Analysis of unfolded protein response during single-chain antibody expression in Saccaromyces cerevisiae reveals different roles for BiP and PDI in folding

Xu, Ping; Raden, David; Doyle, Francis J.; Robinson, Anne S.

doi:10.1016/j.ymben.2005.04.002

Cited by 67 publications

(71 citation statements)

References 28 publications

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“…4, 8, 9 and 10) and its interaction (Table 2, Fig. 3) with the ER luminal protein-folding chaperone and binding protein, BiP (Bertolotti et al, 2000;Xu et al, 2005), further indicated a role of PDI2 in protein folding.…”

Section: Discussionmentioning

confidence: 88%

Protein Disulfide Isomerase-2 of Arabidopsis Mediates Protein Folding and Localizes to Both the Secretory Pathway and Nucleus, Where It Interacts with Maternal Effect Embryo Arrest Factor

Cho

Yuen

Kang

et al. 2011

Molecules and Cells

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Protein disulfide isomerase (PDI) is a thiodisulfide oxidoreductase that catalyzes the formation, reduction and rearrangement of disulfide bonds in proteins of eukaryotes. The classical PDI has a signal peptide, two CXXCcontaining thioredoxin catalytic sites (a,a′), two noncatalytic thioredoxin fold domains (b,b′), an acidic domain (c) and a C-terminal endoplasmic reticulum (ER) retention signal. Although PDI resides in the ER where it mediates the folding of nascent polypeptides of the secretory pathway, we recently showed that PDI5 of Arabidopsis thaliana chaperones and inhibits cysteine proteases during trafficking to vacuoles prior to programmed cell death of the endothelium in developing seeds. Here we describe Arabidopsis PDI2, which shares a primary structure similar to that of classical PDI. Recombinant PDI2 is imported into ER-derived microsomes and complements the E. coli protein-folding mutant, dsbA. PDI2 interacted with proteins in both the ER and nucleus, including ER-resident protein folding chaperone, BiP1, and nuclear embryo transcription factor, MEE8. The PDI2-MEE8 interaction was confirmed to occur in vitro and in vivo. Transient expression of PDI2-GFP fusions in mesophyll protoplasts resulted in labeling of the ER, nucleus and vacuole. PDI2 is expressed in multiple tissues, with relatively high expression in seeds and root tips. Immunoelectron microscopy with GFP-and PDI2-specific antisera on transgenic seeds (PDI2-GFP) and wild type roots demonstrated that PDI2 was found in the secretory pathway (ER, Golgi, vacuole, cell wall) and the nuclei. Our results indicate that PDI2 mediates protein folding in the ER and has new functional roles in the nucleus.

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Section: Discussionmentioning

confidence: 88%

Protein Disulfide Isomerase-2 of Arabidopsis Mediates Protein Folding and Localizes to Both the Secretory Pathway and Nucleus, Where It Interacts with Maternal Effect Embryo Arrest Factor

Cho

Yuen

Kang

et al. 2011

Molecules and Cells

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“…Data indicate that ER function is frequently a major bottleneck in production of secreted heterologous proteins, thus providing a target for optimization (37,49). This will provide the focus of data discussed here.…”

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confidence: 90%

“…Kar2p is the major hsp70 chaperone present in the ER lumen and participates in protein translocation and folding, ER-associated degradation (ERAD), and regulation of unfolded protein response (UPR) signaling (1,9,15,18,19,25,45,48,49). Essential to all of these activities is the ATPdependent cycle of protein binding and release (8,34).…”

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confidence: 99%

Modulation of Chaperone Gene Expression in Mutagenized Saccharomyces cerevisiae Strains Developed for Recombinant Human Albumin Production Results in Increased Production of Multiple Heterologous Proteins

Payne

Finnis

Evans

et al. 2008

Appl Environ Microbiol

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The yeast Saccharomyces cerevisiae has been successfully established as a commercially viable system for the production of recombinant proteins. Manipulation of chaperone gene expression has been utilized extensively to increase recombinant protein production from S. cerevisiae, focusing predominantly on the products of the protein disulfide isomerase gene PDI1 and the hsp70 gene KAR2. Here we show that the expression of the genes SIL1, LHS1, JEM1, and SCJ1, all of which are involved in regulating the ATPase cycle of Kar2p, is increased in a proprietary yeast strain, developed by several rounds of random mutagenesis and screening for increased production of recombinant human albumin (rHA). To establish whether this expression contributes to the enhanced-production phenotype, these genes were overexpressed both individually and in combination. The resultant strains showed significantly increased shake-flask production levels of rHA, granulocyte-macrophage colony-stimulating factor, and recombinant human transferrin.

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“…In some cases, this burden on the cell is translated into the activation of the UPR, and this can both positively and negatively influence the recombinant protein yield. For instance, in yeast, it was found that the overexpression of heterologous 4-4-20 scFv variants activated the UPR response, which in turn led to a reduction of the scFv yield (Kauffman et al, 2002;Xu et al, 2005;Xu and Robinson, 2009). On the other hand, disrupting the endoplasmic reticulum-associated degradation (ERAD) pathway in filamentous fungi led to an enhanced heterologous protein expression by delaying protein degradation (Carvalho et al, 2011).…”

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confidence: 99%

Recombinant Antibody Production in Arabidopsis Seeds Triggers an Unfolded Protein Response

et al. 2012

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Among the many plant-based production systems that are being tested for molecular farming, seeds are very attractive, as they provide a stable environment in which the accumulating recombinant proteins can be stored. However, it is not known exactly how high production levels of recombinant antibodies influence the endogenous transcriptome and proteome of the developing seed. To address this question, we studied the transcriptomic status in developing Arabidopsis (Arabidopsis thaliana) seeds 13 d post anthesis of three transgenic lines, producing varying levels of recombinant VHH or single-chain Fv antibody fragments fused to the human immunoglobulin G1-derived Fc fragment under the control of the b-PHASEOLIN seed-specific promoter. Using genome-wide Tiling arrays, we demonstrated that only a small proportion of the transcriptome was significantly changed in each of the lines compared with the wild type. Strikingly, in all three lines, we found a large overlap of up-regulated genes corresponding to protein folding, glycosylation/modification, translocation, vesicle transport, and protein degradation, suggestive of a state of cellular stress called the unfolded protein response. Moreover, the gene up-regulation amplitude was similar in all three lines. We hypothesize that the production of recombinant antibodies in the endoplasmic reticulum triggers endoplasmic reticulum stress, causing a disturbance of the normal cellular homeostasis.

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Analysis of unfolded protein response during single-chain antibody expression in Saccaromyces cerevisiae reveals different roles for BiP and PDI in folding

Cited by 67 publications

References 28 publications

Protein Disulfide Isomerase-2 of Arabidopsis Mediates Protein Folding and Localizes to Both the Secretory Pathway and Nucleus, Where It Interacts with Maternal Effect Embryo Arrest Factor

Protein Disulfide Isomerase-2 of Arabidopsis Mediates Protein Folding and Localizes to Both the Secretory Pathway and Nucleus, Where It Interacts with Maternal Effect Embryo Arrest Factor

Modulation of Chaperone Gene Expression in Mutagenized Saccharomyces cerevisiae Strains Developed for Recombinant Human Albumin Production Results in Increased Production of Multiple Heterologous Proteins

Recombinant Antibody Production in Arabidopsis Seeds Triggers an Unfolded Protein Response

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