Stoller T J, Shields D
Department of Anatomy, Albert Einstein College of Medicine, Bronx, New York 10461.
J Biol Chem. 1989 Apr 25;264(12):6922-8.
Many small peptide hormones are synthesized as larger precursors in which the mature hormone sequence is flanked by pairs of basic amino acids. These precursors often undergo extensive post-translational modifications; a critical step in this process is proteolytic excision of the hormone at the paired basic residues. To determine the role of paired basic amino acids as recognition signals for cleavage by processing enzymes, we investigated the heterologous expression of prosomatostatin (the pro-somatotropin release inhibiting factor (pro-SRIF). Pro-SRIF is one of the simplest peptide hormone precursors, possessing a single copy of the 14-residue SRIF peptide at its carboxyl terminus preceded by the least common pair of basic amino acids, Arg-Lys. Employing site-directed mutagenesis, we altered the paired basic cleavage site to the more common Arg-Arg and Lys-Arg residues. The native and mutated precursors were expressed in rat pituitary GH3 cells and mouse 3T3 cells using a retroviral vector. Alteration of the paired basic residues had no effect on the specificity of proteolytic cleavage as both the native and mutant precursors were processed with 70 to 80% efficiency in GH3 cells. Surprisingly, when the mutant pro-SRIFs were expressed in 3T3 cells, which do not process the native precursor, the Arg-Arg and Lys-Arg precursors were processed with 16 and 20% efficiency, respectively. The role of an acidic compartment in mediating pro-SRIF cleavage was also investigated using low concentrations of the lysosomotrophic drug Chloroquine. Twenty-five microM Chlorquine completely inhibited pro-SRIF cleavage and intracellular storage; the unprocessed precursor was secreted into the medium. We conclude that (i) exposure to an acidic compartment is required for pro-SRIF maturation, and (ii) the conformation of the processing site, rather than the composition of the basic amino acids, defines cleavage specificity by prohormone processing enzymes.
许多小肽激素都是作为较大的前体合成的，在这些前体中，成熟激素序列两侧是成对的碱性氨基酸。这些前体通常会经历广泛的翻译后修饰；这一过程中的关键步骤是在成对的碱性残基处对激素进行蛋白水解切割。为了确定成对碱性氨基酸作为加工酶切割识别信号的作用，我们研究了前生长抑素（促生长激素释放抑制因子前体（pro-SRIF））的异源表达。Pro-SRIF是最简单的肽激素前体之一，在其羧基末端有一个14个残基的SRIF肽单拷贝，前面是最不常见的一对碱性氨基酸，即精氨酸-赖氨酸。利用定点诱变，我们将成对的碱性切割位点改变为更常见的精氨酸-精氨酸和赖氨酸-精氨酸残基。使用逆转录病毒载体在大鼠垂体GH3细胞和小鼠3T3细胞中表达天然和突变的前体。成对碱性残基的改变对蛋白水解切割的特异性没有影响，因为天然和突变前体在GH3细胞中的加工效率均为70%至80%。令人惊讶的是，当突变的pro-SRIFs在不加工天然前体的3T3细胞中表达时，精氨酸-精氨酸和赖氨酸-精氨酸前体的加工效率分别为16%和20%。还使用低浓度的溶酶体营养药物氯喹研究了酸性区室在介导pro-SRIF切割中的作用。25微摩尔氯喹完全抑制pro-SRIF切割和细胞内储存；未加工的前体分泌到培养基中。我们得出结论：（i）pro-SRIF成熟需要暴露于酸性区室，（ii）加工位点的构象而非碱性氨基酸的组成决定了激素原加工酶切割的特异性。
