Function and regulation of the BAG3 chaperone network under mechanical stress

协同伴侣蛋白BAG3在机械应力下平衡基因表达、蛋白质翻译和蛋白质降解。降解是通过伴侣辅助选择性自噬（CASA）介导的，基于BAG3与细胞骨架接头SYNPO2和小热休克蛋白HSPB8的合作。在第一笔资助中，我们确定了BAG3中的磷酸化位点，这些位点调节BAG3与其伴侣蛋白的相互作用，并调节CASA在机械应力下的活性。此外，还检测到一种力调节磷酸酶与BAG3相关。此外，通过电脉冲刺激小鼠肌管施加不同强度的机械应力，然后系统分析蛋白质平衡因子、信号蛋白和细胞骨架成分的表达和自噬降解，采用有针对性和无偏倚的转录组学和蛋白质组学方法。这揭示了在肌管分化和对长时间轻度机械刺激和急性机械应力的适应过程中，bag3介导的蛋白酶平衡发生了深刻的变化。分化导致核心CASA机制的诱导和激活，但也导致除HSPB8以外的与bag3相互作用的shsp的表达增加，包括HSPB1、HSPB5和HSPB7。我们证明分化肌管中自噬降解途径是活跃的，这与传统的CASA途径不同，分别由sHSPs和丝蛋白相互作用蛋白FILIP1介导。事实上，对急性机械应力的保护涉及传统CASA的关闭，这似乎使HSPB8参与降解无关的功能，而其他自噬途径保持活跃甚至被诱导。我们的工作揭示了bag3相关的sHSP网络在机械应力保护中的核心作用，并展示了由机械应力触发的自噬途径的意想不到的多样性。这将是计划工作计划的一个主要目标，描绘不同的自噬途径，涉及执行和调节因素和受影响的客户。为此，我们将进行系统的sirna介导的介质和调节因子耗竭，分别在机械应力诱导的自噬过程中建立潜在的合作或独立功能。执行因子的复合体将被分离并通过质谱法进行表征，以确定相互作用因子和受影响的客户。已经建立了实验条件来诱导力调节的BAG3网络的定义状态，将有可能将观察到的适应性变化与BAG3和BAG3相关蛋白磷酸化状态的改变联系起来。研究单位内的密切合作将使我们能够验证观察到的机制是否代表了在不同细胞类型和组织中保守的机械应力保护的共同原则。

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.