Abstract
Protein clearing pathways named autophagy (ATG) and ubiquitin proteasome (UP) control homeostasis within eukaryotic cells, while their dysfunction produces neurodegeneration. These pathways are viewed as distinct biochemical cascades occurring within specific cytosolic compartments owing pathway-specific enzymatic activity. Recent data strongly challenged the concept of two morphologically distinct and functionally segregated compartments. In fact, preliminary evidence suggests the convergence of these pathways to form a novel organelle named autophagoproteasome. This is characterized in the present study by using a cell line where, mTOR activity is upregulated and autophagy is suppressed. This was reversed dose-dependently by administering the mTOR inhibitor rapamycin. Thus, we could study autophagoproteasomes when autophagy was either suppressed or stimulated. The occurrence of autophagoproteasome was shown also in non-human cell lines. Ultrastructural morphometry, based on the stochiometric binding of immunogold particles allowed the quantitative evaluation of ATG and UP component within autophagoproteasomes. The number of autophagoproteasomes increases following mTOR inhibition. Similarly, mTOR inhibition produces overexpression of both LC3 and P20S particles. This is confirmed by the fact that the ratio of free vs. autophagosome-bound LC3 is similar to that measured for P20S, both in baseline conditions and following mTOR inhibition. Remarkably, within autophagoproteasomes there is a slight prevalence of ATG compared with UP components for low rapamycin doses, whereas for higher rapamycin doses UP increases more than ATG. While LC3 is widely present within cytosol, UP is strongly polarized within autophagoproteasomes. These fine details were evident at electron microscopy but could not be deciphered by using confocal microscopy. Despite its morphological novelty autophagoproteasomes appear in the natural site where clearing pathways (once believed to be anatomically segregated) co-exist and they are likely to interact at molecular level. In fact, LC3 and P20S co-immunoprecipitate, suggesting a specific binding and functional interplay, which may be altered by inhibiting mTOR. In summary, ATG and UP often represent two facets of a single organelle, in which unexpected amount of enzymatic activity should be available. Thus, autophagoproteasome may represent a sophisticated ultimate clearing apparatus.
Highlights
We described that clearing systems such as the Ubiquitine Proteasome (UP) and Autophagy (ATG) may occur within single morphological entities we named autophagoproteasome (Isidoro et al, 2009; Pasquali et al, 2009; Ferrucci et al, 2011; Natale et al, 2011; Klionsky et al, 2016)
Plain Transmission Electron Microscopy of Autophagy-Like Vacuoles The typical ultrastructure of U87MG cells in baseline conditions is represented in Figure 1 showing depressed autophagy activity as expected from mTOR up-regulation, which occurs in this cell line, which in turn produces only a few ATG-like vacuoles
Such an increase was dose-dependent, to that measured for ATG-like vacuoles for the highest dose of rapamycin (185.68 ± 6.06 compared with 38.54 ± 2.40 for baseline conditions, Figure 2A)
Summary
We described that clearing systems such as the Ubiquitine Proteasome (UP) and Autophagy (ATG) may occur within single morphological entities we named autophagoproteasome (Isidoro et al, 2009; Pasquali et al, 2009; Ferrucci et al, 2011; Natale et al, 2011; Klionsky et al, 2016). When writing the autophagy glossary in the guidelines we contributed to the hypothesis that ‘‘The autophagoproteasome may be derived from the inclusion of ubiquitin-proteasome structures within either early or late autophagosomes containing cytoplasmic material at various stages of degradation’’ (Klionsky et al, 2016) This was based on recent unpublished data. Despite we coined the term autophagoproteasome in the last decade (Pasquali et al, 2009), no specific study was designed to characterize in depth such an organelle This is crucial since biomedical research is producing increasing evidence on the seminal role played by all clearing pathways in modulating cell survival and disease mechanisms. ATG and/or UP are involved in a wide range of disorders encompassing tumors (Liang et al, 1999; Qu et al, 2003; Mani and Gelmann, 2005; Bazzaro et al, 2006; Takamura et al, 2011; Huang and Dixit, 2016; Liu and Debnath, 2016), cardiovascular diseases (Nakai et al, 2007; Willis and Patterson, 2013; Wang and Robbins, 2014; Willis et al, 2014; Delbridge et al, 2015), and neurodegenerative disorders (Nedelsky et al, 2008; Bedford et al, 2009; Chu et al, 2009; Madeo et al, 2009; Dantuma and Bott, 2014; Menzies et al, 2015)
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