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Ca2+-dependent redox modulation of SERCA 2b by ERp57.

Li Y, Camacho P - J. Cell Biol. (2003)

Bottom Line: Work from other laboratories demonstrated that CRT also interacts with the ER oxidoreductase, ER protein 57 (also known as ER-60, GRP58; ERp57) during folding of nascent glycoproteins.Interestingly, ERp57 does not affect the activity of SERCA 2a or SERCA 2b mutants lacking the CRT binding site.Our results suggest that ERp57 modulates the redox state of ER facing thiols in SERCA 2b in a Ca2+-dependent manner, providing dynamic control of ER Ca2+ homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Physiology, MSC 7756, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA.

ABSTRACT
We demonstrated previously that calreticulin (CRT) interacts with the lumenal COOH-terminal sequence of sarco endoplasmic reticulum (ER) calcium ATPase (SERCA) 2b to inhibit Ca2+ oscillations. Work from other laboratories demonstrated that CRT also interacts with the ER oxidoreductase, ER protein 57 (also known as ER-60, GRP58; ERp57) during folding of nascent glycoproteins. In this paper, we demonstrate that ERp57 overexpression reduces the frequency of Ca2+ oscillations enhanced by SERCA 2b. In contrast, overexpression of SERCA 2b mutants defective in cysteines located in intralumenal loop 4 (L4) increase Ca2+ oscillation frequency. In vitro, we demonstrate a Ca2+-dependent and -specific interaction between ERp57 and L4. Interestingly, ERp57 does not affect the activity of SERCA 2a or SERCA 2b mutants lacking the CRT binding site. Overexpression of CRT domains that disrupt the interaction of CRT with ERp57 behave as dominant negatives in the Ca2+ oscillation assay. Our results suggest that ERp57 modulates the redox state of ER facing thiols in SERCA 2b in a Ca2+-dependent manner, providing dynamic control of ER Ca2+ homeostasis.

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Enzymatic activity of ERp57 and mutants measured in vitro. (A) ERp57 mutants lacking thioredoxin motifs. Two conserved WCGHCK motifs of ERp57 are thought to be the active sites for thiol-dependent oxidoreductase activity. Cysteines in each motif are indicated in black circles. Mutagenesis of relevant cysteines into serines is indicated by white circles. (B) Thiol-dependent catalytic activity of GST-PDI (positive control) and GST-ERp57 or mutant GST-ERp57 fusion proteins measured in vitro by the insulin turbidity assay at 300 μM [Ca2+]. GST alone and lack of enzyme input are used as negative controls in this assay. Triplicate absorbanceOD 650 values were taken in three independent experiments and plotted as a function of time. Input amount of GST fusion proteins was 0.8 μM except for GST (22.04 μM). (C) Coomassie blue staining of GST and GST fusion proteins as labeled after one-step affinity purification. Proteins (5 μg each) were resolved through 12% SDS-PAGE.
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fig3: Enzymatic activity of ERp57 and mutants measured in vitro. (A) ERp57 mutants lacking thioredoxin motifs. Two conserved WCGHCK motifs of ERp57 are thought to be the active sites for thiol-dependent oxidoreductase activity. Cysteines in each motif are indicated in black circles. Mutagenesis of relevant cysteines into serines is indicated by white circles. (B) Thiol-dependent catalytic activity of GST-PDI (positive control) and GST-ERp57 or mutant GST-ERp57 fusion proteins measured in vitro by the insulin turbidity assay at 300 μM [Ca2+]. GST alone and lack of enzyme input are used as negative controls in this assay. Triplicate absorbanceOD 650 values were taken in three independent experiments and plotted as a function of time. Input amount of GST fusion proteins was 0.8 μM except for GST (22.04 μM). (C) Coomassie blue staining of GST and GST fusion proteins as labeled after one-step affinity purification. Proteins (5 μg each) were resolved through 12% SDS-PAGE.

Mentions: Two conserved thioredoxin motifs in ERp57 are thought to be responsible for catalytic activity of the enzyme (Hirano et al., 1995). One motif is positioned near the NH2-terminus (T1) and the other is near the COOH-terminus (T2). Two single mutants, ERp57-T1 and ERp57-T2, and a double mutant, ERp57-T1T2, were generated by mutagenesis of the relevant cysteines into serines in each motif (Fig. 3 A). To characterize the mutant ERp57 proteins, we generated GST fusion proteins and measured in vitro the catalytic activity using an insulin turbidity assay (Holmgren, 1979; Hirano et al., 1995). Protein disulfide isomerase (PDI), another ER resident oxidoreductase, was used as a positive control for this assay. We cloned the rat isoform and made purified GST-PDI. Both GST-PDI and GST-ERp57 exhibited strong enzymatic activity, although the latter exhibited slower kinetics. In comparison to ERp57, the ERp57-T2 mutant exhibited ∼70% activity, whereas the ERp57-T1 mutant exhibited only ∼30% activity. The double mutation ERp57-T1T2 completely abolished ERp57 enzymatic activity. There was no activity when GST alone or enzyme was absent in the assay (Fig. 3 B). Purified GST and GST fusion proteins are shown (Fig. 3 C). Together, these results suggest that the T1 motif plays a critical role in ERp57 enzymatic activity.


Ca2+-dependent redox modulation of SERCA 2b by ERp57.

Li Y, Camacho P - J. Cell Biol. (2003)

Enzymatic activity of ERp57 and mutants measured in vitro. (A) ERp57 mutants lacking thioredoxin motifs. Two conserved WCGHCK motifs of ERp57 are thought to be the active sites for thiol-dependent oxidoreductase activity. Cysteines in each motif are indicated in black circles. Mutagenesis of relevant cysteines into serines is indicated by white circles. (B) Thiol-dependent catalytic activity of GST-PDI (positive control) and GST-ERp57 or mutant GST-ERp57 fusion proteins measured in vitro by the insulin turbidity assay at 300 μM [Ca2+]. GST alone and lack of enzyme input are used as negative controls in this assay. Triplicate absorbanceOD 650 values were taken in three independent experiments and plotted as a function of time. Input amount of GST fusion proteins was 0.8 μM except for GST (22.04 μM). (C) Coomassie blue staining of GST and GST fusion proteins as labeled after one-step affinity purification. Proteins (5 μg each) were resolved through 12% SDS-PAGE.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171954&req=5

fig3: Enzymatic activity of ERp57 and mutants measured in vitro. (A) ERp57 mutants lacking thioredoxin motifs. Two conserved WCGHCK motifs of ERp57 are thought to be the active sites for thiol-dependent oxidoreductase activity. Cysteines in each motif are indicated in black circles. Mutagenesis of relevant cysteines into serines is indicated by white circles. (B) Thiol-dependent catalytic activity of GST-PDI (positive control) and GST-ERp57 or mutant GST-ERp57 fusion proteins measured in vitro by the insulin turbidity assay at 300 μM [Ca2+]. GST alone and lack of enzyme input are used as negative controls in this assay. Triplicate absorbanceOD 650 values were taken in three independent experiments and plotted as a function of time. Input amount of GST fusion proteins was 0.8 μM except for GST (22.04 μM). (C) Coomassie blue staining of GST and GST fusion proteins as labeled after one-step affinity purification. Proteins (5 μg each) were resolved through 12% SDS-PAGE.
Mentions: Two conserved thioredoxin motifs in ERp57 are thought to be responsible for catalytic activity of the enzyme (Hirano et al., 1995). One motif is positioned near the NH2-terminus (T1) and the other is near the COOH-terminus (T2). Two single mutants, ERp57-T1 and ERp57-T2, and a double mutant, ERp57-T1T2, were generated by mutagenesis of the relevant cysteines into serines in each motif (Fig. 3 A). To characterize the mutant ERp57 proteins, we generated GST fusion proteins and measured in vitro the catalytic activity using an insulin turbidity assay (Holmgren, 1979; Hirano et al., 1995). Protein disulfide isomerase (PDI), another ER resident oxidoreductase, was used as a positive control for this assay. We cloned the rat isoform and made purified GST-PDI. Both GST-PDI and GST-ERp57 exhibited strong enzymatic activity, although the latter exhibited slower kinetics. In comparison to ERp57, the ERp57-T2 mutant exhibited ∼70% activity, whereas the ERp57-T1 mutant exhibited only ∼30% activity. The double mutation ERp57-T1T2 completely abolished ERp57 enzymatic activity. There was no activity when GST alone or enzyme was absent in the assay (Fig. 3 B). Purified GST and GST fusion proteins are shown (Fig. 3 C). Together, these results suggest that the T1 motif plays a critical role in ERp57 enzymatic activity.

Bottom Line: Work from other laboratories demonstrated that CRT also interacts with the ER oxidoreductase, ER protein 57 (also known as ER-60, GRP58; ERp57) during folding of nascent glycoproteins.Interestingly, ERp57 does not affect the activity of SERCA 2a or SERCA 2b mutants lacking the CRT binding site.Our results suggest that ERp57 modulates the redox state of ER facing thiols in SERCA 2b in a Ca2+-dependent manner, providing dynamic control of ER Ca2+ homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Physiology, MSC 7756, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA.

ABSTRACT
We demonstrated previously that calreticulin (CRT) interacts with the lumenal COOH-terminal sequence of sarco endoplasmic reticulum (ER) calcium ATPase (SERCA) 2b to inhibit Ca2+ oscillations. Work from other laboratories demonstrated that CRT also interacts with the ER oxidoreductase, ER protein 57 (also known as ER-60, GRP58; ERp57) during folding of nascent glycoproteins. In this paper, we demonstrate that ERp57 overexpression reduces the frequency of Ca2+ oscillations enhanced by SERCA 2b. In contrast, overexpression of SERCA 2b mutants defective in cysteines located in intralumenal loop 4 (L4) increase Ca2+ oscillation frequency. In vitro, we demonstrate a Ca2+-dependent and -specific interaction between ERp57 and L4. Interestingly, ERp57 does not affect the activity of SERCA 2a or SERCA 2b mutants lacking the CRT binding site. Overexpression of CRT domains that disrupt the interaction of CRT with ERp57 behave as dominant negatives in the Ca2+ oscillation assay. Our results suggest that ERp57 modulates the redox state of ER facing thiols in SERCA 2b in a Ca2+-dependent manner, providing dynamic control of ER Ca2+ homeostasis.

Show MeSH