Function and regulation of podin proteins under mechanical stress in muscle

肌肉机械应力下podin蛋白的功能和调节

• 批准号: 401379771
• 负责人: Professor Dr. Dieter O. Fürst
• 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/401379771?language=en
• 关键词: Function; regulation; podin; proteins; under

Podin proteins (SYNPO, SYNPO2, SYNPO2L) are multi-adapter proteins involved in multiple cellular activities related to mechanical stress: by binding to filamin, alpha-actinin and actin a subset of podin proteins contributes to actin cytoskeleton architecture, while others are involved in filamin proteostasis via chaperone-assisted selective autophagy (CASA) and signaling under mechanical stress. In the first funding period we established cell type-specific podin isoform expression patterns and identified important novel podin protein interactions: (1) a SYNPO2 PDZ domain interaction with YAP/TAZ transcription factors, (2) binding of SYNPO2 and SYNPO2L to the calcineurin inhibitor calsarcin, (3) phosphorylation-regulated LC3-binding and (4) a phosphoinositide recognition motif in SYNPO2. Bimolecular fluorescence complementation showed that a SYNPO2 isoform-specific interaction directs BAG3 to myofibrillar Z-discs or contraction-induced lesions. Electrical pulse stimulation (EPS) was exploited to study differential protein expression, changes in the phosphoproteome and effects on protein dynamics in response to mechanical stress. Here, the PDZ domain-containing SYNPO2b variant showed slower dynamics in FRAP assays than the PDZ-less SYNPO2 variants and mechanical stress had an impact on the short but not the long SYNPO2 isoforms. By contrast, the recovery of SYNPO2b is strongly depending on protein synthesis. Our data imply that lesions are a degradation-independent compartment stabilising local myofibrillar damage induced by mechanical stress. The role of podins in this process is further emphasised by the fulminant increase in lesion formation in SYNPO2 knockdown cells. Phosphoproteome analyses revealed complex contraction-induced phosphorylation patterns in myotubes, and for some of these we showed an impact on protein-protein interactions. The overall importance of the podin protein family is further highlighted by the identification of mutations in the SYNPO2 gene in nephrotic syndrome patients. A major focus of the planned work program will be to confine distinct and overlapping functions of the podin proteins. In particular, we will establish how contraction-induced (de)phosphorylations regulate protein interactions of podin proteins and what their respective functional impact is. Biochemical analyses of protein complexes will be complemented with light microscopical analyses including FRAP and BiFC in conjunction with EPS and podin protein knockdown cells. Of particular interest will further be to elucidate the role of podin proteins regarding the newly dicovered links to phosphoinositide signaling and calcineurin signaling, as well as the direct link to YAP/TAZ signaling. Our collaborations in this research unit will enable us to unravel the key functions of podin proteins for the regulation of protein homeostasis and signalling under conditions of mechanical stress in muscle.

Podin蛋白（SYNPO, SYNPO2, SYNPO2L）是参与与机械应力相关的多种细胞活动的多接头蛋白：通过与丝蛋白，α -肌动蛋白和肌动蛋白结合，Podin蛋白的一个亚群有助于肌动蛋白的细胞骨架结构，而其他Podin蛋白通过伴侣辅助选择性自噬（CASA）和机械应力下的信号传导参与丝蛋白的蛋白质稳态。在第一个资助期，我们建立了细胞类型特异性的podin异构体表达模式，并鉴定了重要的新的podin蛋白相互作用：(1)SYNPO2 PDZ结构域与YAP/TAZ转录因子的相互作用，(2)SYNPO2和SYNPO2L与钙调神经磷酸酶抑制剂calsarcin的结合，(3)磷酸化调节的lc3结合，(4)SYNPO2中的磷酸肌肽识别基序。双分子荧光互补显示，SYNPO2亚型特异性相互作用将BAG3导向肌纤维z盘或收缩性病变。利用电脉冲刺激（EPS）研究机械应力下蛋白质的差异表达、磷酸化蛋白质组的变化以及对蛋白质动力学的影响。在FRAP实验中，含有PDZ结构域的SYNPO2b突变体比不含PDZ结构域的SYNPO2突变体表现出更慢的动力学，机械应力对SYNPO2短亚型有影响，而对长亚型没有影响。相比之下，SYNPO2b的恢复强烈依赖于蛋白质合成。我们的数据表明，病变是一种降解无关的室稳定局部肌纤维损伤引起的机械应力。在SYNPO2敲低的细胞中，损伤形成的急剧增加进一步强调了pod在这一过程中的作用。磷酸化蛋白质组分析揭示了肌管中复杂的收缩诱导磷酸化模式，其中一些我们显示了对蛋白质-蛋白质相互作用的影响。在肾病综合征患者中发现的SYNPO2基因突变进一步强调了podin蛋白家族的总体重要性。计划的工作计划的一个主要焦点将是限制不同的和重叠的功能的podin蛋白。特别是，我们将确定收缩诱导（去）磷酸化如何调节足素蛋白的蛋白质相互作用以及它们各自的功能影响。蛋白质复合物的生化分析将辅以光镜分析，包括FRAP和BiFC，以及EPS和podin蛋白敲低细胞。特别感兴趣的将是进一步阐明podin蛋白在新发现的与磷酸肌苷信号和钙调磷酸酶信号的联系方面的作用，以及与YAP/TAZ信号的直接联系。我们在这个研究单位的合作将使我们能够解开podin蛋白在肌肉机械应力条件下调节蛋白质稳态和信号传导的关键功能。