Functional Proteomics Establishes the Interaction of SIRT7 with Chromatin Remodeling Complexes and Expands Its Role in Regulation of RNA Polymerase I Transcription
2012; Elsevier BV; Volume: 11; Issue: 5 Linguagem: Inglês
10.1074/mcp.a111.015156
ISSN1535-9484
AutoresYuan-Chin Tsai, Todd M. Greco, Apaporn Boonmee, Yana Miteva, Ileana M. Cristea,
Tópico(s)RNA regulation and disease
ResumoAmong mammalian sirtuins, SIRT7 is the only enzyme residing in nucleoli where ribosomal DNA is transcribed. Recent reports established that SIRT7 associates with RNA Pol I machinery and is required for rDNA transcription. Although defined by its homology to the yeast histone deacetylase Sir2, current knowledge suggests that SIRT7 itself has little to no deacetylase activity. Because only two SIRT7 interactions have been thus far described: RNA Pol I and upstream binding factor, identification of proteins and complexes associating with SIRT7 is critical to understanding its functions. Here, we present the first characterization of SIRT7 interaction networks. We have systematically investigated protein interactions of three EGFP-tagged SIRT7 constructs: wild type, a point mutation affecting rDNA transcription, and a deletion mutant lacking the predicted coiled-coil domain. A combinatorial proteomics and bioinformatics approach was used to integrate gene ontology classifications, functional protein networks, and normalized abundances of proteins co-isolated with SIRT7. The resulting refined proteomic data set confirmed SIRT7 interactions with RNA Pol I and upstream binding factor and highlighted association with factors involved in RNA Pol I- and II-dependent transcriptional processes and several nucleolus-localized chromatin remodeling complexes. Particularly enriched were members of the B-WICH complex, such as Mybbp1a, WSTF, and SNF2h. Prominent interactions were validated by a selected reaction monitoring-like approach using metabolic labeling with stable isotopes, confocal microscopy, reciprocal immunoaffinity precipitation, and co-isolation with endogenous SIRT7. To extend the current knowledge of mechanisms involved in SIRT7-dependent regulation of rDNA transcription, we showed that small interfering RNA-mediated SIRT7 knockdown leads to reduced levels of RNA Pol I protein, but not messenger RNA, which was confirmed in diverse cell types. The down-regulation of RNA Pol I protein levels placed in the context of SIRT7 interaction networks led us to propose that SIRT7 plays a crucial role in connecting the function of chromatin remodeling complexes to RNA Pol I machinery during transcription. Among mammalian sirtuins, SIRT7 is the only enzyme residing in nucleoli where ribosomal DNA is transcribed. Recent reports established that SIRT7 associates with RNA Pol I machinery and is required for rDNA transcription. Although defined by its homology to the yeast histone deacetylase Sir2, current knowledge suggests that SIRT7 itself has little to no deacetylase activity. Because only two SIRT7 interactions have been thus far described: RNA Pol I and upstream binding factor, identification of proteins and complexes associating with SIRT7 is critical to understanding its functions. Here, we present the first characterization of SIRT7 interaction networks. We have systematically investigated protein interactions of three EGFP-tagged SIRT7 constructs: wild type, a point mutation affecting rDNA transcription, and a deletion mutant lacking the predicted coiled-coil domain. A combinatorial proteomics and bioinformatics approach was used to integrate gene ontology classifications, functional protein networks, and normalized abundances of proteins co-isolated with SIRT7. The resulting refined proteomic data set confirmed SIRT7 interactions with RNA Pol I and upstream binding factor and highlighted association with factors involved in RNA Pol I- and II-dependent transcriptional processes and several nucleolus-localized chromatin remodeling complexes. Particularly enriched were members of the B-WICH complex, such as Mybbp1a, WSTF, and SNF2h. Prominent interactions were validated by a selected reaction monitoring-like approach using metabolic labeling with stable isotopes, confocal microscopy, reciprocal immunoaffinity precipitation, and co-isolation with endogenous SIRT7. To extend the current knowledge of mechanisms involved in SIRT7-dependent regulation of rDNA transcription, we showed that small interfering RNA-mediated SIRT7 knockdown leads to reduced levels of RNA Pol I protein, but not messenger RNA, which was confirmed in diverse cell types. The down-regulation of RNA Pol I protein levels placed in the context of SIRT7 interaction networks led us to propose that SIRT7 plays a crucial role in connecting the function of chromatin remodeling complexes to RNA Pol I machinery during transcription. The correlation between histone acetylation status and transcriptional regulation is well established in that hyperacetylation is commonly associated with transcriptional activation, whereas hypoacetylation is associated with transcriptional repression (1Kuo M.H. Brownell J.E. Sobel R.E. Ranalli T.A. Cook R.G. Edmondson D.G. Roth S.Y. Allis C.D. Transcription-linked acetylation by Gcn5p of histones H3 and H4 at specific lysines.Nature. 1996; 383: 269-272Crossref PubMed Scopus (506) Google Scholar, 2de Ruijter A.J. van Gennip A.H. Caron H.N. Kemp S. van Kuilenburg A.B. Histone deacetylases (HDACs): Characterization of the classical HDAC family.Biochem. J. 2003; 370: 737-749Crossref PubMed Scopus (2454) Google Scholar, 3Taunton J. Hassig C.A. Schreiber S.L. A mammalian histone deacetylase related to the yeast transcriptional regulator Rpd3p.Science. 1996; 272: 408-411Crossref PubMed Scopus (1530) Google Scholar). In eukaryotes, Sir2-like proteins form a family of enzymes known as sirtuins (4Sauve A.A. Wolberger C. Schramm V.L. Boeke J.D. The biochemistry of sirtuins.Annu. Rev. Biochem. 2006; 75: 435-465Crossref PubMed Scopus (582) Google Scholar). Distinct from class I and II histone deacetylases (HDACs), which facilitate hydrolysis of acetyl-lysine as a bimolecular reaction (5Finnin M.S. Donigian J.R. Cohen A. Richon V.M. Rifkind R.A. Marks P.A. Breslow R. Pavletich N.P. Structures of a histone deacetylase homologue bound to the TSA and SAHA inhibitors.Nature. 1999; 401: 188-193Crossref PubMed Scopus (1498) Google Scholar), sirtuins consume an equimolar amount of nicotinamide adenine dinucleotide (NAD+) for each hydrolysis reaction and generate nicotinamide, O-acetyl ADP-ribose, and deacetylated substrate as end products (4Sauve A.A. Wolberger C. Schramm V.L. Boeke J.D. The biochemistry of sirtuins.Annu. Rev. Biochem. 2006; 75: 435-465Crossref PubMed Scopus (582) Google Scholar). Based on homology of the sirtuin core domain, there are seven sirtuins (SIRT1–7) in human cells with functions not limited to histone deacetylation, because many additional cellular proteins have been identified as substrates (4Sauve A.A. Wolberger C. Schramm V.L. Boeke J.D. The biochemistry of sirtuins.Annu. Rev. Biochem. 2006; 75: 435-465Crossref PubMed Scopus (582) Google Scholar, 6Frye R.A. Phylogenetic classification of prokaryotic and eukaryotic Sir2-like proteins.Biochem. Biophys. Res. Commun. 2000; 273: 793-798Crossref PubMed Scopus (1153) Google Scholar). In addition, the localizations of human sirtuins are distributed across multiple organelles including nucleolus, nucleus, cytoplasm, and mitochondria (7Michishita E. Park J.Y. Burneskis J.M. Barrett J.C. Horikawa I. Evolutionarily conserved and nonconserved cellular localizations and functions of human SIRT proteins.Mol. Biol. Cell. 2005; 16: 4623-4635Crossref PubMed Scopus (1064) Google Scholar). Systematic monitoring of the cellular localizations of all human sirtuins has demonstrated that sirtuin 7 (SIRT7) is the only enzyme that localizes to nucleoli, similar to that of the founding family member Sir2 in yeast (7Michishita E. Park J.Y. Burneskis J.M. Barrett J.C. Horikawa I. Evolutionarily conserved and nonconserved cellular localizations and functions of human SIRT proteins.Mol. Biol. Cell. 2005; 16: 4623-4635Crossref PubMed Scopus (1064) Google Scholar). Studies of the yeast histone deacetylase Sir2 have shown its involvement in several biological processes, the majority of these functions being related to transcriptional silencing (8Blander G. Guarente L. The Sir2 family of protein deacetylases.Annu. Rev. Biochem. 2004; 73: 417-435Crossref PubMed Scopus (1299) Google Scholar). However, unlike yeast Sir2, there have been no reports to indicate involvement of SIRT7 in transcriptional silencing; instead, several lines of evidence have shown that SIRT7 plays a positive role in activating rDNA transcription (9Ford E. Voit R. Liszt G. Magin C. Grummt I. Guarente L. Mammalian Sir2 homolog SIRT7 is an activator of RNA polymerase I transcription.Genes Dev. 2006; 20: 1075-1080Crossref PubMed Scopus (485) Google Scholar, 10Grob A. Roussel P. Wright J.E. McStay B. Hernandez-Verdun D. Sirri V. Involvement of SIRT7 in resumption of rDNA transcription at the exit from mitosis.J. Cell Sci. 2009; 122: 489-498Crossref PubMed Scopus (122) Google Scholar). It has been proposed that SIRT7 physically interacts with RNA polymerase I (denoted as Pol I) machinery and that its deacetylation activity is crucial for maintaining the elongation phase of Pol I (9Ford E. Voit R. Liszt G. Magin C. Grummt I. Guarente L. Mammalian Sir2 homolog SIRT7 is an activator of RNA polymerase I transcription.Genes Dev. 2006; 20: 1075-1080Crossref PubMed Scopus (485) Google Scholar). However, several important issues remain unresolved in this model because in vivo substrates have not been identified to date, and the evidence for deacetylation activity based on in vitro assays is conflicting (7Michishita E. Park J.Y. Burneskis J.M. Barrett J.C. Horikawa I. Evolutionarily conserved and nonconserved cellular localizations and functions of human SIRT proteins.Mol. Biol. Cell. 2005; 16: 4623-4635Crossref PubMed Scopus (1064) Google Scholar, 9Ford E. Voit R. Liszt G. Magin C. Grummt I. Guarente L. Mammalian Sir2 homolog SIRT7 is an activator of RNA polymerase I transcription.Genes Dev. 2006; 20: 1075-1080Crossref PubMed Scopus (485) Google Scholar, 11Vakhrusheva O. Smolka C. Gajawada P. Kostin S. Boettger T. Kubin T. Braun T. Bober E. Sirt7 increases stress resistance of cardiomyocytes and prevents apoptosis and inflammatory cardiomyopathy in mice.Circ. Res. 2008; 102: 703-710Crossref PubMed Scopus (505) Google Scholar). In addition, there appears to be some uncertainty as to whether SIRT7 directly associates with Pol I, because one study showed that instead of interacting with subunits within the Pol I machinery, SIRT7 preferentially interacts with the nucleolar upstream-binding factor (UBF) (10Grob A. Roussel P. Wright J.E. McStay B. Hernandez-Verdun D. Sirri V. Involvement of SIRT7 in resumption of rDNA transcription at the exit from mitosis.J. Cell Sci. 2009; 122: 489-498Crossref PubMed Scopus (122) Google Scholar). Moreover, the disruption of SIRT7 nucleolar localization followed by inhibition of rDNA transcription is not a generally observed effect across different cell lines (9Ford E. Voit R. Liszt G. Magin C. Grummt I. Guarente L. Mammalian Sir2 homolog SIRT7 is an activator of RNA polymerase I transcription.Genes Dev. 2006; 20: 1075-1080Crossref PubMed Scopus (485) Google Scholar, 10Grob A. Roussel P. Wright J.E. McStay B. Hernandez-Verdun D. Sirri V. Involvement of SIRT7 in resumption of rDNA transcription at the exit from mitosis.J. Cell Sci. 2009; 122: 489-498Crossref PubMed Scopus (122) Google Scholar). Therefore, the mechanisms by which SIRT7 regulates rDNA transcription remain unclear. Based on phylogenetic analysis of sirtuin core domains, sirtuins are further divided into five separate classes (6Frye R.A. Phylogenetic classification of prokaryotic and eukaryotic Sir2-like proteins.Biochem. Biophys. Res. Commun. 2000; 273: 793-798Crossref PubMed Scopus (1153) Google Scholar). Although yeast Sir2 and human SIRT7 share the same nucleolar localization, they belong to different phylogenetic classes, I and IV, respectively (6Frye R.A. Phylogenetic classification of prokaryotic and eukaryotic Sir2-like proteins.Biochem. Biophys. Res. Commun. 2000; 273: 793-798Crossref PubMed Scopus (1153) Google Scholar). This may explain why SIRT1, which belongs to the same class (class I) as Sir2, has a well established deacetylase activity and numerous substrates (4Sauve A.A. Wolberger C. Schramm V.L. Boeke J.D. The biochemistry of sirtuins.Annu. Rev. Biochem. 2006; 75: 435-465Crossref PubMed Scopus (582) Google Scholar), whereas SIRT6 and SIRT7, which are the only two members of class IV, have 1000-fold less or undetectable deacetylase activities (12Pan P.W. Feldman J.L. Devries M.K. Dong A. Edwards A.M. Denu J. M. Structure and biochemical functions of SIRT6. Structure and biochemical functions of SIRT6.J. Biol. Chem. 2011; 286: 14575-14587Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Because it has been reported that SIRT7 has no ADP-ribosylation activity, it is necessary to identify its interacting proteins to understand the role of this enzyme in rDNA transcription and other cellular processes. Here, we have carried out, to our knowledge, the first proteomic study aimed at establishing the interacting proteins of SIRT7. We have systematically investigated the protein networks of EGFP-tagged SIRT7 from three different constructs including a full-length wild type form and two mutant forms containing either a point mutation affecting rDNA transcription or a deletion of exon 2 within the predicted coiled-coil domain. Candidate interactions that showed significant enrichment in SIRT7 isolations relative to EGFP control isolations were subjected to gene ontology classification and functional network analysis, revealing a subset of nucleolar-enriched proteins, chromatin remodeling and modification factors, and transcriptional regulators. By correlating normalized immunoisolated protein abundance with these prominent functional categories, we refined the proteomic data set into targets of significant biological interest. These included Pol I- and II-dependent transcriptional processes as well as several nucleolar chromatin remodeling complexes, in particular the B-WICH complex. The interactions were then validated using a combinatorial approach integrating confocal microscopy, reciprocal immunoaffinity precipitation, metabolic labeling with stable isotopes, and co-isolation with endogenous SIRT7. We further demonstrated that knockdown of SIRT7 leads to down-regulation of Pol I machinery at the protein level. This down-regulation was SIRT7-specific, being partially rescued in a siRNA-resistant SIRT7 cell line. This observation indicates that a loss of SIRT7 functions may lead to arrest or halting of the Pol I complexes and subsequent Pol I degradation. We propose a model in which SIRT7 regulates RNA Pol I transcription through interaction with chromatin remodeling complexes. Antibodies used were in-house developed rabbit polyclonal anti-GFP (13Cristea I.M. Williams R. Chait B.T. Rout M.P. Fluorescent proteins as proteomic probes.Mol. Cell. Proteomics. 2005; 4: 1933-1941Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar), rabbit polyclonal anti-SIRT7 (a generous gift from Dr. Izumi Horikawa of NCI, National Institutes of Health) (7Michishita E. Park J.Y. Burneskis J.M. Barrett J.C. Horikawa I. Evolutionarily conserved and nonconserved cellular localizations and functions of human SIRT proteins.Mol. Biol. Cell. 2005; 16: 4623-4635Crossref PubMed Scopus (1064) Google Scholar), mouse monoclonal anti-GFP (Roche Applied Science), mouse monoclonal anti-UBF (F-9; Santa Cruz), mouse monoclonal anti-RPA194 (C-1; Santa Cruz), rabbit polyclonal anti-hSNF2H (H-300; Santa Cruz), rabbit polyclonal anti-RIF1 (Bethyl Laboratories Inc.), rabbit polyclonal anti-ATRX (Bethyl Laboratories Inc.), rabbit polyclonal anti-Mybbp1a (Bethyl Laboratories Inc.), and rabbit polyclonal anti-c-Myc (Cell Signaling). All of the oligonucleotide primers designed for this study were ordered from Integrated DNA Technologies and are listed in supplemental Table S1. Human SIRT7 siRNA and a universal siRNA negative control were purchased from Sigma-Aldrich. Protein A/G Plus-agarose was purchased from Santa Cruz. All other reagents were purchased from Sigma-Aldrich unless otherwise specified. HEK293 (Human embryonic kidney 293), HeLa (cervical carcinoma cell), A549 (human lung adenocarcinoma epithelial cell line), U2OS (human osteosarcoma cell line), and ZR-75–1 (human breast carcinoma cell line) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum in 37 °C with 5% CO2. HL60 (human promyelocytic leukemia cells) were cultured in RPMI supplemented with 10% fetal bovine serum. The plasmids containing SIRT7 cDNAs with full-length (plasmid 20268), S111A point mutant (plasmid 20269), and exon 2 deletion (plasmid 13818) were purchased from Addgene. SIRT7 cDNAs were amplified by PCR using SIRT7-specific primers (supplemental Table S1), purified, and digested with EcoRI and XhoI restriction enzymes. The digestion products were ligated to the 5′ end of EGFP cDNA (pEGFP-N1; Clontech) that we cloned into a pLXSN retroviral vector (Clontech), thus generating pLXSN-SIRT7-EGFP-FLAG retroviral plasmids. RNA interference-resistant clones were generated by site-directed mutagenesis using primers listed in supplemental Table S1. HEK293 cell lines stably expressing EGFP-FLAG or SIRT7-EGFP-FLAG full-length wild type or mutants were generated using the PhoenixTM retrovirus expression system (Orbigen, San Diego, CA). Briefly, the different pLXSN-SIRT7-EGFP-FLAG plasmids were transfected into Phoenix cells using FuGENE (Roche Applied Science), the cells were grown to 90% confluency, and the resulting retrovirus was collected and used to transduce HEK293 cells. The cells stably expressing SIRT7-EGFP-FLAG and derivatives were selected with 400 μg/liter G418 (EMD, Gibbstown, NJ) for 2 weeks and sorted by fluorescence-activated cell sorting (Vantage S.E. with TurboSort II; Becton Dickinson, Franklin Lakes, NJ). The expression of all EGFP fusion proteins was further confirmed by confocal microscopy and immunoblotting. siRNA-resistant clones were generated based on site-directed mutagenesis procedures. In brief, the primer set (supplemental Table S1) containing several silent mutations at third bases of codons (wobble position) was used in PCR amplification using pLXSN-SIRT7-EGFP-Flag plasmid as template. The reaction mixture was digested with DpnI restriction enzyme to remove the wild type strain. Aliquots of the reaction containing siRNA-resistant clones were transformed into DH5α strain and selected by ampicillin. The cell lines containing siRNA-resistant clones were generated according to the protocol mentioned above. SIRT7 and control immunoaffinity purifications on magnetic beads were performed via EGFP, as previously described (13Cristea I.M. Williams R. Chait B.T. Rout M.P. Fluorescent proteins as proteomic probes.Mol. Cell. Proteomics. 2005; 4: 1933-1941Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 14Greco T.M. Yu F. Guise A.J. Cristea I.M. Nuclear import of histone deacetylase 5 by requisite nuclear localization signal phosphorylation.Mol. Cell. Proteomics. 2010; 10 (10.1074/mcp.M110.004317)Google Scholar). HEK293 cell lines stably expressing EGFP alone, SIRT7WT-EGFP-FLAG (WT), SIRT7S111A-EGFP-FLAG (S111A), and SIRT7dE2-EGFP-FLAG (where dE2 indicates deletion of exon 2; see Fig. 1A) were washed with 10 ml of ice-cold D-PBS (Invitrogen) per 15-cm plate, then harvested by scraping with a plastic spatula (Fisher Scientific) on ice, pelleted at 200 × g at 4 °C for 5 min, and washed with ice-cold D-PBS (Invitrogen). The washed cell pellet was gently mixed with 100 μl/1 g of cells of 20 mm HEPES-NaOH, pH 7.5, containing 1.2% polyvinylpyrrolidone (w/v) and 1:100 (v/v) protease inhibitor mixture (Sigma), then frozen in liquid nitrogen, and subjected to cryogenic lysis using a Retsch MM 301 Mixer Mill (10 steps × 2 min at 30 Hz) (Retsch, Newtown, PA). All of the subsequent steps were performed at 4 °C unless otherwise noted. The cell powder (0.8 g) was suspended in 10 ml of ice-cold optimized lysis buffer (20 mm HEPES-KOH, pH 7.4, containing 0.1 m potassium acetate, 2 mm MgCl2, 0.1% Tween 20, 1 μm ZnCl2, 1 μm CaCl2, 0.5% Triton X-100, 250 mm NaCl, 4 μg/ml DNase, 1/100 (v/v) protease, and phosphatase inhibitor cocktails). The optimization of the lysis buffer for efficient solubilization and isolation of SIRT7-EGFP was performed as reported (15Cristea I.M. Chait B.T. Affinity purification of protein complexes.Cold Spring Harb. Protoc. 2011; : 5Google Scholar) and is illustrated in supplemental Figs. S3–S5 and described in the supplemental text. The resulting cell suspension was subjected to homogenization using a Polytron (2 × 15 s) (Kinematica), and the insoluble material was removed by centrifugation at 8,000 × g for 10 min. The cell lysates (supernatant) were incubated for 45 min with 7 mg of magnetic beads (M270 Epoxy Dynabeads; Invitrogen) conjugated with rabbit anti-GFP antibody, as described (16Cristea I.M. Chait B.T. Conjugation of magnetic beads for immunopurification of protein complexes.Cold Spring Harb. Protoc. 2011; : 5Google Scholar). The magnetic beads were then washed six times with lysis buffer (without protease and phosphatase inhibitors) and once with distilled H2O. The washed beads were incubated with 40 μl of 1× LDS sample buffer (Invitrogen) for 10 min at 70 °C, followed by shaking for 10 min at room temperature. Eluted proteins were reduced with DTT (50 mm for 10 min at 70 °C) and alkylated with iodoacetamide (100 mm, 30 min at room temperature). The samples were either stored at −20 °C or immediately processed for proteomic analysis, as described below. Reciprocal immunoaffinity purifications were performed using 0.1 g of cell pellet from SIRT7WT-EGFP-FLAG (WT) expressing cells for each reaction and the lysis buffer (20 mm HEPES-KOH, pH 7.4, containing 0.1 m potassium acetate, 1 mm MgCl2, 0.1% Tween 20, 0.5% Triton X-100, 150 mm NaCl, 4 μg/ml DNase, 1/100 (v/v) protease inhibitor mixture (Sigma)). The proteins were immunoisolated with 4 μg of antibody to either control IgG or identified SIRT7 protein interactions (e.g. UBF, RPA194, and Mybbp1a) for 1 h, followed by 2 h of incubation with 30 μl of protein A/G Plus-agarose beads (Santa Cruz). The agarose beads were subsequently washed twice with lysis buffer and twice with D-PBS, resuspended in 50 μl of 6× Laemmli sample buffer and heated to 95 °C for 10 min. The identities of the co-isolated proteins were analyzed by Western blot analysis. HEK293 cell lines stably expressing different SIRT7-EGFP-FLAG fusion proteins were cultured on glass-bottomed dishes, pretreated with poly-d-lysine (Sigma). After 48 h, the cells were fixed with 2% paraformaldehyde, washed with D-PBS, incubated with 1 μg/ml 4′,6′-diamino-2-phenylindole in D-PBS (PBST) for 15 min, and visualized by confocal microscopy on a PerkinElmer Life Sciences RS3 spinning disk using a 60× oil immersion lens. For co-localization analysis, the cells were cultured and fixed as above, permeabilized with 0.1% Triton X-100 in PBST for 15 min, and blocked in 2% (w/v) bovine serum albumin, 0.2% (v/v) Tween 20 in PBST at room temperature for 60 min. Incubation with a primary antibody was performed at room temperature for 60 min in blocking buffer. The cells were then washed with PBST and incubated with secondary antibodies conjugated to Alexa 546 or 633 (Invitrogen). Finally, the cells were washed and incubated with 1 μg/ml TO-PRO-3 iodide (Invitrogen) in PBST for 15 min, then washed again, and mounted on glass slides with a drop of Aqua Poly/Mount media (Polysciences) to proceed with confocal imaging. Primary eluates from SIRT7 immunoisolations were partially resolved (∼2 cm) on 4–12% Bis-Tris NuPAGE gels and stained with SimplyBlue Coomassie stain (Invitrogen). Each lane corresponding to a single immunoisolation was excised, cut into 1-mm slices, and pooled into six equal fractions. The gel pieces were destained in 50 mm ammonium bicarbonate, 50% acetonitrile (ACN), followed by two rounds of dehydration, and rehydration in 100% ACN and 50 mm ammonium bicarbonate, respectively. Washed gel pieces were dehydrated a final time in 100% ACN and then incubated with 12.5 ng/μl of sequencing grade trypsin (Promega, Madison, WI) overnight at 37 °C. The peptides were extracted in 0.5% formic acid for 4 h at room temperature, followed by a second extraction in 0.5% formic acid, 50% ACN for 2 h. The peptides were concentrated by vacuum centrifugation to ∼10 μl, and half of the sample was analyzed by nLC-MS/MS on a Dionex Ultimate 3000 RSLC coupled directly to an LTQ-Orbitrap Velos ETD mass spectrometer (ThermoFisher Scientific). The peptides were washed onto the trap column (Magic C18 AQ, 3 μm, 100 μm × 2.5 cm; Michrom Bioresources, Inc.) in 0.5% TFA, 1% ACN, 98.5% water and desalted for 5 min at 5 μl/min and then separated by reverse phase chromatography (Acclaim PepMap RSLC, 1.8 μm, 75 μm × 25 cm) at a flow rate of 250 nl/min using a 90-min discontinuous gradient of ACN as follows: 4% to 20% B over 50 min, 20% to 40% B over 40 min (Mobile phase A: 0.1% formic acid, 0.1% acetic acid in water, Mobile phase B: 0.1% formic acid, 0.1% acetic acid in 97% ACN). The mass spectrometer was operated in data-dependent acquisition mode with FT preview scan disabled and predictive AGC and dynamic exclusion enabled (repeat count, 1; exclusion duration, 70 s). A single acquisition cycle comprised a single full scan mass spectrum (m/z = 350–1700) in the Orbitrap (r = 30,000 at m/z = 400), followed by CID fragmentation on the top 20 most intense precursor ions (minimum signal = 1E3) in the dual pressure linear ion trap. The following instrument parameters were used: FT MS and IT MSn (tandem MS) target values of 1E6 and 5E3, respectively; FT MS and IT MSn maximum injection time of 300 and 100 ms, respectively. CID fragmentation was performed at an isolation width of 2.0 Th, normalized collision energy of 30, and an activation time of 10 ms. MS/MS spectra from raw files corresponding to single biological samples (one gel lanes, n = 6 fractions) were extracted by Proteome Discoverer (version 1.3; ThermoFisher Scientific) and submitted to SEQUEST (version 1.20) for database searching against the UniProt SwissProt sequence database (downloaded November 2010) containing the subset of human, herpesvirus, and common contaminant sequences (21,570 entries). Spectra were searched against indexed peptide databases, generated from the forward and reverse protein sequence entries, using the following settings: full enzyme specificity, maximum of two missed cleavages, parent and fragment ion mass tolerances of 10 ppm and 0.5 Da, respectively, static modification of carbamidomethylcysteine (+57 Da), variable modifications of methionine oxidation (+16 Da), phosphoserine, threonine, and tyrosine (+80 Da), and acetyl-lysine (+42 Da). Peptide spectrum matches were loaded into Scaffold (ver. 3.2; Proteome Software, Inc.) for post-search validation by sequential searching using PeptideProphet and ProteinProphet (17Keller A. Nesvizhskii A.I. Kolker E. Aebersold R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.Anal. Chem. 2002; 74: 5383-5392Crossref PubMed Scopus (3886) Google Scholar, 18Nesvizhskii A.I. Keller A. Kolker E. Aebersold R. A statistical model for identifying proteins by tandem mass spectrometry.Anal. Chem. 2003; 75: 4646-4658Crossref PubMed Scopus (3621) Google Scholar) followed by X!Tandem (GPM 2010.12.1.1) re-search of the peptide spectrum matches using the subset database parameter and additional variable modification of deamidation of asparagine and glutamine (+1 Da) and pyro-Glu formation of peptide N-terminal glutamate (+17 Da). The high mass accuracy search option was enabled. Peptide spectrum matches across all conditions (see Fig. 2B) were loaded into a single Scaffold session, and the following confidence filters were selected to reduce peptide and protein global false discovery rate to <1%: 99% protein confidence, 95% peptide confidence, minimum of two unique peptides/protein in at least one biological samples. Proteins passing these filters were exported with their respective "unweighted spectrum counts" to Excel for further processing. To determine co-isolated proteins that were enriched in SIRT7 derivatives versus the EGFP control, a spectral counting approach was performed as follows: 1) a spectral count of 1 was added to all total spectral counts to facilitate calculation of fold enrichment; 2) spectral counts from biological replicates were averaged; 3) proteins with <8 average spectral counts in at least one condition were excluded; 4) spectral counts for co-isolated proteins from the S111A and dE2 conditions were normalized by the factor: (SIRT7WT spectral counts/SIRT7mutant spectral counts); and finally 5) proteins with ≥3-fold enrichment in SIRT7-EGFP isolation (versus EGFP) were retained for further gene ontology analysis (supplemental Table S2). Proteins identified across the SIRT7 immunoisolations were imported into Cytoscape, with nodes representing protein identifications and edges connecting to the respective immunoisolation (WT, S111A, or dE2) in which it was identified. Full gene ontologies (OBO v1.2) and human gene annotations were downloaded from online and respective ontologies were imported into Cytoscape (19Smoot M.E. Ono K. Ruscheinski J. Wang P.L. Ideker T. Cytoscape 2.8: New features for data integration and network visualization.Bioinformatics. 2011; 27: 431-432Crossref PubMed Scopus (3477) Google Scholar) as node attributes. Proteins classified by a "cytoplasmic only" cellular component were excluded (supplemental Table S4). The remaining "nuclear" or "unassigned" proteins (supplemental Table S5) were classified into functional subgroups according to biological processes (supplemental Table S7). All STRING network analyses were performed u
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