Yerlikaya Azmi, Stanley Bruce A
Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA.
J Biol Chem. 2004 Mar 26;279(13):12469-78. doi: 10.1074/jbc.M312625200. Epub 2004 Jan 12.
The short-lived enzyme S-adenosylmethionine decarboxylase uses a covalently bound pyruvoyl cofactor to catalyze the formation of decarboxylated S-adenosylmethionine, which then donates an aminopropyl group for polyamine biosynthesis. Here we demonstrate that S-adenosylmethionine decarboxylase is ubiquitinated and degraded by the 26 S proteasome in vivo, a process that is accelerated by inactivation of S-adenosylmethionine decarboxylase by substrate-mediated transamination of its pyruvoyl cofactor. Proteasome inhibition in COS-7 cells prevents the degradation of S-adenosylmethionine decarboxylase antigen; however, even brief inhibition of the 26 S proteasome caused substantial losses of S-adenosylmethionine decarboxylase activity despite accumulation of S-adenosylmethionine decarboxylase antigen. Levels of the enzyme's substrate (S-adenosylmethionine) increased rapidly after 26 S proteasome inhibition, and this increase in substrate level is consistent with the observed loss of activity arising from an increased rate of inactivation by substrate-mediated transamination. Evidence is also presented that this substrate-mediated transamination accelerates normal degradation of S-adenosylmethionine decarboxylase, as the rate of degradation of the enzyme was increased in the presence of AbeAdo (5'-([(Z)-4-amino-2-butenyl]methylamino]-5'-deoxyadenosine) (a substrate analogue that transaminates the enzyme); conversely, when the intracellular substrate level was reduced by methionine deprivation, the rate of degradation of the enzyme was decreased. Ubiquitination of S-adenosylmethionine decarboxylase is demonstrated by isolation of His-tagged AdoMetDC (S-adenosylmethionine decarboxylase) from COS-7 cells co-transfected with hemagglutinin-tagged ubiquitin and showing bands that were immunoreactive to both anti-AdoMetDC antibody and anti-hemagglutinin antibody. This is the first study to demonstrate that AdoMetDC is ubiquitinated and degraded by the 26 S proteasome, and substrate-mediated acceleration of degradation is a unique finding.
寿命较短的S-腺苷甲硫氨酸脱羧酶利用共价结合的丙酮酰辅因子催化形成脱羧的S-腺苷甲硫氨酸，然后该物质为多胺生物合成提供一个氨丙基。在此我们证明，S-腺苷甲硫氨酸脱羧酶在体内被26S蛋白酶体泛素化并降解，该过程会因丙酮酰辅因子的底物介导转氨作用导致S-腺苷甲硫氨酸脱羧酶失活而加速。在COS-7细胞中抑制蛋白酶体可防止S-腺苷甲硫氨酸脱羧酶抗原的降解；然而，即使对26S蛋白酶体进行短暂抑制，尽管S-腺苷甲硫氨酸脱羧酶抗原会积累，但仍会导致S-腺苷甲硫氨酸脱羧酶活性大幅丧失。26S蛋白酶体抑制后，该酶底物（S-腺苷甲硫氨酸）的水平迅速升高，底物水平的这种升高与观察到的因底物介导转氨作用导致失活速率增加而引起的活性丧失一致。同时也有证据表明，这种底物介导的转氨作用会加速S-腺苷甲硫氨酸脱羧酶的正常降解，因为在存在AbeAdo（5'-([(Z)-4-氨基-2-丁烯基]甲基氨基]-5'-脱氧腺苷）（一种使该酶转氨的底物类似物）时，该酶的降解速率会增加；相反，当通过蛋氨酸剥夺降低细胞内底物水平时，该酶的降解速率会降低。通过从与血凝素标记的泛素共转染的COS-7细胞中分离出组氨酸标记的AdoMetDC（S-腺苷甲硫氨酸脱羧酶），并显示出对抗AdoMetDC抗体和抗血凝素抗体均有免疫反应性的条带，证明了S-腺苷甲硫氨酸脱羧酶的泛素化。这是第一项证明AdoMetDC被26S蛋白酶体泛素化并降解的研究，而底物介导的降解加速是一个独特的发现。
