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| Status |
Public on Dec 01, 2011 |
| Title |
Balancing Protein Folding and Disulfide Bond Formation Rates is Key to Mitigating Secretory Stress |
| Platform organisms |
Schizosaccharomyces pombe; Saccharomyces cerevisiae |
| Sample organism |
Saccharomyces cerevisiae |
| Experiment type |
Expression profiling by array
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| Summary |
The protein secretory pathway must maintain homoeostasis while producing a wide assortment of proteins in different conditions. It is also used extensively to produce many useful proteins in biotechnology. As such, secretory pathway dysfunction can be highly detrimental to the cell, resulting in the molecular basis for many human diseases, and can drastically inhibit product titers in biochemical production. Because the secretory pathway is a highly-integrated, multi-organelle system, dysfunction can happen at many levels and dissecting the root cause can be challenging.
To better understand some of these dysfunctions, we measured multiple systems-level states of the cell (physiology, transcriptome, metabolism) while secreting a small protein (insulin precursor) or a large protein (α-amylase). This was carried out in the presence and absence of HAC1, a key transcription factor in maintaining secretory homeostasis. Clear trends in cellular stress were apparent across multiple data resulting from our perturbations. In particular, processes involving (1) degradation of protein / recycling amino acids, (2) overall transcription/translation repression, and (3) oxidative stress.
Apparent runaway oxidative radical production was explained by a thermodynamic model that we put forward for disulfide formation in the endoplasmic reticulum. This model predicts that balancing the relative rates of protein folding and disulfide bond formation are key to easing oxidative stress. These predictions have direct implications in how to engineer a broad range of recombinant proteins for secretion and provide potential hypotheses for the root causes of several secretory-associated diseases.
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| Overall design |
Yeast strains were constructed that produce and secrete (a) IP or (b) α-amylase and were compared to yeast strains containing (c) an empty vector in both wild-type and HAC1 deletion backgrounds.
These strains are named WN (WT with empty vector), WI (WT secreting IP), WA (WT secreting α-amylase), dN (Δhac1 with empty vector), dI (Δhac1 secreting IP), and dA (Δhac1 secreting α-amylase). Strains were characterized in batch fermentation and samples were taken in mid-exponential phase. Triplicate fermentations were carried out for each strain.
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| Contributor(s) |
Tyo KE, Liu Z, Petranovic D, Nielsen J |
| Citation(s) |
22380681 |
| |
| Submission date |
Feb 03, 2011 |
| Last update date |
Feb 21, 2017 |
| Contact name |
Keith EJ Tyo |
| E-mail(s) |
k-tyo@northwestern.edu
|
| Phone |
847-868-0319
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| Organization name |
Chalmers University of Technology
|
| Department |
Chemical and Biological Engineering
|
| Lab |
Systems and Synthetic Biology Group
|
| Street address |
Kemivägen 10
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| City |
Göteborg |
| ZIP/Postal code |
SE-412 96 |
| Country |
USA |
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| Platforms (1) |
| GPL2529 |
[Yeast_2] Affymetrix Yeast Genome 2.0 Array |
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| Samples (18)
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| Relations |
| BioProject |
PRJNA137605 |
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