Both PDI and PDIp can attack the native disulfide bonds in thermally-unfolded RNase and form stable disulfide-linked complexes.
Protein disulfide isomerase (PDI) and its pancreatic homolog (PDIp) are folding catalysts for the formation, reduction, and/or isomerization of disulfide bonds in substrate proteins. However, the question as to whether PDI and PDIp can directly attack the native disulfide bonds in substrate proteins is still not answered, which is the subject of the present study. We found that RNase can be thermally unfolded at 65°C under non-reductive conditions while its native disulfide bonds remain intact, and the unfolded RNase can refold and reactivate during cooling. Co-incubation of RNase with PDI or PDIp during thermal unfolding can inactivate RNase in a PDI/PDIp concentration-dependent manner. The alkylated PDI and PDIp, which are devoid of enzymatic activities, cannot inactivate RNase, suggesting that the inactivation of RNase results from the disruption of its native disulfide bonds catalyzed by the enzymatic activities of PDI/PDIp. In support of this suggestion, we show that both PDI and PDIp form stable disulfide-linked complexes only with thermally-unfolded RNase, and RNase in the complexes can be released and reactivated dependently of the redox conditions used. The N-terminal active site of PDIp is essential for the inactivation of RNase. These data indicate that PDI and PDIp can perform thiol-disulfide exchange reactions with native disulfide bonds in unfolded RNase via formation of stable disulfide-linked complexes, and from these complexes RNase is further released.
Department of Pharmacology, Toxicology and Therapeutics School of Medicine, University of Kansas Medical Center, Kansas City, KS 66160, USA.
Xin-Miao
XM
Fu
Bao Ting
BT
Zhu
D000478:Alkylation; D000818:Animals; D020134:Catalytic Domain; D002417:Cattle; D004220:Disulfides / Q000378:metabolism*; D004789:Enzyme Activation; D004795:Enzyme Stability; D019803:Glutathione Disulfide / Q000378:metabolism; D010084:Oxidation-Reduction; D010179:Pancreas / Q000201:enzymology*; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*; D058767:Protein Unfolding; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors
D004220:Disulfides; D012259:Ribonuclease, Pancreatic; D019704:Protein Disulfide-Isomerases; D019803:Glutathione Disulfide
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