SIRT1 mediates FOXA2 breakdown by deacetylation in a nutrient-dependent manner

The Forkhead transcription factor FOXA2 plays a fundamental role in controlling metabolic homeostasis in the liver during fasting. The precise molecular regulation of FOXA2 in response to nutrients is not fully understood. Here, we studied whether FOXA2 could be controlled at a post-translational level by acetylation. By means of LC-MS/MS analyses, we identified five acetylated residues in FOXA2. Sirtuin family member SIRT1 was found to interact with and deacetylate FOXA2, the latter process being dependent on the NAD +-binding catalytic site of SIRT1. Deacetylation by SIRT1 reduced protein stability of FOXA2 by targeting it towards proteasomal degradation, and inhibited transcription from the FOXA2-driven G6pase and CPT1a promoters. While mutation of the five identified acetylated residues weakly affected protein acetylation and stability, mutation of at least seven additional lysine residues was required to abolish acetylation and reduce protein levels of FOXA2. The importance of acetylation of FOXA2 became apparent upon changes in nutrient levels. The interaction of FOXA2 and SIRT1 was strongly reduced upon nutrient withdrawal in cell culture, while enhanced Foxa2 acetylation levels were observed in murine liver in vivo after starvation for 36 hours. Collectively, this study demonstrates that SIRT1 controls the acetylation level of FOXA2 in a nutrient-dependent manner and in times of nutrient shortage the interaction between SIRT1 and FOXA2 is reduced. As a result, FOXA2 is protected from degradation by enhanced acetylation, hence enabling the FOXA2 transcriptional program to be executed to maintain metabolic homeostasis

Hsp70 and Hsp40 inhibit an inter-domain interaction necessary for transcriptional activity in the androgen receptor.

Molecular chaperones such as Hsp40 and Hsp70 hold the androgen receptor (AR) in an inactive conformation. They are released in the presence of androgens, enabling transactivation and causing the receptor to become aggregation-prone. Here we show that these molecular chaperones recognize a region of the AR N-terminal domain (NTD), including a FQNLF motif, that interacts with the AR ligand-binding domain (LBD) upon activation. This suggests that competition between molecular chaperones and the LBD for the FQNLF motif regulates AR activation. We also show that, while the free NTD oligomerizes, binding to Hsp70 increases its solubility. Stabilizing the NTD-Hsp70 interaction with small molecules reduces AR aggregation and promotes its degradation in cellular and mouse models of the neuromuscular disorder spinal bulbar muscular atrophy. These results help resolve the mechanisms by which molecular chaperones regulate the balance between AR aggregation, activation and quality control

Increased immune cell infiltration in patient-derived tumor explants treated with Traniplatin: an original Pt(iv) pro-drug based on Cisplatin and Tranilast

Elevated intra-tumoral immune infiltrate is associated with an improved prognosis in cancer of distinct origins. Traniplatin (TPT) is a novel platinum(IV) pro-drug based on Cisplatin (CDDP) and the marketed drug Tranilast. When compared in vitro to Cisplatin, TPT showed increased cytotoxic activity against colon and lung cancer cells but decreased activity against immune cells. In addition, TPT efficiency was evaluated in tumor explants derived from colorectal cancer samples from patients subjected to intended curative surgery. TPT induced strong intra-tumoral cytotoxic activity yet was associated with an elevated presence of immune cell infiltrate, suggesting a reduced cytotoxic activity against immune cells in colorectal cancer

Role of Hog1 and Yaf9 in the transcriptional response of Saccharomyces cerevisiae to cesium chloride

We analyzed the global transcriptional response of Saccharomyces cerevisiae cells exposed to different concentrations of CsCl in the growth medium and at different times after addition. Early responsive genes were mainly involved in cell wall structure and biosynthesis. About half of the induced genes were previously shown to respond to other alkali metal cations in a Hog1-dependent fashion. Western blot analysis confirmed that cesium concentrations as low as 100 mM activate Hog1 phosphorylation. Another important fraction of the cesium-modulated genes requires Yaf9p for full responsiveness as shown by the transcriptome of a yaf9-deleted strain in the presence of cesium. We showed that a cell wall-restructuring process promptly occurs in response to cesium addition, which is dependent on the presence of both Hog1 and Yaf9 proteins. Moreover, the sensitivity to low concentration of cesium of the yaf9-deleted strain is not observed in a strain carrying the hog1/yaf9 double deletion. We conclude that the observed early transcriptional modulation of cell wall genes has a crucial role in S. cerevisiae adaptation to cesium

SIRT1 interacts with and deacetylates FOXA2.

<p>(A) Increase in FOXA2 acetylation by NAM. FLAG-FOXA2-transfected HEK293T cells were treated with class I/II HDAC inhibitor TSA (5 µM), class III HDAC inhibitor NAM (20 mM) or both for 16 hours. Acetylation level of immunoprecipitated FLAG-FOXA2 was assessed on Western Blot probed with an anti-acetylated-lysine specific antibody. (B) SIRT1 co-transfection affects FOXA2 acetylation. HEK293T cells were transfected with FLAG-FOXA2 alone or together with MYC-SIRT1 in the presence of 20 mM NAM. Acetylation was assessed as in (A). (C) SIRT1 interacts with FOXA2. HEK293T cells were co-transfected as in (B) followed by co-immunoprecipitation with anti-FLAG M2 beads. Interaction of FLAG-FOXA2 and MYC-SIRT1 was assessed by Western Blot analysis with indicated antibodies. (D) Endogenous FOXA2 and SIRT1 interact in the nucleus. HepG2 cells were subjected to immunofluorescence-based proximity ligation assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098438#pone.0098438-Plas1" target="_blank">[28]</a>. Nuclei were counterstained with DAPI.</p

SIRT1-mediated deacetylation targets FOXA2 towards proteasomal degradation.

<p>(A) SIRT1 affects FOXA2 protein stability. HEK293T cells were transfected with FLAG-FOXA2 in the presence of 20 mM NAM or vehicle for 16 hours prior to cell lysis. Western Blots were probed for FLAG, and actin as a loading control. (B) Wild type SIRT1 and mutant SIRT1 H363Y similarly interact with FOXA2. HEK293T cells were transfected with FLAG-FOXA2 alone or in combination with either wild-type (wt) or the catalytically impaired MYC-SIRT1-H363Y mutant. FOXA2 and SIRT1 were co-immunoprecipitated with anti-FLAG-M2 beads and protein levels were assessed by Western Blot analysis probed with the indicated antibodies. (C) SIRT1 catalytic activity regulates FOXA2 stability. HEK293T cells were transfected and subjected to co-immunoprecipitation as in (B). Protein expression and FOXA2 acetylation level were assessed by Western Blot using the indicated antibodies. (D) Acetylation-impaired FLAG-12K-R-FOXA2 mutant showed reduced acetylation and protein stability. HEK293T cells were transfected with either FLAG-FOXA2 or FLAG-12K-R-FOXA2 and cultured as in (A). After FLAG-immunoprecipitation, protein levels were assessed by Western Blot probed with the indicated antibodies. (E) FOXA2 is subjected to proteasomal degradation in a time-dependent manner. HEK293T cells were transfected with FLAG-FOXA2 and supplemented with proteasome inhibitor MG132 (20 µM) or vehicle-containing medium for 3 or 6 hours. FOXA2 protein levels were assessed by Western Blot, and actin as a loading control. (F) SIRT1 mediates endogenous FOXA2 breakdown. HepG2 cells were transfected with MYC-SIRT1 and supplemented with 20 µM MG132- or vehicle-containing medium for 3 hours. (G) NAM preserves stability of FOXA2 upon abrogation of protein translation. HEK293T cells were transfected with FLAG-FOXA2 and supplemented 20 mM NAM or vehicle for 16 hours, followed by addition of cycloheximide (5 µg/ml) or vehicle-containing medium for 4 or 8 hours. (E–G) show a representative experiment from three independent experiments, and quantification of FOXA2 protein levels was performed by normalizing to actin protein levels. (H) 12K-R FOXA2 mutant has a higher level of poly-ubiquitylation. HEK293T cells were transfected with FLAG-FOXA2 or FLAG-12K-R-FOXA2. After culture of 20 µM MG132 for 3 hours, FOXA2 was immunoprecipitated with FLAG-M2 beads and immunoprecipitated proteins were assessed by Western Blot probed with the indicated antibodies. (I) Nutrient-dependent regulation of Foxa2 acetylation level in murine livers. Murine Foxa2 was immunoprecipitated from whole liver protein lysates from mice fed <i>ad libitum</i> (AL) or starved (ST) to 25% of <i>ad libitum</i> levels. Western Blots were probed with the indicated antibodies. The asterisk (*) marks a co-eluting acetylated protein of unknown identity.</p

Nutrient withdrawal reduces SIRT1 and FOXA2 interaction.

<p>(A) Nutrient withdrawal reduced SIRT1-FOXA2 interaction. HEK293T cells were co-transfected with indicated constructs and starved from glucose and serum for 16 hours. FOXA2 and SIRT1 were co-immunoprecipitated with anti-FLAG-M2 beads and protein levels were assessed by Western Blot analysis probed with the indicated antibodies. Relative interaction is defined as the fraction of FOXA2 that binds to SIRT1. (B) Reduced endogenous interaction of FOXA2 and SIRT1 in the nucleus upon nutrient withdrawal as in A for 2 or 4 hours. Interaction between endogenously expressed SIRT1 and FOXA2 was assessed in HepG2 cells by PLA. Nuclei were counterstained with DAPI.</p