Function and regulation of podin proteins under mechanical stress in muscle

Podin蛋白(SYNPO、SYNPO2、SYNPO2L)是与机械应激相关的多种细胞活动的多接头蛋白：通过与细丝蛋白、α-肌动蛋白和肌动蛋白结合，参与肌动蛋白的细胞骨架结构，而其他蛋白通过伴侣辅助的选择性自噬(CASA)和机械应激下的信号转导参与丝蛋白蛋白的稳定。在第一个资助时期，我们建立了特定细胞类型的PODIN亚型表达模式，并确定了重要的新的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，以及(4)SYNPO2中的磷脂酰肌醇识别基序。双分子荧光互补实验表明，SYNPO2异构体特异性相互作用将BAG3定向到肌原纤维Z盘或收缩诱导的损伤。利用电脉冲刺激(EPS)研究机械应激对蛋白质表达的影响、蛋白质组的变化及其对蛋白质动力学的影响。在这里，含有PDZ结构域的SYNPO2b变体在FRAP分析中表现出比不含PDZ的SYNPO2变体更慢的动力学，机械应力对短的SYNPO2异构体有影响，但对长的SYNPO2异构体没有影响。相比之下，SYNPO2b的恢复很大程度上依赖于蛋白质合成。我们的数据表明，损伤是一个不依赖于降解的间隔，稳定了由机械应力引起的局部肌纤维损伤。在SYNPO2基因敲除细胞中，病变形成的急剧增加进一步强调了Podins在这一过程中的作用。磷蛋白质组分析揭示了复杂的收缩诱导的肌管磷酸化模式，对于其中的一些模式，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SYNPO2基因突变，进一步强调了PODIN蛋白家族的整体重要性。计划中的工作计划的一个主要焦点将是限制PODIN蛋白的不同和重叠功能。特别是，我们将建立收缩诱导(去)磷酸化如何调节PODIN蛋白的蛋白质相互作用以及它们各自的功能影响。蛋白质复合体的生化分析将与光学显微镜分析相补充，包括FRAP和BIFC以及EPS和PODIN蛋白质敲除细胞。特别有兴趣的是进一步阐明PODIN蛋白在新发现的与肌醇磷脂信号和钙调神经磷酸酶信号的联系以及与YAP/TAZ信号的直接联系中所起的作用。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动态平衡和信号传递的关键功能。

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.