Cadiñanos Juan, Schmidt Walter K, Fueyo Antonio, Varela Ignacio, López-Otín Carlos, Freije José M P
Departamento de Bioqumica y Biología Molecular, Instituto Universitario de Oncología, Universidad de Oviedo, Campus del Cristo, 33006 Oviedo, Spain.
Biochem J. 2003 Mar 15;370(Pt 3):1047-54. doi: 10.1042/BJ20021514.
Post-translational processing of proteins such as the Ras GTPases, which contain a C-terminal CaaX motif (where C stands for cysteine, a for aliphatic and X is one of several amino acids), includes prenylation, proteolytic removal of the C-terminal tripeptide and carboxy-methylation of the isoprenyl-cysteine residue. In the present study, we report the presence of two distinct CaaX-proteolytic activities in membrane extracts from Caenorhabditis elegans, which are sensitive to EDTA and Tos-Phe-CH(2)Cl (tosylphenylalanylchloromethane; 'TPCK') respectively. A protein similar to the mammalian and yeast farnesylated-proteins converting enzyme-1 (FACE-1)/Ste24p CaaX metalloprotease, encoded by a hypothetical gene (CeFACE-1/C04F12.10) found in C. elegans chromosome I, probably accounts for the EDTA-sensitive activity. An orthologue of FACE-2/Rce1p, the enzyme responsible for the proteolytic maturation of Ras oncoproteins and other prenylated substrates, probably accounts for the Tos-Phe-CH(2)Cl-sensitive activity, even though the gene for FACE-2/Rce1 has not been previously identified in this model organism. We have identified a previously overlooked gene in C. elegans chromosome V, which codes for a 266-amino-acid protein (CeFACE-2) with 30% sequence identity to human FACE-2/Rce1. We show that both CeFACE-1 and CeFACE-2 have the ability to promote production of the farnesylated yeast pheromone a -factor in vivo and to cleave a farnesylated peptide in vitro. These results indicate that CeFACE-1 and CeFACE-2 are bona fide CaaX proteases and support the evolutionary conservation of this proteolytic system in eukaryotes.
蛋白质的翻译后加工，如含有C末端CaaX基序（其中C代表半胱氨酸，a代表脂肪族氨基酸，X是几种氨基酸之一）的Ras GTP酶，包括异戊二烯化、C末端三肽的蛋白水解去除以及异戊二烯基半胱氨酸残基的羧甲基化。在本研究中，我们报告了秀丽隐杆线虫膜提取物中存在两种不同的CaaX蛋白水解活性，它们分别对EDTA和甲苯磺酰苯丙氨酰氯甲烷（tosylphenylalanylchloromethane；“TPCK”）敏感。一种与哺乳动物和酵母法尼基化蛋白转化酶-1（FACE-1）/Ste24p CaaX金属蛋白酶相似的蛋白质，由秀丽隐杆线虫I号染色体上一个假定基因（CeFACE-1/C04F12.10）编码，可能是对EDTA敏感的活性的原因。FACE-2/Rce1p的一个直系同源物，即负责Ras癌蛋白和其他异戊二烯化底物蛋白水解成熟的酶，可能是对甲苯磺酰苯丙氨酰氯甲烷敏感的活性的原因，尽管FACE-2/Rce1的基因此前在这种模式生物中尚未被鉴定。我们在秀丽隐杆线虫V号染色体上鉴定出一个先前被忽视的基因，它编码一种266个氨基酸的蛋白质（CeFACE-2），与人类FACE-2/Rce1有30%的序列同一性。我们表明，CeFACE-1和CeFACE-2都有能力在体内促进法尼基化酵母信息素α-因子的产生，并在体外切割法尼基化肽。这些结果表明CeFACE-1和CeFACE-2是真正的CaaX蛋白酶，并支持了真核生物中这种蛋白水解系统的进化保守性。
