Brooks Darren R, Hooper Nigel M, Isaac R Elwyn
Molecular and Cellular Biosciences, Faculty of Biological Sciences, University of Leeds, Miall Bldg., Leeds, West Yorkshire LS2 9JT, United Kingdom.
J Biol Chem. 2003 Oct 31;278(44):42795-801. doi: 10.1074/jbc.M306216200. Epub 2003 Aug 20.
Mammals possess membrane-associated and cytosolic forms of the puromycin-sensitive aminopeptidase (PSA; EC 3.4.11.14). Increasing evidence suggests the membrane PSA is involved in neuromodulation within the central nervous system and in reproductive biology. The functional roles of the cytosolic PSA are less clear. The genome of the nematode Caenorhabditis elegans encodes an aminopeptidase, F49E8.3 (PAM-1), that is orthologous to PSA, and sequence analysis predicts it to be cytosolic. We have determined the spatio/temporal gene expression pattern of pam-1 by using the promoter region of F49E8.3 to control expression in the nematode of a second exon translational fusion of the aminopeptidase to green fluorescent protein. Cytosolic fluorescence was observed throughout development in the intestine and nerve cells of the head. Neuronal expression was also observed in the tail of adult males. Recombinant PAM-1, expressed and purified from Escherichia coli, hydrolyzed the N-terminal amino acid from peptide substrates. Favored substrates had positively charged or small neutral amino acids in the N-terminal position. Peptide hydrolysis was inhibited by the metal-chelating agent 1,10-phenanthroline and by the aminopeptidase inhibitors actinonin, amastatin, and leuhistin. However, the enzyme was approximately 100-fold less sensitive toward puromycin (IC50, 135 mum) than other PSA homologues. Following inactivation of the enzyme, aminopeptidase activity was recovered with Zn2+, Co2+, and Ni2+. Silencing expression of pam-1 by RNA interference resulted in 30% embryonic lethality. Surviving adult hermaphrodites deposited large numbers of oocytes throughout the self-fertile period. The overall brood size was, however, unaffected. We conclude that pam-1 encodes an aminopeptidase that clusters phylogenetically with the PSAs, despite attenuated sensitivity toward puromycin, and that it functions in embryo development and reproduction of the nematode.
哺乳动物拥有嘌呤霉素敏感氨基肽酶（PSA；EC 3.4.11.14）的膜相关形式和胞质形式。越来越多的证据表明，膜PSA参与中枢神经系统内的神经调节以及生殖生物学过程。胞质PSA的功能作用尚不清楚。线虫秀丽隐杆线虫的基因组编码一种氨基肽酶F49E8.3（PAM-1），它与PSA是直系同源物，序列分析预测它是胞质的。我们通过使用F49E8.3的启动子区域来控制氨基肽酶与绿色荧光蛋白的第二个外显子翻译融合体在秀丽隐杆线虫中的表达，从而确定了pam-1的时空基因表达模式。在整个发育过程中，在头部的肠道和神经细胞中观察到胞质荧光。在成年雄性的尾部也观察到神经元表达。从大肠杆菌中表达并纯化的重组PAM-1从肽底物上水解N端氨基酸。偏好的底物在N端位置带有带正电荷或小的中性氨基酸。肽水解受到金属螯合剂1,10-菲咯啉以及氨基肽酶抑制剂放线菌酮、抑氨肽酶素和亮抑酶肽的抑制。然而，该酶对嘌呤霉素的敏感性（IC50为135 μmol）比其他PSA同源物低约100倍。酶失活后，用Zn2+、Co2+和Ni2+可恢复氨基肽酶活性。通过RNA干扰使pam-1的表达沉默导致30%的胚胎致死率。存活的成年雌雄同体在整个自体受精期都产下大量卵母细胞。然而，总产卵量未受影响。我们得出结论，pam-1编码一种氨基肽酶，尽管对嘌呤霉素的敏感性减弱，但在系统发育上与PSA聚类，并且它在秀丽隐杆线虫的胚胎发育和繁殖中发挥作用。
