基于TALEN技术敲除的ttc36小鼠研究高酪氨酸血症关键代谢酶HPD的分子调控机制

4-Hydroxyphenylpyruvate dioxygenase (HPD) is one of pivotal enzymes in the process of tyrosine metabolism while the mechanism of molecular regulation remains unclear. Hypertyrosinemia was unexpectedly detected in the homozygous knockout mouse (ttc36-/-) generated by the transcription activator-like effector nucleases (TALENs) strategy, along with a significant decline of hpd. Thus, the goal of this study is to clarify the molecular mechanism of ttc36 influencing tyrosine metabolism via hpd. First of all, to determine whether ttc36 mediating hypertyrosinemia is caused by hpd deficiency, we are going to overexpress exogenous hpd in the ttc36-deficient mouse to rescue hypertyrosinemia. In the absence of ttc36, our previous data showed that there was no significant change in the mRNA level of hpd compared with the wild mice, suggesting that ttc36 doesn’t influence the transcription regulation of hpd. According to the bioinformatics analysis, ttc36 was predicted to interact with hpd, suggesting that ttc36 could inhibit the degradation of hpd. We have successfully generated the rabbit polyclonal antibody of ttc36 against mouse. In this study, we plan to apply immunoprecipitation , mass spectrometry and pull-down to verify the direct interaction between ttc36 and hpd, meanwhile predict other proteins that may interact with ttc36. Eventually, we design to elucidate the specific posttranslational modification of ttc36 towards hpd thoroughly by the experiments of ubiquitin-proteasome, autophagy as well as lysosomal degradation.

4-羟基苯丙酮酸双加氧酶（HPD）为酪氨酸代谢过程的关键酶之一，其分子调节机制不清楚。我们用TALEN技术构建的ttc36基因敲除小鼠纯合子中意外发现高酪氨酸血症，且伴随hpd表达水平的显著降低。本课题研究ttc36基因通过何种分子机制调节HPD表达影响酪氨酸代谢。拟在该小鼠中过表达hpd，检测高酪氨酸血症是否能得到纠正，明确ttc36是通过hpd这个环节来影响酪氨酸。我们前期实验已表明，ttc36并不是以转录调控方式影响hpd表达；生物信息学分析ttc36和hpd之间潜在相互作用，提示其可能通过影响hpd翻译后蛋白修饰方式来进行调控。我们已经成功制备ttc36多克隆抗体，将用免疫沉淀加质谱分析，以及pull-down首先验证ttc36和hpd之间是否真实存在直接相互作用；其次分析和ttc36可能相互作用的其他蛋白。做泛素化、自噬、以及溶酶体降解实验，探明ttc36对hpd蛋白修饰方式。

4-羟基苯基丙酮酸双加氧酶（HPD）（酪氨酸代谢的关键酶）的表达降低是人酪氨酸血症的病因。但是，调控HPD表达的分子机制尚未阐明。本研究证明了在肝脏中高表达的分子伴侣TTC36与HPD相关，其通过减少蛋白激酶STK33与HPD的结合，进而抑制了STK33介导的HPD T382磷酸化。HPD T382磷酸化的减少导致含FHA结构域的E3 泛素连接酶PELI1和PELI1介导的HPD泛素化降解途径受阻。相反，TTC36的缺乏会导致STK33介导的HPD T382磷酸化增多，PELI1与HPD的结合增多以及PELI1介导的HPD下调。Ttc36-/-小鼠肝脏中HPD表达降低，并表现出酪氨酸血症，海马神经元损害以及学习记忆能力减退。这些发现揭示了TTC36与HPD相互作用以及调控STK33、PELI1降解HPD的分子机制，首次系统阐释了TTC36-STK33-PELI1信号轴在酪氨酸代谢及其中间产物对神经损伤的重要生理意义。