Human Sirtuin 2 Localization, Transient Interactions, and Impact on the Proteome Point to Its Role in Intracellular Trafficking
2016; Elsevier BV; Volume: 15; Issue: 10 Linguagem: Inglês
10.1074/mcp.m116.061333
ISSN1535-9484
AutoresHanna G. Budayeva, Ileana M. Cristea,
Tópico(s)Autophagy in Disease and Therapy
ResumoHuman sirtuin 2 (SIRT2) is an NAD+-dependent deacetylase that primarily functions in the cytoplasm, where it can regulate α-tubulin acetylation levels. SIRT2 is linked to cancer progression, neurodegeneration, and infection with bacteria or viruses. However, the current knowledge about its interactions and the means through which it exerts its functions has remained limited. Here, we aimed to gain a better understanding of its cellular functions by characterizing SIRT2 subcellular localization, the identity and relative stability of its protein interactions, and its impact on the proteome of primary human fibroblasts. To assess the relative stability of SIRT2 interactions, we used immunoaffinity purification in conjunction with both label-free and metabolic labeling quantitative mass spectrometry. In addition to the expected associations with cytoskeleton proteins, including its known substrate TUBA1A, our results reveal that SIRT2 specifically interacts with proteins functioning in membrane trafficking, secretory processes, and transcriptional regulation. By quantifying their relative stability, we found most interactions to be transient, indicating a dynamic SIRT2 environment. We discover that SIRT2 localizes to the ER-Golgi intermediate compartment (ERGIC), and that this recruitment requires an intact ER-Golgi trafficking pathway. Further expanding these findings, we used microscopy and interaction assays to establish the interaction and coregulation of SIRT2 with liprin-β1 scaffolding protein (PPFiBP1), a protein with roles in focal adhesions disassembly. As SIRT2 functions may be accomplished via interactions, enzymatic activity, and transcriptional regulation, we next assessed the impact of SIRT2 levels on the cellular proteome. SIRT2 knockdown led to changes in the levels of proteins functioning in membrane trafficking, including some of its interaction partners. Altogether, our study expands the knowledge of SIRT2 cytoplasmic functions to define a previously unrecognized involvement in intracellular trafficking pathways, which may contribute to its roles in cellular homeostasis and human diseases. Human sirtuin 2 (SIRT2) is an NAD+-dependent deacetylase that primarily functions in the cytoplasm, where it can regulate α-tubulin acetylation levels. SIRT2 is linked to cancer progression, neurodegeneration, and infection with bacteria or viruses. However, the current knowledge about its interactions and the means through which it exerts its functions has remained limited. Here, we aimed to gain a better understanding of its cellular functions by characterizing SIRT2 subcellular localization, the identity and relative stability of its protein interactions, and its impact on the proteome of primary human fibroblasts. To assess the relative stability of SIRT2 interactions, we used immunoaffinity purification in conjunction with both label-free and metabolic labeling quantitative mass spectrometry. In addition to the expected associations with cytoskeleton proteins, including its known substrate TUBA1A, our results reveal that SIRT2 specifically interacts with proteins functioning in membrane trafficking, secretory processes, and transcriptional regulation. By quantifying their relative stability, we found most interactions to be transient, indicating a dynamic SIRT2 environment. We discover that SIRT2 localizes to the ER-Golgi intermediate compartment (ERGIC), and that this recruitment requires an intact ER-Golgi trafficking pathway. Further expanding these findings, we used microscopy and interaction assays to establish the interaction and coregulation of SIRT2 with liprin-β1 scaffolding protein (PPFiBP1), a protein with roles in focal adhesions disassembly. As SIRT2 functions may be accomplished via interactions, enzymatic activity, and transcriptional regulation, we next assessed the impact of SIRT2 levels on the cellular proteome. SIRT2 knockdown led to changes in the levels of proteins functioning in membrane trafficking, including some of its interaction partners. Altogether, our study expands the knowledge of SIRT2 cytoplasmic functions to define a previously unrecognized involvement in intracellular trafficking pathways, which may contribute to its roles in cellular homeostasis and human diseases. Human sirtuin 2 (SIRT2) 1The abbreviations used are: SIRT2sirtuin 2ACLYATP citrate lyaseEGFPenhanced Green Fluorescent ProteinERendoplasmic reticulumERGICER-Golgi intermediate compartmentFHL2four and a half LIM-2FOXO3Aforkhead box protein O3GOBPgene ontology biological processHDAChistone deacetylaseI-DIRTdifferentiation of interactions as random or targetedIPimmunoaffinity purificationMRC5human fibroblastsNAD+nicotinamide adenine dinucleotideNFκΒnuclear factor kBNSAFnormalized spectral abundance factorNUDT21cleavage and polyadenylation specificity factor subunit 5PAXprotein abundance valuesPGAMphosphoglycerate mutasePPFiBP1liprin-beta-1RER1retention in endoplasmic reticulum sorting receptor 1RTN4reticulon 4SAINTsignificance analysis of interactome computational toolSFRP1secreted frizzled-related protein 1SILACstable isotope labeling with amino acids in cell cultureSSR1translocon-associated protein 1TUBA1Atubulin alpha-1A chainWTwild type. is one of seven NAD+-dependent deacylases (SIRT1–7) that were originally discovered as homologues of S. Cerevisiae Sir2 regulator of gene silencing in mating-type loci and telomeres (1.Frye R.A. Phylogenetic classification of prokaryotic and eukaryotic Sir2-like proteins.Biochem. Biophys. Res. Commun. 2000; 273: 793-798Crossref PubMed Scopus (1053) Google Scholar). As the enzymatic activity of sirtuins is dependent on the presence of a major metabolic molecule, NAD+, these enzymes can act as sensors of intracellular energy states. Sirtuins are distributed throughout major intracellular compartments to deliver this information to nuclear (SIRT1, SIRT6), nucleolar (SIRT7), cytoplasmic (SIRT2), and mitochondrial (SIRT3, SIRT4, SIRT5) processes (2.Michishita 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 (916) Google Scholar). The prominent enzymatic activity of sirtuins is thought to be deacetylation, i.e. removal of acetylation from lysine residues of protein substrates. However, studies from several research groups have demonstrated that sirtuins also possess other enzymatic activities. SIRT4 and SIRT6 can act as ADP-ribosyltransferases (3.Liszt G. Ford E. Kurtev M. Guarente L. Mouse Sir2 homolog SIRT6 is a nuclear ADP-ribosyltransferase.J. Biol. Chem. 2005; 280: 21313-21320Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar, 4.Haigis M.C. Mostoslavsky R. Haigis K.M. Fahie K. Christodoulou D.C. Murphy A.J. Valenzuela D.M. Yancopoulos G.D. Karow M. Blander G. Wolberger C. Prolla T.A. Weindruch R. Alt F.W. Guarente L. SIRT4 inhibits glutamate dehydrogenase and opposes the effects of calorie restriction in pancreatic beta cells.Cell. 2006; 126: 941-954Abstract Full Text Full Text PDF PubMed Scopus (828) Google Scholar), and more recently SIRT4 was identified as an efficient lipoamidase (5.Mathias R.A. Greco T.M. Oberstein A. Budayeva H.G. Chakrabarti R. Rowland E.A. Kang Y. Shenk T. Cristea I.M. Sirtuin 4 is a lipoamidase regulating pyruvate dehydrogenase complex activity.Cell. 2014; 159: 1615-1625Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). SIRT2 and SIRT6 display demyristoylation activity (6.Jiang H. Khan S. Wang Y. Charron G. He B. Sebastian C. Du J. Kim R. Ge E. Mostoslavsky R. Hang H.C. Hao Q. Lin H. SIRT6 regulates TNF-alpha secretion through hydrolysis of long-chain fatty acyl lysine.Nature. 2013; 496: 110-113Crossref PubMed Scopus (402) Google Scholar, 7.Teng Y.B. Jing H. Aramsangtienchai P. He B. Khan S. Hu J. Lin H. Hao Q. Efficient demyristoylase activity of SIRT2 revealed by kinetic and structural studies.Sci. Rep. 2015; 5: 8529Crossref PubMed Scopus (93) Google Scholar), whereas SIRT5 acts as demalonylase and desuccinylase (8.Du J. Zhou Y. Su X. Yu J.J. Khan S. Jiang H. Kim J. Woo J. Kim J.H. Choi B.H. He B. Chen W. Zhang S. Cerione R.A. Auwerx J. Hao Q. Lin H. Sirt5 is a NAD-dependent protein lysine demalonylase and desuccinylase.Science. 2011; 334: 806-809Crossref PubMed Scopus (754) Google Scholar). Given the wide range of histone and nonhistone substrates of sirtuins, these enzymes have been studied in the context of cancer, viral infection, neurological disorders, and lifespan (9.Bosch-Presegue L. Vaquero A. The dual role of sirtuins in cancer.Genes Cancer. 2011; 2: 648-662Crossref PubMed Scopus (217) Google Scholar, 10.Chopra V. Quinti L. Kim J. Vollor L. Narayanan K.L. Edgerly C. Cipicchio P.M. Lauver M.A. Choi S.H. Silverman R.B. Ferrante R.J. Hersch S. Kazantsev A.G. The sirtuin 2 inhibitor AK-7 is neuroprotective in Huntington's disease mouse models.Cell Rep. 2012; 2: 1492-1497Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 11.Koyuncu E. Budayeva H.G. Miteva Y.V. Ricci D.P. Silhavy T.J. Shenk T. Cristea I.M. Sirtuins are evolutionarily conserved viral restriction factors.MBio. 2014; : 5Google Scholar). For instance, a recent study has demonstrated that sirtuins, including SIRT2, have broad-spectrum antiviral functions in primary human fibroblasts upon infection with several DNA or RNA viruses (11.Koyuncu E. Budayeva H.G. Miteva Y.V. Ricci D.P. Silhavy T.J. Shenk T. Cristea I.M. Sirtuins are evolutionarily conserved viral restriction factors.MBio. 2014; : 5Google Scholar). sirtuin 2 ATP citrate lyase enhanced Green Fluorescent Protein endoplasmic reticulum ER-Golgi intermediate compartment four and a half LIM-2 forkhead box protein O3 gene ontology biological process histone deacetylase differentiation of interactions as random or targeted immunoaffinity purification human fibroblasts nicotinamide adenine dinucleotide nuclear factor kB normalized spectral abundance factor cleavage and polyadenylation specificity factor subunit 5 protein abundance values phosphoglycerate mutase liprin-beta-1 retention in endoplasmic reticulum sorting receptor 1 reticulon 4 significance analysis of interactome computational tool secreted frizzled-related protein 1 stable isotope labeling with amino acids in cell culture translocon-associated protein 1 tubulin alpha-1A chain wild type. SIRT2 is primarily known as a cytoplasmic NAD+-dependent deacetylase. Previous studies reported that two SIRT2 isoforms are present in human cells, where isoform 1 represents the full-length protein, whereas isoform 2 results from alternative splicing and lacks the first 38 amino acids (12.North B.J. Verdin E. Mitotic regulation of SIRT2 by cyclin-dependent kinase 1-dependent phosphorylation.J. Biol. Chem. 2007; 282: 19546-19555Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Both isoforms are catalytically active, but differ in their tissue expression patterns. Isoform 1 is abundant in skeletal muscle, whereas isoform 2 is predominant in the brain (13.Maxwell M.M. Tomkinson E.M. Nobles J. Wizeman J.W. Amore A.M. Quinti L. Chopra V. Hersch S.M. Kazantsev A.G. The Sirtuin 2 microtubule deacetylase is an abundant neuronal protein that accumulates in the aging CNS.Hum. Mol. Genet. 2011; 20: 3986-3996Crossref PubMed Scopus (115) Google Scholar). This suggests cell type-specific functions and contribution to different human diseases that remain to be fully understood. So far, most studies on SIRT2 functions within intracellular pathways have been focused on characterization of its substrates. The first identified substrate was α-tubulin, which is deacetylated by SIRT2 at Lys40 (14.North B.J. Marshall B.L. Borra M.T. Denu J.M. Verdin E. The human Sir2 ortholog, SIRT2, is an NAD+-dependent tubulin deacetylase.Mol. Cell. 2003; 11: 437-444Abstract Full Text Full Text PDF PubMed Scopus (1100) Google Scholar). This deacetylation process is thought to play a role in the progression of mitosis and in neuronal cell motility (15.Dryden S.C. Nahhas F.A. Nowak J.E. Goustin A.S. Tainsky M.A. Role for human SIRT2 NAD-dependent deacetylase activity in control of mitotic exit in the cell cycle.Mol. Cell. Biol. 2003; 23: 3173-3185Crossref PubMed Scopus (365) Google Scholar, 16.Inoue T. Nakayama Y. Yamada H. Li Y.C. Yamaguchi S. Osaki M. 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SIRT2, a tubulin deacetylase, acts to block the entry to chromosome condensation in response to mitotic stress.Oncogene. 2007; 26: 945-957Crossref PubMed Scopus (173) Google Scholar, 20.North B.J. Verdin E. Interphase nucleo-cytoplasmic shuttling and localization of SIRT2 during mitosis.PLoS ONE. 2007; 2: e784Crossref PubMed Scopus (182) Google Scholar, 21.Vaquero A. Scher M.B. Lee D.H. Sutton A. Cheng H.L. Alt F.W. Serrano L. Sternglanz R. Reinberg D. SirT2 is a histone deacetylase with preference for histone H4 Lys 16 during mitosis.Genes Dev. 2006; 20: 1256-1261Crossref PubMed Scopus (408) Google Scholar). Another SIRT2 histone substrate, H3Lys18 was shown to regulate gene expression during bacterial infection with Listeria (22.Eskandarian H.A. Impens F. Nahori M.A. Soubigou G. Coppee J.Y. Cossart P. Hamon M.A. A role for SIRT2-dependent histone H3K18 deacetylation in bacterial infection.Science. 2013; 341: 1238858Crossref PubMed Scopus (144) Google Scholar). The roles of SIRT2 were also explored in connection to metabolic diseases, in part via its deacetylation of ATP citrate lyase (ACLY) (23.Lin R. Tao R. Gao X. Li T. Zhou X. Guan K.L. Xiong Y. Lei Q.Y. Acetylation stabilizes ATP-citrate lyase to promote lipid biosynthesis and tumor growth.Mol. Cell. 2013; 51: 506-518Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar) and phosphoglycerate mutase (PGAM) (24.Tsusaka T. Guo T. Yagura T. Inoue T. Yokode M. Inagaki N. Kondoh H. Deacetylation of phosphoglycerate mutase in its distinct central region by SIRT2 down-regulates its enzymatic activity.Genes Cells. 2014; 19: 766-777Crossref PubMed Scopus (12) Google Scholar). Overall, these studies of SIRT2 substrates indicate its involvement in a variety of intracellular processes. In addition to regulating substrates, it is becoming evident that sirtuins and other histone deacetylases (HDACs) can also modulate protein functions through the formation of diverse protein-protein interactions. For instance, a global HDAC interactome study revealed previously undefined HDAC11 functions in mRNA splicing through its interaction with the SMN complex (25.Joshi P. Greco T.M. Guise A.J. Luo Y. Yu F. Nesvizhskii A.I. Cristea I.M. The functional interactome landscape of the human histone deacetylase family.Mol. Syst. Biol. 2013; 9: 672Crossref PubMed Google Scholar). Activity-dependent interactions of SIRT6 with the stress response factor G3BP1 pointed at its involvement in regulation of cellular stress responses (26.Miteva Y.V. Cristea I.M. A proteomic perspective of Sirtuin 6 (SIRT6) phosphorylation and interactions and their dependence on its catalytic activity.Mol. Cell. 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Additionally, a large-scale study aimed at gaining insights into the broad interaction network of over 2000 human proteins was performed in HEK293T cells, and identified several associations when using FLAG-HA-tagged SIRT2 as one of the baits (29.Huttlin E.L. Ting L. Bruckner R.J. Gebreab F. Gygi M.P. Szpyt J. Tam S. Zarraga G. Colby G. Baltier K. Dong R. Guarani V. Vaites L.P. Ordureau A. Rad R. Erickson B.K. Wuhr M. Chick J. Zhai B. Kolippakkam D. Mintseris J. Obar R.A. Harris T. Artavanis-Tsakonas S. Sowa M.E. De Camilli P. Paulo J.A. Harper J.W. Gygi S.P. The BioPlex Network: a systematic exploration of the human interactome.Cell. 2015; 162: 425-440Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar). However, the functional significance of these interactions requires further investigation, and there is a need to understand SIRT2 interactions in primary human cells. Sirtuins can additionally impact cellular pathways through the ability of some of their substrates and interactions to regulate gene expression. For example, SIRT7 was found to play a role in RNA Pol I-dependent transcription through its interactions with the B-WICH chromatin remodeling complex (30.Tsai Y.C. Greco T.M. Boonmee A. Miteva Y. Cristea I.M. Functional proteomics establishes the interaction of SIRT7 with chromatin remodeling complexes and expands its role in regulation of RNA polymerase I transcription.Mol. Cell. Proteomics. 2012; 11: 60-76Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Both SIRT1 and SIRT2 were shown to modulate gene expression through deacetylation of several transcription factors, including nuclear factor κΒ (NFκΒ) (31.Rothgiesser K.M. Erener S. Waibel S. Luscher B. Hottiger M.O. SIRT2 regulates NF-kappaB dependent gene expression through deacetylation of p65 Lys310.J. 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SIRT2 deacetylates FOXO3a in response to oxidative stress and caloric restriction.Aging Cell. 2007; 6: 505-514Crossref PubMed Scopus (420) Google Scholar, 36.Wang F. Chan C.H. Chen K. Guan X. Lin H.K. Tong Q. Deacetylation of FOXO3 by SIRT1 or SIRT2 leads to Skp2-mediated FOXO3 ubiquitination and degradation.Oncogene. 2012; 31: 1546-1557Crossref PubMed Scopus (122) Google Scholar), and coactivator β-catenin (37.Nguyen P. Lee S. Lorang-Leins D. Trepel J. Smart D.K. SIRT2 interacts with beta-catenin to inhibit Wnt signaling output in response to radiation-induced stress.Mol. Cancer Res. 2014; 12: 1244-1253Crossref PubMed Scopus (33) Google Scholar, 38.Firestein R. Blander G. Michan S. Oberdoerffer P. Ogino S. Campbell J. Bhimavarapu A. Luikenhuis S. de Cabo R. Fuchs C. Hahn W.C. Guarente L.P. Sinclair D.A. The SIRT1 deacetylase suppresses intestinal tumorigenesis and colon cancer growth.PLoS ONE. 2008; 3: e2020Crossref PubMed Scopus (474) Google Scholar). In addition, the neuroprotective effects of SIRT2 inhibition in Huntington's disease was associated with the SIRT2-dependent regulation of the SREBP-2 transcription factor trafficking from the endoplasmic reticulum to the nucleus (39.Luthi-Carter R. Taylor D.M. Pallos J. Lambert E. Amore A. Parker A. Moffitt H. Smith D.L. Runne H. Gokce O. Kuhn A. Xiang Z. Maxwell M.M. Reeves S.A. Bates G.P. Neri C. Thompson L.M. Marsh J.L. Kazantsev A.G. SIRT2 inhibition achieves neuroprotection by decreasing sterol biosynthesis.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 7927-7932Crossref PubMed Scopus (236) Google Scholar). These studies highlight the importance of continuing the evaluation of sirtuin impact on gene expression and total protein levels. The limited knowledge regarding the nonenzymatic roles of SIRT2 via either protein interactions or gene expression, as well as its likely context-dependent functions, may contribute to the different observations for SIRT2 functions in cancer progression. For instance, SIRT2 may act as a tumor suppressor, promoter, or both. In SIRT2 knockout mice, tumor development was observed to increase with age (40.Kim H.S. Vassilopoulos A. Wang R.H. Lahusen T. Xiao Z. Xu X. Li C. Veenstra T.D. Li B. Yu H. Ji J. Wang X.W. Park S.H. Cha Y.I. Gius D. Deng C.X. SIRT2 maintains genome integrity and suppresses tumorigenesis through regulating APC/C activity.Cancer Cell. 2011; 20: 487-499Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar) or when challenged with mutagenic agents (41.Serrano L. Martinez-Redondo P. Marazuela-Duque A. Vazquez B.N. Dooley S.J. Voigt P. Beck D.B. Kane-Goldsmith N. Tong Q. Rabanal R.M. Fondevila D. Munoz P. Kruger M. Tischfield J.A. Vaquero A. The tumor suppressor SirT2 regulates cell cycle progression and genome stability by modulating the mitotic deposition of H4K20 methylation.Genes Dev. 2013; 27: 639-653Crossref PubMed Scopus (156) Google Scholar). It was also noted that SIRT2 expression is decreased in gliomas (42.Hiratsuka M. Inoue T. Toda T. Kimura N. Shirayoshi Y. Kamitani H. Watanabe T. Ohama E. Tahimic C.G. Kurimasa A. Oshimura M. Proteomics-based identification of differentially expressed genes in human gliomas: down-regulation of SIRT2 gene.Biochem. Biophys. Res. Commun. 2003; 309: 558-566Crossref PubMed Scopus (228) Google Scholar). On the other hand, disruption of SIRT2 activity was recently reported to have anticancer effects in nonsmall cell lung cancer (43.Hoffmann G. Breitenbucher F. Schuler M. Ehrenhofer-Murray A.E. A novel sirtuin 2 (SIRT2) inhibitor with p53-dependent pro-apoptotic activity in non-small cell lung cancer.J. Biol. 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Here, we addressed these questions by generating a refined network of SIRT2 protein interactions in primary human fibroblasts. We integrated label-free and metabolic labeling mass spectrometry to assess the relative stability of SIRT2 interactions. These analyses, in conjunction with microscopy and reciprocal isolation studies, led to the discovery of a previously unrecognized localization for SIRT2 at an intersection of intracellular trafficking routes and its interaction with proteins that function in these pathways. Specifically, we find SIRT2 to localize at the endoplasmic reticulum-Golgi intermediate compartment (ERGIC). This localization and its interaction with trafficked proteins, including the focal adhesion adaptor protein liprin-β1 (PPFiBP1), were dependent on the presence of an intact ER-Golgi trafficking pathway. Furthermore, we assessed the impact of SIRT2 knockdown on global protein levels, identifying an up-regulation of selected proteins in the endoplasmic reticulum and membrane organization pathways. Altogether, our results point to a role for SIRT2 in regulation of cellular trafficking through formation of protein interactions and regulation of protein levels. The following primary antibodies and stains were used for Western blotting and/or confocal imaging: anti-SIRT2 (Abcam, Cambridge, MA), anti-PPFiBP1 (Abcam for Western blotting and Bethyl Laboratories for immunoaffinity purification), anti-ERGIC53 (Sigma Aldrich, Allentown, PA), DAPI (ThermoFisher Scientific, Waltham, MA). Anti-rabbit or anti-mouse light chain specific HRP-conjugated (Jackson ImmunoResearch, West Grove, PA) secondary antibodies were used for Western blotting. Alexa Fluor (Invitrogen Life Technology, Grand Island, NY) secondary antibodies were used for confocal imaging. ER tracker Red (BODIPY TR Glibenclamide, Life Technologies) and Golgi tracker red (BODIPY TR Ceramide) were used for confocal imaging of endoplasmic reticulum and golgi, respectively. Brefeldin A was obtained from Sigma and used at 3 μg/ml. Human MRC5 fibroblasts, HEK293T, and Phoenix cells were obtained from ATCC and grown in Dulbecco's Modified Eagle medium (DMEM, Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum at 37 °C and in 5% CO2. For the I-DIRT (30.Tsai Y.C. Greco T.M. Boonmee A. Miteva Y. Cristea I.M. Functional proteomics establishes the interaction of SIRT7 with chromatin remodeling complexes and expands its role in regulation of RNA polymerase I transcription.Mol. Cell. Proteomics. 2012; 11: 60-76Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 46.Tackett A.J. DeGrasse J.A. Sekedat M.D. Oeffinger M. Rout M.P. Chait B.T. I-DIRT, a general method for distinguishing between specific and nonspecific protein interactions.J. Proteome Res. 2005; 4: 1752-1756Crossref PubMed Scopus (109) Google Scholar) experiments, metabolic labeling was achieved by culturing MRC5 cells in SILAC DMEM (ThermoFisher Scientific, Waltham, MA) with the addition of "light" arginine and lysine (12C6) or "heavy" arginine and lysine (13C6) from Cambridge Isotope (Tewksbury, MA) and supplemented with dialyzed FBS (Gemini Bio-Products, West Sacramento, CA). Cell culture was allowed to proceed for five passages to ensure over 95% incorporation rate. Dulbecco's Phosphate-Buffered Saline (DPBS) buffer was obtained from ThermoFisher Scientific. EGFP open reading frame (ORF) was obtained from pEGFP-N1 (Clontech, Mountain View, CA) and inserted into pLXSN retroviral vector (Clontech, Mountain View, CA) alone or in fusion with FLAG at the 3′ end (EGFP-FLAG). SIRT2 isoform2-containing constructs were cloned from pCDNA3.1 plasmid (Addgene, Cambridge, MA) into pLXSN EGFP plasmid by PCR amplification of SIRT2 isoform-2 ORF. Phoenix cells were transfected with the resulting SIRT2-EGFP or EGFP-FLAG plasmid using Lipofectamin 2000 (Invitrogen Life Technology) in accordance with manufacturer's protocol. Generated retrovirus-containing media was filtered with 0.45 μm Acrodisc syringe filter (Pall, Port Washington, NY), supplemented with 4 μg/ml polybrene (Millipore, Billerica, MA) and used to transduce MRC5 fibroblasts. Cells stably expressing transduced construct were selected with 400 ng/ml G418 (ThermoFisher Scientific) for 7 days. Total mRNA was isolated with RNeasy mini kit (Qiagen, Manchester, UK). 1 μg of total mRNA was used for cDNA synthesis using RETROscript First Strand Synthesis Kit for RT-PCR (Ambion Life Technologies, ThermoFisher Scientific). Primers used for qPCR analysis were as followed: SIRT2: Fwd: 5′-ACCCGCTAAGCTGGATGAAAGAG-3′; Rev: 5′-AGTCTTCACACTTGGGCGTCAC-3′; β-actin: Fwd: 5′-TCCTCCTGAGCGCAAGTACTC-3′; Rev: 5′-CGGACTCGTCATACTCCTGCT-3′. ABI 7900 or ABI 384 thermocyclers (Applied Biosystems, Foster City, CA) were used for qPCR reaction and data collection. SDS 2.3 (Applied Biosystems) software was used for data processing. SIRT2-EGFP and EGFP-FLAG immunoaffinity purification was performed as described previously (47.Cristea 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 (195) Google Scholar). MRC5 cells stably expressing SIRT2-EGFP or EGFP-FLAG were washed with DPBS, resuspended in PVP-HEPES buffer (20 mm Na-HEPES, 1.2% polyvinylpyrrolidone (w/v) pH 7.4 with protease inhibitor mixture (Sigma Aldrich), and frozen as droplets in liquid nitrogen with frozen lysis buffer for cryogenic grinding. Lysis buffer was based on common TBT buffer components (200 mm K-HEPS pH7.4, 1.1 m KOac, 1% Tween-20(v/v)) with varying concentrations of NaCl and Triton X-100 used for optimization. Frozen pellets were ground using Retsch MM301 Mixer Mill (Retsch, Newtown, P
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