Biological processing of the cocaine and amphetamine-regulated transcript precursors by prohormone convertases, PC2 and PC1/3
Biological processing of the cocaine and amphetamine-regulated transcript precursors by prohormone convertases, PC2 and PC1/3
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DOI：
10.1074/jbc.m212128200
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发表时间：
2003-04-25
影响因子：
4.8
通讯作者：
Steiner, DF
中科院分区：
文献类型：
--
作者：
Dey, A;Xhu, XR;Steiner, DF
可卡因和苯丙胺调节转录物(CART)是一种影响奖赏、摄食/食欲和应激反应的神经内分泌肽，由129和116个氨基酸(AA)组成的两个肽前体通过在大鼠和小鼠中的单个CART基因选择性剪接而产生。信号肽组成第一个27个氨基酸，导致前CART分子102或89个氨基酸。在目前的研究中，我们已经证明PRO-CART是神经内分泌枯草杆菌/可信素样激素转换酶PC2(SPC2)和PC1/3(SPC3)的底物。通过使用不同的神经内分泌细胞系，无论是否内源性表达PC2或PC1/3或两种酶，我们通过瞬时转基因研究证明，长前CART产生一种中间肽，残基33102，以及两种主要的生物活性CART形式，残基分别为55-102(1)和62-102(11)。同样，短亲CART还产生三个肽，一个中间体，残基10-89，两个相同的生物活性CART形成。我们已经通过显微测序和/或高效液相色谱和质谱仪确认了生物活性分子和中间体CART分子的身份。我们已经证明，与PC1/3相比，PC2在产生生物活性购物车I方面更有效，而更小的生物活性购物车11的生产只由PC2进行。PC1/3主要负责从长和短PRO-CART分别生成中间CART片段33-102和10-89。为了比较前CART的体外和体内处理，我们研究了它在PC2、7132和PC1/3基因敲除小鼠下丘脑提取物中的处理，结果表明，在体外，PC2比PC1/3更有效地产生生物活性CART I，而生物活性CART II只由PC2产生。此外，在体内，我们已经证明PC1/3在释放两个中间CART片段33-102和10-方面占主导地位。这些发现证实了PC2和PC1/3在活体中共同或单独作用于选定的位点进行CART加工的关键作用。
Cocaine and amphetamine-regulated transcript (CART), a neuroendocrine peptide influencing reward, feeding/appetite, and stress responses is derived from two peptide precursors of 129 and 116 amino acid (aa) residues that arise via alternative splicing from a single Cart gene in rats and mice. The signal peptide constitutes the first 27 aa resulting in pro-CART molecules of either 102 or 89 aa. In the present study, we have shown that pro-CART is a substrate for the neuroendocrine subtilisin/kexin-like prohormone convertases, PC2 (SPC2) and PC1/3 (SPC3). By using different neuroendocrine cell lines, with or without endogenous expression of either PC2 or PC1/3 or both enzymes, we have demonstrated through transient transfection studies that long pro-CART gives rise to an intermediate peptide, residues 33102, and the two major bioactive CART forms, residues 55-102 (1) and 62-102 (11), respectively. Likewise, short pro-CART also generates three peptides, an intermediate, residues 10-89, and the two identical bioactive CART forms. We have confirmed the identities of the bioactive and intermediate CART molecules by microsequencing and/or high performance liquid chromatography and mass spectrometry. We have shown that PC2 is more efficient in generating bioactive CART I compared with PC1/3, whereas the production of the smaller bioactive CART 11 is exclusively carried out by PC2. PC1/3 is predominantly responsible for generating the intermediate CART fragments, 33-102 and 10-89, from long and short pro-CART, respectively. To compare in vitro and in vivo processing of pro-CART, we have examined its processing in PC2, 7132, and PC1/3 knock-out mouse hypothalamic extracts and demonstrated that, as in vitro, PC2 is more potent than PC1/3 in generating bioactive CART I whereas bioactive CART II is solely generated by PC2. Also, in vivo, we have shown that PC1/3 is predominantly active in liberating the two intermediate CART fragments, 33-102 and 10-89. These findings confirm the key roles of PC2 and PC1/3 acting together or separately to carry out CART processing in selected sites in vivo.