Dataset Information

Protein Folding Modulation in Cells Subject to Differentiation and Stress.

ABSTRACT: Cytomimetic media are used to mimic the physicochemical properties of the cellular milieu in an in vitro experiment. The motivation is that compared to entire cells, they can be used efficiently in combination with a broad range of experimental techniques. However, the development and use of cytomimetic media is hampered by the lack of in-cell data that could be used as a hallmark to directly evaluate and improve the performance of cytomimetic media in different applications. Such data must include the study of specific biomolecular reactions in different cell types, different compartments of a single cells and different cellular conditions. In previous studies, model systems such as cancer cell lines, bacteria or oocytes were used. Here we studied how the environment of cells that undergo neuronal differentiation or proteostasis stress modulates the protein folding equilibrium. We found that NGF induced differentiation leads to a decrease of the melting temperature and a change of the folding mechanism. Proteomic changes that occur upon differentiation could explain this effect, however, we found that the crowding effect remained unchanged. Using MG132, a common proteasome inhibitor and inducer of the unfolded protein response, we show that changes to the quality control machinery modulate the folding equilibrium, leading to protein destabilization at prolonged stress exposure. Our study explores the range of protein folding modulation within cells subject to differentiation or stress that must be encountered in the development of cytomimetic media.

SUBMITTER: Gnutt D

PROVIDER: S-EPMC6544126 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

Protein Folding Modulation in Cells Subject to Differentiation and Stress.

Gnutt David D Sistemich Linda L Ebbinghaus Simon S

Frontiers in molecular biosciences 20190524

Cytomimetic media are used to mimic the physicochemical properties of the cellular milieu in an <i>in vitro</i> experiment. The motivation is that compared to entire cells, they can be used efficiently in combination with a broad range of experimental techniques. However, the development and use of cytomimetic media is hampered by the lack of in-cell data that could be used as a hallmark to directly evaluate and improve the performance of cytomimetic media in different applications. Such data mu ...[more]

PMID: 31179287

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Project description: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 (?-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. Yeast strains were constructed that produce and secrete (a) IP or (b) ?-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 ?-amylase), dN (?hac1 with empty vector), dI (?hac1 secreting IP), and dA (?hac1 secreting ?-amylase). Strains were characterized in batch fermentation and samples were taken in mid-exponential phase. Triplicate fermentations were carried out for each strain.

Dataset Information

Protein Folding Modulation in Cells Subject to Differentiation and Stress.

Publications

Protein Folding Modulation in Cells Subject to Differentiation and Stress.

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