UCHL1调控TrkB去泛素化的机制及其功能研究

It has been well known that Brain derived neurotrophic factor (BDNF) plays an important role in regulation of neuronal growth, survival and synaptic plasticity via activating its receptor TrkB and downstream signaling pathways. By binding with BDNF TrkB could be activated by phosphorylation, and consequently activating several signaling pathways. It has been reported that BDNF could induce dramatic increase of another posttranslational modification-ubiquitination of TrkB. However how TrkB ubiquitination is regulated and its biological functions remain unknown. In the preliminary studies we have found that Ubiquitin C-terminal hydrolase L1 (UCHL1) could interact with TrkB in vitro, and overexpression of UCHL1 leads to significant reduction of TrkB ubiquitination; furthermore inhibition of UCHL1 activity blocks BDNF induced TrkB internalization and impairs TrkB activation and its downstream signaling pathways activity, indicating that UCHL1 as a deubiquitinating enzyme for TrkB regulates its ubiquitination level and functions. In this project we aim to explore the mechanisms underlying UCHL1 regulated TrkB ubiquitination and its biological functions. The detailed domain/motif of TrkB and UCHL1 that required for their interaction will be mapped, and the UCHL1 regulated deubiquitination sites of TrkB will also be investigated. The effects of UCHL1 regulated TrkB ubiquitination on TrkB internalization, recycling, degradation and BDNF regulated synaptic plasticity will be further examined. Moreover, the transgenic mice with conditional knockdown of UCHL1 and heterozygous TrkB will be used to investigate the role of UCHL1 in central nervous system, confirming the in vitro studies in which UCHL1 perform its biological functions by regulation of TrkB ubiquitination. In summary this project will the first study as we know demonstrate that UCHL1 as a deubiquitinating enzyme for TrkB, and deepen our understanding in TrkB ubiquitination and UCHL1 functions in nervous system.

脑源神经营养因子BDNF通过受体TrkB在调控神经元生长发育、突触可塑性等方面发挥重要作用，TrkB的翻译后修饰直接影响其功能。已知TrkB可被泛素化，但是其调控分子和功能仍不清楚。申请者在前期实验发现羧基末端水解酶UCHL1可与TrkB结合，过表达UCHL1使TrkB泛素化水平降低，而且抑制其去泛素化酶活性可抑制BDNF诱导的TrkB内吞和下游信号通路的活性，提示UCHL1作为TrkB的去泛素化酶调控TrkB的泛素化水平和功能。本课题将在细胞模型中深入研究UCHL1调控TrkB去泛素化的机制，以及对TrkB的胞内运输、定位和BDNF调控的神经元突触可塑性的影响。同时还将利用UCHL1条件敲低小鼠和TrkB+/-小鼠模型，阐明UCHL1在中枢神经系统中的作用，验证UCHL1通过调控TrkB的泛素化发挥功能。本课题将首次阐明TrkB泛素化的调控机制和功能，深化UCHL1在神经系统功能的认识。

脑源神经营养因子BDNF通过受体TrkB在调控神经元生长发育、突触可塑性等方面发挥重要作用，TrkB的翻译后修饰直接影响其功能。已知TrkB可被泛素化，但是其调控分子，泛素化的生物学功能仍不清楚。本项目重点研究了TrkB泛素化的调控机制及其生物学功能。我们发现体外和体内羧基末端水解酶UCHL1都可与TrkB结合，并且两者的结合与细胞活性相关，当BDNF刺激体外培养神经元或者小鼠经过CFC训练后，伴随TrkB泛素化水平升高，两者结合水平也显著升高。我们进一步证明UCHL1作为TrkB的去泛素化酶调控TrkB的泛素化水平，调控位点主要在TrkB胞内近膜区K460位点。当利用特异性多肽Tat-UCHL75-85抑制TrkB与UCHL1的结合时，与对照组相比，BDNF或者CFC训练诱导的TrkB的泛素化水平显著升高，证明此多肽的有效性。进一步利用此多肽我们发现，特异性抑制UCHL1对TrkB泛素化的调控会导致BDNF诱导的TrkB内吞增加，内吞后更多的TrkB在胞内被降解，从而导致BDNF-TrkB信号通路活性降低。此结果提示TrkB泛素化调控其内吞和胞内运输，抑制其去泛素化酶活性可使TrkB泛素化水平升高，导致TrkB内吞增加，降解升高，膜表面TrkB水平下降，抑制BDNF诱导的TrkB内吞和下游信号通路的活性。在CFC学习记忆模型中，此多肽抑制海马依赖的学习记忆，进一步阐明了UCHL1和TrkB在体内的重要作用。本项目首次报道UCHL1作为TrkB的去泛素化酶，参与调控BDNF-TrkB信号通路的调控，扩展了UCHL1在中枢神经系统中的作用，对我们深入理解TrkB泛素化的调控机制和功能，今后以UCHL1和TrkB为靶点研发神经系统相关药物打下了理论基础。