Function and regulation of the BAG3 chaperone network under mechanical stress

辅伴侣蛋白BAG3在机械应力下平衡基因表达、蛋白质翻译和蛋白质降解。降解是通过分子伴侣辅助的选择性自噬（CASA）介导的，基于BAG3与细胞骨架接头SYNPO2和小的热休克蛋白HSPB8的合作。在第一次资助中，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，并在机械应力下调节CASA活性。此外，检测到与BAG3相关的力调节磷酸酶。此外，小鼠肌管进行不同强度的机械应力，通过电脉冲刺激，然后通过有针对性的和公正的转录组学和蛋白质组学方法的蛋白质稳态因子，信号蛋白和细胞骨架成分的表达和自噬降解的系统分析。这揭示了肌管分化过程中BAG3介导的蛋白质稳态的深刻变化以及对长时间轻度机械刺激和急性机械应力的适应。分化导致核心CASA机制的诱导和激活，但也导致BAG3相互作用的除HSPB8以外的sHSP（包括HSPB1、HSPB5和HSPB7）的表达增加。我们证明，在分化的肌管自噬降解途径是活跃的，这是不同于传统的CASA途径，并介导的sHSPs和细丝蛋白相互作用蛋白FILIP1，分别。事实上，对急性机械应力的保护涉及关闭常规CASA，这似乎使HSPB8能够参与降解独立的功能，而其他自噬途径保持活性甚至被诱导。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，并证明了机械应力引发的自噬途径的意外多样性。这将是计划工作计划的一个主要目标，以描绘不同的自噬途径，涉及执行和监管因素和受影响的客户端。为此，我们将进行系统的siRNA介导的介导的介质和调节剂的消耗，以建立一个潜在的合作或独立的功能，分别在机械应力诱导的自噬。执行因素的复杂性将被分离出来，并通过质谱法进行表征，以确定相互作用者和受影响的客户端。在建立了诱导力调节的BAG3网络的定义状态的实验条件后，将有可能将观察到的适应性变化与BAG3和BAG3相关蛋白的磷酸化状态的改变相关联。研究单位内的密切合作将使我们能够验证所观察到的机制是否代表在不同细胞类型和组织中保存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.