Regulation of unfolded protein response modulator XBP1s by acetylation and deacetylation

Abstract

XBP1 (X-box-binding protein 1) is a key modulator of the UPR (unfolded protein response), which is involved in a wide range of pathological and physiological processes. The mRNA encoding the active spliced form of XBP1 (XBP1s) is generated from the unspliced form by IRE1 (inositol-requiring enzyme 1) during the UPR. However, the post-translational modulation of XBP1s remains largely unknown. In the present study, we demonstrate that XBP1s is a target of acetylation and deacetylation mediated by p300 and SIRT1 (sirtuin 1) respectively. p300 increases the acetylation and protein stability of XBP1s, and enhances its transcriptional activity, whereas SIRT1 deacetylates XBP1s and inhibits its transcriptional activity. Deficiency of SIRT1 enhances XBP1s-mediated luciferase reporter activity in HEK (human embryonic kidney)-293 cells and the up-regulation of XBP1s target gene expression under ER (endoplasmic reticulum) stress in MEFs (mouse embryonic fibroblasts). Consistent with XBP1s favouring cell survival under ER stress, Sirt1-/- MEFs display a greater resistance to ER-stress-induced apoptotic cell death compared with Sirt1+/+ MEFs. Taken together, these results suggest that acetylation/deacetylation constitutes an important post-translational mechanism in controlling protein levels, as well as the transcriptional activity, of XBP1s. The present study provides a novel insight into the molecular mechanisms by which SIRT1 regulates UPR signalling.

Figure 1. XBP1s is a target of acetylation

(A) HEK293 cells were transfected with Flag-XBP1s and increasing amount of p300. Cell lysates were analyzed by immunoprecipitation and Western blotting. Ace-K, acetylated lysine. *, non-specific band. (B) Equal amount of XBP1s was cotransfected with p300 or a control vector into HEK293 cells. Cells were harvested for lysates after cycloheximide (100 μg/ml) treatment for indicated time period. Cell lysates were analyzed by Western blotting.

Figure 2. XBP1s is deacetylated by SIRT1

(A and B) HEK293 cells were transfected with Flag-XBP1s and p300. Cells were incubated for 4 hours in the absence or presence of TSA (5μM), NAM (10 mM), or EX-527 (1 μM). Cell lysates were analyzed by immunoprecipitation and Western blotting. *, non-specific band. The extent of acetylation was defined by the ratio of acetylated vs. total XBP1s from duplicate experiments (Fig.2A, lower panel). (C) HEK293 cells were transfected with Flag-XBP1s and p300 together with a pSuper-shRNA vector targeting SIRT1 or its empty vector. Cell lysates were analyzed by immunoprecipitation and Western blotting. *, non-specific band. (D) HEK293 cells were transfected with Flag-XBP1s and p300 together with or without SIRT1 expressing construct. O/E, overexpression. Cell lysates were analyzed by immunoprecipitation and Western blotting. *, non-specific band. (E) The acetylated Flag-XBP1s was purified from 293T cells transfected with Flag-XBP1s and p300. The Flag-SIRT1 was purified from 293T cells separately transfected with Flag-SIRT1. Purified acetylated XBP1s was incubated in the presence or absence of SIRT1 and/or NAD⁺. The acetylated and total amounts of XBP1s were determined by Western blotting and the extent of acetylation was defined by the ratio of acetylated vs. total XBP1s from duplicate experiments.

Figure 3. Physical interaction between XBP1s and SIRT1

(A) Western blotting showing the presence of endogenous SIRT1 and XBP1s in the nuclear extracts HEK293 cells treated with thapsigargin (TG). (B) Cos7 cells were transfected with untagged XBP1s and Flag-SIRT1. Immunofluorescent staining for XBP1s and Flag-SIRT1 was performed using the anti-XBP1 and anti-Flag antibodies respectively. Confocal microscopy images at 60× magnification were presented to indicate the co-localization of XBP1s and SIRT1. (C) HEK293 cells were transfected with Flag-XBP1s or an empty vector. After immunoprecipitation using an anti-FLAG^® M2 affinity gel, Western blotting was performed to detect endogenous SIRT1. *, non-specific band.

Figure 4. SIRT1 negatively regulates the transcriptional activity of XBP1s

(A, B, and C) A 5×UPRE luciferase reporter was used to evaluate the transcriptional activity of XBP1s in HEK293 cells. To normalize the level of the experimental reporter activity, firefly luciferase value was divided by Renilla luciferase value from the same sample. (A) Cells were transfected with XBP1s or an empty vector together with SIRT1 at indicated doses. Asterisk indicates statistical significance determined by Student’s t test (*p<0.01). (B) Cells were transfected with XBP1s and SIRT1 either in the presence or the absence of p300. Asterisks indicate statistical significance determined by Student’s t test (*p < 0.05; **p < 0.01). (C) Cells were transfected with XBP1s and an SIRT1 shRNA expression plasmid. Asterisk indicates statistical significance determined by Student’s t test (*p < 0.01). (D) Both Sirt1 WT and KO MEFs were treated with 300 nM thapsigargin, or 2.5 μg/mL tunicamycin, or their corresponding vehicle for 5 hours. The mRNA levels of UPR target genes were evaluated by quantitative PCR. UPR-mediated Xbp1 mRNA splicing in Sirt1 WT and KO MEFs treated with ER stress agents was calculated based on the quantitative PCR results. Asterisks indicate statistical significance determined by Student’s t test (*p < 0.05, **p < 0.01).

Figure 5. SIRT1 sensitizes ER stress-induced cell death

(A, B, and C) Sirt1^+/+ and Sirt1^−/− MEFs were treated with 2.5 μg/mL tunicamycin or its vehicle (DMSO) for 24 hours. (A) Cell culture images were taken under a phase contrast microscope. Scale Bar, 200 μm. (B) The relative fold of cell mortality was assessed after trypan blue staining. Asterisk indicates statistical significance determined by Student’s t test (*p < 0.01). (C) Parallel cell lysates were analyzed for expression levels of SIRT1, XBP1s, Caspase-3 and β-actin by Western blotting. (D) Working model illustrating the role of SIRT1 in regulating XBP1s during UPR.