黑素细胞TGN内Rab33B在ABCB6-RUTBC1下游调控黑素合成酶转运的机制研究

Melanosomes are members of the well-documented family of LROs. Melanogenic enzymes are required for melanosome mature. There is much knowledge about melanogenic enzymes trafficking from the TGN to melanosomes in melanocytes, but they transport within the TGN is still poorly understood. ABCB6, a novel agent involved in the process of skin pigmentation, was identified from patients with dyschromatosis universalis hereditaria by our team in 2013. Our recent experimental findings indicated that ABCB6 regulates the anterograde transport of melanogenic enzymes through its interaction with RUTBC1 in the TGN. Some data show that RUTBC1 functions as a GTPase-acitvating Protein for Rab33B and Rab33B is present in the TGN. So, these data suggest that Rab33B might regulate the trafficking of melanogenic enzymes through its interaction with RUTBC1 in the TGN. To verify this hypothesis, we will demonstrate that Rab33B acts downstream of ABCB6-RUTBC1 in regulating the trafficking of melanogenic enzymes in the TGN using confocal laser scanning microscope and co-immunoprecipitation technology. At last, we will find out the Rab33B ineracting protein in the TGN by co-immunoprecipitation technology and mass spectrometric analysis. The mechanism of melanogenic enzymes transport in the TGN will be further understood and it will shed new light on other LROs when we accomplish this project.

黑素合成酶是黑素小体成熟所必需蛋白。它们从TGN转运到黑素小体的机制已较明确，但在TGN内转运并启动分拣的机制仍不清楚。ABCB6是我们于2013年率先鉴定的一个色素代谢相关蛋白。近期我们又发现：在黑素细胞TGN内ABCB6通过与下游RUTBC1相互作用而调控黑素合成酶顺行转运。RUTBC1是一个Rab33B的GTP酶活化蛋白，Rab33B存在于TGN。因此推测：TGN内Rab33B可能通过与RUTBC1相互作用而调控黑素合成酶转运。本项目拟采用CLSM技术等对Rab33b/Rutbc1/Abcb6稳定沉默的黑素细胞进行分组研究，明确TGN内Rab33B在RUTBC1下游调控黑素合成酶转运；探明TGN内Rab33B互作蛋白，阐明其作用机制。本项目的完成将丰富ABCB6-RUTBC1调控黑素合成酶转运的机制，对系统理解TGN功能具有重要的理论意义，并为其它LROs研究提供参考依据。

黑素代谢是黑素细胞的特有功能，充分认识对人类健康具有重要意义。本项目首先成功构建了野生型Rab33B和RaB33B shRNA质粒，转染至B16-F1细胞系。与对照组相比，Rab33B shRNA质粒转染组Rab33B mRNA和蛋白表达均明显下降，Tyrp1/Tyrp2/Tyr mRNA表达下降。NaOH裂解法检测发现野生型Rab33B质粒转染组黑素含量明显升高，Rab33B shRNA质粒转染组黑素含量显著下降，其中shRAB33B#3下降最明显。. 随后构建了RAB33B过表达和敲减慢病毒，感染B16-F1细胞系。Real-time PCR发现RAB33B过表达细胞TYRP2 mRNA表达明显升高，RAB33B敲减细胞TYRP1 mRNA表达明显下降；采用CLSM技术分析Tyrp1/Tyrp2/Tyr亚细胞定位，发现抑制RAB33B表达对其影响甚微，在TGN也一样。. 再次构建人ABCB6敲减慢病毒，感染MNT-1细胞系。分组分析黑素细胞Tyrp1/Tyrp2/Tyr/RUTBC1/Rab33B表达水平，发现3种黑素合成酶的蛋白表达有一定的下调，而对RUTBC1和Rab33B表达影响不明显。采用CLSM技术分析稳转MNT-1细胞3种黑素合成酶亚细胞定位，未发现明显差异，TGN也呈类似表现。采用NaOH裂解法发现抑制ABCB6对黑素含量有一定抑制作用。. 随后，采用转录组测序技术对shControl和shABCB6感染的MNT-1细胞分组测序，对差异基因KEGG通路富集分析显示ABCB6与Wnt通路强相关。经Real-time PCR、双向Co-IP、Western Blot和Rescue实验证实ABCB6与GSK3-β相互作用，通过Wnt/β-catenin信号通路调控黑素合成。该发现对系统理解黑素细胞生理功能具有重要的科学价值和理论意义。