Thiol-independent interaction of protein disulphide isomerase with type X collagen during intra-cellular folding and assembly (original) (raw)
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Journal of Biological Chemistry, 1994
The complexity of protein folding is often aggravated reticulum (Marquardt, T., and Helenius, A. (1992) J. by the low solubility of the denatured state. The ineffi-Cell. Biol. 117, 506-513). ciency of the oxidative refolding of reduced, denatured lysozyme results from a kinetic partitioning of the unfolded Protein between Pathways leading to aggrega-The uncatalyzed in vitro refolding of denatured proteins fretion and Pathways leading to the native structure. Proquently yields low levels of native protein and often is kinetitein disulfide isomerase (PDI), a resident foldase of the cally incompetent when compared with the in vivo reaction. endoplasmic reticulum, catalyzes the in vitro oxidative one of the major obstacles to the folding of many 'luding lysozyme' Depending On the results in hydrophobic aggregation (1). The efficacy of folding trations of foldase and denatured substrate and the ordepends on the partitioning of the unfolded protein between der in which they are added to initiate folding, PDI can exhibit either a chaperone activity or an anti-chaper-productive pathways leading to the native structure and non-Chem 269, 7764-7771). PDI,s chaperone activity leads to vivo folding and assembly of newly synthesized proteins often quantitative recovery of native lysozyme. Its anti-chapinvolves a variety of cellular folding enzymes and molecular erOne activity diverts substrate away from productive chaperones (2). Cellular foldases increase the rate and yield of folding and facilitates disulfide cross-linking of lyproductive folding by catalyzing slow chemical steps, such as sozyme into large, inactive aggregates that specifically disulfide formation and proline isomerization, that accompany incorporate PDI. A mutant PDI (N,C,.pDI), in which the correct folding and maturation of proteins (3, 4). On the both the Nand C-terminal active site cysteines have other hand, molecular chaperones bind to unfolded proteins been changed to serines, loses all chaperone activity and prevent nonproductive folding and aggregation (2, 5). In and behaves as an anti-chaperone at all substrate and uivo, the proper interplay between unfolded substrates, fol-PDI concentrations tested. The dithiolldisulfide sites of dases, and molecular chaperones normally ensures a proper PDI are essential for the chaperone activity observed balance between productive and nonproductive pathways. at high PDI concentrations, but they are not required Protein disulfide isomerase (PDI)' is a 55-kDa protein that for the anti-chaperone activity found at low PDI conresides in the endoplasmic reticulum (ER). PDI catalyzes the in centrations. Inactivation of PDI's peptide/protein bindvitro formation and rearrangement of disulfide bonds (6) and ing site by a specific Photoaffinity label (Noiva, R.9 has two functionally independent active sites, each of which Freedman, R. and Lennarz, J* (lgg3) J. Bioz. contains a dithiolldisulfide center. Both sites are present in Chem. 268, 19210-19217) inhibits the disulfide isomerthioredoxin-like domains, one near the N terminus and the * Supported by National Institutes of Health Grant GM40379 (to H. ~ F. G.) and by a grant from the National Science Foundation EPSCoR The abbreviations used are: PDI, protein disulfide isomerase; GSH, program and the South Dakota Futures Fund (to R. N.). The costs of glutathione; GSSG, glutathione disulfide; wt, wild-type; N,C,-PDI, publication of this article were defrayed in part by the payment of page mutant of PDI in which the N-and C-terminal active site cysteines have in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tide binding site; N U , monoiodo-N"-3-(4-hydroxyphenylpropionyl)-charges. This article must therefore be hereby marked "aduertisement" been changed to serines; pal-PDI, PDI photoaffinity labeled at the pep-1 To whom correspondence should be addressed: Dept. of Biochemis-Asn-Lys-(N-€-p-azidobenzoy1)-Ala-NH,; ER, endoplasmic reticulum; try,
Seminars in Cell and Developmental Biology, 1999
Procollagen assembly occurs within the endoplasmic reticulum, where the C-propeptide domains of three polypeptide ␣-chains fold individually, and then interact and trimerise to initiate folding of the triple helical region. This highly complex folding and assembly pathway requires the co-ordinated action of a large number of endoplasmic reticulum-resident enzymes and molecular chaperones. Disease-causing mutations in the procollagens disturb folding and assembly and lead to prolonged interactions with molecular chaperones, retention in the endoplasmic reticulum, and intracellular degradation. This review focuses predominantly on prolyl 4-hydroxylase, an essential collagen modifying enzyme, and HSP47, a collagen-specific binding protein, and their proposed roles as molecular chaperones involved in fibrillar procollagen folding and assembly, quality control, and secretion.
Journal of Biological Chemistry, 2007
Type XIII collagen is a type II transmembrane protein with three collagenous (COL1-3) and four noncollagenous domains (NC1-4). The human ␣1(XIII) chain contains altogether eight cysteine residues. We introduced point mutations to six of the most N-terminal cysteine residues, and we show here that the two cysteines 117 and 119 at the end of the N-terminal noncollagenous domain (NC1) are responsible for linking the three ␣1(XIII) chains together by means of interchain disulfide bonds. In addition, the intracellular and transmembrane domains have an impact on trimer formation, whereas the cysteines in the transmembrane domain and the COL1, the NC2, and the C-terminal NC4 domains do not affect trimer formation. We also suggest that the first three noncollagenous domains (NC1-3) harbor repeating heptad sequences typical of ␣-helical coiledcoils, whereas the conserved NC4 lacks a coiled-coil probability. Prevention of the coiled-coil conformation in the NC3 domain is shown here to result in labile type XIII collagen molecules. Furthermore, a new subgroup of collagenous transmembrane proteins, the Rattus norvegicus, Drosophila melanogaster, and Caenorhabditis elegans colmedins, is enlarged to contain also Homo sapiens collomin, and Pan troglodytes, Mus musculus, Tetraodon nigroviridis, and Dano rerio proteins. We suggest that there is a structurally varied group of collagenous transmembrane proteins whose biosynthesis is characterized by a coiled-coil motif following the transmembrane domain, and that these trimerization domains appear to be associated with each of the collagenous domains. In the case of type XIII collagen, the trimeric molecule has disulfide bonds at the junction of the NC1 and COL1 domains, and the type XIII collagen-like molecules (collagen types XXIII and XXV) and the colmedins are similar in that they all have a pair of cysteines in the same location. Moreover, furin cleavage at the NC1 domain can be expected in most of the proteins.
Febs Letters, 2004
We have reported that human protein disulfide isomerase-related protein (hPDIR) has isomerase and chaperone activities that are lower than those of the human protein disulfide isomerase (hPDI), and that the b domain of hPDIR is critical for its chaperone activity [J. Biol. Chem. 279 (2004) 4604]. To investigate the basis of the differences between hPDI and hPDIR, and to determine the functions of each hPDIR domain in detail, we constructed several hPDIR domain mutants. Interestingly, when the b domain of hPDIR was replaced with the b 0 domain of hPDI, a dramatic increase in chaperone activity that was close to that of hPDI itself was observed. However, this mutant showed decreased oxidative refolding of a1-antitrypsin. The replacement of the b domain of hPDIR with the c domain of hPDI also increased its chaperone activity. These observations suggest that putative peptide-binding sites of hPDI determine both its chaperone activity and its substrate specificity. (M. Kikuchi).
Journal of Biological Chemistry, 2001
Protein-disulfide isomerase (PDI) catalyzes the formation, rearrangement, and breakage of disulfide bonds and is capable of binding peptides and unfolded proteins in a chaperone-like manner. In this study we examined which of these functions are required to facilitate efficient refolding of denatured and reduced proinsulin. In our model system, PDI and also a PDI mutant having only one active site increased the rate of oxidative folding when present in catalytic amounts. PDI variants that are completely devoid of isomerase activity are not able to accelerate proinsulin folding, but can increase the yield of refolding, indicating that they act as a chaperone. Maximum refolding yields, however, are only achieved with wild-type PDI. Using genistein, an inhibitor for the peptide-binding site, the ability of PDI to prevent aggregation of folding proinsulin was significantly suppressed. The present results suggest that PDI is acting both as an isomerase and as a chaperone during folding and disulfide bond formation of proinsulin.
The Essential Function of Protein-disulfide Isomerase Is to Unscramble Non-native Disulfide Bonds
Journal of Biological Chemistry, 1995
Protein-disulfide isomerase (PDI) is an abundant protein of the endoplasmic reticulum that catalyzes dithiol oxidation and disulfide bond reduction and isomerization using the active site CGHC. Haploid pdi1⌬ Saccharomyces cerevisiae are inviable, but can be complemented with either a wild-type rat PDI gene or a mutant gene coding for CGHS PDI (shufflease). In contrast, pdi1⌬ yeast cannot be complemented with a gene coding for SGHC PDI. In vitro, shufflease is an efficient catalyst for the isomerization of existing disulfide bonds but not for dithiol oxidation or disulfide bond reduction. SGHC PDI catalyzes none of these processes. These results indicate that in vivo protein folding pathways contain intermediates with non-native disulfide bonds, and that the essential role of PDI is to unscramble these intermediates.
Protein Expression and Purification, 2001
1). Hsp47, 2 a 47-kDa heat-shock protein, is a recent Hsp47 is regarded as a collagen-specific chaperone addition to the list of proteins that act as molecular with several suggested roles in collagen biosynthesis chaperones during the biosynthesis of collagen, the under normal and disease conditions. We describe here most abundant protein in vertebrates. The synthesis of a procedure for the expression and purification of Hsp47 is seen to parallel that of collagen in many tis-Hsp47 in Escherichia coli using the IMPACT expression sues (2). This, and the association of Hsp47 with procolsystem (New England Biolabs) where the guest gene lagen at different stages of its biosynthesis, point to is fused to the adduct, intein, with a chitin-binding its functioning as a collagen-specific chaperone (2, 3). domain. Use of this system resulted in relatively high Hsp47 from chick embryo fibroblasts shows a high delevels of soluble Hsp47 compared to other available gree of sequence identity to human and rat gp46 and protocols, especially when the bacterial cells were to J6 from mouse teratocarcinoma cells (4). The term induced at 14؇C instead of 37؇C. The cell lysate was Hsp47 is now used to represent this group of collagenpassed through a chitin-Sepharose affinity column and binding proteins, the differences being accounted for by Hsp47 was cleaved from intein using -mercaptoethaspecies variation (5).
Journal of Cellular Biochemistry, 1994
Hps47, Grp78, have been implicated with procellagen maturation events. In particular Hps47 has been shown to blind to nascent procellagen α1(I) chains in the course of synthesis and/or translocation into the endoplasmic reticulum (ER). Although, Hsp47 binding to gelatin and collgen has previsously been suggested to mechanism. The early association of Hps47 with procollagen and its relatively late relese suggested that other chaperones, Grp78 and Grp94, interact successively or concurrently with Hps47. Herein, we examined how these events occurs in cells metabolically stressed by depletion of ATP. In cells depleted of ATP, the releses of Hps47, Grp78, and Grp94 from maturing procollange is delayed. Thus, in cell experiencing metabolic stress, newly synthesized procollagen unable to property fold became stable bound to a complex of molecular chaperones. In that Hps47, Grp78, and Grp98 could be recovered with nascent procollagen and as oligomer in ATP depleted cells suggests that these chaperones function in a series of coupled or successive reactions.
The Journal of Cell Biology, 2000
Triple helix formation of procollagen after the assembly of three ␣ -chains at the C-propeptide is a prerequisite for refined structures such as fibers and meshworks. Hsp47 is an ER-resident stress inducible glycoprotein that specifically and transiently binds to newly synthesized procollagens. However, the real function of Hsp47 in collagen biosynthesis has not been elucidated in vitro or in vivo. Here, we describe the establishment of Hsp47 knockout mice that are severely deficient in the mature, propeptide-processed form of ␣ 1(I) collagen and fibril structures in mesenchymal tissues. The molecular form of type IV collagen was also affected, and basement membranes were discontinuously disrupted in the homozygotes. The homozygous mice did not survive beyond 11.5 days postcoitus (dpc), and displayed abnormally orientated epithelial tissues and ruptured blood vessels. When triple helix formation of type I collagen secreted from cultured cells was monitored by protease digestion, the collagens of Hsp47 ϩ / ϩ and Hsp47 ϩ / Ϫ cells were resistant, but those of Hsp47 Ϫ / Ϫ cells were sensitive. These results indicate for the first time that type I collagen is unable to form a rigid triple-helical structure without the assistance of molecular chaperone Hsp47, and that mice require Hsp47 for normal development.