Abstract
Proteolytic systems and the aggresome pathway contribute to preventing accumulation of cytotoxic aggregation-prone proteins. Although polyubiquitylation is usually required for degradation or aggresome formation, several substrates are processed independently of ubiquitin through a poorly understood mechanism. Here, we found that p62/SQSTM1, a multifunctional adaptor protein, was involved in the selective autophagic clearance of a non-ubiquitylated substrate, namely an aggregation-prone isoform of STAT5A (STAT5A_ΔE18). By using a cell line that stably expressed STAT5A_ΔE18, we investigated the properties of its aggregation and degradation. We found that STAT5A_ΔE18 formed non-ubiquitylated aggresomes and/or aggregates by impairment of proteasome functioning or autophagy. Transport of these aggregates to the perinuclear region was inhibited by trichostatin A or tubacin, inhibitors of histone deacetylase (HDAC), indicating that the non-ubiquitylated aggregates of STAT5A_ΔE18 were sequestered into aggresomes in an HDAC6-dependent manner. Moreover, p62 was bound to STAT5A_ΔE18 through its PB1 domain, and the oligomerization of p62 was required for this interaction. In p62-knockdown experiments, we found that p62 was required for autophagic clearance of STAT5A_ΔE18 but not for its aggregate formation, suggesting that the binding of p62 to non-ubiquitylated substrates might trigger their autophagic clearance.
Highlights
Misfolded or aggregated proteins are usually refolded with molecular chaperones or degraded by the proteolytic machinery consisting of two main systems, designated the ubiquitin– proteasome system (UPS) and the autophagy–lysosome pathway (Goldberg, 2003)
To understand the properties of the resulting aggregates, we verified whether this abnormal protein was degraded by proteolytic systems, such as the UPS or the autophagy–lysosome pathway, using a HeLa cell line stably expressing STAT5A_E18–EGFP at a very low level (Fig. 1A)
When HeLa cells stably expressing STAT5A_⌬E18–EGFP were transfected with short interfering RNAs against ATG5 or ATG7, starvation-induced autophagy was inhibited in both cell types (Fig. 2A,B)
Summary
Misfolded or aggregated proteins are usually refolded with molecular chaperones or degraded by the proteolytic machinery consisting of two main systems, designated the ubiquitin– proteasome system (UPS) and the autophagy–lysosome pathway (Goldberg, 2003). The formation of aggregates and inclusion bodies in the brain is a pathognomonic feature of many neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease and Huntington’s disease, suggesting that dysfunctions of the UPS and the autophagy–lysosome pathway are involved in these diseases (Ross and Poirier, 2005; Rubinsztein, 2006) Impairment of these proteolytic systems causes neurodegeneration. It was recently reported that parkin, which is the causal gene of familial Parkinson’s disease and an E3 ubiquitin ligase, is selectively recruited to damaged or uncoupled mitochondria for clearance by the autophagy–lysosome pathway (Narendra et al, 2008; Vives-Bauza et al, 2010) These proteolytic systems are very important for quality control of proteins or organelles, some proteins that escape degradation often accumulate in the cytoplasm. This pathway involves histone deacetylase (HDAC) 6-mediated transport, which recruits the aggregates to dynein motors for transport to aggresomes located in close proximity to the microtubule-organizing center (MTOC) (Kawaguchi et al, 2003)
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