BAG3-mediated rerouting of proteasomal clients towards autophagy upon proteasomal impairment

The accumulation of misfolded, mutant proteins is a common basis for many adult onset neurodegenerative diseases. Cells have evolved an elaborate protein quality control system, which acts to facilitate the folding or refolding of misfolded protein species by molecular chaperones. When folding is unsuccessful, chaperones can also target the misfolded proteins for degradation, thereby preventing protein aggregation. Intracellular degradation is primarily mediated by two proteolytic systems: the ubiquitin proteasomal systems and the autophagy. Proteasomal degradation has high selectivity for short-lived proteins and only ubiquitinated substrates are recognized. Macroautophagy is responsible mainly for the degradation of long-lived proteins, protein aggregates and entire (damaged) organelles. Macroautophagy was initially perceived as an ‘‘in bulk’’ process. Nevertheless, macroautophagy also sense for degradation of misfolded, aggregated and/or ubiquitinated proteins. Thus autophagy acts as compensatory system for proteasomal degradation, when the latter is impaired. Numerous studies have shown that changes in both the ubiquitin proteasomal system and the autophagy-lysosome system occur with age. This suggests that a proper balance between these protein quality-control systems is required for protein homeostasis, and that its alteration may contribute to ageing and disease. However, little is known about mechanisms controlling autophagic degradation of ubiquitinated and/or damaged substrates nor that of their re-routing from proteasomal to autophagosomal degradation (the proteasome-autophagy switch). Recent studies implicated several members of the BAG (Bcl-2-associated athanogene) protein family in cellular protein quality control. BAG1 has been suggested to bridge HSP70-bound clients to the proteasome through its ubiquitin-like domain. BAG3 was found to stimulate the selective degradation of several disease-associated proteins such as polyQ huntingtin and superoxide dismutase-1 (SOD1) by the autophagic machinery. To do so, BAG3 cooperates with HSP70s and/or HSPB8, a member of the family of small heat shock proteins and with the macroautophagy receptor protein p62/SQSTM1. Interestingly, during ageing, a switch in the expression of BAG1 and BAG3 seems to occur: whilst BAG1 is expressed at relatively higher levels in young tissue, BAG3 is expressed at relatively higher levels in aged tissue and this has been correlated to a higher proteasomal activity in young tissues and a more intensive use of the autophagic system in aged tissue. These correlative data suggested that the BAG3/BAG1 expression ratio may play a crucial role in the balance between proteasomal and autophagosomal degradation. But, direct evidence for and insights in the precise mechanism of this hypothesis is yet lacking. Here, we found in our cellular model (HEK393T) that treatment with the proteasome inhibitors leads to an upregulation of BAG3 and HSPB8. Moreover, using different approaches, we found that overexpression of BAG3 (and/or HSPB8) increases the total amount of polyubiquitinated proteins but it does not lead to a direct inhibition of the proteasome. Full length BAG3 can pull-down polyubiquitinated clients and its binding to HSC70/HSP70 is required for the interaction with these polyubiquitinated proteins. BAG3, via its interaction with HSC70/HSP70, competes in a concentration dependent manner with BAG1 to bind and to process polyubiquitinated proteins in order to re-route them from the proteasome to the autophagy for degradation. All these data suggest that after proteasome inhibition, BAG-3 is upregulated and is a major executor of the proteasome-autophagy switch. Bibliografia: (Feldman and Frydman 2000); (Goldberg 2003); (Hartl 2009); (Ellgaard 2003); (Meusser 2005); (Rubinsztein, 2006); (Yang and Klionsky 2010); (Ding and Yin, 2008); (Gamerdinger 2011b); (Arndt 2005); Luders 2000); (Gamerdinger 2009); (Kalia 2004); (Luders 2000); (Crippa 2010); (Carra 2008a); (Carra 2008b); (Gamerdinger 2011a); (Hua-Qin Wang 2008); (Du ZX 2009); (Ward 2002); (Martinez-Vicente 2005); (Powers 2009).