Protein aggregation, which is associated with the impairment of the ubiquitin proteasome system, is a hallmark of many neurodegenerative diseases. To better understand the contribution of proteasome inhibition in aggregation, we analyzed which proteins may potentially localize in chemically induced aggregates in human neuroblastoma tissue culture cells. We enriched for proteins in high-density structures by using a sucrose gradient in combination with stable isotope labeling with amino acids in cell culture (SILAC). The quantitative analysis allowed us to distinguish which proteins were specifically affected by the proteasome inhibition. We identified 642 potentially aggregating proteins, including the p62/sequestosome 1 and NBR1 ubiquitin-binding proteins involved in aggregation. We also identified the ubiquitin-associated protein 2 like (UBAP2L). We verified that it cofractionated with ubiquitin in the high-density fraction and that it was colocalized in the ubiquitin-containing aggregates after proteasome inhibition. In addition, we identified several chaperone proteins and used data from protein interaction networks to show that they potentially interact with distinct subgroups of proteins within the aggregating structures. Several other proteins associated with neurodegenerative diseases, like UCHL1, were identified, further underlining the potential of our analysis to better understand the aggregation process and proteotoxic stress caused by proteasome inhibition.
BackgroundProtein aggregation is a hallmark of many neurodegenerative diseases and has been linked to the failure to degrade misfolded and damaged proteins. In the cell, aberrant proteins are degraded by the ubiquitin proteasome system that mainly targets short-lived proteins, or by the lysosomes that mostly clear long-lived and poorly soluble proteins. Both systems are interconnected and, in some instances, autophagy can redirect proteasome substrates to the lysosomes.Principal FindingsTo better understand the interplay between these two systems, we established a neuroblastoma cell population stably expressing the GFP-ubiquitin fusion protein. We show that inhibition of the proteasome leads to the formation of large ubiquitin-containing inclusions accompanied by lower solubility of the ubiquitin conjugates. Strikingly, the formation of the ubiquitin-containing aggregates does not require ectopic expression of disease-specific proteins. Moreover, formation of these focused inclusions caused by proteasome inhibition requires the lysine 63 (K63) of ubiquitin. We then assessed selected compounds that stimulate autophagy and found that the antihelmintic chemical niclosamide prevents large aggregate formation induced by proteasome inhibition, while the prototypical mTORC1 inhibitor rapamycin had no apparent effect. Niclosamide also precludes the accumulation of poly-ubiquitinated proteins and of p62 upon proteasome inhibition. Moreover, niclosamide induces a change in lysosome distribution in the cell that, in the absence of proteasome activity, may favor the uptake into lysosomes of ubiquitinated proteins before they form large aggregates.ConclusionsOur results indicate that proteasome inhibition provokes the formation of large ubiquitin containing aggregates in tissue culture cells, even in the absence of disease specific proteins. Furthermore our study suggests that the autophagy-inducing compound niclosamide may promote the selective clearance of ubiquitinated proteins in the absence of proteasome activity.
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