Differential cooperative enzymatic activities of protein disulfide isomerase family in protein folding - PubMed (original) (raw)

Comparative Study

. 2005 Autumn;10(3):211-20.

doi: 10.1379/csc-109r.1.

Affiliations

Comparative Study

Differential cooperative enzymatic activities of protein disulfide isomerase family in protein folding

Mamoru Satoh et al. Cell Stress Chaperones. 2005 Autumn.

Abstract

Endoplasmic reticulum (ER)p61, ERp72, and protein disulfide isomerase (PDI), which are members of the PDI family protein, are ubiquitously present in mammalian cells and are thought to participate in disulfide bond formation and isomerization. However, why the 3 different members need to be colocalized in the ER remains an enigma. We hypothesized that each PDI family protein might have different modes of enzymatic activity in disulfide bond formation and isomerization. We purified PDI, ERp61, and ERp72 proteins from rat liver microsomes and compared the effects of each protein on the folding of bovine pancreatic trypsin inhibitor (BPTI). ERp61 and ERp72 accelerated the initial steps more efficiently than did PDI. ERp61 and ERp72, however, accelerated the rate-limiting step less efficiently than did PDI. PDI or ERp72 did not impede the folding of BPTI by each other but rather catalyzed the folding reaction cooperatively with each other. These data suggest that differential enzymatic activities of ERp proteins and PDI represent a complementary contribution of these enzymes to protein folding in the ER.

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Figures

Fig 2.

Fig 2.

Time course of folding of reduced BPTI in the presence of ERp protein with both 2.0 mM GSH and 0.5 mM GSSG. N′ [5-SSG; 55-SSG] denotes the double-mixed disulfide derivative of N′, in which Cys 5 and Cys 55 are each disulfide bonded to glutathione. The reactions with no enzyme (A), with PDI (B), with ERp61 (C), and with ERp72 (D) are indicated. BPTI, bovine pancreatic trypsin inhibitor; GSSG, glutathione disulfide; PDI, protein disulfide isomerase; ER, endoplasmic reticulum

Fig 3.

Fig 3.

Catalysis by ERp protein of the intramolecular rearrangement of the native 2-disulfide intermediate, N′, with both 2.0 mM GSH and 0.5 mM GSSG. The reaction with no enzyme (A), with PDI (B), with ERp72 (C), and with ERp61 (D) are indicated. (E) The kinetics of the native form:total BPTI ratio. The area under the curve of elution profiles corresponding to intermediates (N′, N*, and

NSHSH

) and native form (N) was measured using National Institutes of Health image. Molecular ratios are indicated in arbitrary values. ERp61 and ERp72 accelerated the conversion of N′ to N, but less efficiently than did PDI, and hardly converted N′ to N*. Open circles, no enzyme; closed circles, 1.5 μM PDI; open squares, 1.5 μM ERp61; closed squares, 1.5 μM ERp72. GSSG, glutathione disulfide; PDI, protein disulfide isomerase; ER, endoplasmic reticulum

Fig 4.

Fig 4.

Catalysis by ERp protein of the intramolecular rearrangement of the native 2-disulfide intermediate, N*, with both 2.0 mM GSH and 0.5 mM GSSG. The reactions with no enzyme (A), with PDI (B), with ERp72 (C), and with ERp61 (D) are indicated. (E) The kinetics of the native form:total BPTI ratio. Values were determined in the same manner as in Figure 2. PDI efficiently catalyzed the conversion of N* to N. ERp61 and ERp72 barely accelerated the conversion of N* to N in comparison with reaction in the absence of enzyme. Open circles, no enzyme; closed circles, 1.5 μM PDI; open squares, 1.5 μM ERp61; closed squares, 1.5 μM ERp72. GSSG, glutathione disulfide; PDI, protein disulfide isomerase; ER, endoplasmic reticulum

Fig 5.

Fig 5.

Catalysis by ERp protein of the intramolecular rearrangement of N′ in the absence of redox reagents. The reactions with PDI (A), with ERp72 (B), and with ERp61 (C) are indicated. PDI, protein disulfide isomerase; ER, endoplasmic reticulum

Fig 6.

Fig 6.

Conversion of fully reduced BPTI to intermediates bearing single or double disulfide bond. (A) Time course of decrease in the amount of starting material R, the fully reduced BPTI. (B) Time course of increase in the amount of intermediates bearing single disulfide bond [30–51] and [5–55]. (C) Time course of increase in the amount of intermediates bearing double disulfide bonds, N′, N*, and N′ [5-SSG, 55-SSG]. Values were determined in the same manner as in Figure 3. Circles, no enzyme; squares, PDI; triangles, ERp61; diamonds, ERp72. BPTI, bovine pancreatic trypsin inhibitor; PDI, protein disulfide isomerase; ER, endoplasmic reticulum

Fig 7.

Fig 7.

Cooperation of PDI family proteins in the folding of BPTI. The ratio of fully folded BPTI to the starting material after the reduced BPTI was incubated for 1 minute. The reactions were initiated with either PDI alone, ERp72 alone, or 3 kinds of the combination of PDI and ERp72. Values were determined in the same manner as in Figure 2. Experiments were done 3 or 5 times in each group. Values are indicated as mean ± SD. 1:0, 1.5 μM PDI; 3:1, 1.125 μM PDI + 0.375 μM ERp72; 1:1, 0.75 μM PDI + 0.75 μM ERp72; 1:3, 0.375 μM PDI + 1.125 μM ERp72; 0:1, 1.5 μM ERp72. **P < 0.01. BPTI, bovine pancreatic trypsin inhibitor; PDI, protein disulfide isomerase; ER, endoplasmic reticulum

Fig 1.

Fig 1.

Preparation of PDI family proteins and the pathway of BPTI folding. (A) PDI, ERp61, and ERp72 were prepared from rat liver microsomes as described in Materials and Methods. The proteins were observed by coomassie brilliant blue R-250 staining. Lane 1: PDI; lane 2: ERp61; lane 3: ERp72

Fig 1.

Fig 1.

(Continued) (B) Schematic representation of the folding pathway for BPTI. PDI, protein disulfide isomerase; BPTI, bovine pancreatic trypsin inhibitor; ER, endoplasmic reticulum

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