CCP3调控造血干细胞发育分化的分子机制研究

CCP3 is an important intracellular deglutamylate enzyme, which mediates the deglutamylation of many substrates, but it’s role in the development and differentiation of hematopoietic stem cells (HSC) has not been elucidated. We have found that in the bone marrow, CCP3 expression is significantly higher than the other cytosolic carboxypeptidase. We prepared CCP3 knockout mice, and found that the mice have spleen enlargement and there are obvious extramedullary hematopoiesis. Flow cytometric analysis shows that the number of tablets and myeloid lineage cells are abnormally elevated. The number of bone marrow stem and progenitor cells LSKs, multipotent progenitor cells MPPs are significantly increased, but the number of HSCs change is not significantly altered. Through pulldown assay using recombinant CCP3, we found that c-Myc is the substrate of CCP3 in vivo. On this basis, we will take use of CCP3 gene deficient mice as experimental model to study the regulatory role of CCP3 in the development and differentiation process of HSC and decipher how c-Myc glutamate modification is modulated by CCP3 and finally decipher the molecular mechanism of HSC development and differentiation regulated by CCP3.

CCP3是细胞内重要的去谷氨酸酶，它介导了众多底物的去谷氨酸化修饰过程，但是它在造血干细胞(HSC)的发育分化中的作用尚未阐明。我们发现，在骨髓中，CCP3的表达量显著高于其他胞内羧肽酶。我们制备了CCP3基因敲除小鼠，发现该小鼠的脾脏肿大，有明显的髓外造血。流式分析发现髓系和粒系细胞数异常升高。骨髓中干祖细胞LSK、多能祖细胞MPP的数量显著升高，但造血干细胞HSC数量变化并不明显。利用重组的CCP3蛋白与小鼠骨髓裂解液进行共孵育并分离差异条带发现，c-Myc蛋白是CCP3的生理性底物。在此基础上，我们将以CCP3基因缺失的小鼠为实验模型，研究在HSC发育分化过程中CCP3是如何通过调控c-Myc的谷氨酸化修饰而影响MPP的发育分化，最终揭示CCP3调节机体造血干细胞发育分化的分子机制。

所有的造血系都来源于有限的造血干细胞。尽管HSC自我更新的机制已被广泛研究，但对蛋白质谷氨酸化和去谷氨酸化在造血中的作用知之甚少。在这里，我们发现羧肽酶CCP成员中的CCP3在BM细胞中高表达。CCP3缺乏会损害HSC的自我更新和造血功能。去泛素化酶BAP1是HSCs中CCP3的底物。BAP1在Glu651处被TTLL5和TTLL7谷氨酸化，BAP1-E651A突变终止影响BAP1谷氨酸化。BAP1-谷氨酸化加速其泛素化，引发其降解。CCP3能清除BAP1的谷氨酸化，促进BAP1的稳定性，增强Hoxa1的表达，促进HSC的自我更新。Bap1E651A小鼠能产生更多的LT-HSC和外周血细胞。此外，TTLL5和TTLL7缺陷维持了BAP1的稳定，促进HSC自我更新和有效造血。因此，BAP1的谷氨酸化和去谷氨酸化调节对HSC的自我更新有着重要作用。