Functional roles and efficiencies of the thioredoxin boxes of calcium-binding proteins 1 and 2 in protein folding

Abstract

The rat luminal endoplasmic-recticulum calcium-binding proteins 1 and 2 (CaBP1 and CaBP2 respectively) are members of the protein disulphide-isomerase (PDI) family. They contain two and three thioredoxin boxes (Cys-Gly-His-Cys) respectively and, like PDI, may be involved in the folding of nascent proteins. We demonstrate here that CaBP1, similar to PDI and CaBP2, can complement the lethal phenotype of the disrupted Saccharomyces cerevisiae PDI gene, provided that the natural C-terminal Lys-Asp-Glu-Leu sequence is replaced by His-Asp-Glu-Leu. Both the in vitro RNase AIII-re-activation assays and in vivo pro-(carboxypeptidase Y) processing assays using CaBP1 and CaBP2 thioredoxin (trx)-box mutants revealed that, whereas the three trx boxes in CaBP2 seem to be functionally equivalent, the first trx box of CaBP1 is significantly more active than the second trx box. Furthermore, only about 65% re-activation of denatured reduced RNase AIII could be obtained with CaBP1 or CaBP2 compared with PDI, and the yield of PDI-catalysed reactions was significantly reduced in the presence of either CaBP1 or CaBP2. In contrast with PDI, neither CaBP1 nor CaBP2 could catalyse the renaturation of denatured glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a redox-independent process, and neither protein had any effect on the PDI-catalysed refolding of GAPDH. Furthermore, although PDI can bind peptides via its b' domain, a property it shares with PDIp, the pancreas-specific PDI homologue, and although PDI can bind malfolded proteins such as 'scrambled' ribonuclease, no such interactions could be detected for CaBP2. We conclude that: (1) both CaBP2 and CaBP1 lack peptide-binding activity for GAPDH attributed to the C-terminal region of the a' domain of PDI; (2) CaBP2 lacks the general peptide-binding activity attributed to the b' domain of PDI; (3) interaction of CaBP2 with substrate (RNase AIII) is different from that of PDI and substrate; and (4) both CaBP2 and CaBP1 may promote oxidative folding by different kinetic pathways.

A role for the thiol isomerase protein ERP5 in platelet function

“…37 To verify that human ERP5 is a functionally active thiol isomerase, we analyzed the recombinant fusion protein in this assay system ( Figure 3A). The protein was found to possess thiol isomerase activity, with activity approximately 70% of that measured for molar equivalents of the PDI recombinant fusion protein.…”

“…On Western blots ( Figure 1B), a protein was detected of the same mobility as that recognized by antibodies to CaBP1, the rat homolog of ERP5 (not shown). 37 These antibodies showed no cross-reactivity with human recombinant and platelet PDI ( Figure 1B).Flow cytometry was employed to confirm cell-surface expression of ERP5 and investigate whether this was a static or dynamic process. Washed platelets were stimulated with agonists convulxin, collagen, or thrombin, and the concentration-and time-dependent patterns of cell-surface exposure for ERP5 were studied (Figure 2).…”

A Developmentally Regulated Chaperone Complex for the Endoplasmic Reticulum of Male Haploid Germ Cells

“…PDI homologues are not functionally equivalent, as shown by differences in their ability to complement a yeast strain deficient for PDI (Gunther et al, 1993;Kramer et al, 2001). Furthermore, the different PDI family members interact with discrete sets of substrates.…”

“…In PDI, these domains have an a-b-bЈ-aЈ organization. PDIp, ERp57, and PDILT share the same domain structure with PDI, but various other configurations of thioredoxin domains exist in other PDI family members, with most PDIs containing at least one a-type domain with an intact active site motif (Ellgaard and Ruddock, 2005).PDI homologues are not functionally equivalent, as shown by differences in their ability to complement a yeast strain deficient for PDI (Gunther et al, 1993;Kramer et al, 2001). Furthermore, the different PDI family members interact with discrete sets of substrates.…”

ERp57 Is a Multifunctional Thiol-Disulfide Oxidoreductase

“…The redox activity of thioredoxin-like domains is provided by two cysteine residues present in a characteristic CXXC sequence motif. For several of the proteins mentioned above, the redox activity has been characterized in vitro (12,14,15), but in many cases the in vivo function is less well studied. Few endogenous substrates have been identified, and it is often not clear whether these PDI homologues serve a role in reduction, oxidation, or disulfide isomerization in the ER.…”