Functions of Intracellular Regulators of Notch Signaling

Mam是Notch信号通路中进化上保守的元件之一。果蝇的遗传分析表明它是该途径的重要正调节因子。我们鉴定了哺乳动物Mam蛋白家族的三个成员（Mam-1、Mam-2和Mam-3），并阐明了Mam蛋白的生化作用机制。所有三种人类和果蝇Mam稳定并参与CSL DNA结合蛋白和Notch细胞内结构域的DNA结合复合物，所述Notch细胞内结构域充当信号传导的中间体。为了阐明Mam在哺乳动物系统中的体内功能，我们通过同源重组产生了Mam-1和Mam-2缺陷的小鼠品系。Mam-1缺陷小鼠表现出生长迟缓。这种表型在围产期很明显，并在出生后加重。小鼠在断奶前死亡，表明Mam-1具有非冗余功能，是一种必需基因。相比之下，我们还没有确定Mam-2缺陷小鼠的主要发育缺陷。此外，在Mam-2缺陷小鼠的包括T和B淋巴细胞的造血系统中没有发现缺陷。这些结果表明Mam基因座在脊椎动物系统中具有独特的功能。先天性糖基化IIc障碍（Congenital Disorder of Glycosylation 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遗传综合征。最近，发现负责CDG IIc的基因编码GDP-岩藻糖转运蛋白。已知GDP-岩藻糖对于N-连接聚糖的岩藻糖基化和O-岩藻糖基化至关重要，并且两种岩藻糖修饰均存在于Notch上。我们发现哺乳动物GFR是哺乳动物培养细胞中Notch信号传导所必需的。因此，减少的Notch信号转导与CDG IIc的病理学有关。

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.