HspB8 Chaperone Activity toward Poly(Q)-containing Proteins Depends on Its Association with Bag3, a Stimulator of Macroautophagy

Abstract

Mutations in HspB8, a member of the B group of heat shock proteins (Hsp), have been associated with human neuromuscular disorders. However, the exact function of HspB8 is not yet clear. We previously demonstrated that overexpression of HspB8 in cultured cells prevents the accumulation of aggregation-prone proteins such as the polyglutamine protein Htt43Q. Here we report that HspB8 forms a stable complex with Bag3 in cells and that the formation of this complex is essential for the activity of HspB8. Bag3 overexpression resulted in the accelerated degradation of Htt43Q, whereas Bag3 knockdown prevented HspB8-induced Htt43Q degradation. Additionally, depleting Bag3 caused a reduction in the endogenous levels of LC3-II, a key molecule involved in macroautophagy, whereas overexpressing Bag3 or HspB8 stimulated the formation LC3-II. These results suggested that the HspB8-Bag3 complex might stimulate the degradation of Htt43Q by macroautophagy. This was confirmed by the observation that treatments with macroautophagy inhibitors significantly decreased HspB8-and Bag3-induced degradation of Htt43Q. We conclude that the HspB8 activity is intrinsically dependent on Bag3, a protein that may facilitate the disposal of doomed proteins by stimulating macroautophagy.The intracellular aggregation of proteins, whether it occurs as a result of proteotoxic stress or genetic mutations, represents a major threat in the crowded environment of the cell. Consequently, efficient mechanisms of protein quality control exist involving molecular chaperones such as Hsp70 and Hsp90, which can recognize and bind to unfolded proteins thereby preventing their aggregation (1-4). The fate of the chaperone substrates is determined by associated co-chaperones. CHIP, a ubiquitin E3 ligase and Bag1, a member of the Bag family of proteins (Bag1-Bag6) that contains a ubiquitin-like domain, associate with Hsp70 and facilitate the targeting of Hsp70 substrates to the ubiquitin-proteasome pathway (5, 6). In contrast, by associating with Hsp70, Bag2 hampers CHIP activity and favors substrate renaturation (7,8), whereas Bag3 prevents Hsp70 substrate degradation and causes ubiquitylated substrate accumulation (9).The role of chaperone complexes containing Hsp70 has been experimentally highlighted in a range of neurodegenerative disorders characterized by the accumulation of protein aggregates (10 -14). However, it is within the HspB group of heat shock proteins (also known as small heat shock proteins) that mutations have first been associated with human diseases (15). We previously showed that HspB8 (H11/Hsp22), a member of this family, has chaperone activity in vivo toward Htt43Q, a pathogenic form of huntingtin that contains an expanded polyglutamine stretch making the protein prone to aggregation. Overexpression of HspB8 but not two closely related family members, HspB1 (Hsp27) or HspB5 (␣B-crystallin), accelerated the degradation of Htt43Q and prevented both aggregation and formation of inclusion bodies (16). A similar chaperone activity was observed usi...

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“…8 BAG3 is a co-chaperone of the BAG family (BAG1-6) and binds to chaperone proteins such as HSP70 and HSPB8, which allows the recognition and binding to misfolded substrates. 9 Furthermore, BAG3 stimulates substrate transfer from HSP70 to the dynein motor complex by acting as a nucleotide-exchange factor, thereby facilitating the dyneinmediated retrograde transport of misfolded proteins to the microtubule organizing center to form aggresomes. 8 In addition, BAG3 promotes selective autophagy via its interaction with p62.…”

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“…◀ of the HSP70 chaperone family of proteins 92 . BAG3 complexes with HSPB8 and stimulates autophagy to clear damaged organelles and protein aggregates 93,94 , including aggregates associated with the DCM causing CRYAB R120G mutant protein 95 . BAG3 not only interacts with HSP70 and HSPBs, but can also bind to tubulin, which is essential for transport of aggregated cargo destined for autophagic degradation 94 .…”

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