Function and regulation of the BAG3 chaperone network under mechanical stress

机械应力下 BAG3 伴侣网络的功能和调节

• 批准号: 466025467
• 负责人: Professor Dr. Jörg Höhfeld
• 金额: --
• 依托单位: Institut für Zellbiologie
• 依托单位国家: 德国
• 项目类别: Research Units
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/466025467?language=en
• 关键词: Function; regulation; BAG3; chaperone; network

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.

辅助伴侣BAG3在机械压力下平衡基因表达、蛋白质翻译和蛋白质降解。降解是通过伴侣辅助的选择性自噬(CASA)介导的，基于BAG3与细胞骨架适配器SYNPO2和小的热休克蛋白HSPB8的合作。在第一笔资金中，我们确定了BAG3中的磷酸化位点，它调控BAG3与其伙伴蛋白的相互作用，并调节机械应力下的CASA活性。此外，还检测到一种与BAG3相关的强制调节的磷酸酶。此外，通过电脉冲刺激，小鼠肌管受到不同强度的机械应力，随后系统地分析了蛋白稳定因子、信号蛋白和细胞骨架成分的表达和自噬降解，并采用靶向和无偏见的转录和蛋白质组学方法。这揭示了BAG3介导的蛋白平衡在肌管分化过程中的深刻变化，以及对持续的轻微机械刺激和急性机械应激的适应。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sHSPs表达增加，包括HSPB1、HSPB5和HSPB7。我们证明在分化的肌管中，自噬降解途径是活跃的，这与传统的CASA途径不同，分别由sHSPs和细丝蛋白相互作用蛋白FILIP1介导。事实上，对急性机械应激的保护包括关闭传统的CASA，这似乎使HSPB8能够参与非降解功能，而其他自噬途径仍然活跃，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意想不到的多样性的自噬途径。这将是计划工作计划的主要目标，描绘涉及执行和监管因素以及受影响客户的不同自噬途径。为此，我们将在机械应力诱导的自噬过程中，进行系统的siRNA介导的介质和调节因子的耗竭，以建立潜在的合作或独立功能。执行因素的复合体将被分离出来，并用质谱仪进行表征，以确定相互作用者和受影响的客户。在建立了诱导力量调节的BAG3网络的特定状态的实验条件后，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变化联系起来。研究单位内部的密切合作将使我们能够验证所观察到的机制是否代表了在不同类型的细胞和组织中保存的机械应力保护的共同原则。