Protein Disulfide-Isomerase: Role in Biosynthesis of Secretory Proteins

Complicated and sophisticated protein homeostasis (proteostasis) networks in the endoplasmic reticulum (ER), comprising disulfide catalysts, molecular chaperones, and their regulators, help to maintain cell viability. Newly synthesized proteins inserted into the ER need to fold and assemble into unique native structures to fulfill their physiological functions, and this is assisted by protein disulfide isomerase (PDI) family. Herein, we focus on recent advances in understanding the detailed mechanisms of PDI family members as guides for client folding and assembly to ensure the efficient production of secretory proteins.

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Functional Relationship between Protein Disulfide Isomerase Family Members during the Oxidative Folding of Human Secretory Proteins

Molecular Biology of the Cell ◽

10.1091/mbc.e10-04-0356 ◽

2010 ◽

Vol 21 (18) ◽

pp. 3093-3105 ◽

Cited By ~ 62

Author(s):

Lori A. Rutkevich ◽

Myrna F. Cohen-Doyle ◽

Ulf Brockmeier ◽

David B. Williams

Keyword(s):

Protein Disulfide Isomerase ◽

High Efficiency ◽

Family Members ◽

Secretory Proteins ◽

Human Hepatoma Cells ◽

Oxidative Folding ◽

Disulfide Isomerase ◽

The Impact ◽

Protein Disulfide ◽

Protein Disulfide Isomerase Family

To examine the relationship between protein disulfide isomerase family members within the mammalian endoplasmic reticulum, PDI, ERp57, ERp72, and P5 were depleted with high efficiency in human hepatoma cells, either singly or in combination. The impact was assessed on the oxidative folding of several well-characterized secretory proteins. We show that PDI plays a predominant role in oxidative folding because its depletion delayed disulfide formation in all secretory proteins tested. However, the phenotype was surprisingly modest suggesting that other family members are able to compensate for PDI depletion, albeit with reduced efficacy. ERp57 also exhibited broad specificity, overlapping with that of PDI, but with preference for glycosylated substrates. Depletion of both PDI and ERp57 revealed that some substrates require both enzymes for optimal folding and, furthermore, led to generalized protein misfolding, impaired export from the ER, and degradation. In contrast, depletion of ERp72 or P5, either alone or in combination with PDI or ERp57 had minimal impact, revealing a narrow substrate specificity for ERp72 and no detectable role for P5 in oxidative protein folding.

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Characterization of a Foldase, Protein Disulfide Isomerase A, in the Protein Secretory Pathway ofAspergillus niger

Applied and Environmental Microbiology ◽

10.1128/aem.66.2.775-782.2000 ◽

2000 ◽

Vol 66 (2) ◽

pp. 775-782 ◽

Cited By ~ 66

Author(s):

Celina Ngiam ◽

David J. Jeenes ◽

Peter J. Punt ◽

Cees A. M. J. J. Van Den Hondel ◽

David B. Archer

Keyword(s):

Protein Disulfide Isomerase ◽

Secretory Pathway ◽

Functional Characterization ◽

Killer Toxin ◽

Rnase A ◽

Secretory Proteins ◽

Mrna Levels ◽

Western Blots ◽

Disulfide Isomerase ◽

Protein Disulfide

ABSTRACT Protein disulfide isomerase (PDI) is important in assisting the folding and maturation of secretory proteins in eukaryotes. A gene,pdiA, encoding PDIA was previously isolated fromAspergillus niger, and we report its functional characterization here. Functional analysis of PDIA showed that it catalyzes the refolding of denatured and reduced RNase A.pdiA also complemented PDI function in aSaccharomyces cerevisiae Δpdi1 mutant in a yeast-based killer toxin assay. Levels of pdiA mRNA and PDIA protein were raised by the accumulation of unfolded proteins in the endoplasmic reticulum. This response of pdiA mRNA levels was slower and lower in magnitude than that of A. niger bipA, suggesting that the induction of pdiA is not part of the primary stress response. An increased level of pdiA transcripts was also observed in two A. niger strains overproducing a heterologous protein, hen egg white lysozyme (HEWL). Although overexpression of PDI has been successful in increasing yields of some heterologous proteins in S. cerevisiae, overexpression of PDIA did not increase secreted yields of HEWL in A. niger, suggesting that PDIA itself is not limiting for secretion of this protein. Downregulation of pdiA by antisense mRNA reduced the levels of microsomal PDIA activity by up to 50%, lowered the level of PDIA as judged by Western blots, and lowered the secreted levels of glucoamylase by 60 to 70%.

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Distribution of protein disulfide isomerase in rat epiphyseal chondrocytes.

Journal of Histochemistry & Cytochemistry ◽

10.1177/37.12.2584692 ◽

1989 ◽

Vol 37 (12) ◽

pp. 1835-1844 ◽

Cited By ~ 5

Author(s):

S Akagi ◽

A Yamamoto ◽

T Yoshimori ◽

R Masaki ◽

R Ogawa ◽

...

Keyword(s):

Golgi Apparatus ◽

Disulfide Bonds ◽

Protein Disulfide Isomerase ◽

Protein A ◽

Secretory Proteins ◽

Disulfide Isomerase ◽

Gold Particles ◽

Golgi Compartment ◽

Preferential Binding ◽

Protein Disulfide

We investigated the intracellular distribution of protein disulfide isomerase (PDI) in rat epiphyseal chondrocytes by immunocytochemistry, using a post-embedding protein A-gold technique. Gold particles were localized primarily in the cisternal space of the rough endoplasmic reticulum (ER) and nuclear envelopes. The ER cisternae of the chondrocytes in all the differentiating epiphyseal zones--resting, proliferative, pre-hypertrophic, and hypertrophic--were equally and highly labeled. The labeling density of the cisternal space of the dilated ER, probably reflecting marked accumulation of secretory proteins such as procollagen, was always higher than that of the non-dilated ER. In the dilated cisternal space, gold particles were freely and evenly distributed, without preferential binding to the luminal surface of the ER membranes. We suggest that PDI catalyzes the formation of disulfide bonds of various secretory proteins, perhaps type II procollagen, in the cisternal space of the ER in epiphyseal chondrocytes. The exclusive localization of gold particles in the cisternal space of the ER and nuclear envelopes and the lack of gold particles in the Golgi apparatus, including cis-Golgi cisternae, indicate that PDI is an ER-soluble protein in the chondrocytes and is presumably sorted out in some pre-Golgi compartment and not transported to the Golgi apparatus.

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A High-Throughput Turbidometric Assay for Screening Inhibitors of Protein Disulfide Isomerase Activity

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057104265292 ◽

2004 ◽

Vol 9 (7) ◽

pp. 614-620 ◽

Cited By ~ 33

Author(s):

Anthony M. Smith ◽

John Chan ◽

Donna Oksenberg ◽

Roman Urfer ◽

David S. Wexler ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Protein Disulfide Isomerase ◽

Secretory Proteins ◽

Screening Assay ◽

Kinetic Assay ◽

Chemical Libraries ◽

Disulfide Isomerase ◽

High Throughput Screening Assay ◽

Protein Disulfide

Protein disulfide isomerase (PDI) plays a key role in protein folding by catalyzing rearrangements of disulfide bonds in substrate proteins following their synthesis in eukaryotic cells. Besides its major role in the processing and maturation of secretory proteins in the endoplasmic reticulum, this enzyme and its homologs have been implicated in multiple important cellular processes; however, they have not served as targets for the development of therapeutic agents. The authors developed a high-throughput screening assay for PDI and its homologous enzymes in 384-well microplates. The method is based on the enzyme-catalyzed reduction of insulin in the presence of dithiothreitol and measures the aggregation of reduced insulin chains at 650 nm. This kinetic assay was converted to an end-point assay by using hydrogen peroxide as a stop reagent. The feasibility of this high-throughput assay for screening chemical libraries was demonstrated in a pilot screen. The authors show that this homogenous turbidometric assay is robust and cost-effective and can be applied to identify PDI inhibitors from chemical libraries, opening this class of enzymes for therapeutic exploration.

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Molecular Bases of Cyclic and Specific Disulfide Interchange between Human ERO1α Protein and Protein-disulfide Isomerase (PDI)

Journal of Biological Chemistry ◽

10.1074/jbc.m111.231357 ◽

2011 ◽

Vol 286 (18) ◽

pp. 16261-16271 ◽

Cited By ~ 52

Author(s):

Shoji Masui ◽

Stefano Vavassori ◽

Claudio Fagioli ◽

Roberto Sitia ◽

Kenji Inaba

Keyword(s):

Family Member ◽

Protein Disulfide Isomerase ◽

Electron Flow ◽

Physiological Role ◽

Secretory Proteins ◽

Hydrophobic Pocket ◽

Disulfide Isomerase ◽

Docking Simulations ◽

Flow Pathways ◽

Protein Disulfide

In the endoplasmic reticulum (ER) of human cells, ERO1α and protein-disulfide isomerase (PDI) constitute one of the major electron flow pathways that catalyze oxidative folding of secretory proteins. Specific and limited PDI oxidation by ERO1α is essential to avoid ER hyperoxidation. To investigate how ERO1α oxidizes PDI selectively among more than 20 ER-resident PDI family member proteins, we performed docking simulations and systematic biochemical analyses. Our findings reveal that a protruding β-hairpin of ERO1α specifically interacts with the hydrophobic pocket present in the redox-inactive PDI b′-domain through the stacks between their aromatic residues, leading to preferred oxidation of the C-terminal PDI a′-domain. ERO1α associated preferentially with reduced PDI, explaining the stepwise disulfide shuttle mechanism, first from ERO1α to PDI and then from oxidized PDI to an unfolded polypeptide bound to its hydrophobic pocket. The interaction of ERO1α with ERp44, another PDI family member protein, was also analyzed. Notably, ERO1α-dependent PDI oxidation was inhibited by a hyperactive ERp44 mutant that lacks the C-terminal tail concealing the substrate-binding hydrophobic regions. The potential ability of ERp44 to inhibit ERO1α activity may suggest its physiological role in ER redox and protein homeostasis.

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Protein disulfide isomerase: Multiple roles in the modification of nascent secretory proteins

Cell ◽

10.1016/0092-8674(89)90043-3 ◽

1989 ◽

Vol 57 (7) ◽

pp. 1069-1072 ◽

Cited By ~ 327

Author(s):

Robert B. Freedman

Keyword(s):

Protein Disulfide Isomerase ◽

Multiple Roles ◽

Secretory Proteins ◽

Disulfide Isomerase ◽

Protein Disulfide

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Export of a Cysteine-Free Misfolded Secretory Protein from the Endoplasmic Reticulum for Degradation Requires Interaction with Protein Disulfide Isomerase

The Journal of Cell Biology ◽

10.1083/jcb.147.7.1443 ◽

1999 ◽

Vol 147 (7) ◽

pp. 1443-1456 ◽

Cited By ~ 136

Author(s):

Pauline Gillece ◽

José Manuel Luz ◽

William J. Lennarz ◽

Francisco Javier de la Cruz ◽

Karin Römisch

Keyword(s):

Quality Control ◽

Protein Disulfide Isomerase ◽

Secretory Protein ◽

Secretory Proteins ◽

Disulfide Isomerase ◽

Thiol Content ◽

Mutant Forms ◽

Protein Disulfide ◽

Chemical Cross Linking ◽

Er Membrane

Protein disulfide isomerase (PDI) interacts with secretory proteins, irrespective of their thiol content, late during translocation into the ER; thus, PDI may be part of the quality control machinery in the ER. We used yeast pdi1 mutants with deletions in the putative peptide binding region of the molecule to investigate its role in the recognition of misfolded secretory proteins in the ER and their export to the cytosol for degradation. Our pdi1 deletion mutants are deficient in the export of a misfolded cysteine-free secretory protein across the ER membrane to the cytosol for degradation, but ER-to-Golgi complex transport of properly folded secretory proteins is only marginally affected. We demonstrate by chemical cross-linking that PDI specifically interacts with the misfolded secretory protein and that mutant forms of PDI have a lower affinity for this protein. In the ER of the pdi1 mutants, a higher proportion of the misfolded secretory protein remains associated with BiP, and in export-deficient sec61 mutants, the misfolded secretory protein remain bounds to PDI. We conclude that the chaperone PDI is part of the quality control machinery in the ER that recognizes terminally misfolded secretory proteins and targets them to the export channel in the ER membrane.

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