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Distinct transcriptional responses elicited by unfolded nuclear or cytoplasmic protein in mammalian cells.

Miyazaki Y, Chen LC, Chu BW, Swigut T, Wandless TJ - Elife (2015)

Bottom Line: The sudden appearance of unfolded protein in mammalian cells elicits a robust transcriptional response, which is distinct from the HSR and other known pathways that respond to unfolded proteins.The cellular response to unfolded protein is strikingly different in the nucleus and the cytosol, although unfolded protein in either compartment engages the p53 network.This response provides cross-protection during subsequent proteotoxic stress, suggesting that it is a central component of protein quality control networks, and like the HSR, is likely to influence the initiation and progression of human pathologies.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Systems Biology, Stanford University, Stanford, United States.

ABSTRACT
Eukaryotic cells possess a variety of signaling pathways that prevent accumulation of unfolded and misfolded proteins. Chief among these is the heat shock response (HSR), which is assumed to respond to unfolded proteins in the cytosol and nucleus alike. In this study, we probe this axiom further using engineered proteins called 'destabilizing domains', whose folding state we control with a small molecule. The sudden appearance of unfolded protein in mammalian cells elicits a robust transcriptional response, which is distinct from the HSR and other known pathways that respond to unfolded proteins. The cellular response to unfolded protein is strikingly different in the nucleus and the cytosol, although unfolded protein in either compartment engages the p53 network. This response provides cross-protection during subsequent proteotoxic stress, suggesting that it is a central component of protein quality control networks, and like the HSR, is likely to influence the initiation and progression of human pathologies.

No MeSH data available.


Related in: MedlinePlus

The effect of transcription factors on cell growth.(A) NIH3T3 cells expressing DD-GFP were co-cultured with unmodified 3T3 cells in the presence of 1 μM S1. The stabilizing S1 was withdrawn for 45, 135, or 405 min and then re-administered to the cells for an additional 17 hr. Analytical flow cytometry was used to quantify the relative populations of each cell type. The experiment was performed in triplicate for populations of cells expressing high levels of DD-GFP experiencing siRNA of p53 (blue) as well as the cell line experiencing non-target siRNA (siNT, black). (B) NIH3T3 cells expressing DD-GFP were co-cultured with unmodified 3T3 cells in the presence of 1 μM S1. The stabilizing S1 was withdrawn for 45, 135, or 405 min and then re-administered to the cells for an additional 17 hr. Analytical flow cytometry was used to quantify the relative populations of each cell type. The experiment was performed in triplicate for populations of cells expressing high levels of DD-GFP experiencing siRNA of HSF1 (red) as well as the cell line experiencing non-target siRNA (siNT, black). (C) NIH3T3 cells expressing DD-GFP were co-cultured with unmodified 3T3 cells in the presence of 1 μM S1. The stabilizing S1 was withdrawn for 45, 135, or 405 min and then re-administered to the cells for an additional 17 hr. Analytical flow cytometry was used to quantify the relative populations of each cell type. The experiment was performed in triplicate for populations of cells expressing high levels of DD-GFP experiencing siRNA of NRF2 (green) as well as the cell line experiencing non-target siRNA (siNT, black). (D) Cells expressing DD-GFP were co-cultured with unmodified 3T3 cells in a 24-well plate. One group of cells was not exposed to stress from either the unfolded DD or 10 μM MG132 (Ctl). The second group was exposed to MG132 only (MG132). The third group experienced the unfolded DD by withdrawal of S1 (shown as ufDD), and the fourth group was exposed to the unfolded DD stress followed by proteotoxic stress (ufDD + MG132). The relative populations of each cell type were quantified using analytical flow cytometry, with the control sample as the reference point for cells expressing DD-GFP. Expected = predicted fraction of DD-GFP cells assuming unfolded DD and MG132 are additive. ** p-value < 0.05 by Welch's test, ** p-value < 0.01 by Welch's test, *** p-value < 0.001 by Welch's test.DOI:http://dx.doi.org/10.7554/eLife.07687.016
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fig7s1: The effect of transcription factors on cell growth.(A) NIH3T3 cells expressing DD-GFP were co-cultured with unmodified 3T3 cells in the presence of 1 μM S1. The stabilizing S1 was withdrawn for 45, 135, or 405 min and then re-administered to the cells for an additional 17 hr. Analytical flow cytometry was used to quantify the relative populations of each cell type. The experiment was performed in triplicate for populations of cells expressing high levels of DD-GFP experiencing siRNA of p53 (blue) as well as the cell line experiencing non-target siRNA (siNT, black). (B) NIH3T3 cells expressing DD-GFP were co-cultured with unmodified 3T3 cells in the presence of 1 μM S1. The stabilizing S1 was withdrawn for 45, 135, or 405 min and then re-administered to the cells for an additional 17 hr. Analytical flow cytometry was used to quantify the relative populations of each cell type. The experiment was performed in triplicate for populations of cells expressing high levels of DD-GFP experiencing siRNA of HSF1 (red) as well as the cell line experiencing non-target siRNA (siNT, black). (C) NIH3T3 cells expressing DD-GFP were co-cultured with unmodified 3T3 cells in the presence of 1 μM S1. The stabilizing S1 was withdrawn for 45, 135, or 405 min and then re-administered to the cells for an additional 17 hr. Analytical flow cytometry was used to quantify the relative populations of each cell type. The experiment was performed in triplicate for populations of cells expressing high levels of DD-GFP experiencing siRNA of NRF2 (green) as well as the cell line experiencing non-target siRNA (siNT, black). (D) Cells expressing DD-GFP were co-cultured with unmodified 3T3 cells in a 24-well plate. One group of cells was not exposed to stress from either the unfolded DD or 10 μM MG132 (Ctl). The second group was exposed to MG132 only (MG132). The third group experienced the unfolded DD by withdrawal of S1 (shown as ufDD), and the fourth group was exposed to the unfolded DD stress followed by proteotoxic stress (ufDD + MG132). The relative populations of each cell type were quantified using analytical flow cytometry, with the control sample as the reference point for cells expressing DD-GFP. Expected = predicted fraction of DD-GFP cells assuming unfolded DD and MG132 are additive. ** p-value < 0.05 by Welch's test, ** p-value < 0.01 by Welch's test, *** p-value < 0.001 by Welch's test.DOI:http://dx.doi.org/10.7554/eLife.07687.016

Mentions: In order to assess how cellular fitness might be affected by the appearance of the unfolded DD, we co-cultured the DD-GFP-expressing cells with unmodified NIH3T3 cells (Sancho et al., 2013). We created unfolded DD by withdrawing S1 for 45, 135, or 405 min. Cells were co-cultured for an additional 17 hr in S1-containing media and then analyzed by flow cytometry to quantify the ratio of DD-GFP-expressing cells to control cells (Figure 7A). We observed that the amount of time cells were exposed to the unfolded DD inversely correlated with replicative fitness. Creating unfolded DD for a period as brief as 45 min caused a significant and reproducible reduction in the population of DD-GFP cells, and 405 min of exposure to unfolded DD caused the original ratio of 61:39 (DD-GFP:control cells) to fall to 51:49. Cells that express threefold lower amounts of the DD do not experience this replicative fitness defect (Figure 7A). In attempt to identify the source of this replicative fitness defect, we used RNAi to knockdown p53, Hsf1, and Nrf2 in separate populations of cells. Loss of p53 reduces the replicative phenotype exhibited by cells challenged with the unfolded DD, whereas loss of Hsf1 or Nrf2 did not reveal any detectable effect in cycling cells (Figure 7—figure supplement 1A–C). Thus, there is a replicative cost for cells challenged with the unfolded DD, and this response appears to be mediated, at least in part, by p53.10.7554/eLife.07687.015Figure 7.The cUPR provides protection against subsequent proteotoxic stress.


Distinct transcriptional responses elicited by unfolded nuclear or cytoplasmic protein in mammalian cells.

Miyazaki Y, Chen LC, Chu BW, Swigut T, Wandless TJ - Elife (2015)

The effect of transcription factors on cell growth.(A) NIH3T3 cells expressing DD-GFP were co-cultured with unmodified 3T3 cells in the presence of 1 μM S1. The stabilizing S1 was withdrawn for 45, 135, or 405 min and then re-administered to the cells for an additional 17 hr. Analytical flow cytometry was used to quantify the relative populations of each cell type. The experiment was performed in triplicate for populations of cells expressing high levels of DD-GFP experiencing siRNA of p53 (blue) as well as the cell line experiencing non-target siRNA (siNT, black). (B) NIH3T3 cells expressing DD-GFP were co-cultured with unmodified 3T3 cells in the presence of 1 μM S1. The stabilizing S1 was withdrawn for 45, 135, or 405 min and then re-administered to the cells for an additional 17 hr. Analytical flow cytometry was used to quantify the relative populations of each cell type. The experiment was performed in triplicate for populations of cells expressing high levels of DD-GFP experiencing siRNA of HSF1 (red) as well as the cell line experiencing non-target siRNA (siNT, black). (C) NIH3T3 cells expressing DD-GFP were co-cultured with unmodified 3T3 cells in the presence of 1 μM S1. The stabilizing S1 was withdrawn for 45, 135, or 405 min and then re-administered to the cells for an additional 17 hr. Analytical flow cytometry was used to quantify the relative populations of each cell type. The experiment was performed in triplicate for populations of cells expressing high levels of DD-GFP experiencing siRNA of NRF2 (green) as well as the cell line experiencing non-target siRNA (siNT, black). (D) Cells expressing DD-GFP were co-cultured with unmodified 3T3 cells in a 24-well plate. One group of cells was not exposed to stress from either the unfolded DD or 10 μM MG132 (Ctl). The second group was exposed to MG132 only (MG132). The third group experienced the unfolded DD by withdrawal of S1 (shown as ufDD), and the fourth group was exposed to the unfolded DD stress followed by proteotoxic stress (ufDD + MG132). The relative populations of each cell type were quantified using analytical flow cytometry, with the control sample as the reference point for cells expressing DD-GFP. Expected = predicted fraction of DD-GFP cells assuming unfolded DD and MG132 are additive. ** p-value < 0.05 by Welch's test, ** p-value < 0.01 by Welch's test, *** p-value < 0.001 by Welch's test.DOI:http://dx.doi.org/10.7554/eLife.07687.016
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fig7s1: The effect of transcription factors on cell growth.(A) NIH3T3 cells expressing DD-GFP were co-cultured with unmodified 3T3 cells in the presence of 1 μM S1. The stabilizing S1 was withdrawn for 45, 135, or 405 min and then re-administered to the cells for an additional 17 hr. Analytical flow cytometry was used to quantify the relative populations of each cell type. The experiment was performed in triplicate for populations of cells expressing high levels of DD-GFP experiencing siRNA of p53 (blue) as well as the cell line experiencing non-target siRNA (siNT, black). (B) NIH3T3 cells expressing DD-GFP were co-cultured with unmodified 3T3 cells in the presence of 1 μM S1. The stabilizing S1 was withdrawn for 45, 135, or 405 min and then re-administered to the cells for an additional 17 hr. Analytical flow cytometry was used to quantify the relative populations of each cell type. The experiment was performed in triplicate for populations of cells expressing high levels of DD-GFP experiencing siRNA of HSF1 (red) as well as the cell line experiencing non-target siRNA (siNT, black). (C) NIH3T3 cells expressing DD-GFP were co-cultured with unmodified 3T3 cells in the presence of 1 μM S1. The stabilizing S1 was withdrawn for 45, 135, or 405 min and then re-administered to the cells for an additional 17 hr. Analytical flow cytometry was used to quantify the relative populations of each cell type. The experiment was performed in triplicate for populations of cells expressing high levels of DD-GFP experiencing siRNA of NRF2 (green) as well as the cell line experiencing non-target siRNA (siNT, black). (D) Cells expressing DD-GFP were co-cultured with unmodified 3T3 cells in a 24-well plate. One group of cells was not exposed to stress from either the unfolded DD or 10 μM MG132 (Ctl). The second group was exposed to MG132 only (MG132). The third group experienced the unfolded DD by withdrawal of S1 (shown as ufDD), and the fourth group was exposed to the unfolded DD stress followed by proteotoxic stress (ufDD + MG132). The relative populations of each cell type were quantified using analytical flow cytometry, with the control sample as the reference point for cells expressing DD-GFP. Expected = predicted fraction of DD-GFP cells assuming unfolded DD and MG132 are additive. ** p-value < 0.05 by Welch's test, ** p-value < 0.01 by Welch's test, *** p-value < 0.001 by Welch's test.DOI:http://dx.doi.org/10.7554/eLife.07687.016
Mentions: In order to assess how cellular fitness might be affected by the appearance of the unfolded DD, we co-cultured the DD-GFP-expressing cells with unmodified NIH3T3 cells (Sancho et al., 2013). We created unfolded DD by withdrawing S1 for 45, 135, or 405 min. Cells were co-cultured for an additional 17 hr in S1-containing media and then analyzed by flow cytometry to quantify the ratio of DD-GFP-expressing cells to control cells (Figure 7A). We observed that the amount of time cells were exposed to the unfolded DD inversely correlated with replicative fitness. Creating unfolded DD for a period as brief as 45 min caused a significant and reproducible reduction in the population of DD-GFP cells, and 405 min of exposure to unfolded DD caused the original ratio of 61:39 (DD-GFP:control cells) to fall to 51:49. Cells that express threefold lower amounts of the DD do not experience this replicative fitness defect (Figure 7A). In attempt to identify the source of this replicative fitness defect, we used RNAi to knockdown p53, Hsf1, and Nrf2 in separate populations of cells. Loss of p53 reduces the replicative phenotype exhibited by cells challenged with the unfolded DD, whereas loss of Hsf1 or Nrf2 did not reveal any detectable effect in cycling cells (Figure 7—figure supplement 1A–C). Thus, there is a replicative cost for cells challenged with the unfolded DD, and this response appears to be mediated, at least in part, by p53.10.7554/eLife.07687.015Figure 7.The cUPR provides protection against subsequent proteotoxic stress.

Bottom Line: The sudden appearance of unfolded protein in mammalian cells elicits a robust transcriptional response, which is distinct from the HSR and other known pathways that respond to unfolded proteins.The cellular response to unfolded protein is strikingly different in the nucleus and the cytosol, although unfolded protein in either compartment engages the p53 network.This response provides cross-protection during subsequent proteotoxic stress, suggesting that it is a central component of protein quality control networks, and like the HSR, is likely to influence the initiation and progression of human pathologies.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Systems Biology, Stanford University, Stanford, United States.

ABSTRACT
Eukaryotic cells possess a variety of signaling pathways that prevent accumulation of unfolded and misfolded proteins. Chief among these is the heat shock response (HSR), which is assumed to respond to unfolded proteins in the cytosol and nucleus alike. In this study, we probe this axiom further using engineered proteins called 'destabilizing domains', whose folding state we control with a small molecule. The sudden appearance of unfolded protein in mammalian cells elicits a robust transcriptional response, which is distinct from the HSR and other known pathways that respond to unfolded proteins. The cellular response to unfolded protein is strikingly different in the nucleus and the cytosol, although unfolded protein in either compartment engages the p53 network. This response provides cross-protection during subsequent proteotoxic stress, suggesting that it is a central component of protein quality control networks, and like the HSR, is likely to influence the initiation and progression of human pathologies.

No MeSH data available.


Related in: MedlinePlus