Human pancreas-specific protein disulfide-isomerase (PDIp) can function as a chaperone independently of its enzymatic activity by forming stable complexes with denatured substrate proteins.
Members of the PDI (protein disulfide-isomerase) family are critical for the correct folding of secretory proteins by catalysing disulfide bond formation as well as by serving as molecular chaperones to prevent protein aggregation. In the present paper, we report that the chaperone activity of the human pancreas-specific PDI homologue (PDIp) is independent of its enzymatic activity on the basis of the following lines of evidence. First, alkylation of PDIp by iodoacetamide fully abolishes its enzymatic activity, whereas it still retains most of its chaperone activity in preventing the aggregation of reduced insulin B chain and denatured GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Secondly, mutation of the cysteine residues in PDIp's active sites completely abolishes its enzymatic activity, but does not affect its chaperone activity. Thirdly, the b-b' fragment of PDIp, which does not contain the active sites and is devoid of enzymatic activity, still has chaperone activity. Mechanistically, we found that both the recombinant PDIp expressed in Escherichia coli and the natural PDIp present in human or monkey pancreas can form stable complexes with thermal-denatured substrate proteins independently of their enzymatic activity. The high-molecular-mass soluble complexes between PDIp and GAPDH are formed in a stoichiometric manner (subunit ratio of 1:3.5-4.5), and can dissociate after storage for a certain time. As a proof-of-concept for the biological significance of PDIp in intact cells, we demonstrated that its selective expression in E. coli confers strong protection of these cells against heat shock and oxidative-stress-induced death independently of its enzymatic activity.
Fu
Xin-Miao
XM
Department of Pharmacology, Toxicology and Therapeutics, School of Medicine, University of Kansas Medical Center, 2146 W. 39th Ave, Kansas City, KS 66160, USA.
Zhu
Bao Ting
BT
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D015415:Biomarkers / Q000378:metabolism; D043424:Carboxypeptidase B / Q000737:chemistry* / Q000378:metabolism*; D002417:Cattle; D016923:Cell Death / Q000502:physiology; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D008253:Macaca mulatta; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D018384:Oxidative Stress; D011485:Protein Binding / Q000502:physiology; D011489:Protein Denaturation / Q000502:physiology; D019704:Protein Disulfide-Isomerases / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D055550:Protein Stability; D011817:Rabbits; D013379:Substrate Specificity / Q000502:physiology
D015415:Biomarkers; C058173:CPB1 protein, human; D018832:Molecular Chaperones; D043424:Carboxypeptidase B; D019704:Protein Disulfide-Isomerases