Oligodendrocytes (OLs) provide the myelin sheath surrounding axons that propagates action potentials in the central nervous system (CNS). The metabolism of myelinated membranes and proteins is strictly regulated in the OLs and is closely associated with OL differentiation and maturation. The ubiquitination-associated proteasome and endosomal system have not yet been well studied during OL differentiation and maturation. Here, we determined the functions of the Lys63-linked ubiquitination (K63Ub) and K63-specific deubiquitination (DUB) systems regulated by BRCA1/BRCA2-containing complex subunit 3 (BRCC3) during OL differentiation. The competitive inhibition of K63Ub by overexpression of mutant ubiquitin (K63R) in oligodendrocyte precursor cells (OPCs) indicated that the two major CNS myelin proteins, myelin basic protein (MBP) and proteolipid protein (PLP), were upregulated in OLs derived from K63R OPCs. In contrast, the knockdown of BRCC3 (BRCC3-KD) through the application of lentivirus-mediated shRNA delivery system into OPCs suppressed OL differentiation by decreasing MBP expression and PLP production. Further immunoprecipitation assays revealed higher levels of sphingolipid GalC, MBP, and PLP, which were associated with K63Ub-immunoprecipitants and detected in endosome/lysosomal compartments, in BRCC3-KD OLs than those in OLs transfected with the scrambled shRNA (scramble OLs). The differentiation of OLs from BRCC3-KD OPCs was impaired in the demyelinating corpus callosum of rats receiving a cuprizone-containing diet. In the demyelinating tissues from human patients suffering from multiple sclerosis, we detected a decreased number of BRCC3-expressing OLs at the lesion site, accompanied by a greater number of OLs expressing EEA1 and K63Ub at high levels. Altogether, the counterbalance of the K63Ub machinery and BRCC3-triggered DUB machinery are important for the cellular trafficking of myelin proteins and OL differentiation.

中文翻译：

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包含BRCA1 / BRCA2的复杂亚基3通过动态调节赖氨酸63连接的泛素化作用来控制少突胶质细胞的分化。

少突胶质细胞（OL）在轴突周围提供髓鞘，在中枢神经系统（CNS）中传播动作电位。OL中严格控制髓鞘膜和蛋白质的代谢，并且与OL分化和成熟密切相关。在OL分化和成熟过程中，与泛素化相关的蛋白酶体和内体系统尚未得到很好的研究。在这里，我们确定了OL分化过程中由含BRCA1 / BRCA2的复合亚基3（BRCC3）调控的Lys63连锁泛素化（K63Ub）和K63特异性脱泛素（DUB）系统的功能。在少突胶质细胞前体细胞（OPC）中过量表达突变泛素（K63R）对K63Ub的竞争抑制作用表明，这两种主要的中枢神经系统髓鞘蛋白，在衍生自K63R OPC的OL中，髓磷脂碱性蛋白（MBP）和蛋白脂蛋白（PLP）被上调。相比之下，通过将慢病毒介导的shRNA递送系统应用于OPC来降低BRCC3（BRCC3-KD）可以通过降低MBP表达和PLP产生来抑制OL分化。进一步的免疫沉淀分析表明，BRCC3-KD OLs中与K63Ub免疫沉淀物相关并在核糖体/溶酶体区室中检测到的鞘脂GalC，MBP和PLP的水平高于在加扰的shRNA（scramble OLs）中转染的OLs中的水平。接受含铜酮饮食的大鼠脱髓鞘call体中，OLs从BRCC3-KD OPCs的分化受到损害。在患有多发性硬化症的人类患者的脱髓鞘组织中，我们在病变部位检测到表达BRCC3的OL数量减少，同时高水平表达EEA1和K63Ub的OL数量增加。总之，K63Ub机制和BRCC3触发的DUB机制的平衡对髓磷脂蛋白的细胞运输和OL分化很重要。