Zambrano Nicola, Bimonte Marida, Arbucci Salvatore, Gianni Davide, Russo Tommaso, Bazzicalupo Paolo
Dipartimento di Biochimica e Biotecnologie Mediche, Università degli Studi di Napoli Federico II, Via S. Pansini, 5, I-80131, Napoli, Italy.
J Cell Sci. 2002 Apr 1;115(Pt 7):1411-22. doi: 10.1242/jcs.115.7.1411.
The multigenic family of mammalian Fe65s encodes three highly similar proteins with the same modular organisation: a WW domain and two phosphotyrosine-binding domains. The PTB2 domain of these proteins binds to the cytosolic domains of the Alzheimer's beta-amyloid precursor protein APP and related proteins APLP1 and APLP2, generating a highly redundant system that is hard to dissect by reverse genetics. By searching potential Fe65-like genes in the nematode Caenorhabditis elegans, we identified a single gene, feh-1 (Fe65 homolog-1), encoding a protein with a high sequence similarity to mammalian Fe65s. FEH-1 is also functionally related to mammalian orthologues; in fact its PTB2 domain binds to APL-1, the product of the C. elegans orthologue of APP. Staining with specific antibodies show that the neuromuscular structures of the pharynx are the sites in which FEH-1 is present at highest levels. Expression studies with reporters indicate that the feh-1 gene is also expressed by a subset of the worm neurons. We generated and isolated a deletion allele of feh-1, and the corresponding homozygous mutants arrest as late embryos or as L1 larvae, demonstrating for the first time an essential role for a Fe65-like gene in vivo. The pharynx of homozygous larvae does not contract and the worms cannot feed. Analysis of pharyngeal pumping in heterozygous worms and in feh-1 RNA-interfered worms indicates that dosage of feh-1 function affects the rate of pharyngeal contraction in C. elegans. Interference with apl-1 double-stranded RNA showed a similar effect on pharyngeal pumping, suggesting that FEH-1 and APL-1 are involved in the same pathway. The non-redundant system of the nematode will prove useful for studying the basic biology of the Fe65-APP interaction and the molecular events regulated by this evolutionarily conserved system of interacting proteins.
哺乳动物Fe65s的多基因家族编码三种高度相似的蛋白质,它们具有相同的模块化结构:一个WW结构域和两个磷酸酪氨酸结合结构域。这些蛋白质的PTB2结构域与阿尔茨海默病β-淀粉样前体蛋白APP以及相关蛋白APLP1和APLP2的胞质结构域结合,形成了一个高度冗余的系统,难以通过反向遗传学进行剖析。通过在秀丽隐杆线虫中搜索潜在的Fe65样基因,我们鉴定出了一个单一基因feh-1(Fe65同源物-1),它编码一种与哺乳动物Fe65s具有高度序列相似性的蛋白质。FEH-1在功能上也与哺乳动物的直系同源物相关;事实上,它的PTB2结构域与APL-1结合,APL-1是秀丽隐杆线虫APP直系同源物的产物。用特异性抗体染色显示,咽部的神经肌肉结构是FEH-1水平最高的部位。用报告基因进行的表达研究表明,feh-1基因也在一部分线虫神经元中表达。我们产生并分离出了feh-1的缺失等位基因,相应的纯合突变体在晚期胚胎或L1幼虫阶段停滞发育,首次证明了Fe65样基因在体内的重要作用。纯合幼虫的咽部不收缩,线虫无法进食。对杂合线虫和经feh-1 RNA干扰的线虫的咽部抽吸分析表明,feh-1功能的剂量会影响秀丽隐杆线虫咽部收缩的速率。干扰apl-1双链RNA对咽部抽吸也有类似影响,表明FEH-1和APL-1参与同一途径。线虫的非冗余系统将被证明有助于研究Fe65-APP相互作用的基础生物学以及由这种进化上保守的相互作用蛋白系统调控的分子事件。