分泌系统内新型激酶的结构和底物识别机制研究

Phosphorylation is a key regulatory mechanism for many cellular processes. The majority of phosphorylation occurs in the nucleus and cytoplasm; however, many secreted proteins are phosphorylated within the ER and Golgi compartments during secretion. Kinases that function specifically in the secretory pathway have only been identified recently. Fam20C is the long-sought genuine Golgi Casein Kinase. It phosphorylates many secreted proteins, and its mutation can cause a lethal form of osteosclerotic bone dysplasia known as Raine syndrome. Fam20B and Fam20A are two paralogues of Fam20C in human; however, they each function in a distinct manner. Though highly similar to Fam20C in the kinase domain, Fam20B is a sugar kinase and phosphorylates a xylose residue within the tetrasaccharide linker of proteoglycans. Mutations of Fam20A cause Amelogenesis imperfecta and Enamel renal syndrome; however, the kinase activity and function mechanism of Fam20A remain to be determined. Sgk196 is another sugar kinase within the secretory system, phosphorylating a mannose residue on alpha-dystroglycan. Interestingly, Sgk196 is long thought to be inactive, since it lacks several canonical kinase active site residues. How such a“pseudo” kinase can catalyze the phosphorylation reaction remains to be understood. To gain fundamental insights into the function of these novel secretory pathway kinases, and to decipher their catalytic and substrate-recognition mechanism, we plan to determine their structures in both apo and substrate-bound states. Based on these results, we will further pinpoint their function by structure-guided mutagenesis combined with biochemical and enzymatic analyses. Successful completion of these experiments will result in a better understanding of this important family of proteins, and shed light on the pathological conditions caused by their malfunction.

磷酸化是调节细胞内各种过程的一个关键机制。分泌型分子在内质网或高尔基体的管腔内被磷酸化，但执行这些反应的激酶直到近几年才被发现。Fam20C磷酸化很多分泌蛋白而调节人体包括骨骼发育在内的生理过程，其功能的失活则引起多种疾病。Fam20B和Fam20A是Fam20C的两个同源蛋白，但功能机制与Fam20C很不相同。Fam20B是一个糖的激酶，磷酸化蛋白聚糖中的一个木糖分子。Fam20A的突变引起人牙齿和肾脏相关疾病，但活性与功能依然未知。分泌系统内另一个糖的激酶是Sgk196，催化阿尔法肌萎缩蛋白聚糖上甘露糖残基的磷酸化。Sgk196的失活导致一种严重的肌肉萎缩症，但其催化机制还是个迷。本项目拟利用X射线晶体学、生物化学和酶动力学等方法对这些分泌系统内的激酶进行研究，解析它们的三维结构和与底物分子的复合物结构，阐明它们识别底物的分子基础和功能机制，为与它们相关的疾病提供诊疗思路。

内质网和高尔基体在细胞中执行很多关键功能。分泌蛋白或细胞表面蛋白在这里折叠修饰，然后被分泌到细胞外或转运到其他细胞器。很多分泌蛋白会被磷酸化修饰，但分泌系统内的激酶近几年才被发现。Fam20C磷酸化上百个其他分泌蛋白而调节包括骨骼发育和血磷代谢等多种生理和病理过程。Fam20A是一个没有激酶活性的假激酶，通过与Fam20C形成复合体而激活其活性。Fam20A基因的突变引起人体牙齿和肾脏发育的疾病。Fam20B是一个磷酸化木糖分子的激酶，在蛋白多糖的合成过程发挥重要调节功能。“假激酶”POMK作为甘露糖激酶调控肌萎缩蛋白聚糖的体内合成。在本项目的资助下，我实验室系统研究了这些新型激酶功能与调控的机制，全面阐明了它们的结构特性及演化关系，也为理解相关疾病的分子机理奠定了基础（Nature Communications, 2018; eLife, 2017; eLife, 2016; Methods in Molecular Biology, 2016）。