Identification of the physiological substrates of PDIp, a pancreas-specific protein-disulfide isomerase family member
Identification of the physiological substrates of PDIp, a pancreas-specific protein-disulfide isomerase family member
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DOI：
10.1074/jbc.ra118.003694
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发表时间：
2018-11-30
影响因子：
4.8
通讯作者：
Kadokura, Hiroshi
中科院分区：
文献类型：
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作者：
Fujimoto, Takushi;Nakamura, Orie;Kadokura, Hiroshi
哺乳动物细胞的内质网中存在大约20个蛋白二硫异构酶（PDI）家族成员。它们被认为催化分泌蛋白或膜蛋白内的硫醇-二硫交换反应，以协助其折叠或调节其功能。PDIp是PDI家族成员，在胰腺中高度表达，已知在体内和体外结合雌激素。然而，PDIp的生理功能尚不清楚。在这项研究中，我们着手确定其生理基础。通过酸猝灭和巯基烷基化相结合，我们稳定并纯化了小鼠胰腺内源性PDIp与其靶蛋白之间形成的复合物。对这些复合物的质谱分析有助于在体内确定二硫化物连接的PDIp靶点，揭示PDIp直接与许多胰腺消化酶相互作用。有趣的是，当胰腺弹性蛋白酶（其中一种已确定的蛋白质）在培养细胞中单独表达时，其前酶在细胞内形成二硫化物连接的聚集体。然而，当胰腺弹性酶与PDIp共表达时，后者阻止了这些聚集体的形成，并以一种可以在胰蛋白酶处理下转化为活性酶的形式增强了前列腺弹性酶的产生和分泌。这些发现表明PDIp的主要靶点是消化酶，PDIp在消化酶的生物合成中起着重要作用，通过协助细胞内原酶的适当折叠。
About 20 members of the protein-disulfide isomerase (PDI) family are present in the endoplasmic reticulum of mammalian cells. They are thought to catalyze thiol-disulfide exchange reactions within secretory or membrane proteins to assist in their folding or to regulate their functions. PDIp is a PDI family member highly expressed in the pancreas and known to bind estrogen in vivo and in vitro. However, the physiological functions of PDIp remained unclear. In this study, we set out to identify its physiological substrates. By combining acid quenching and thiol alkylation, we stabilized and purified the complexes formed between endogenous PDIp and its target proteins from the mouse pancreas. MS analysis of these complexes helped identify the disulfide-linked PDIp targets in vivo, revealing that PDIp interacts directly with a number of pancreatic digestive enzymes. Interestingly, when pancreatic elastase, one of the identified proteins, was expressed alone in cultured cells, its proenzyme formed disulfide-linked aggregates within cells. However, when pancreatic elastase was co-expressed with PDIp, the latter prevented the formation of these aggregates and enhanced the production and secretion of proelastase in a form that could be converted to an active enzyme upon trypsin treatment. These findings indicate that the main targets of PDIp are digestive enzymes and that PDIp plays an important role in the biosynthesis of a digestive enzyme by assisting with the proper folding of the proenzyme within cells.