Function and regulation of the BAG3 chaperone network under mechanical stress

• Applicant: Professor Dr. Jörg Höhfeld
• Subject Area: Cell Biology
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 388932620

The cochaperone BAG3 balances gene expression, protein translation and protein degradation under mechanical stress. Degradation is mediated through chaperone-assisted selective autophagy (CASA), based on the cooperation of BAG3 with the cytoskeleton adaptor SYNPO2 and the small heat shock protein HSPB8. In the first funding we identified phosphorylation sites in BAG3, which modulate the interaction of BAG3 with its partner proteins and regulate CASA activity under mechanical stress. In addition, a force-regulated phosphatase was detected in association with BAG3. Furthermore, murine myotubes were subjected to mechanical stress of different intensity through electrical pulse stimulation, followed by a systematic analysis of the expression and autophagic degradation of proteostasis factors, signalling proteins and cytoskeleton components in targeted and unbiased transcriptomic and proteomic approaches. This revealed profound changes of BAG3-mediated proteostasis during myotube differentiation and the adaptation to prolonged mild mechanical stimulation and acute mechanical stress. Differentiation leads to the induction and activation of the core CASA machinery but also causes an increased expression of BAG3-interacting sHSPs other than HSPB8, including HSPB1, HSPB5 and HSPB7. We demonstrate that in differentiated myotubes autophagic degradation pathways are active, which are distinct from the conventional CASA pathway and are mediated by sHSPs and the filamin interacting protein FILIP1, respectively. Indeed, protection against acute mechanical stress involves a shut-off of conventional CASA, which seems to enable HSPB8 to engage in degradation-independent functions, whereas other autophagy pathways remain active or are even induced. Our work reveals the central role of the BAG3-associated sHSP network for mechanical stress protection and demonstrates an unexpected diversity of autophagy pathways triggered by mechanical stress. It will be a main objective of the planned work program to delineate the diverse autophagy pathways regarding involved executing and regulatory factors and affected clients. To this end, we will perform a systematic siRNA-mediated depletion of mediators and regulators to establish a potential cooperation or independent functions, respectively, during mechanical stress induced autophagy. Complexes of executing factors will be isolated and characterized by mass spectrometry to identify interactors and affected clients. Having established experimental conditions to induce defined states of the force-regulated BAG3 network, it will be possible to correlate observed adaptive changes with alterations of the phosphorylation status of BAG3 and BAG3-associated proteins. Close cooperation within the research unit will allow us to verify whether observed mechanisms represent common principles of mechanical stress protection conserved across diverse cells types and tissues.

• DFG Programme: Research Units
• Subproject of: FOR 2743: Mechanical Stress Protection