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Luman contributes to brefeldin A-induced prion protein gene expression by interacting with the ERSE26 element

View Article: PubMed Central - PubMed

ABSTRACT

The cellular prion protein (PrP) is essential for transmissible prion diseases, but its exact physiological function remains unclear. Better understanding the regulation of the human prion protein gene (PRNP) expression can provide insight into this elusive function. Spliced XBP1 (sXBP1) was recently shown to mediate endoplasmic reticulum (ER) stress-induced PRNP expression. In this manuscript, we identify Luman, a ubiquitous, non-canonical unfolded protein response (UPR), as a novel regulator of ER stress-induced PRNP expression. Luman activity was transcriptionally and proteolytically activated by the ER stressing drug brefeldin A (BFA) in human neurons, astrocytes, and breast cancer MCF-7 cells. Over-expression of active cleaved Luman (ΔLuman) increased PrP levels, while siRNA-mediated Luman silencing decreased BFA-induced PRNP expression. Site-directed mutagenesis and chromatin immunoprecipitation demonstrated that ΔLuman regulates PRNP expression by interacting with the ER stress response element 26 (ERSE26). Co-over-expression and siRNA-mediated silencing experiments showed that sXBP1 and ΔLuman both up-regulate ER stress-induced PRNP expression. Attempts to understand the function of PRNP up-regulation by Luman excluded a role in atorvastatin-induced neuritogenesis, ER-associated degradation, or proteasomal inhibition-induced cell death. Overall, these results refine our understanding of ER stress-induced PRNP expression and function.

No MeSH data available.


PrP does not influence the degradation of ERAD substrate TransthyretinD18G (TTRD18G).(a) Western blot for TTRD18G-eGFP, PrP and β-Actin of MCF-7 cells co-transfected with pBud-eGFP (Vector) or pBud-eGFP-PrP (PrP) and pcDNA3.1(−)-TTRD18G-GFP, following a cycloheximide chase (75 μg/mL), with antibodies against eGFP (B-2), PrP (3F4) and β-Actin (AC15). (b) Schematic representation of quantified TTRD18G-GFP levels shown in (a) Data represent the mean ± SEM of three independent experiments. (c) Western blot for TTRD18G-eGFP, PrP and β-Actin of wild type or the PrP KO#1 and KO#2 CR7 cell lines transfected with pcDNA3.1(−)-TTRD18G-GFP, following a cycloheximide chase (75 μg/mL). (d) Schematic representation of quantified TTRD18G-GFP levels shown in (c) Data represent the relative intensity of a single experiment involving two independent mutant cell lines. Full-length images of blots are presented in Supplementary Information.
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f5: PrP does not influence the degradation of ERAD substrate TransthyretinD18G (TTRD18G).(a) Western blot for TTRD18G-eGFP, PrP and β-Actin of MCF-7 cells co-transfected with pBud-eGFP (Vector) or pBud-eGFP-PrP (PrP) and pcDNA3.1(−)-TTRD18G-GFP, following a cycloheximide chase (75 μg/mL), with antibodies against eGFP (B-2), PrP (3F4) and β-Actin (AC15). (b) Schematic representation of quantified TTRD18G-GFP levels shown in (a) Data represent the mean ± SEM of three independent experiments. (c) Western blot for TTRD18G-eGFP, PrP and β-Actin of wild type or the PrP KO#1 and KO#2 CR7 cell lines transfected with pcDNA3.1(−)-TTRD18G-GFP, following a cycloheximide chase (75 μg/mL). (d) Schematic representation of quantified TTRD18G-GFP levels shown in (c) Data represent the relative intensity of a single experiment involving two independent mutant cell lines. Full-length images of blots are presented in Supplementary Information.

Mentions: To better understand the physiological relevance of the regulation of PRNP expression by Luman and XBP1 during ER stress, and because both XBP1 and Luman regulate several genes involved in ERAD, the impact of PrP over-expression on the degradation rate of the ERAD substrate TTRD18G-GFP was assessed. The results show that PrP over-expression in the low PrP-expressing MCF-7 cell line did not facilitate TTRD18G-GFP degradation during a cycloheximide chase (Fig. 5a,b). Conversely, the degradation rate of the TTRD18G-GFP substrate was assessed in high PrP-expressing CR7 glioblastoma cells that underwent CRISPR/Cas9-mediated PrP knockout (KO) (Fig. 5c). No difference was observed between the degradation rate of TTRD18G-GFP in WT and PrP KO CR7 cells (Fig. 5c,d). In summary, these findings argue against a role of PrP in modulating ERAD.


Luman contributes to brefeldin A-induced prion protein gene expression by interacting with the ERSE26 element
PrP does not influence the degradation of ERAD substrate TransthyretinD18G (TTRD18G).(a) Western blot for TTRD18G-eGFP, PrP and β-Actin of MCF-7 cells co-transfected with pBud-eGFP (Vector) or pBud-eGFP-PrP (PrP) and pcDNA3.1(−)-TTRD18G-GFP, following a cycloheximide chase (75 μg/mL), with antibodies against eGFP (B-2), PrP (3F4) and β-Actin (AC15). (b) Schematic representation of quantified TTRD18G-GFP levels shown in (a) Data represent the mean ± SEM of three independent experiments. (c) Western blot for TTRD18G-eGFP, PrP and β-Actin of wild type or the PrP KO#1 and KO#2 CR7 cell lines transfected with pcDNA3.1(−)-TTRD18G-GFP, following a cycloheximide chase (75 μg/mL). (d) Schematic representation of quantified TTRD18G-GFP levels shown in (c) Data represent the relative intensity of a single experiment involving two independent mutant cell lines. Full-length images of blots are presented in Supplementary Information.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5304227&req=5

f5: PrP does not influence the degradation of ERAD substrate TransthyretinD18G (TTRD18G).(a) Western blot for TTRD18G-eGFP, PrP and β-Actin of MCF-7 cells co-transfected with pBud-eGFP (Vector) or pBud-eGFP-PrP (PrP) and pcDNA3.1(−)-TTRD18G-GFP, following a cycloheximide chase (75 μg/mL), with antibodies against eGFP (B-2), PrP (3F4) and β-Actin (AC15). (b) Schematic representation of quantified TTRD18G-GFP levels shown in (a) Data represent the mean ± SEM of three independent experiments. (c) Western blot for TTRD18G-eGFP, PrP and β-Actin of wild type or the PrP KO#1 and KO#2 CR7 cell lines transfected with pcDNA3.1(−)-TTRD18G-GFP, following a cycloheximide chase (75 μg/mL). (d) Schematic representation of quantified TTRD18G-GFP levels shown in (c) Data represent the relative intensity of a single experiment involving two independent mutant cell lines. Full-length images of blots are presented in Supplementary Information.
Mentions: To better understand the physiological relevance of the regulation of PRNP expression by Luman and XBP1 during ER stress, and because both XBP1 and Luman regulate several genes involved in ERAD, the impact of PrP over-expression on the degradation rate of the ERAD substrate TTRD18G-GFP was assessed. The results show that PrP over-expression in the low PrP-expressing MCF-7 cell line did not facilitate TTRD18G-GFP degradation during a cycloheximide chase (Fig. 5a,b). Conversely, the degradation rate of the TTRD18G-GFP substrate was assessed in high PrP-expressing CR7 glioblastoma cells that underwent CRISPR/Cas9-mediated PrP knockout (KO) (Fig. 5c). No difference was observed between the degradation rate of TTRD18G-GFP in WT and PrP KO CR7 cells (Fig. 5c,d). In summary, these findings argue against a role of PrP in modulating ERAD.

View Article: PubMed Central - PubMed

ABSTRACT

The cellular prion protein (PrP) is essential for transmissible prion diseases, but its exact physiological function remains unclear. Better understanding the regulation of the human prion protein gene (PRNP) expression can provide insight into this elusive function. Spliced XBP1 (sXBP1) was recently shown to mediate endoplasmic reticulum (ER) stress-induced PRNP expression. In this manuscript, we identify Luman, a ubiquitous, non-canonical unfolded protein response (UPR), as a novel regulator of ER stress-induced PRNP expression. Luman activity was transcriptionally and proteolytically activated by the ER stressing drug brefeldin A (BFA) in human neurons, astrocytes, and breast cancer MCF-7 cells. Over-expression of active cleaved Luman (ΔLuman) increased PrP levels, while siRNA-mediated Luman silencing decreased BFA-induced PRNP expression. Site-directed mutagenesis and chromatin immunoprecipitation demonstrated that ΔLuman regulates PRNP expression by interacting with the ER stress response element 26 (ERSE26). Co-over-expression and siRNA-mediated silencing experiments showed that sXBP1 and ΔLuman both up-regulate ER stress-induced PRNP expression. Attempts to understand the function of PRNP up-regulation by Luman excluded a role in atorvastatin-induced neuritogenesis, ER-associated degradation, or proteasomal inhibition-induced cell death. Overall, these results refine our understanding of ER stress-induced PRNP expression and function.

No MeSH data available.