALS mtUBQLN2 proteins caused interference in proteasomal degradation, including perturbation of endoplasmic reticulum-associated degradation (ERAD) (13,(28)(29)(30)(31). The interference in degradation appears to stem from an inability of the mutants to deliver polyubiquitinated proteins to the proteasome for degradation (30,32).…”

Differential Contributions of Ubiquitin-Modified APOBEC3G Lysine Residues to HIV-1 Vif-Induced Degradation

“…Work by Zhao et al suggests that proteasomal degradation requires multifaceted coordination between deubiquitinases, substrate unfolding, ATP hydrolysis and interactions between proteins for substrate recognition [42]. The conformational structure of modified proteins has been proposed to prevent the substrate from tethering to the 19S regulatory subunit of the proteasome or from entering into the 20S catalytic core [43]. Alternatively, the translocation of substrates to the proteasome may be impeded.…”