Overexpression of SIRT3 disrupts mitochondrial proteostasis and cell cycle progression

As a mitochondrial deacetylase, SIRT3 deacetylates many enzymes involved in central metabolism and maintains mitochondrial proteostasis (Verdin et al., 2010; Papa and Germain, 2014). Substrates of SIRT3 include components of the respiratory complexes, proteins involved in fatty acid oxidation and TCA cycle (Yu et al., 2012). SIRT3 activates MnSOD to maintain reactive oxygen species (ROS) home-ostasis and a loss of SIRT3 contributes to the age-associated diseases (McDonnell et al., 2015; Qiu et al., 2010). SIRT3 plays dual roles functioning as a tumor suppressor or a promoter in tumorigenesis and progression (Alhazzazi et al., 2011). On one hand, SIRT3 regulates the cellular ROS level and maintains genomic stability, and mediates metabolic reprogramming to prevent tumorigenesis (Finley and Haigis, 2012). As a result, the low expression of SIRT3 has been found in breast cancer, glioblastoma, colon cancer, osteosarcoma, prostate, and ovarian cancers (Kim et al., 2010; Finley and Haigis, 2012). On the other hand, SIRT3 is a prosurvival factor that modulates p53 activities and is upregulated in oral cancer, the node-positive breast cancer, and bladder cancer (Ashraf et al., 2006; Alhazzazi et al., 2011). These results suggest that SIRT3 possesses the tumor-type dependent function and its precise role needs to be elucidated in the context of a specific cancer. Clear cell renal cell carcinoma (ccRCC) is the most common histo-logical subtype of renal cancer (Cohen and McGovern, 2005). The aims of the present study were to examine the expression of SIRT3 in ccRCC and to characterize effects of SIRT3 on tumorigenesis and progression using 293T human embryonic kidney cells as the model system that has cancer stem cell-like features (Debeb et al., 2010). Equal amounts of proteins extracted from 18 paired ccRCC lesions and associated pericarcinous tissue samples were analyzed by Western blotting and the representative Western blot images of eight paired samples were shown in Fig. 1A, indicating that the expression levels of SIRT3 were lower in ccRCC than those in normal tissues. The gray scale analysis of the Western blot data for all eighteen paired samples showed that the SIRT3 expression was statistically down-regulated in ccRCC tissues (Fig. 1B), suggesting that the low expression of SIRT3 is important for ccRCC progression. To understand the role of SIRT3 in tumorigenesis and progression of ccRCC, stable cells overexpressing SIRT3 were established in 293T cells. The overexpression of SIRT3 in 293T cells (SIRT3-OE) was examined by Western blotting (Fig. S1), confirming that the …

overexpression cells (100 μg) were reduced with 5 mM dithiothreitol (DTT) and alkylated with 12.5 mM iodoacetamide (IAA). Samples were diluted with PBS to 1.5 M Urea followed by digestion with trypsin of a 1:100 protease/protein ratio at 37°C overnight. Samples were desalted using Sep-Pak C18 cartridges. Purified peptides were labeled with TMT reagents (Thermo, Pierce Biotechnology, Rockford, IL) according to the manufacture's instruction. Briefly, the TMT labeling reagents was dissolved in anhydrous acetonitrile and added to each digested products, then the reaction was incubated at room temperature for 1 hour, and quenched by 5% hydroxylamine for 15 min. The TMT labeled peptides were mixed and desalted by desalted by Sep-Pak C18 cartridges. The peptides were fractionated by a UPLC3000 system (Dionex, CA) with a XBridge TMBEH300 C18 column (Waters, MA). Mobile phase A is H2O with ammonium hydroxide, pH 10; and mobile phase B is acetonitrile in ammonium hydroxide pH 10. Peptides were separated with the following gradients: 8% to 18% phase B, 30 min; 18% to 32% phase B, 22 min;48 fractions were collected, dried by a speedvac, combined into 12 fractions, and redissolved in 0.1% formic acid for the following nano-LC-MS/MS analysis.
For quantitative proteomics analysis, the peptides were separated by a C18 column (75 µm inner-diameter, 150 mm length; Upchurch, Oak Harbor, USA) at a flow rate of 250 nL/min. Mobile phase A consisted of 0.1% formic acid, and mobile phase B consisted of 100% acetonitrile and 0.1% formic acid. The Q-Exactive mass spectrometer was operated in the data-dependent acquisition mode using Xcalibur 3.0 software: there was a single full-scan mass spectrum in the orbitrap (300 -1800 m/z, 70,000 resolution) followed by 20 data-dependent MS/MS scans at 35% normalized collision energy.
The generated MS/MS spectra were searched against the Uniprot Human database (January 10, 2015; 89105 sequences) using the SEQUEST searching engine of Proteome Discoverer software (version 1.4). The search criteria were as follows: full tryptic specificity was required; one missed cleavage was allowed; carbamidomethylation (C) and TMT sixplex (K and N-terminal) were set as the fixed modifications; the oxidation (M) was set as the variable modification; precursor ion mass tolerances were set at 10 ppm for all MS acquired in an Orbitrap mass analyzer; and the fragment ion mass tolerance was set at 20 mmu for all MS2 spectra acquired.
The peptide false discovery rate was calculated using Percolator provided by PD. When the q value was smaller than 1%, the peptide spectrum match was considered to be correct. False discovery was determined based on peptide spectrum match when searched against the reverse, decoy database. Peptides only assigned to a given protein group were considered as unique. The false discovery rate was also set to 0.01 for protein identifications. Relative protein quantification was performed using Proteome Discoverer software (Version 1.4) according to manufacturer's instructions on the six reporter ion intensities per peptide. Protein ratios were calculated as the median of all peptide hits belonging to a protein. Quantitative precision was expressed as protein ratio variability. Differentially expressed proteins were further confirmed by qPCR or western blotting.

Pull down assay
Cells were lysed in ice-cold 1% NP-40 lysis buffer. The lysates were incubated with anti-FLAG beads for 8h. The precipitated protein complexes were washed three times with lysis buffer without NP-40 and then suspended in protein loading buffer (contained Dithiothreitol). The samples were subjected to SDS-PAGE separation followed by LC-MS/MS analysis.

Western blotting
Cells were harvested and lysed on ice with RIPA lysis buffer. The supernatants were collected after centrifugation at 14,000 ×g for 10 min at 4C. Protein concentrations were measured with the BCA protein assay kit. Proteins were separated in 12% SDS-PAGE gel and transferred onto a PVDF transfer membrane with electroblotting. After blocking with 5% nonfat milk for 1 h at room temperature, the membrane was incubated overnight at 4C with primary antibody, washed with TBST buffer for 3 times, then incubated with anti-mouse or anti-rabbit secondary antibody labeled with HRP at room temperature for 1 h. The membrane was further washed with TBST buffer 3 times and developed with ECL reagents (Engreen, China). β-actin was detected with anti-β-actin antibody as an internal control. BioRad Image Lab software was used to analyze the images.

Real-time Quantitative PCR (qPCR)
The control cells and SIRT3 overexpression cells were cultured in DMEM medium.
Total RNA was extracted using RNAprep pure Cell / Bacteria Kit. cDNA was synthesized from 3 μg total RNA with the Reverse transcription kit. Quantitative realtime PCR was performed the Roche LightCycler® 480II Detection System with SYBR green incorporation according to the manufacturer's instructions and β-actin was detected as an internal control. The primers were either designed by using the Primer Premier 5 software or from Primer Bank (http://pga.mgh.harvard.edu/primerbank/).
The specific PCR products were confirmed by melting curve analysis. Relative expression was analyzed using the 2 -ΔΔCt method. Primer sequences for qPCR are listed in supplemental Table S3.

Statistical Method
Statistical analysis was carried out with GraphPad Prism 5.0 software.
Significant differences in the data were determined by Student's t-test. P values of <0.05 were considered significant.