E6-AP Promotes Misfolded Polyglutamine Proteins for Proteasomal Degradation and Suppresses Polyglutamine Protein Aggregation and Toxicity

Abstract

The accumulation of intracellular protein deposits as inclusion bodies is the common pathological hallmark of most agerelated neurodegenerative disorders including polyglutamine diseases. Appearance of aggregates of the misfolded mutant disease proteins suggest that cells are unable to efficiently degrade them, and failure of clearance leads to the severe disturbances of the cellular quality control system. Recently, the quality control ubiquitin ligase CHIP has been shown to suppress the polyglutamine protein aggregation and toxicity. Here we have identified another ubiquitin ligase, called E6-AP, which is able to promote the proteasomal degradation of misfolded polyglutamine proteins and suppress the polyglutamine protein aggregation and polyglutamine protein-induced cell death. E6-AP interacts with the soluble misfolded polyglutamine protein and associates with their aggregates in both cellular and transgenic mouse models. Partial knockdown of E6-AP enhances the rate of aggregate formation and cell death mediated by the polyglutamine protein. Finally, we have demonstrated the up-regulation of E6-AP in the expanded polyglutamine protein-expressing cells as well as cells exposed to proteasomal stress. These findings suggest that E6-AP is a critical mediator of the neuronal response to misfolded polyglutamine proteins and represents a potential therapeutic target in the polyglutamine diseases.

Identification and Proteomic Analysis of Distinct UBE3A/E6AP Protein Complexes

“…HUL5 has recently been shown to play a major role in the ubiquitylation and turnover of cytosolic misfolded proteins as part of the heat shock stress response, whereas in unstressed cells it is involved in the ubiquitylation of low-solubility proteins but without affecting the overall ubiquitylation levels in the cells (25). Since E6AP has also been associated with the degradation of misfolded proteins (20,63,64), perhaps a contribution of E6AP to the proteasome function becomes more significant under conditions that produce proteotoxic stress. Another possibility is that E6AP might affect the subcellular distribution of associated proteasomes, perhaps in a manner similar to that of CaMKII␣, which can act as a scaffold in neurons to recruit proteasomes to dendritic spines (8).…”

Distinguishing aggregate formation and aggregate clearance using cell-based assays

“…First, we introduce a novel method by which we can distinguish how a given pathway or modifier affects protein accumulation. Different degradation pathways have been implicated in affecting the accumulation of aggregation-prone proteins, from proteasome-mediated degradation (Mishra et al, 2008;Iwata et al, 2009;Zhang et al, 2010;Urushitani et al, 2010;Ortega and Lucas, 2014;Schipper-Krom et al, 2014) to the lysosome-mediated pathway or macroautophagy as being the predominant pathway for aggregate clearance (Ravikumar et al, 2002;Iwata et al, 2005b;Yamamoto et al, 2006;Pankiv et al, 2007;Lamark et al, 2009;Fan et al, 2010;Lamark and Johansen, 2012;Odagiri et al, 2012;Castillo et al, 2014;Bae et al, 2014). Our ability to interrogate how each pathway might contribute to aggregate turnover has been limited by our inability to temporally characterize aggregate formation and clearance in cells.…”

Malin Regulates Wnt Signaling Pathway through Degradation of Dishevelled2

“…as described earlier (35). Bound proteins were eluted from the beads with 1ϫ SDS sample buffer, vortexed, boiled for 6 min, and analyzed by immunoblotting.…”