Inhibition of nucleolar stress response by Sirt1: A potential mechanism of acetylation‐independent regulation of p53 accumulation

Abstract The mammalian Sirt1 deacetylase is generally thought to be a nuclear protein, but some pilot studies have suggested that Sirt1 may also be involved in orchestrating nucleolar functions. Here, we show that nucleolar stress response is a ubiquitous cellular reaction that can be induced by different types of stress conditions, and Sirt1 is an endogenous suppressor of nucleolar stress response. Using stable isotope labeling by amino acids in cell culture approach, we have identified a physical interaction of between Sirt1 and the nucleolar protein nucleophosmin, and this protein–protein interaction appears to be necessary for Sirt1 inhibition on nucleolar stress, whereas the deacetylase activity of Sirt1 is not strictly required. Based on the reported prerequisite role of nucleolar stress response in stress‐induced p53 protein accumulation, we have also provided evidence suggesting that Sirt1‐mediated inhibition on nucleolar stress response may represent a novel mechanism by which Sirt1 can modulate intracellular p53 accumulation independent of lysine deacetylation. This process may represent an alternative mechanism by which Sirt1 regulates functions of the p53 pathway.


SILAC and mass spectrometry analysis
Stable isotope labeling by amino acids in cell culture (SILAC) technique was used to identify Sirt1-binding partners (Gruhler & Kratchmarova, 2008). Labeled HeLa cells were continuously passaged for 6 times in SILAC DMEM medium supplemented with 13 C/ 15 N-labeled (heavy) arginine (Thermo). Non-labeled cells were transfected with Flag-Sirt1 and maintained in normal DMEM. Labeled and non-labeled cells of equal number were mixed and homogenized using an ultrasonic disruptor. Total protein was extracted and immunoprecipitated with anti-DDDDK agarose beads. Digested peptides were separated by nanoscale liquid chromatography, followed by mass spectrometry analysis using an ESI-LTQ-Orbitrap Velos Pro system (Thermo Fisher). The proteomic data was processed using MASCOT Distiller (Matrix Science, Boston, MA, USA).

Purification of nucleoli
Nucleoli purification was performed according to the protocol descried by Lam and Lamond (Lam & Lamond, 2006). Briefly, 10 7 HeLa cells were harvested by trypsin digestion and 3 washed with cold PBS. The cells were resuspended in 5 ml of Buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl 2 , 0.5 mM DTT) for a hypotonic shock treatment for 30 min. Then the cells were disrupted using a Dounce homogenizer till more than 90% of the cells were broken by checking under a phase contrast microscope. The nuclei were collected by centrifugation (200×g for 5 min) and resuspended in 3 ml of S1 solution (0.25 M sucrose, 10 mM MgCl 2 ). The crude nuclei fraction was then layered on 3 ml of S2 solution (0.35 M sucrose, 0.5 mM MgCl 2 ), centrifuged at 1400×g for 5 min. The pelleted nuclei were resuspended in 3 ml of S2 solution and sonicated for 6 intermittent 10-second bursts at 60% power. The homogenate was layered on 3 ml of S3 solution (0.88 M sucrose, 0.5 mM MgCl 2 ) and centrifuged at 2000×g for 10min to pellet the nucleoli, which were washed and finally collected in 0.5 ml of S2 solution. All procedures were carried out on ice.

Immunofluorescence and confocal microscopy
Immunofluorescence was performed as described previously (Liu et al., 2015). Cells cultured on Lab-Tek II chamber slides were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 2% BSA for 30 min. Cells were then incubated with primary antibodies overnight followed by conjugated anti-IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h. DAPI was used for counterstaining. Images were taken with a laser-scanning confocal microscope (Model LSM710, Zeiss, Jena, Germany). To assess NSR, fluorescent images for NPM were reviewed by an independent investigator and the nuclei were classified as either nucleoli-intact or nucleoli-disrupted in a blind manner. We also measured the average florescence intensity of nucleoli and nucleoplasm using Image J software (NIH), and used the nucleoplasm to nucleoli florescence intensity ratio as an index of nucleolar disruption. For each experiment, 10 random high-power fields or at least 50 cells from different 4 areas were examined.

Immunoprecipitation and western blot
Cells were homogenized by sonication in cold lysis buffer. The lysates were precleared using non-immune IgG, and incubated with 2 µg of capture antibody and 20 µl of 50% protein A/G-agarose bead slurry (Pierce Biotechnology, Rockford, IL, USA) overnight at 4 °C with gentle rotation. The beads were washed and boiled in 2 × Laemmli buffer. Western blot was performed as described (Yan et al., 2014).

Real-time quantitative PCR
Total RNA was extracted using TRIzol (Thermo) and quantified with NanoDrop 2000 (Thermo). Reverse transcription and quantitative PCR (qPCR) were performed using PrimeScript and SYBR Premix Ex Taq kits respectively (all from Takara, Dalian, China). The primer sequences for human pre-rRNA were GCCTTCTCTAGCGATCTGAGAG (forward) and CCATAACGGAGGCAGAGACA (reverse) as described (Liao et al., 2014). The mRNA of βactin was used as the house-keeping gene. The 2 -∆∆Ct method was used to assess the relative RNA expression levels.

Preparation of recombinant NPM
Human NPM cDNA as PCR amplified and cloned into pGEX-4T-1 at the BamHI site, and used to transformed BL21 competent cells. Single clones were recovered and the correct insertion was verified by sequencing. Cells were cultured and expression induced by IPTG for 1 hr. The fusion protein was purified using glutathione spin columns (from Thermo) and verified by SDS-PAGE and Coomassie Brilliant Blue staining.
The beads were incubated at 30 o C for 3 hr with rotation, and washed with PBS. Half of the beads were recovered and then incubated with 5 µg of recombinant human Sirt1 protein in a deacetylation reaction buffer containing 1 mM oxidized nicotinamide adenine dinucleotide (NAD + ), 50 mM Tris (pH9.0), 50 mM NaCl, 4 mM MgCl 2 , 0.5 mM DTT, 0.2 mM PMSF, 0.02% NP-40 and 5% glycerol, at 30 o C for 3 hr. The bound fusion protein was eluted using reduced glutathione. The acetylation status of different lysine residues was characterized by mass spectrometry.

Live cell imaging
HeLa cells expressing eGFP-NPM were continuously monitored with a spinning-disk confocal live cell imaging system (Cell Observer SD, Zeiss) equipped with a top cage incubator.
Fluorescent photographs were taken every 15 min for a total period of 24 hr, with a laser excitation at 490 nm.

Flow cytometry analysis
Cell cycle was analyzed using a FACSCalibur cytometer (BD Biosciences, Mountain View, CA, USA). Cells were detached with trypsin and fixed overnight in cold ethanol. Propidium iodide staining was performed using a kit from Abcam (ab139418) according to the manufacturer's instructions.

Statistics
Data are presented as mean ± standard error of the mean (SEM). Data analysis was performed using unpaired t-test or one-way analysis of variance (ANOVA) followed by post hoc