Abstract
The ubiquitin-proteasome system and the autophagy-lysosome system are two major intracellular proteolytic pathways in eukaryotes. Although several biochemical mechanisms underlying the crosstalk between them have been suggested, little is known about the effect of enhanced proteasome activity on autophagic flux. Here, we found that upregulation of proteasome activity, which was achieved through the inhibition of USP14, significantly impaired cellular autophagic flux, especially at the autophagosome-lysosome fusion step. UVRAG appeared to function as a crucial checkpoint for the proper progression of autophagic flux. Although proteasome activation through USP14 inhibition facilitated the clearance of microtubule-associated protein tau (MAPT) and reduced the amount of its oligomeric forms, the same conditions increased the formation of inclusion bodies from nonproteasomal substrates such as huntingtin with long polyglutamine repeats. Our results collectively indicate that USP14 may function as a common denominator in the compensatory negative feedback between the two major proteolytic processes in the cell.
Highlights
The ubiquitin (Ub)-proteasome system (UPS) and the autophagy-lysosome system are the two primary proteolytic mechanisms in eukaryotic cells and control the amounts of various regulatory proteins and damaged or misfolded proteins in a spatiotemporal manner
Inhibition of USP14 Perturbs Cellular Autophagic Flux To determine how the enhanced proteasome activity affects the flux of autophagy, we first knocked down USP14 through small interfering RNA; this protein is known to function as an endogenous inhibitor of proteasomal activity both catalytically and noncatalytically (Peth et al, 2009)
We found that a partial knockdown of USP14 led to the significant accumulation of specific autophagic substrate LC3-II in HEK293 cells (Figure 1A), indicating that USP14 depletion may affect autophagic flux
Summary
The ubiquitin (Ub)-proteasome system (UPS) and the autophagy-lysosome system (hereinafter referred to as autophagy) are the two primary proteolytic mechanisms in eukaryotic cells and control the amounts of various regulatory proteins and damaged or misfolded proteins in a spatiotemporal manner. Extensive evidence has shown that proteasome inhibition leads to compensatory induction of autophagy to restore cellular proteolytic functions (Ding et al, 2007; Milani et al, 2009; Zhu et al, 2010) Several mechanisms of this autophagic induction have been suggested, which include the accumulation of unfolded proteins in the endoplasmic reticulum (ER)—or ER stress—and the subsequent unfolded protein response (HøyerHansen and Jaa ̈ attela , 2007), p53 stabilization and PRKAAmediated mTOR inhibition (Vousden and Ryan, 2009), and HDAC6’s participation in the transport of proteasome substrates to the autophagy machinery (Pandey et al, 2007). Pharmacological and genetic inhibition of autophagy, results in more complex and disputable consequences Both downregulation of normal proteasomal degradation (because of excessively accumulated p62, known as SQSTM1) and upregulation of proteasomal activity have been observed (Korolchuk et al, 2009; Wang et al, 2013). To completely elucidate the general cross-talk between the UPS and autophagy, autophagic flux should be monitored after the activation of UPS flux, whose concept has only recently become valid due to the identification of inhibitory components of the proteasome function
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