TGFbR1调控动力肌球蛋白Myo3A在遗传性听力疾病中的机制和功能的研究

Myo3A gene mutation is associated with nonsyndromic recessive progressive HL （DFNB30）. Furthermore, ATP enzyme activity in Myo3A regulates the formation and length of stereocilia; Even so, it is still unclear regarding the mechanism of Myo3A ATP enzyme activity and the mechanism of HL associated with genetic mutation. To this end, we found in our pro-phase experiment that tyrosine kinase receptor of TGFbR1 in TGFbeta signaling pathway was expressed in the cochlea, besides, Myo3A and TGFbR1 were co-localized and interacted with each other in the cochlea, TGFbR1 also regulated the expression of Myo3A protein and the activity of ATP; Meanwhile, T204D, the kinase mutant of TGFbR1, could abolish such regulatory effect, expression of TGFbR1 was thus changed in the ciliated cell line, and the length of cilia changed accordingly. Our study will continue to further study along this clue to verify the universality and specificity of the interaction between Myosin family members and TGFbR1, and hence to explore the specific mechanism of TGFbR1 regulating the activity of Myo3A. With the establishment of TGFbR1-specific knockout mouse model of inner ear, it was discovered that TGFbR1 and TGFbeta might have potential impact on the structure and function of cilia. It will be the first time in the world to find the role of TGFbeta signaling pathway and TGFbR1 in the ciliary bundles, and to identify more important regulation mechanism of Myo3A activity and the cause of HL caused by Myo3A mutation, so as to provide a scientific foundation for the treatment ofand related diseases.

Myo3A基因突变与遗传性渐进性听力损失DFNB30相关。Myo3A的ATP酶活调控耳纤毛的形成与长度。尽管如此，Myo3A ATP酶活性机制及突变导致DFNB30机制不清。我们前期实验发现TGFbeta信号通路中丝苏氨酸激酶TGFbR1在耳蜗中表达，TGFbR1与Myo3A在耳蜗内共定位并发生相互作用，TGFbR1调节Myo3A的表达水平以及ATP酶活性，TGFbR1的激酶突变体T204D可废除该调控效应，过表达TGFbR1纤毛的长度发生变化。本项目将深入研究，通过TGFbR1内耳基因特异性敲除小鼠模型和纤毛形成的细胞系发现TGFbR1以及TGFbeta1对耳纤毛结构和功能具体机制。这将是国际上首次发现TGFbeta信号通路以及TGFbR1参与耳纤毛束的功能，发现Myo3A更为重要的活性调控机制以及Myo3A突变致DFNB30机制，为听力损失疾病的治疗提供靶标。

TGF-β家族对多种细胞生物的胚胎发生过程和组织稳态至关重要。TGF-β信号的调控与包括癌症在内的多种人类疾病有关。然而，TGF-β信号是否在听力功能中起调控作用以及是否与听力疾病相关的机制尚不清楚。本研究表明，TGF-β信号通路主要的1型受体TGFBR1是耳蜗毛细胞中与Myo3A和Myo3B相互作用的重要蛋白。此外，TGFBR1的激酶位点K232特异性识别Myo3A内的Motor结构域，TGFBR1可以通过Ser614和Ser703磷酸化增加Myo3A和Myo3B的蛋白表达的稳定性。最后，它抑制Smurf2对其的多聚泛素化降解。其中，作为DFNB30主要突变体的S614F(见于渐进性耳聋疾病)不受TGFBR1磷酸化的影响，并被Smurf2促进泛素化降解。这样，TGF-β1和TGFBR1正向调控纤毛的形成，而TGF-β信号通路的拮抗剂SB431542则抑制纤毛的形成。更重要的是，研究表明Atoh1-Tgfbr1-/-小鼠表现出异常的静纤毛和毛细胞以及进行性听力丧失。此外，通过Atoh1-Tgfbr1-/-小鼠的TMT定量蛋白质组学分析，发现了多种核糖体蛋白和细胞器相关蛋白的缺陷。综上所述，本研究的数据揭示了TGFβ信号通路通过重要家族成员TGFBR1磷酸化和HECTE3-Smurf2多聚泛素化促进调节Myo3A和Myo3B稳定性以及对听力功能的必要性。由于TGFβ信号通路中的TGFbr1是影响耳蜗正常静纤毛、毛细胞结构和听力功能的关键基因，研究结果还确定了TGF-β1对遗传性耳聋疾病DFNB30(隐性非综合征性听力损失30疾病)和其他未发现的听力疾病的潜在治疗方法。