Abstract
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.
Highlights
About 20 members of the protein-disulfide isomerase (PDI) family are present in the endoplasmic reticulum of mammalian cells
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
The ER of mammalian cells houses more than 20 PDI family members [2]
Summary
About 20 members of the protein-disulfide isomerase (PDI) family are present in the endoplasmic reticulum of mammalian cells. The two cysteines in the active-site motifs of PDI family members are mostly redox-active They can exist in an oxidized (disulfide-bonded) or reduced (free) form. Physiological substrates of PDIp mouse tissues in the presence of 10% TCA using a homogenizer immediately after excising the tissues from a mouse (Fig. 1C) We used this method because protonation of thiols of free cysteines by strong acid rapidly quenches thiol– disulfide exchange reactions in tissue samples [15]. We show that PDIp interacts with a number of digestive enzymes via intermolecular disulfide bonds and can prevent the formation of the disulfide-linked aggregates of one of the identified proteins These findings led to our proposal that another important function of PDIp is to assist in the production of digestive enzymes
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