Chaperone‐Like Activity of Protein Disulfide‐Isomerase in the Refolding Of Rhodanese

Abstract

Protein disulfide-isomerase (PDI) in near stoichiometric concentrations promotes reactivation and prevents aggregation of guanidine-hydrochloride-denatured rhodanese during refolding upon dilution. PDI also suppresses aggregation of rhodanese during thermal inactivation. The above-mentioned properties displayed by PDI completely satisfy the definition of chaperone and provide additional evidence to confirm the hypothesis proposed previously [Wang, C. C. & Tsou, C. L. (1993) FASEB J. 7, 1515-15171 that PDI is both an enzyme and a chaperone. Since rhodanese contains no disulfide bonds, the chaperonelike activity of PDI acting on rhodanese is independent of its disulfide-isomerase activity.Keywords. Protein disulfide-isomerase ; chaperone ; rhodanese ; refolding ; aggregation.Protein disulfide isomerase (PDI) is one of the two characterized foldases [I] and is believed to assist nascent peptide folding by catalyzing the formation of disulfide bonds during protein biogenesis [2]. In view of its catalysis of the joining of thiols distantly situated in the peptide sequences and its peptide-binding property with low specificity [3], we have hypothesized that PDI may act both as an enzyme and a chaperone in protein folding [4]. Recently, we have shown that PDI assists the refolding of Gdn . HC1 (guanidine hydrochloride)-denatured GraP-DH (~-glyceraldehyde-3-phosphate dehydrogenase), a protein containing no disulfide bonds, to its native state and increases its reactivation yield by suppressing aggregation in a way closely similar to the action of a chaperone [5]. It has also been suggested by a few authors that PDI may have chaperone function reported the chaperone and antichaperone activity of PDI in the oxidative refolding of lysozyme. However, Lilie et al. [lo] found no chaperone-like effect of PDI on the refolding of the antibody Fab (antigen-binding antibody fragment consisting of the entire light chain and the two N-terminal domains of the heavy chain) with intact disulfide bonds at pH 7.0 under oxidizing conditions. LaMantia and Lennarz [I I] indicated that the isomerase activity is not essential for cell viability, which may require instead some other functions of PDI.In order to provide more evidence on the chaperone activity of PDI with a target protein that has been well studied in its chaperone-assisted folding 112, 131, we have studied the PDIassisted refolding of Gdn . HC1-denatured rhodanese, which consists of a single chain folded into two domains of equal size containing four cysteine residues [14]. The sequence retains all the information required for targeting, transport, folding, and activity [15], which makes rhodanese a valuable model to study the folding processes of single chain and multidomain proteins. As rhodanese has no disulfide bonds, it is possible to analyze the chaperone activity of PDI independent of its isomerase activity. With target proteins containing disulfide bonds like lysozyme or immunoglobulin, it is difficult to exclude the possibility of disulfide isomerization as the prim...

The proteasomal system

“…Since virtually every thiol of a protein can form a disulfide bridge with another one, that formation represents important steps in proper protein maturation and post-translational modification. The chaperone activity of PDI has already been shown in the ability of PDI to avoid the formation of protein aggregates (Song and Wang, 1995;Winter et al, 2002). The affinity of PDI to unfolded or non-native, proteins is both high and unspecific.…”

Evaluation of the transcription level of the protein disulfide isomerase in different stages from Ancylostoma caninum with a real-time PCR assay

“…The proposed function is a catalyzator for the folding process and association of proteins, supporting the oxidation of free sulfhydryl groups for building of disulfide bridges (Wilkinson and Gilbert 2004). Other functions such as the chaperone-like activity of PDI (Song and Wang 1995;Wang and Tsou 1993) or the redox isomerase function (Darby et al 1998) are discussed and are described elsewhere (Pohl 2006). In trematodes such as Schistosoma mansoni, PDI genes are described to encode one or more PDI isoforms, whereas in nematodes such as Onchocerca volvulus, the PDI is a single-copy gene (Wilson et al 1994).…”

Bacitracin is not a specific inhibitor of protein disulfide isomerase

“…In addition to disulfide bond formation, PDI has been shown to have chaperone‐like activity [34]. As rhodanese contains no disulfide bonds, and is prone to aggregation during refolding, it can be used as a model with which to study chaperone activity in folding.…”

“…The molecular chaperone‐like activity of PDI was monitored using a slightly modified version of the rhodanese assay used by Song and Wang [34]. Rhodanese from bovine liver (Sigma‐Aldrich) was denatured to a final concentration of 45 μ m in 0.2 m sodium phosphate buffer (pH 7.2) containing 6 m guanidine hydrochloride and 10 m m dithiothreitol for 45 min at room temperature.…”