Site-specific analysis of protein S-acylation by resin-assisted capture
Protein S-acylation is a major posttranslational modification whereby a cysteine thiol is converted to a thioester. A prototype is S-palmitoylation (fatty acylation), in which a protein undergoes acylation with a hydrophobic 16 carbon lipid chain. Although this modification is a well-recognized determinant of protein function and localization, current techniques to study cellular S-acylation are cumbersome and/or technically demanding. We recently described a simple and robust methodology to rapidly identify S-nitrosylation sites in proteins via resin-assisted capture (RAC) and provided an initial description of the applicability of the technique to S-acylated proteins (acyl-RAC). Here we expand on the acyl-RAC assay, coupled with mass spectrometry-based proteomics, to characterize both previously reported and novel sites of endogenous S-acylation. Acyl-RAC should therefore find general applicability in studies of both global and individual protein S-acylation in mammalian cells. Protein S-acylation is a major posttranslational modification whereby a cysteine thiol is converted to a thioester. A prototype is S-palmitoylation (fatty acylation), in which a protein undergoes acylation with a hydrophobic 16 carbon lipid chain. Although this modification is a well-recognized determinant of protein function and localization, current techniques to study cellular S-acylation are cumbersome and/or technically demanding. We recently described a simple and robust methodology to rapidly identify S-nitrosylation sites in proteins via resin-assisted capture (RAC) and provided an initial description of the applicability of the technique to S-acylated proteins (acyl-RAC). Here we expand on the acyl-RAC assay, coupled with mass spectrometry-based proteomics, to characterize both previously reported and novel sites of endogenous S-acylation. Acyl-RAC should therefore find general applicability in studies of both global and individual protein S-acylation in mammalian cells. Protein cysteine residues undergo a wide variety of chemical reactions owing to thiol nucleophilicity and redox reactivity. These reactions include S-nitrosylation (1Benhar M. Forrester M.T. Stamler J.S. Protein denitrosylation: enzymatic mechanisms and cellular functions.Nat. Rev. Mol. Cell Biol. 2009; 10: 721-732Crossref PubMed Scopus (406) Google Scholar, 2Foster M.W. Hess D.T. Stamler J.S. Protein S-nitrosylation in health and disease: a current perspective.Trends Mol. Med. 2009; 15: 391-404Abstract Full Text Full Text PDF PubMed Scopus (600) Google Scholar), S-prenylation (3Wright L.P. Philips M.R. Thematic review series: lipid posttranslational modifications. CAAX modification and membrane targeting of Ras.J. Lipid Res. 2006; 47: 883-891Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 4Zhang F.L. Casey P.J. Protein prenylation: molecular mechanisms and functional consequences.Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1743) Google Scholar), and S-acylation (5Schultz A.M. Henderson L.E. Oroszlan S. Fatty acylation of proteins.Annu. Rev. Cell Biol. 1988; 4: 611-647Crossref PubMed Scopus (159) Google Scholar, 6Smotrys J.E. Linder M.E. Palmitoylation of intracellular signaling proteins: regulation and function.Annu. Rev. Biochem. 2004; 73: 559-587Crossref PubMed Scopus (486) Google Scholar), which involve the adduction of nitroso, isoprenyl (thioether), and acyl (thioester) moieties, respectively. Within mammalian cells, an important type of S-acylation involves S-palmitoylation (the attachment of a 16 carbon fully saturated lipid moiety). S-palmitoylation has been shown to significantly impact protein function and localization, largely via modulating membrane affinity and protein stability (7Nadolski M.J. Linder M.E. Protein lipidation.FEBS J. 2007; 274: 5202-5210Crossref PubMed Scopus (213) Google Scholar, 8Linder M.E. Deschenes R.J. Palmitoylation: policing protein stability and traffic.Nat. Rev. Mol. Cell Biol. 2007; 8: 74-84Crossref PubMed Scopus (773) Google Scholar, 9Resh M.D. Trafficking and signaling by fatty-acylated and prenylated proteins.Nat. Chem. Biol. 2006; 2: 584-590Crossref PubMed Scopus (437) Google Scholar). In contrast to the stable thioether linkage of S-prenylation, the thioester linkage of S-acylation confers a reversible and dynamic nature on this modification, and many recent efforts are shedding light on how this modification is regulated (8Linder M.E. Deschenes R.J. Palmitoylation: policing protein stability and traffic.Nat. Rev. Mol. Cell Biol. 2007; 8: 74-84Crossref PubMed Scopus (773) Google Scholar, 9Resh M.D. Trafficking and signaling by fatty-acylated and prenylated proteins.Nat. Chem. Biol. 2006; 2: 584-590Crossref PubMed Scopus (437) Google Scholar, 10Dekker F.J. Rocks O. Vartak N. Menninger S. Hedberg C. Balamurugan R. Wetzel S. Renner S. Gerauer M. Scholermann B. et al.Small-molecule inhibition of APT1 affects Ras localization and signaling.Nat. Chem. Biol. 2010; 6: 449-456Crossref PubMed Scopus (284) Google Scholar, 11Rocks O. Gerauer M. Vartak N. Koch S. Huang Z.P. Pechlivanis M. Kuhlmann J. Brunsveld L. Chandra A. Ellinger B. et al.The palmitoylation machinery is a spatially organizing system for peripheral membrane proteins.Cell. 2010; 141: 458-471Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). There are a variety of methodologies to detect protein S-acylation/palmitoylation in intact cells. A well-established method involves incubating cells with 3H-labeled palmitate, followed by autoradiography to visualize the degree of isotopic incorporation. However, this approach requires high levels of [3H]palmitate (as many as several mCi per sample) and exposure times on the order of weeks (12Magee A.I. Wootton J. de Bony J. Detecting radiolabeled lipid-modified proteins in polyacrylamide gels.Methods Enzymol. 1995; 250: 330-336Crossref PubMed Scopus (22) Google Scholar, 13Bizzozero O.A. Chemical analysis of acylation sites and species.Methods Enzymol. 1995; 250: 361-379Crossref PubMed Scopus (47) Google Scholar). More recent methods have cleverly circumvented these issues by using nonradioactive derivatives of palmitate, which can be enriched or detected via cycloaddition reactions ( “click chemistry”) (14Charron G. Zhang M.M. Yount J.S. Wilson J. Raghavan A.S. Shamir E. Hang H.C. Robust fluorescent detection of protein fatty-acylation with chemical reporters.J. Am. Chem. Soc. 2009; 131: 4967-4975Crossref PubMed Scopus (234) Google Scholar, 15Martin B.R. Cravatt B.F. Large-scale profiling of protein palmitoylation in mammalian cells.Nat. Methods. 2009; 6: 135-138Crossref PubMed Scopus (365) Google Scholar, 16Yap M.C. Kostiuk M.A. Martin D.D. Perinpanayagam M.A. Hak P.C. Siddam A. Majjigapu J.R. Rajaiah G. Keller B.O. Prescher J.A. Wu P. Bertozzi C.R. Falck J.R. Berthiaume L.G. Rapid and selective detection of fatty acylated proteins using ω-alkynyl-fatty acids and click chemistry.J. Lipid Res. 2010; 51: 1566-1580Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 17Hannoush R.N. Arenas-Ramirez N. Imaging the lipidome: omega-alkynyl fatty acids for detection and cellular visualization of lipid-modified proteins.ACS Chem. Biol. 2009; 4: 581-587Crossref PubMed Scopus (105) Google Scholar). Nonetheless, these “palmitate-centric” approaches are encumbered by i) the need for radioactive or chemically modified palmitate analogs; ii) the likely bias for proteins that undergo rapid palmitate turnover versus proteins that are more stably palmitoylated; iii) difficulty in detecting individual S-acylated proteins or their specific sites of S-acylation; and iv) the inability to detect proteins that are acylated with moieties other than palmitate (e.g., shorter, longer, or unsaturated lipid chains). Recently, a “cysteine-centric” approach to identify S-acylated proteins was introduced that uses the conversion of the protein thioester to a disulfide-linked biotin (18Drisdel R.C. Green W.N. Labeling and quantifying sites of protein palmitoylation.Biotechniques. 2004; 36: 276-285Crossref PubMed Google Scholar, 19Roth A.F. Wan J. Bailey A.O. Sun B. Kuchar J.A. Green W.N. Phinney B.S. Yates III, J.R. Davis N.G. Global analysis of protein palmitoylation in yeast.Cell. 2006; 125: 1003-1013Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). This assay, known as acyl-biotin exchange (ABE), is readily adapted to immunoblotting techniques and is also adaptable to mass spectrometric-based identification of individual S-acylated proteins (19Roth A.F. Wan J. Bailey A.O. Sun B. Kuchar J.A. Green W.N. Phinney B.S. Yates III, J.R. Davis N.G. Global analysis of protein palmitoylation in yeast.Cell. 2006; 125: 1003-1013Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar, 20Huang K. Sanders S. Singaraja R. Orban P. Cijsouw T. Arstikaitis P. Yanai A. Hayden M.R. El-Husseini A. Neuronal palmitoyl acyl transferases exhibit distinct substrate specificity.FASEB J. 2009; 23: 2605-2615Crossref PubMed Scopus (117) Google Scholar, 21Kang R. Wan J. Arstikaitis P. Takahashi H. Huang K. Bailey A.O. Thompson J.X. Roth A.F. Drisdel R.C. Mastro R. et al.Neural palmitoyl-proteomics reveals dynamic synaptic palmitoylation.Nature. 2008; 456: 904-909Crossref PubMed Scopus (429) Google Scholar, 22Yang W. Di Vizio D. Kirchner M. Steen H. Freeman M.R. Proteome scale characterization of human S-acylated proteins in lipid raft-enriched and non-raft membranes.Mol. Cell. Proteomics. 2010; 9: 54-70Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). However, the detection of biotinylated proteins requires expensive reagents and complicated procedures (e.g., repeated protein precipitations, SDS neutralization, and avidin pull down). We recently provided an initial description of a simple and robust alternative to ABE that uses the detection of S-acylated species via resin-assisted capture (acyl-RAC) in lieu of biotinylation (23Forrester M.T. Thompson J.W. Foster M.W. Nogueira L. Moseley M.A. Stamler J.S. Proteomic analysis of S-nitrosylation and denitrosylation by resin-assisted capture.Nat. Biotechnol. 2009; 27: 557-559Crossref PubMed Scopus (310) Google Scholar). The method is rapid (the entire procedure can be completed in several hours) and is readily adapted to mass spectrometry techniques for identifying sites of S-acylation. Here we provide a detailed validation and expansion of the acyl-RAC method and demonstrate its efficacy in detecting S-acylated protein substrates and sites of modification. All materials were obtained from Sigma Chemicals (St. Louis, MO), unless otherwise indicated. Sources of antibodies were mouse MAb α-HA (code 2367; Cell Signaling Technology); and rabbit polyclonal antibody α-H-Ras (code sc-520; Santa Cruz Biotechnology). Bovine brain membranes were isolated as described previously (24Sternweis P.C. Robishaw J.D. Isolation of two proteins with high affinity for guanine nucleotides from membranes of bovine brain.J. Biol. Chem. 1984; 259: 13806-13813Abstract Full Text PDF PubMed Google Scholar). All cells were cultured at 37°C in a 5% Co2 atmosphere. Cell lines were obtained from the Duke Cell Culture Facility and grown in DMEM (HEK293 cells) or McCoy's 5A medium (T24 cells) supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were transfected with Superfect (Qiagen) per the manufacturer's instructions. In general, HEK293 cells were grown in 10 cm dishes to 70%–80% confluency and transfected with 12 μg of the indicated DNA and 48 μl of Superfect (Qiagen). Approximately 24 h later, cells were harvested with cold PBS and used immediately. All PCR procedures were performed with Advantage Taq DNA polymerase (Clontech), and products were verified by DNA sequencing (Duke DNA Sequencing Facility). The pCDNA3.1-3xHA-H-Ras construct was acquired from Missouri S and T cDNA Resource Center (product no. RASH00TN00). Integrated Molecular Analysis of Genomics and Expression (IMAGE) clones containing cDNAs for human Sec61B (BC001734), Rps11 (BC070224), and MGST3 (BC005964) were acquired from OpenBiosystems (shown in parentheses are the corresponding Genbank accession numbers). These three cDNAs were amplified by PCR and subcloned into pIRES-puro3 (Clontech) at the 5′-NheI and 3′-EcoRI sites to generate mammalian expression vectors containing an N- or C-terminal hemagglutinin (HA)-tagged cDNA. Primers used for generating pIRES-puro3-Sec61B-HA were 5′-TATTAGCTAGCACCATGGCTGGTCCGACCCCCAGTG-3′ and 5′-TTAAGAATTCTTAAGCGTAGTCTGGGACGTCGTATGGGTACGAACGAGTGTACTTGCCCCAAATG-3′; primers for pIRES-puro3-Rps11-HA were 5′-TATTAGCTAGCACCATGGCGGACATTCAGACTGAG-3′ and 5′-TTAAGAATTCTTAAGCGTAGTCTGGGACGTCGTATGGGTAGAACTTCTGGAACTGCTTCTTGGTGCC-3′; and primers for pIRES-puro3-HA-MGST3 were 5′-TATTAGCTAGCACCATGGTTTACCCATACGACGTCCCAGACTACGCTGCTGTCCTCTCTAAGG-3′ and 5′-TTAAGAATTCTTAATGGCAGCATTTGGGTCC-3′. Point mutations in MGST3 were generated via PCR as above, except reverse 3′- primer 5′-ATTAGAATTCTTAATGGCAGCTTTTGGGTCCACTGC-3′ was used for C150S; primer 5′-ATTAGAATTCTTAATGGCTGCATTTGGGTCCACTGC-3′ was used for C151S; and primer 5′-ATTAGAATTCTTAATGGCTGCTTTTGGGTCCACTGC-3′ was used for C150/151S. Other Cys-to-Ser point mutations were generated with a QuikChange XL II kit (Stratagene) according to the manufacturer's instructions, using primers 5′-CGGCAGCATGAGCAGCAAGTGTG’3- and 5′-CACACTTGCTGCTCATGCTGCCG-3′ for pDNA3.1-3xHA-H-Ras C181/184S; primers 5′-AATGCCAGCAGTGGGACAAGGAGTGC-3′ and 5′-GCACTCCTTGTCCCACTGCTGGCATT-3′ for Sec61B C39S; and primers 5′-CATTGACAAGAAAAGCCCCTTCACTGG-3′ and 5′-CCAGTGAAGGGGCTTTTCTTGTCAATG-3′ for Rps11 C60S. Following the indicated treatments/transfections, cells were collected and washed in cold PBS. After undergoing a freeze-thaw cycle, cells were lysed in lysis buffer (25 mM HEPES, 25 mM NaCl, 1 mM EDTA, pH 7.5) containing protease inhibitor cocktail (Roche). Lysis was improved by repeated passaging through a 28 gauge needle. For enrichment of membranes, lysates were depleted of nuclei via centrifugation at 800 g for 5 min. The supernatant was then centrifuged at 20,000 g for 30 min, and the pellet was resuspended in lysis buffer containing 0.5% Triton X-100. Total protein was quantified with a bicinchononic acid (BCA) assay (Pierce) using BSA as the standard. Methodology for acyl-RAC, including blocking of free thiols with methyl methanethiosulfonate (MMTS), cleavage of thioester linkages, and capture of nascent thiols on was as described previously (23Forrester M.T. Thompson J.W. Foster M.W. Nogueira L. Moseley M.A. Stamler J.S. Proteomic analysis of S-nitrosylation and denitrosylation by resin-assisted capture.Nat. Biotechnol. 2009; 27: 557-559Crossref PubMed Scopus (310) Google Scholar). In of protein for and for mass spectrometry were to a of in blocking buffer mM HEPES, mM EDTA, pH 7.5) and at for 10 with of cold were and proteins were to at for min. Following centrifugation of the at g for 10 min, the pellet was washed with resuspended in μl of buffer mM HEPES, mM EDTA, pH 7.5) and to μl of this was μl of in from and to pH with or reactions were on a at for Approximately μl of supernatant was as the were washed at times with For was performed using μl of buffer containing mM at for min. were and with to for 5 min, and via on a This procedure was performed as described previously (23Forrester M.T. Thompson J.W. Foster M.W. Nogueira L. Moseley M.A. Stamler J.S. Proteomic analysis of S-nitrosylation and denitrosylation by resin-assisted capture.Nat. Biotechnol. 2009; 27: 557-559Crossref PubMed Scopus (310) Google is fully detailed in the at The acyl-RAC assay is chemically to the ABE assay, the with the of to containing This is for is rapid and and the proteins to be with chemical or except shown in acyl-RAC was to S-acylated proteins in bovine brain membranes, which are known to be in A of proteins were readily detected by acyl-RAC in a via of proteins by In two proteins known to be in J. Wilson membrane localization of requires two Biol. Cell. 9: PubMed Scopus Google and D. M.C. Palmitoylation protein of by J. PubMed Scopus Google Scholar), were readily detected by analysis of acyl-RAC and the been with to endogenous In which is a substrate for was detected by the acyl-RAC technique can be to the and identification of S-acylated proteins in the of acyl-RAC to detect S-acylation in an intact mammalian HEK293 cells were transfected with vectors which is known to undergo S-palmitoylation on and A.I. J.E. All proteins are are Full Text PDF PubMed Scopus Google and on (3Wright L.P. Philips M.R. Thematic review series: lipid posttranslational modifications. CAAX modification and membrane targeting of Ras.J. Lipid Res. 2006; 47: 883-891Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 4Zhang F.L. Casey P.J. Protein prenylation: molecular mechanisms and functional consequences.Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1743) Google Scholar). The modified of human is shown in shown in acyl-RAC readily detected S-palmitoylation of in a the which undergo was the to undergo on H. R. S. M. E. H. J.R. and membrane targeting of the of in mammalian 36: PubMed Scopus Google Scholar), these the that acyl-RAC detect proteins the thioether linkage is to that the protein species by acyl-RAC are S-acylated was provided by the that the degree of S-palmitoylation was by with a known inhibitor of S-palmitoylation also be readily detected in the in which the of is known to the E. O. K. M. M. of the is to a acid PubMed Scopus Google Scholar). Although is a prototype of S-acylated a more system was to the general applicability of that a from HEK293 cells was with buffer or followed by analysis via acyl-RAC and visualization of proteins via and of cellular proteins was both by and on the assay, that acyl-RAC can detect a of We also the of acyl-RAC in identification of specific sites of S-acylation on by using and of a from HEK293 cells were to the acyl-RAC procedure in the and of followed by of proteins and with mass or containing the proteins from both were and the were by a of the human sites of S-acylation on were including a of sites previously known to undergo S-palmitoylation the at (the was to and therefore provided an obtained from the of the which is on the M.E. P. J.R. R. Lipid of proteins: are S A. PubMed Scopus Google Scholar, C. regulation of S A. PubMed Scopus Google Scholar), are shown This containing was by acyl-RAC, of the other from were in the These the of acyl-RAC for identifying sites of S-palmitoylation in intact cells. other sites of S-acylation that were by using acyl-RAC are shown in These sites include and the of the A. M.A. A.I. M. G. The of palmitoylation of the guanine protein in with the J. 1995; PubMed Scopus Google Scholar), and of and A.I. J.E. All proteins are are Full Text PDF PubMed Scopus Google as as residues of which are in its a known protein D.T. Wilson M.C. The 25 protein is the major in rapid and a major substrate for palmitoylation in PubMed Google Scholar). was the of 1 This protein is known to a thioester with the C-terminal of at H. into and to 2008; Full Text Full Text PDF PubMed Scopus Google Scholar), which is for to Although acyl-RAC detected the from the that acyl-RAC is of identifying of S-acylation. the of identification of S-acylation sites from three proteins that were previously in the of S-acylation were for the of the protein system protein and These three and mutations of in which the S-acylated been to a were in HEK293 cells. Cells were transfected with the and then by acyl-RAC with shown in acyl-RAC detected three proteins in a mutations of the acylation sites their detection by These the of acyl-RAC in detecting both known and novel sites of S-acylation in intact cells. Fatty acylation of proteins is as a of protein localization and a of signaling from cellular membranes (8Linder M.E. Deschenes R.J. Palmitoylation: policing protein stability and traffic.Nat. Rev. Mol. Cell Biol. 2007; 8: 74-84Crossref PubMed Scopus (773) Google Scholar, 9Resh M.D. Trafficking and signaling by fatty-acylated and prenylated proteins.Nat. Chem. Biol. 2006; 2: 584-590Crossref PubMed Scopus (437) Google Scholar, 10Dekker F.J. Rocks O. Vartak N. Menninger S. Hedberg C. Balamurugan R. Wetzel S. Renner S. Gerauer M. Scholermann B. et al.Small-molecule inhibition of APT1 affects Ras localization and signaling.Nat. Chem. Biol. 2010; 6: 449-456Crossref PubMed Scopus (284) Google Scholar, 11Rocks O. Gerauer M. Vartak N. Koch S. Huang Z.P. Pechlivanis M. Kuhlmann J. Brunsveld L. Chandra A. Ellinger B. et al.The palmitoylation machinery is a spatially organizing system for peripheral membrane proteins.Cell. 2010; 141: 458-471Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, Ras proteins: from Rev. Mol. Cell Biol. 4: PubMed Scopus Google Scholar). the of proteins that are known to undergo S-acylation (e.g., and has been on the mechanisms and of S-acylation. These efforts be significantly by the of for detecting S-acylated proteins and sites of modification. Here we have described in and the acyl-RAC which endogenous S-acylation. the acyl-RAC procedure can be completed in an and is fully with methodologies to identify proteins and their sites of modification. The acyl-RAC methodology should provide into the of S-acylation. The major of acyl-RAC, is the with which can be with the ABE assay, acyl-RAC uses the of at which as acyl-RAC is to the detection of proteins by resin-assisted capture acyl-RAC to be with to to the ABE assay for approaches acyl-RAC and are likely (23Forrester M.T. Thompson J.W. Foster M.W. Nogueira L. Moseley M.A. Stamler J.S. Proteomic analysis of S-nitrosylation and denitrosylation by resin-assisted capture.Nat. Biotechnol. 2009; 27: 557-559Crossref PubMed Scopus (310) Google Scholar). In with approaches (e.g., click acyl-RAC can provide a approach to the analysis of dynamic fatty should be that acyl-RAC (as in the of of the of and characterize the nature of the endogenous acyl For the of undergoes S-acylation with a is the acyl with radiolabeled palmitate or chemically palmitate (14Charron G. Zhang M.M. Yount J.S. Wilson J. Raghavan A.S. Shamir E. Hang H.C. Robust fluorescent detection of protein fatty-acylation with chemical reporters.J. Am. Chem. Soc. 2009; 131: 4967-4975Crossref PubMed Scopus (234) Google Scholar, 15Martin B.R. Cravatt B.F. Large-scale profiling of protein palmitoylation in mammalian cells.Nat. Methods. 2009; 6: 135-138Crossref PubMed Scopus (365) Google Scholar, 16Yap M.C. Kostiuk M.A. Martin D.D. Perinpanayagam M.A. Hak P.C. Siddam A. Majjigapu J.R. Rajaiah G. Keller B.O. Prescher J.A. Wu P. Bertozzi C.R. Falck J.R. Berthiaume L.G. Rapid and selective detection of fatty acylated proteins using ω-alkynyl-fatty acids and click chemistry.J. Lipid Res. 2010; 51: 1566-1580Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 17Hannoush R.N. Arenas-Ramirez N. Imaging the lipidome: omega-alkynyl fatty acids for detection and cellular visualization of lipid-modified proteins.ACS Chem. Biol. 2009; 4: 581-587Crossref PubMed Scopus (105) Google Scholar), as as of palmitoylation (e.g., can the nature of the acyl The acyl-RAC method should analysis of cellular protein S-acylation and The are to W. E. and E. for and with acyl-biotin exchange S-acylation by resin-assisted capture detection of proteins by resin-assisted capture