Abstract
Simple SummaryThe physiological functions of proteins are destined by their unique three-dimensional structures. Almost all biological kingdoms share conserved disulfide-catalysts and chaperone networks that assist in correct protein folding and prevent aggregation. Disruption of these networks is implicated in pathogenesis, including neurodegenerative disease. In the mammalian endoplasmic reticulum (ER), more than 20 members of the protein disulfide isomerase family (PDIs) are believed to cooperate in the client folding pathway, but it remains unclear whether complex formation among PDIs via non-covalent interaction is involved in regulating their enzymatic and chaperone functions. Herein, we report novel functional hetero complexes between PDIs that promote oxidative folding and inhibit aggregation along client folding. The findings provide insight into the physiological significance of disulfide-catalyst and chaperone networks and clues for understanding pathogenesis associated with disruption of the networks.P5 is one of protein disulfide isomerase family proteins (PDIs) involved in endoplasmic reticulum (ER) protein quality control that assists oxidative folding, inhibits protein aggregation, and regulates the unfolded protein response. P5 reportedly interacts with other PDIs via intermolecular disulfide bonds in cultured cells, but it remains unclear whether complex formation between P5 and other PDIs is involved in regulating enzymatic and chaperone functions. Herein, we established the far-western blot method to detect non-covalent interactions between P5 and other PDIs and found that PDI and ERp72 are partner proteins of P5. The enzymatic activity of P5-mediated oxidative folding is up-regulated by PDI, while the chaperone activity of P5 is stimulated by ERp72. These findings shed light on the mechanism by which the complex formations among PDIs drive to synergistically accelerate protein folding and prevents aggregation. This knowledge has implications for understanding misfolding-related pathology.
Highlights
Numerous amount of secretory and membrane proteins that are inserted into the mammalian endoplasmic reticulum (ER) must undergo disulfide bond formation-coupled protein folding, oxidative folding, to form unique functional three-dimensional structures [1,2,3]
There was no signal for P5 bound to the secretory proteins lactoferrin and albumin, but positive signals were observed for the ER stress sensor IRE1, which is regulated by P5 [36,39] (Figure 1b)
The Isothermal titration calorimetry (ITC) analyses revealed that the change in enthalpy (∆H) of P5 binding to protein disulfide isomerase family (PDIs) and ERp72 was −4.4 ± 0.2 and −10.7 ± 0.8 kcal mol−1, respectively
Summary
Numerous amount of secretory and membrane proteins that are inserted into the mammalian endoplasmic reticulum (ER) must undergo disulfide bond formation-coupled protein folding, oxidative folding, to form unique functional three-dimensional structures [1,2,3]. Non-native disulfide-bonded misfolded proteins frequently lead to the formation of aggregates [4,5,6,7]. To reduce the risk of accumulation of aberrant proteins, almost all biological kingdoms have evolved both disulfide-catalyst and chaperone networks that assist proper folding [8,9,10]. More than 20 members of the protein disulfide isomerase family (PDIs), e.g., PDI, ERp57, ERp72, ERp46, and P5, and other ER-resident chaperones constitute a protein homeostasis network that catalyzes oxidative folding and inhibits aggregation to ensure proper protein production in the mammalian ER [11,12,13,14,15]. Many researchers have vigorously investigated the disulfide-catalyst and molecular chaperone activities of PDI [21,22,23,24,25,26,27,28,29,30], the underlying mechanism of how other PDIs modulate the assistance of oxidative folding and the inhibition of client aggregation remains unclear [31]
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