蛋白质二硫键异构酶家族的结构与功能

蛋白质二硫键异构酶（protein disulfide isomerase,PDI）家族是一类在内质网中起作用的巯基-二硫键氧化还原酶.它们通常含有CXXC（Cys-Xaa-Xaa-Cys,CXXC）活性位点,活性位点的两个半胱氨酸残基可催化底物二硫键的形成、异构及还原.所有PDI家族成员包含至少一个约100个氨基酸残基的硫氧还蛋白同源结构域.PDI家族的主要职能是催化内质网中新生肽链的氧化折叠,另外在内质网相关的蛋白质降解途径（ERAD）、蛋白质转运、钙稳态、抗原提呈及病毒入侵等方面也起重要作用.     

Autodegradation of Protein Disulfide Isomerase

Protein disulfide isomerase (PDI) and its degradation products were found in HepG2, COS-1, and CHO-K1 cells. Whether or not the products were formed through autodegradation of PDI was examined, since PDI contains the CGHC motif, which is the active center of proteolytic activity in ER-60 protease. Commercial bovine PDI was autodegraded to produce a trimmed PDI. In addition, human recombinant PDI also had autodegradation activity. Mutant recombinant PDIs with CGHC motifs of which cysteine residues were replaced with serine or alanine residues were prepared. However, they were not autodegraded, suggesting the cysteine residues of motifs are necessary for autodegradation.     

Crystal Structure and Functional Analysis of the Protein Disulfide Isomerase-Related Protein ERp29

The protein disulfide isomerase-related protein ERp29 is a putative chaperone involved in processing and secretion of secretory proteins. Until now, however, both the structure and the exact nature of interacting substrates remained unclear. We provide for the first time a crystal structure of human ERp29, refined to 2.9 Å, and show that the protein has considerable structural homology to its Drosophila homolog Wind. We show that ERp29 binds directly not only to thyroglobulin and thyroglobulin-derived peptides in vitro but also to the Wind client protein Pipe and Pipe-derived peptides, although it fails to process Pipe in vivo. A monomeric mutant of ERp29 and a D domain mutant in which the second peptide binding site is inactivated also bind protein substrates, indicating that the monomeric thioredoxin domain is sufficient for client protein binding. Indeed, the b domains of ERp29 or Wind, expressed alone, are sufficient for binding proteins and peptides. Interacting peptides have in common two or more aromatic residues, with stronger binding for sequences with overall basic character. Thus, the data allow a view of the two putative peptide binding sites of ERp29 and indicate that the apparent, different processing activity of the human and Drosophila proteins in vivo does not stem from differences in peptide binding properties.     

The Protein-disulfide Isomerase ERp57 Regulates the Steady-state Levels of the Prion Protein

Although the accumulation of a misfolded and protease-resistant form of the prion protein (PrP) is a key event in prion pathogenesis, the cellular factors involved in its folding and quality control are poorly understood. PrP is a glycosylated and disulfide-bonded protein synthesized at the endoplasmic reticulum (ER). The ER foldase ERp57 (also known as Grp58) is highly expressed in the brain of sporadic and infectious forms of prion-related disorders. ERp57 is a disulfide isomerase involved in the folding of a subset of glycoproteins in the ER as part of the calnexin/calreticulin cycle. Here, we show that levels of ERp57 increase mainly in neurons of Creutzfeldt-Jacob patients. Using gain- and loss-of-function approaches in cell culture, we demonstrate that ERp57 expression controls the maturation and total levels of wild-type PrP and mutant forms associated with human disease. In addition, we found that PrP physically interacts with ERp57, and also with the closest family member PDIA1, but not ERp72. Furthermore, we generated a conditional knock-out mouse for ERp57 in the nervous system and detected a reduction in the steady-state levels of the mono- and nonglycosylated forms of PrP in the brain. In contrast, ERp57 transgenic mice showed increased levels of endogenous PrP. Unexpectedly, ERp57 expression did not affect the susceptibility of cells to ER stress in vitro and in vivo. This study identifies ERp57 as a new modulator of PrP levels and may help with understanding the consequences of ERp57 up-regulation observed in human disease.     

Yeast Mpd1p reveals the structural diversity of the protein disulfide isomerase family

Oxidoreductases belonging to the protein disulfide isomerase (PDI) family promote proper disulfide bond formation in substrate proteins in the endoplasmic reticulum. In plants and metazoans, new family members continue to be identified and assigned to various functional niches. PDI-like proteins typically contain tandem thioredoxin-fold domains. The limited information available suggested that the relative orientations of these domains may be quite uniform across the family, and structural models based on this assumption are appearing. However, the X-ray crystal structure of the yeast PDI family protein Mpd1p, described here, demonstrates the radically different domain orientations and surface properties achievable with multiple copies of the thioredoxin fold. A comparison of Mpd1p with yeast Pdi1p expands our perspective on the contexts in which redox-active motifs are presented in the PDI family.     

Expression of sialyl Le, sialyl Le, Le and Le glycotopes in secreted human ovarian cyst glycoproteins

Human blood group A, B, H, Ii, Le^a and Le^b antigens and their determinants expressed on ovarian cyst glycoproteins have been studied for over five decades. However, little is known about sialyl Le^x and sialyl Le^a glycotopes, which play essential roles in normal immunity, inflammation, and cancer cell metastasis. Furthermore, Le^x and Le^y were classified as glycotopes of unknown genes. Identification of these Lewis epitopes was hampered by the lack of specific antibodies. In this study, the occurrence of sialyl Le^x, sialyl Le^a, Le^x and Le^y reactivities in cyst glycoproteins was characterized by enzyme-linked immunosorbent assays. The results indicated that most human ovarian cyst glycoproteins carried Le^x (8/25) and/or Le^y (17/25) glycotopes. The expression (epitopes) of the new genes described in previous reports are Le^x and Le^y glycotopes; the reactivities of sialyl Le^x and sialyl Le^a glycotopes in secreted cyst glycoproteins may be affected by the conditions of purification; the relationship between Le^y and human blood group ABH was confirmed; recognition profiles of sialyl Le^x, sialyl Le^a, Le^x and Le^y present in the carbohydrate chains of water-soluble cyst glycoproteins were illustrated; possible attachments of glycotopes to the internal carbohydrate complex of cyst glycoproteins have been reconstructed; proposed biosynthetic pathways for the formation of sialyl Le^a, sialyl Le^x, Le^x, Le^y, ALe^y and BLe^y determinant structures on Type I and Type II core structures of human ovarian cyst glycoproteins are also included in this study.     

ERp57 interacts with conserved cysteine residues in the MHC class I peptide-binding groove

The oxidoreductase ERp57 is a component of the major histocompatibility complex (MHC) class I peptide-loading complex. ERp57 can interact directly with MHC class I molecules, however, little is known about which of the cysteine residues within the MHC class I molecule are relevant to this interaction. MHC class I molecules possess conserved disulfide bonds between cysteines 101-164, and 203-259 in the peptide-binding and alpha3 domain, respectively. By studying a series of mutants of these conserved residues, we demonstrate that ERp57 predominantly associates with cysteine residues in the peptide-binding domain, thus indicating ERp57 has direct access to the peptide-binding groove of MHC class I molecules during assembly.     

Human Eosinophil Leukocytes Express Protein Disulfide Isomerase in Secretory Granules and Vesicles: Ultrastructural Studies

Protein disulfide isomerase (PDI) has fundamental roles in the oxidative folding of proteins in the endoplasmic reticulum (ER) of eukaryotic cells. The study of this molecule has been attracting considerable attention due to its association with other cell functions and human diseases. In leukocytes, such as neutrophils, PDI is involved with cell adhesion, signaling and inflammation. However, the expression of PDI in other leukocytes, such as eosinophils, important cells in inflammatory, allergic and immunomodulatory responses, remains to be defined. Here we used different approaches to investigate PDI expression within human eosinophils. Western blotting and flow cytometry demonstrated high PDI expression in both unstimulated and CCL11/eotaxin-1-stimulated eosinophils, with similar levels in both conditions. By using an immunogold electron microscopy technique that combines better epitope preservation and secondary Fab-fragments of antibodies linked to 1.4-nm gold particles for optimal access to microdomains, we identified different intracellular sites for PDI. In addition to predictable strong PDI labeling at the nuclear envelope, other unanticipated sites, such as secretory granules, lipid bodies and vesicles, including large transport vesicles (eosinophil sombrero vesicles), were also labeled. Thus, we provide the first identification of PDI in human eosinophils, suggesting that this molecule may have additional/specific functions in these leukocytes.     

A Protein Disulfide Isomerase Controls Neuronal Migration through Regulation of Wnt Secretion

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Protein Disulfide Isomerase 2 of Chlamydomonas reinhardtii Is Involved in Circadian Rhythm Regulation

Protein disulfide isomerases （PDIs） are known to play important roles in the folding of nascent proteins and in the formation of disulfide bonds. Recently, we identified a PDI from Chlamydomonas reinhardtii （CrPDI2） by a mass spectrometry approach that is specifically enriched by heparin affinity chromatography in samples taken during the night phase. Here, we show that the recombinant CrPDI2 is a redox-active protein. It is reduced by thioredoxin reductase and catalyzes itself the reduction of insulin chains and the oxidative refolding of scrambled RNase A. By immunoblots, we confirm a high-amplitude change in abundance of the heparin-bound CrPDI2 during subjective night. Interestingly, we find that CrPDI2 is present in protein complexes of different sizes at both day and night. Among three identified interac- tion partners, one （a 2-cys peroxiredoxin） is present only during the night phase. To study a potential function of CrPDI2 within the circadian system, we have overexpressed its gene. Two transgenic lines were used to measure the rhythm of phototaxis~ In the transgenic strains, a change in the acrophase was observed. This indicates that CrPDI2 is involved in the circadian signaling pathway and, together with the night phase-specific interaction of CrPDI2 and a peroxiredoxin, these findings suggest a close coupling of redox processes and the circadian clock in C. reinhardtii.     

Novel Roles of the Non-catalytic Elements of Yeast Protein-disulfide Isomerase in Its Interplay with Endoplasmic Reticulum Oxidoreductin 1

The formation of disulfide bonds in the endoplasmic reticulum (ER) of eukaryotic cells is catalyzed by the sulfhydryl oxidase, ER oxidoreductin 1 (Ero1), and protein-disulfide isomerase (PDI). PDI is oxidized by Ero1 to continuously introduce disulfides into substrates, and feedback regulates Ero1 activity by manipulating the regulatory disulfides of Ero1. In this study we find that yeast Ero1p is enzymatically active even with its regulatory disulfides intact, and further activation of Ero1p by reduction of the regulatory disulfides requires the reduction of non-catalytic Cys⁹⁰-Cys⁹⁷ disulfide in Pdi1p. The principal client-binding site in the Pdi1p b′ domain is necessary not only for the functional Ero1p-Pdi1p disulfide relay but also for the activation of Ero1p. We also demonstrate by complementary activation assays that the regulatory disulfides in Ero1p are much more stable than those in human Ero1α. These new findings on yeast Ero1p-Pdi1p interplay reveal significant differences from our previously identified mode of human Ero1α-PDI interplay and provide insights into the evolution of the eukaryotic oxidative protein folding pathway.