A Mutant Truncated Protein Disulfide Isomerase with No Chaperone Activity

Dai, Yong; Wang, Chih-chen

doi:10.1074/jbc.272.44.27572

Cited by 40 publications

(33 citation statements)

References 38 publications

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“…Although deletion of this sequence does not disrupt the redox activity of the protein [98], elimination of the terminal 51 amino acids from PDI results in an inability to refold denatured lysozyme and a reduced rate of refolding of certain disulphide-containing substrates (lysozyme, acidic phospholipase A # ), indicating the presence of a distinct peptide-binding site and the co-operation of peptide-binding and disulphide redox activities in the folding process [99].…”

Section: Peptide Bindingmentioning

confidence: 99%

The protein disulphide-isomerase family: unravelling a string of folds

Ferrari

Söling

1999

Biochemical Journal

399

359

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The mammalian protein disulphide-isomerase (PDI) family encompasses several highly divergent proteins that are involved in the processing and maturation of secretory proteins in the endoplasmic reticulum. These proteins are characterized by the presence of one or more domains of roughly 95-110 amino acids related to the cytoplasmic protein thioredoxin. All but the PDI-D subfamily are composed entirely of repeats of such domains, with at least one domain containing and one domain lacking a redox-active -Cys-Xaa-Xaa-Cys-tetrapeptide. In addition to their known roles as redox catalysts and isomerases, the last few years have revealed additional functions of the PDI proteins,

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Section: Peptide Bindingmentioning

confidence: 99%

The protein disulphide-isomerase family: unravelling a string of folds

Ferrari

Söling

1999

Biochemical Journal

399

359

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“…The mutant PDI, with its C-terminal 51 amino acid residues responsible for peptide binding deleted, shows neither peptide binding ability nor chaperone activity in assisting the refolding of denatured GAPDH, but keeps most of its catalytic activities [19]. The above provides a straightforward demonstration that the peptide binding site is directly responsible for 0014-5793/98/$19.00 ß 1998 Federation of European Biochemical Societies.…”

Section: Chaperone Activity Of Protein Disul¢de Isomerasementioning

confidence: 82%

“…PDI alkylated at the active site thiols shows little isomerase activity but retains nearly full ability for increasing reactivation and decreasing aggregation during refolding of denatured GAPDH as that of native PDI [17], suggesting that the chaperone activity of PDI is independent of its -CGHC-active sites. It has also been found that mutant PDI with no isomerase activity is still in many in vivo cases functional for cell viability [18], assisting folding of other proteins [11], and assembly of prolyl-4-hydroxylase [12] and microsomal triglyceride transfer protein complex [13] as essential subunits.The mutant PDI, with its C-terminal 51 amino acid residues responsible for peptide binding deleted, shows neither peptide binding ability nor chaperone activity in assisting the refolding of denatured GAPDH, but keeps most of its catalytic activities [19]. The above provides a straightforward demonstration that the peptide binding site is directly responsible for 0014-5793/98/$19.00 ß 1998 Federation of European Biochemical Societies.…”

mentioning

confidence: 82%

“…For the maximal refolding and reactivation of denatured and reduced acidic phospholipase A P , a protein composed of 124 amino acid residues and seven disul¢de bonds, 90% of the native PDI in the refolding bu¡er can be replaced by the alkylated PDI with only chaperone but no isomerase activity [17], indicating that the in vitro action of PDI as a foldase consists of both its isomerase and chaperone activities [10]. The simultaneous presence of the C-terminal truncated mutant PDI with no peptide binding site [19] and the alkylated PDI with a peptide binding site but no isomerase activity [17] also shows a collaborative action to promote the reactivation of acidic phospholipase A P [19].…”

Section: Chaperone Activity Of Protein Disul¢de Isomerasementioning

confidence: 99%

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Enzymes as chaperones and chaperones as enzymes

Wang

Tsou

1998

FEBS Letters

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Chaperones and foldases are two groups of accessory proteins which assist maturation of nascent peptides into functional proteins in cells. Protein disulfide isomerase, a foldase, and ATP-dependent proteases, responsible for degradation of misfolded proteins in cells, both have intrinsic chaperone activities. Trigger factor and DnaJ, well known Escherichia coli chaperones, show peptidyl prolyl isomerase and protein disulfide isomerase activities respectively. It is suggested that the combination of chaperone and enzyme activities in one molecule is the result of evolution to increase molecular efficiency.z 1998 Federation of European Biochemical Societies.Key words: Foldase; Chaperone; Protein disul¢de isomerase ; Trigger factor; ATP-dependent protease; Protein folding From spontaneous to assisted foldingIt is now widely accepted that a large number of proteins do not fold and assemble spontaneously in vivo to form the biologically active molecules but require the help of other proteins. This results in a conceptional transition for protein folding: from the classical`self-assembly' to the new`assisted assembly' principle [1]. Most in vitro experiments employed conditions considerably di¡erent from the in vivo situation of high protein concentrations with a high risk of aggregation [2] and relatively high temperature for warm-blooded animals. The evolution of higher organisms must create a mechanism to overcome problems resulting from the crowded protein solution and the elevated temperature.Two groups of proteins have been found to function as accessory proteins for folding: molecular chaperones and foldases which catalyze the necessary covalent reactions directly involved in protein folding [3]. Only two foldases, protein disul¢de isomerase (PDI) and peptidyl prolyl cis-trans isomerase (PPI), have so far been characterized as foldases. Based on the de¢nition of molecular chaperones as proteins assisting correct folding without covalent changes the foldases were therefore excluded as chaperones [1]. Chaperone activity of protein disul¢de isomerasePDI, a multifunctional protein present in the endoplasmic reticulum at high concentration [4], is remarkable in its capacity of non-speci¢c peptide binding, an important feature for a protein to be a molecular chaperone. In addition, for PDI to promote the joining of thiol groups distantly situated in the peptide sequence to form correct disul¢des the peptide chain has to be folded at least to some extent to bring the thiol groups together. The spontaneous folding of the peptide chain and the oxidation of thiol groups in vitro are often slow processes and the PDI-catalyzed folding does not need the presence of other chaperones. We therefore put forward the hypothesis that PDI is both an enzyme and a molecular chaperone [5]. Many in vitro and in vivo data have now accumulated to support the hypothesis that in addition to its isomerase activity in the catalysis of native disul¢de formation PDI does have intrinsic activity as a chaperone [6^13].The folding of a p...

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“…Although the hypothesis is attractive, the domain associated with peptide binding has not been completely elucidated. Initially it was reported that deletion of 51 amino acid residues of the C-terminal domain totally prevented peptide binding and chaperone activity (6). Subsequent studies have shown that the deletion includes amino acid residues pertaining to the Cterminal domain and a fraction of the aЈ domain (7).…”

Section: Reactivation Of Partially Folded Mmdh-sincementioning

confidence: 99%

Recognition of Protein Substrates by Protein-disulfide Isomerase

Cheung

Churchich

1999

Journal of Biological Chemistry

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Refolding of partially folded mitochondrial malate dehydrogenase (mMDH) is assisted by protein-disulfide isomerase (PDI).It is shown that the fluorescence probe is covalently attached to a SH residue located in the b domain. Based on the fluorescence measurements of native and derivatized PDI, it is suggested that recognition of the unfolded substrate involves conformational changes propagated to several domains of PDI. Protein-disulfide isomerase (PDI)1 is a multifunctional enzyme that both catalyzes the formation of disulfide bonds (1, 2) and acts as a subunit of prolyl-4-hydroxylase (3). PDI has been proposed to function as a molecular chaperone by binding to unfolded protein species, thereby preventing aggregation and misfolding (4, 5). Despite these interesting studies, the situation is complicated because PDI, unlike other chaperones, catalyzes disulfide bond formation and reduction.Several laboratories have attempted to show the site of binding of small molecular weight polypeptides that compete with misfolded protein substrates. Mutated PDI with the carboxyl terminus deleted shows neither peptide binding nor chaperone activity in assisting the refolding of denatured D-glyceraldehyde-3-phosphate dehydrogenase (6). On the other hand, it has been reported that deletion of the carboxyl-terminal domain (C domain) has no inhibitory effect on the assembly of recombinant prolyl-4-hydroxylase (7). Other investigators have reported that small molecular weight peptides bind to the bЈ domain of PDI (8).The possibility exists that more than one site in the structure of PDI is involved in recognition and refolding of protein substrates. The binding and hydrolysis of ATP by PDI has been reported by Guthapfel et al. (9); strikingly, the ATPase reaction is stimulated in the presence of denatured polypeptides, whereas the disulfide oxidation of PDI is not influenced by ATP. However, the functional role played by ATP hydrolysis during the refolding of denatured proteins has not been investigated in detail.The aim of the present work is 2-fold: first, to study regions of the primary structure of PDI involved in recognition of unfolded protein substrates and, second, to investigate whether the free energy of hydrolysis of ATP is required for unfolding of misfolded protein substrates. EXPERIMENTAL PROCEDURESPurification of Proteins-PDI was purified by the method described in Ref. 10 with small modifications. Fresh porcine livers (600 g) were homogenized in 0.1 M phosphate (pH 7.5) containing 1% Triton X-100 and 5 mM EDTA. After centrifugation, the supernatant was treated with ammonium sulfate, and the fractions obtained between 55-85% saturation were suspended in 25 mM citrate buffer (pH 5.3), dialyzed against the same buffer (buffer A), applied to CM-Sephadex C-50 column and eluted with the same buffer. Fractions displaying PDI activity were pooled, dialyzed against 20 mM sodium phosphate (pH 6.3) (buffer B), and applied to a DEAE-Sepharose fast flow column, which was eluted using a linear gradient of 0 -0.7 M NaCl in buffer...

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A Mutant Truncated Protein Disulfide Isomerase with No Chaperone Activity

Cited by 40 publications

References 38 publications

The protein disulphide-isomerase family: unravelling a string of folds

The protein disulphide-isomerase family: unravelling a string of folds

Enzymes as chaperones and chaperones as enzymes

Recognition of Protein Substrates by Protein-disulfide Isomerase

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