Functions of Intracellular Regulators of Notch Signaling

主脑(Mam)是Notch信号在进化上保守的元件之一。对果蝇的遗传分析表明，它是该途径的重要正向调节因子。我们已经鉴定了哺乳动物Mam蛋白家族，该家族由三个成员(Mam-1、Mam-2和Mam-3)组成，并阐明了Mam蛋白的生化作用机制。这三个人和果蝇的Mam都稳定并参与了CSL DNA结合蛋白的DNA结合复合体和作为信号中间物的Notch胞内域。为了阐明Mam在哺乳动物系统中的体内功能，我们通过同源重组获得了Mam-1和Mam-2缺失的小鼠品系。MAM-1基因缺陷的小鼠表现出生长迟缓。这种表型在围产期很明显，在出生后加重。小鼠在断奶前就屈服了，这表明Mam-1具有非冗余功能，是一个必不可少的基因。相反，我们还没有发现MAM-2缺陷小鼠的主要发育缺陷。此外，未发现MAM-2基因缺陷小鼠的T、B淋巴细胞等造血系统存在缺陷。这些结果表明Mam基因座在脊椎动物系统中具有明显的功能。先天性糖基化紊乱IIc(CDG IIc)是一种以生长缓慢、智力低下和严重免疫缺陷为特征的隐性综合征。最近，负责CDG IIc的基因被发现编码一种GDP-岩藻糖转运蛋白。已知GDP-岩藻糖对于N-连接的多糖岩藻糖化和O-岩藻糖化是必不可少的，并且这两种岩藻糖修饰都存在于Notch上。我们发现哺乳动物培养的细胞中Notch信号转导需要哺乳动物的GFR。因此，Notch信号的减少参与了CDG IIc的病理过程。

Mastermind (Mam) is one of the evolutionarily conserved elements of Notch signaling. Genetic analyses in Drosophila implicated it as an essential positive regulator of the pathway. We had identified mammalian Mam family of proteins that consists of three members (Mam-1,Mam-2 and Mam-3), and elucidated biochemical mechanism of action of the Mam proteins. All three human and Drosophila Mam stabilize and participate in the DNA binding complex of a CSL DNA-binding protein and the Notch intracellular domains that serve as intermediates of the signaling. All the Mam proteins enhanced the activation of transcription from target promoters by Notch signaling.In order to elucidate the in vivo function of the Mam in a mammalian system, we have generated mouse strains deficient in Mam-1 and Mam-2 by homologous recombination. Mam-1-deficient mice exhibit growth retardation. This phenotype is apparent at the perinatal stage and exacerbated in their postnatal period. The mice succumb before weaning, indicating that Mam-1 has a non-redundant function and is an essential gene. In contrast, we have not identified major developmental defect in Mam-2-deficient mice. Furthermore, no defect has been found in hematopoietic system including T and B lymphocytes of the Mam-2-deficient mice. These results indicate that the Mam loci have distinct function in the vertebrate system.Congenital disorder of glycosylation IIc (CDG IIc) is a recessive syndrome characterized by slowed growth, mental retardation, and severe immunodeficiency. Recently, the gene responsible for CDG IIc was found to encode a GDP-fucose transporter. GDP-fucose is known to be essential for the fucosylation of N-linked glycans and for O-fucosylation, and both fucose modifications are present on Notch. We found that mammalian Gfr is required for Notch signaling in mammalian cultured cells. Therefore, reduced Notch signaling is implicated in the pathology of CDG IIc.