2-Bromopalmitate and 2-(2-hydroxy-5-nitro-benzylidene)-benzo[b]thiophen-3-one inhibit DHHC-mediated palmitoylation in vitroстатья из журнала
Аннотация: Pharmacologic approaches to studying palmitoylation are limited by the lack of specific inhibitors. Recently, screens have revealed five chemical classes of small molecules that inhibit cellular processes associated with palmitoylation (Ducker, C. E., L. K. Griffel, R. A. Smith, S. N. Keller, Y. Zhuang, Z. Xia, J. D. Diller, and C. D. Smith. 2006. Discovery and characterization of inhibitors of human palmitoyl acyltransferases. Mol. Cancer Ther. 5: 1647–1659). Compounds that selectively inhibited palmitoylation of N-myristoylated vs. farnesylated peptides were identified in assays of palmitoyltransferase activity using cell membranes. Palmitoylation is catalyzed by a family of enzymes that share a conserved DHHC (Asp-His-His-Cys) cysteine-rich domain. In this study, we evaluated the ability of these inhibitors to reduce DHHC-mediated palmitoylation using purified enzymes and protein substrates. Human DHHC2 and yeast Pfa3 were assayed with their respective N-myristoylated substrates, Lck and Vac8. Human DHHC9/GCP16 and yeast Erf2/Erf4 were tested using farnesylated Ras proteins. Surprisingly, all four enzymes showed a similar profile of inhibition. Only one of the novel compounds, 2-(2-hydroxy-5-nitro-benzylidene)-benzo[b]thiophen-3-one [Compound V (CV)], and 2-bromopalmitate (2BP) inhibited the palmitoyltransferase activity of all DHHC proteins tested. Hence, the reported potency and selectivity of these compounds were not recapitulated with purified enzymes and their cognate lipidated substrates. Further characterization revealed both compounds blocked DHHC enzyme autoacylation and displayed slow, time-dependent inhibition but differed with respect to reversibility. Inhibition of palmitoyltransferase activity by CV was reversible, whereas 2BP inhibition was irreversible. Pharmacologic approaches to studying palmitoylation are limited by the lack of specific inhibitors. Recently, screens have revealed five chemical classes of small molecules that inhibit cellular processes associated with palmitoylation (Ducker, C. E., L. K. Griffel, R. A. Smith, S. N. Keller, Y. Zhuang, Z. Xia, J. D. Diller, and C. D. Smith. 2006. Discovery and characterization of inhibitors of human palmitoyl acyltransferases. Mol. Cancer Ther. 5: 1647–1659). Compounds that selectively inhibited palmitoylation of N-myristoylated vs. farnesylated peptides were identified in assays of palmitoyltransferase activity using cell membranes. Palmitoylation is catalyzed by a family of enzymes that share a conserved DHHC (Asp-His-His-Cys) cysteine-rich domain. In this study, we evaluated the ability of these inhibitors to reduce DHHC-mediated palmitoylation using purified enzymes and protein substrates. Human DHHC2 and yeast Pfa3 were assayed with their respective N-myristoylated substrates, Lck and Vac8. Human DHHC9/GCP16 and yeast Erf2/Erf4 were tested using farnesylated Ras proteins. Surprisingly, all four enzymes showed a similar profile of inhibition. Only one of the novel compounds, 2-(2-hydroxy-5-nitro-benzylidene)-benzo[b]thiophen-3-one [Compound V (CV)], and 2-bromopalmitate (2BP) inhibited the palmitoyltransferase activity of all DHHC proteins tested. Hence, the reported potency and selectivity of these compounds were not recapitulated with purified enzymes and their cognate lipidated substrates. Further characterization revealed both compounds blocked DHHC enzyme autoacylation and displayed slow, time-dependent inhibition but differed with respect to reversibility. Inhibition of palmitoyltransferase activity by CV was reversible, whereas 2BP inhibition was irreversible. Protein palmitoylation is the posttranslational attachment of palmitate or other long-chain fatty acids to cysteine residues in proteins via a thioester linkage (as reviewed in Refs.1Resh M.D Palmitoylation of ligands, receptors, and intracellular signaling molecules.Sci. STKE. 2006; 2006: re14Crossref PubMed Scopus (341) Google Scholar, 2Linder M.E Deschenes R.J. Palmitoylation: policing protein stability and traffic.Nat. Rev. Mol. Cell Biol. 2007; 8: 74-84Crossref PubMed Scopus (757) Google Scholar). The functional consequences of protein palmitoylation are diverse and include effects on protein localization, trafficking, and stability. In contrast to other lipid modifications, palmitoylation is reversible. Consequently several cellular processes use palmitoylation-depalmitoylation cycles to affect the localization and function of key proteins. Protein acyltransferases (PATs) catalyze the addition of palmitate (3Mitchell D.A Vasudevan A. Linder M.E. Deschenes R.J. Protein pamitoylation by a family of DHHC protein S-acyltransferases..J. Lipid Res. 2006; 47: 1118-1127Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). Genetic and biochemical studies in yeast uncovered a family of integral membrane enzymes that mediate palmitate addition to proteins that are modified on the cytoplasmic face of cell membranes. The hallmark of this family of proteins is the DHHC (Asp-His-His-Cys) cysteine-rich domain. A family of seven DHHC proteins is present in S. cerevisiae, and homology searches have identified at least 23 genes that encode DHHC proteins in mammals. PAT activity is dependent upon the DHHC domain and mutation of the cysteine of the DHHC motif abolishes catalytic activity of the enzyme. Palmitoylated proteins are prominent in cell-signaling pathways and are particularly abundant in the nervous system (4el-Husseini Ael D. Bredt D.S. Protein palmitoylation: a regulator of neuronal development and function.Nat. Rev. Neurosci. 2002; 3: 791-802Crossref PubMed Scopus (269) Google Scholar). Signal transducers, receptors, ion channels, and scaffolds are among the targets of palmitoyltransferases. Finding the enzymes responsible for palmitoylation has accelerated our understanding of the role of palmitoylation in native and disease states. An example is HIP14 (DHHC17), a DHHC protein initially identified as huntingtin (htt)-interacting protein 14 (5Singaraja R.R Hadano S. Metzler M. Givan S. Wellington C.L. Warby S. Yanai A. Gutekunst C.A. Leavitt B.R. Yi H. et al.HIP14, a novel ankyrin domain-containing protein, links huntingtin to intracellular trafficking and endocytosis.Hum. Mol. Genet. 2002; 11: 2815-2828Crossref PubMed Scopus (178) Google Scholar). HIP14 has been linked to palmitoylation of htt, SNAP-25, cysteine string protein, and other neuronal substrates (6Huang K. Yanai A. Kang R. Arstikaitis P. Singaraja R.R. Metzler M. Mullard A. Haigh B. Gauthier-Campbell C. Gutekunst C.A. et al.Huntingtin-interacting protein HIP14 is a palmitoyl transferase involved in palmitoylation and trafficking of multiple neuronal proteins.Neuron. 2004; 44: 977-986Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). Palmitoylation of htt by HIP14 influences htt localization and protects it from aggregation (7Yanai A. Huang K. Kang R. Singaraja R.R. Arstikaitis P. Gan L. Orban P.C. Mullard A. Cowan C.M. Raymond L.A. et al.Palmitoylation of huntingtin by HIP14is essential for its trafficking and function.Nat. Neurosci. 2006; 9: 824-831Crossref PubMed Scopus (238) Google Scholar). In flies, HIP14 is essential for presynaptic function and plays a role in neurotransmitter release, likely through palmitoylation of SNAP-25 and cysteine string protein (8Stowers R.S Isacoff E.Y. Drosophila huntingtin-interacting protein 14 is a presynaptic protein required for photoreceptor synaptic transmission and expression of the palmitoylated proteins synaptosome-associated protein 25 and cysteine string protein.J. Neurosci. 2007; 27: 12874-12883Crossref PubMed Scopus (50) Google Scholar, 9Ohyama T. Verstreken P. Ly C.V. Rosenmund T. Rajan A. Tien A.C. Haueter C. Schulze K.L. Bellen H.J. Huntingtin-interacting protein 14, a palmitoyl transferase required for exocytosis and targeting of CSP to synaptic vesicles.J. Cell Biol. 2007; 179: 1481-1496Crossref PubMed Scopus (84) Google Scholar). Oncogenic Ras proteins are associated with numerous human tumors. Palmitoylation of H- and N-Ras plays a key role in trafficking between the plasma membrane and Golgi apparatus (3Mitchell D.A Vasudevan A. Linder M.E. Deschenes R.J. Protein pamitoylation by a family of DHHC protein S-acyltransferases..J. Lipid Res. 2006; 47: 1118-1127Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar), as well as facilitating the oncogenic potential of activating Ras mutations (10Willumsen B.M Cox A.D. Solski P.A. Der C.J. Buss J.E. Novel determinants of H-Ras plasma membrane localization and transformation.Oncogene. 1996; 13: 1901-1909PubMed Google Scholar). Several DHHC proteins have been linked to cancer. Human HIP14 is oncogenic, promoting colony formation in soft agar and tumor formation in nude mice (11Ducker C.E Stettler E.M. French K.J. Upson J.J. Smith C.D. Huntingtin interacting protein 14 is an oncogenic human protein: palmitoyl acyltransferase.Oncogene. 2004; 23: 9230-9237Crossref PubMed Scopus (93) Google Scholar). HIP14 mRNA is upregulated in numerous tumors (12Ducker C.E Griffel L.K. Smith R.A. Keller S.N. Zhuang Y. Xia Z. Diller J.D. Smith C.D. Discovery and characterization of inhibitors of human palmitoyl acyltransferases.Mol. Cancer Ther. 2006; 5: 1647-1659Crossref PubMed Scopus (77) Google Scholar). DHHC9, a PAT for H- and N-Ras in vitro, is upregulated in microsatellite stable tumors (13Mansilla F. Birkenkamp-Demtroder K. Kruhoffer M. Sorensen F.B. Andersen C.L. Laiho P. Aaltonen L.A. Verspaget H.W. Orntoft T.F. Differential expression of DHHC9 in microsatellite stable and instable human colorectal cancer subgroups.Br. J. Cancer. 2007; 96: 1896-1903Crossref PubMed Scopus (56) Google Scholar), whereas DHHC2 is a putative tumor suppressor (14Oyama T. Miyoshi Y. Koyama K. Nakagawa H. Yamori T. Ito T. Matsuda H. Arakawa H. Nakamura Y. Isolation of a novel gene on 8p21.3–22 whose expression is reduced significantly in human colorectal cancers with liver metastasis.Genes Chromosomes Cancer. 2000; 29: 9-15Crossref PubMed Scopus (0) Google Scholar). The importance of palmitoylation in physiology and pathophysiology suggest that palmitoylation inhibitors could be beneficial for the treatment of diseases, as well as tools for probing the role of palmitoylation in cellular processes. Inhibitors of palmitoylation have been limited to 2-bromopalmitate (2BP), cerulenin, and tunicamycin. The most commonly used, 2BP, inhibits palmitoylation in cells (15Webb Y. Hermida-Matsumoto L. Resh M.D. Inhibition of protein palmitoylation, raft localization, and T cell signaling by 2-bromopalmitate and polyunsaturated fatty acids.J. Biol. Chem. 2000; 275: 261-270Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar) and PAT activity of DHHC proteins in vitro (16Fukata M. Fukata Y. Adesnik H. Nicoll R.A. Bredt D.S. Identification of PSD-95 palmitoylating enzymes.Neuron. 2004; 44: 987-996Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar). However, 2BP also inhibits fatty acid CoA ligase (17Chase J.F Tubbs P.K. Specific inhibition of mitochondrial fatty acid oxidation by 2-bromopalmitate and its coenzyme A and carnitine esters.Biochem. J. 1972; 129: 55-65Crossref PubMed Scopus (118) Google Scholar) and other enzymes involved in lipid metabolism (18Coleman R.A Rao P. Fogelsong R.J. Bardes E.S. 2-Bromopalmitoyl-CoA and 2-bromopalmitate: promiscuous inhibitors of membrane-bound enzymes.Biochim. Biophys. Acta. 1992; 1125: 203-209Crossref PubMed Scopus (80) Google Scholar). Similarly, cerulenin and tunicamycin inhibit palmitoylation within cells but also inhibit other cellular process including fatty acid synthesis (19Omura S. The antibiotic cerulenin, a novel tool for biochemistry as an inhibitor of fatty acid synthesis.Bacteriol. Rev. 1976; 40: 681-697Crossref PubMed Google Scholar) and N-glycosylation (20DeJesus G. Bizzozero O.A. Effect of 2-fluoropalmitate, cerulenin and tunicamycin on the palmitoylation and intracellular translocation of myelin proteolipid protein.Neurochem. Res. 2002; 27: 1669-1675Crossref PubMed Scopus (19) Google Scholar, 21Patterson S.I Skene J.H. Inhibition of dynamic protein palmitoylation in intact cells with tunicamycin.Methods Enzymol. 1995; 250: 284-300Crossref PubMed Scopus (40) Google Scholar), respectively. Consequently, there is a need to identify specific inhibitors of palmitoylation. Ducker et al. (12Ducker C.E Griffel L.K. Smith R.A. Keller S.N. Zhuang Y. Xia Z. Diller J.D. Smith C.D. Discovery and characterization of inhibitors of human palmitoyl acyltransferases.Mol. Cancer Ther. 2006; 5: 1647-1659Crossref PubMed Scopus (77) Google Scholar) have developed high throughput screens for palmitoylation inhibitors. The screens yielded compounds that fell into five chemical classes, and a representative compound from each class was further characterized. Cell membranes from MCF-7 cells were used as a source of PAT activity to evaluate the representative compounds' ability to inhibit palmitoylation in vitro. Fluorescently labeled peptides mimicking either N-myristoylated, palmitoylated proteins, such as Gα subunits and Src-related tyrosine kinases, or mimicking C-terminally farnesylated, palmitoylated proteins, like N- and H-Ras, were used as substrates. Four of the representative compounds showed a preference for inhibiting palmitoylation of the Ras-like peptide (average of 76% inhibition) but not the myristoylated peptide (15% inhibited). Conversely, the fifth compound displayed the reverse, inhibiting palmitoylation of the myristoylated peptide (74%) but not the Ras-like peptide (17%). Thus, it appears these compounds not only inhibit palmitoylation but also display substrate specificity. Additionally, IC50 values were reported in low- to submicromolar range (11.8–0.5 μM) (12Ducker C.E Griffel L.K. Smith R.A. Keller S.N. Zhuang Y. Xia Z. Diller J.D. Smith C.D. Discovery and characterization of inhibitors of human palmitoyl acyltransferases.Mol. Cancer Ther. 2006; 5: 1647-1659Crossref PubMed Scopus (77) Google Scholar). We sought to further investigate the properties of these compounds. Because cell membranes were used as a source of PAT activity, it is unclear whether the inhibitors reduced palmitoylation by directly blocking DHHC proteins. Here we tested if these compounds inhibit DHHC-mediated palmitoylation of protein substrates. Additionally, by using DHHC proteins that display different substrate preferences, the ability of these inhibitor compounds to specifically inhibit palmitoylation of farnesylated substrates vs. N-myristoylated substrates was examined. Compounds that inhibited DHHC-mediated palmitoylation were further characterized. Inhibitor compounds (Fig. 1) were purchased from ChemBridge Corporation (San Diego, CA). [3H]palmitoyl-CoA ([3H]palmCoA) was synthesized using [3H]palmitate (45 Ci/mmol, PerkinElmer Life Sciences), CoA (Sigma), and acyl CoA synthase (Sigma) as described (22Dunphy J.T Greentree W.K. Manahan C.L. Linder M.E. G-protein palmitoyltransferase activity is enriched in plasma membranes.J. Biol. Chem. 1996; 271: 7154-7159Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar) with the following modification. Following synthesis, [3H]palmCoA was separated from palmitate by chloroform/methanol extraction (23Duncan J.A Gilman A.G. Autoacylation of G protein alpha subunits.J. Biol. Chem. 1996; 271: 23594-23600Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) and subsequently purified on a C8 reversed phase cartridge (22Dunphy J.T Greentree W.K. Manahan C.L. Linder M.E. G-protein palmitoyltransferase activity is enriched in plasma membranes.J. Biol. Chem. 1996; 271: 7154-7159Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Standard molecular biology techniques were used to manipulate DNA. All plasmid constructs were verified by DNA sequence analysis. DHHC2 was expressed using recombinant baculovirus. DHHC2 was prone to proteolysis and several constructs were made with affinity tags at both ends to generate a virus that would yield intact DHHC2 protein. Oligonucleotide sequences used to generate plasmids are available upon request. Human DHHC2 was amplified from Image Clone 4398300 (GenBank ID BF938682) and subcloned as a KpnI fragment into pBlueBacHis2B (Invitrogen; Carlsbad, CA). This yielded pML850, encoding His6Express-DHHC2. pML850 was digested with HindIII and SalI to permit ligation with a double-stranded oligonucleotide linker that encodes a HindIII site, the FLAG epitope, an EcoRI site, a His6 epitope, a stop codon, and a SalI site. This yielded pML892, encoding His6Express-DHHC2-FLAG-His6. pML892 was cut with BglII and AgeI, purified, and ligated with double-stranded oligonucleotides encoding the calmodulin binding peptide (CBP) followed by an XbaI site to generate His6Express-CBP-DHHC2-FLAG-His6 (pML943). Catalytically inactive DHHS2 (pML1023) was generated by site-directed mutagenesis (Stratagene) of pML943. Plasmids expressing the N-terminal 226 amino acids of Lck (LckNT) and LckNT(C3,5S) were constructed as follows. The murine Lck cDNA encoding residues 1-226 was amplified as a NdeI-XhoI fragment and subcloned into the bacterial expression vector pET23a(+) yielding pML1008. This construct was used as template to generate the C3,5S mutant (pML1175) using a mutagenic primer to amplify the 5′ end of the coding sequence. Yeast N-myristoyltransferase was expressed using pBB131 (24Duronio R.J Jackson-Machelski E. Heuckeroth R.O. Olins P.O. Devine C.S. Yonemoto W. Slice L.W. Taylor S.S. Gordon J.I. Protein N-myristoylation in Escherichia coli: reconstitution of a eukaryotic protein modification in bacteria.Proc. Natl. Acad. Sci. USA. 1990; 87: 1506-1510Crossref PubMed Scopus (200) Google Scholar). The plasmid pML1067 was constructed to express human NMT1 by excising the yeast NMT1 gene from pBB131 with BglII and EcoR1 and replacing it with human NMT1 flanked by pBB131 vector sequences. Human NMT1 was amplified from pBB218 and overlap extension PCR was used to generate flanking pBB131 vector sequences (25Duronio R.J Reed S.I. Gordon J.I. Mutations of human myristoyl-CoA:protein N-myristoyltransferase cause temperature-sensitive myristic acid auxotrophy in Saccharomyces cerevisiae..Proc. Natl. Acad. Sci. USA. 1992; 89: 4129-4133Crossref PubMed Scopus (110) Google Scholar). Sf9 insect cells were purchased from ATCC and grown in suspension culture medium [IPL-41 (Gibco) supplemented with 10% heat inactivated bovine growth serum, yeastolate, Pluronic F68, 50 μg/ml gentamycin, and 250 ng/ml fungizone] at 27°C with rotation at 110 rpm. Recombinant baculoviruses were generated using Invitrogen's Bac-n-Blue™ transfection kit with pML943 and pML1023 and plaque purified. For DHHC2, Sf9 insect cells were inoculated with baculovirus expressing human DHHC2 N-terminally tagged with His6-Express-CBP and C-terminally tagged with FLAG-His6. Infected cells were collected by centrifugation and washed with cold PBS 61 h post infection. Cell pellets were stored at −80°C until purification. All purification steps were performed at 4°C, and all buffers contained the protease inhibitors 1 mM PMSF, 1–5 μg/ml pepstatin A, 1.4 μg/ml aprotinin, 1.6 μg/ml leupeptin, and 1.6 μg/ml lima bean trypsin inhibitor. A cell pellet of ∼5 ml (from 335 ml Sf9 culture) was quickly thawed at 37°C and suspended in 35 ml cavitation buffer (50 mM Tris pH 7.4, 150 mM NaCl, 10 mM β-ME, 1 mM EDTA). Cells were lysed by nitrogen cavitation (30 min at 700 psi). The lysate was centrifuged at 700 g for 10 min to remove nuclei and unbroken cells. The postnuclear supernatant was centrifuged at 100,000 g for 30 min to generate P100 and S100 fractions. P100 membranes were suspended in 9 ml extract buffer (50 mM Tris pH 7.4, 200 mM NaCl, 10 mM β-ME, and 10% glycerol) by sequential passage through 14, 18, and 25 gauge needles. Total protein concentration was determined using Bio-Rad's Bradford protein assay (Hercules, CA). Membranes were diluted with extract buffer and 10% n-dodecyl-β-D-maltoside detergent (DDM; Dojindo Laboratories, Japan) to give a final protein concentration of 2 mg/ml in 1% DDM. The extract was again passed through a 25 G needle and incubated 80 min with end-over-end rotation. The extract was cleared at 100,000 g for 30 min, diluted 1:1 with extract buffer (no DDM), and gravity-flowed twice through a column of 3.4 ml Ni2+-nitrilotriacetic acid-agarose resin (Ni-NTA; Qiagen) equilibrated in wash buffer (50 mM Tris pH 7.4, 100 mM NaCl, 3 mM β-ME, 10% glycerol, 0.1% DDM, and 20 mM imidazole). The resin was washed with 60 ml wash buffer and eluted with wash buffer containing 200 mM imidazole (2 × 3 ml) and wash buffer containing 500 mM imidazole (3 × 3 ml). Ni elutions 1–4 were pooled, diluted 1:1 with buffer A (50 mM Tris pH 7.4, 100 mM NaCl, 10% glycerol, 0.1% DDM, 1 mM EDTA, and 0.2 mM β-ME), and passed thrice through a column of 300 μl ANTI-FLAG® M2-agarose affinity gel (Sigma) equilibrated in buffer A. The resin was washed with 14 ml buffer A and eluted with 5 × 250 μl buffer A containing 0.23 mg/ml FLAG peptide (Sigma) with a 10 min incubation for each elution. The concentration of enzyme was determined by extrapolation from a linear curve with known concentrations of BSA using Sypro Ruby protein gel stain (Molecular Probes) and quantitation with a Storm™ 860 (Amersham Biosciences). DHHS2 purification paralleled that of DHHC2 through nickel affinity chromatography. The identity of purified DHHC2 or DHHS2 was confirmed by immunoblots using antibodies at the following dilutions: anti-FLAG 1:3,000 (Stratagene), anti-Express 1:1,500 (Invitrogen), and goat anti-mouse IgG secondary conjugated to HRP at 1:2,000 (MP Biomedicals, OH). For human DHHC9/GCP16, Sf9 cells were coinfected with baculoviruses expressing DHHC9-myc-His6 and FLAG-GCP16 (26Swarthout J.T Lobo S. Farh L. Croke M.R. Greentree W.K. Deschenes R.J. Linder M.E. DHHC9 and GCP16 constitute a human protein fatty acyltransferase with specificity for H- and N-Ras..J. Biol. Chem. 2005; 280: 31141-31148Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar) and cultured for 73 h before harvesting. DHHC9/GCP16 was purified similarly to DHHC2. The purification of Erf2/Erf4p was performed as previously described (27Budde C. Schoenfish M.J. Linder M.E. Deschenes R.J. Purification and characterization of recombinant protein acyltransferases.Methods. 2006; 40: 143-150Crossref PubMed Scopus (12) Google Scholar). For partially purified yeast Pfa3, YPH499 was transformed with a Pfa3-His6-Flag pESC expression construct. Yeast were cultured and cell membranes prepared as described (28Smotrys J.E Schoenfish M.J. Stutz M.A. Linder M.E. The vacuolar DHHC-CRD protein Pfa3p is a protein acyltransferase for Vac8p..J. Cell Biol. 2005; 170: 1091-1099Crossref PubMed Scopus (65) Google Scholar). Pfa3-His6-Flag was extracted from cell membranes in 1% Triton X-100 at 5 mg/ml total protein in extraction buffer (50 mM Tris pH 8.0, 100 mM NaCl, 10% glycerol, 5 mM β-ME, and 0.1 mM PMSF) rotating for 1 h at 4°C. Insoluble material was pelleted at 200,000 g for 20 min. The detergent extract (7.5 mg total protein) was diluted 1:1 in extraction buffer and allowed to bind to 2 ml Ni-NTA resin for 1 h at 4°C. The resin was reconstituted in a column and washed with 20 ml wash buffer [50 mM Tris pH 8.0, 100 mM NaCl, 10% glycerol, 5 mM β-ME, 0.1% Triton X-100, 0.1 mg/ml bovine liver lipids (Avanti Polar Lipids), and 0.1 mM PMSF]. Bound proteins were eluted with elution buffer (50 mM Tris pH 8.0, 100 mM NaCl, 10% glycerol, 200 mM imidazole, 1 mM β-ME, 0.1% Triton X-100, and 0.1 mg/ml bovine liver lipids), and four 2 ml fractions were collected. Fractions one and two were pooled, aliquoted, and stored at −80°C. Mouse N-myristoylated LckNT (myrLckNT) was purified from ER2566 E.coli (New England Biolabs) harboring pBB131 (24Duronio R.J Jackson-Machelski E. Heuckeroth R.O. Olins P.O. Devine C.S. Yonemoto W. Slice L.W. Taylor S.S. Gordon J.I. Protein N-myristoylation in Escherichia coli: reconstitution of a eukaryotic protein modification in bacteria.Proc. Natl. Acad. Sci. USA. 1990; 87: 1506-1510Crossref PubMed Scopus (200) Google Scholar) and pML1008. Cells were grown in Luria-Bertani broth containing 50 μg/ml kanamycin and 50 μg/ml ampicillin at 37°C with shaking at 250 rpm for 4 h. Isopropyl β-D-1-thiogalactopyranoside was added to 0.6 mM final concentration, and the temperature was reduced to 30°C for 5 h. Cells were harvested by centrifugation at 3,080 g for 20 min at 4°C. Cell pellets were washed with cold PBS, collected by centrifugation at 3,800 g for 17 min at 4°C, flash frozen in liquid N2, and stored at −80°C until needed. Purification steps were performed on ice or in a 4°C cold room. Cell pellets representing 400 ml culture were quickly thawed at 30°C, suspended in 11 ml lysis buffer (50 mM Tris pH 7.4, 8 mM β-ME, 150 mM NaCl, 1 μg/ml pepstatin A, 1.6 μg/ml leupeptin, 1.6 μg/ml lima bean trypsin inhibitor, and 1.4 μg/ml aprotinin), and passed thrice through a French press cell. The soluble fraction was collected by centrifugation at 76,500 g for 30 min. To enrich for myristoylated-LckNT, this soluble fraction was extracted twice with 1.3 ml 10% Triton X-114 detergent (Sigma) essentially as described (29Osanai K. Takahashi K. Nakamura K. Takahashi M. Ishigaki M. Sakuma T. Toga H. Suzuki T. Voelker D.R. Expression and characterization of Rab38, a new member of the Rab small G protein family.Biol. Chem. 2005; 386: 143-153Crossref PubMed Scopus (34) Google Scholar) except incubations were for 5 min at 37°C. The detergent phases were pooled, diluted to 0.2% Triton X-114 (135 ml total), and gravity loaded onto a 3 ml Ni-NTA column equilibrated in wash buffer (50 mM Tris pH 8.0, 300 mM NaCl, 10 mM imidazole, and 8 mM β-ME). The column was washed with 150 ml wash buffer and eluted 5 × 3 ml with elution buffer (50 mM Tris pH 7.4, 100 mM NaCl, 300 mM imidazole, 8 mM β-ME, and 10% glycerol). Elution 2 was used for subsequent PAT assays. The concentration of myrLckNT was determined by extrapolation from a linear curve with known concentrations of BSA using Coomassie Blue gel stain and quantitation with ImageJ software (NIH). C3,5S myrLckNT was expressed in BL21(DE3) E.coli transformed with pML1067 and pML1175 and purified as described for wild-type. The identity of purified protein was confirmed by immunoblotting with mouse monoclonal anti-Lck (3A5, sc-433) purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Yeast N-myristoylated Vac8 was purified by ion-exchange and hydroxylapatite chromatography as described (27Budde C. Schoenfish M.J. Linder M.E. Deschenes R.J. Purification and characterization of recombinant protein acyltransferases.Methods. 2006; 40: 143-150Crossref PubMed Scopus (12) Google Scholar). Prenylated Ras proteins were expressed in yeast from the galactose-inducible vector pEG(KG) and purified as described (27Budde C. Schoenfish M.J. Linder M.E. Deschenes R.J. Purification and characterization of recombinant protein acyltransferases.Methods. 2006; 40: 143-150Crossref PubMed Scopus (12) Google Scholar, 30Lobo S. Greentree W.K. Linder M.E. Deschenes R.J. Identification of a Ras palmitoyltransferase in Saccharomyces cerevisiae.J. Biol. Chem. 2002; 277: 41268-41273Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). The glutathione S-transferase (GST)-Ras2(HV) construct consists of the C-terminal 35 amino acid residues of yeast Ras2 fused to GST. Full-length mammalian H-Ras was fused to GST. Inhibitors were dissolved and serially diluted to 100× stocks in 100% DMSO. Stocks were diluted to 5× with buffer containing 20 mM Tris pH 7.4, 1 mM EDTA, and 1 mM 1,4- dithiothreitol (DTT). Equal volumes (5 μl) of DHHC enzyme and 5× inhibitor solutions were preincubated for various times and temperatures as indicated in figure legends. Protein substrates diluted in buffer containing 20 mM Tris pH 7.4, 1 mM EDTA, and 1 mM DTT (5 μl) were premixed with reaction hot mix [RHM; 10 μl; 250 mM MES pH 6.4, 1 mM DTT, 1.2-3.4 μM [3H]palmCoA and incubated at the same temperature as enzyme/inhibitor mix. This substrate mixture (15 μl) was added to the enzyme/inhibitor mix to start the reaction and incubated as indicted for each figure. The reactions (25 μl) were stopped by addition of 5× gel loading buffer with final 2 mM DTT (6.25 μl). Except for assays of reversibility, all inhibitor concentrations shown are for the final 25 μl reaction. Stopped reactions were heated for 60 s at 95°C before resolution by SDS-PAGE. Gels were stained with Coomassie Blue, destained, and scanned. Protein substrate bands were excised from the gel, placed into scintillation vials, solubilized in 500 μl Soluene 350 (PerkinElmer) at 50°C for at least 3 h, and quantitated by liquid scintillation spectroscopy. Enzyme autoacylation assays without inhibitors (Fig. 2, right panel) were performed similar to inhibitor profile assays described above. Briefly, 500 fmol of enzyme was incubated in 0.82 μM [3H]palmCoA for 10 min at 25°C in RHM. Reactions were stopped with gel loading buffer and resolved by SDS-PAGE. Gels were stained, destained, soaked in 1 M sodium salicylate/15% methanol for 20 min, dried onto filter paper, and exposed to film at −80°C for 8 days. Enzyme autoacylation assays with inhibitors (Fig. 5) were performed similar to inhibitor profile assay except without protein substrate. Briefly, 250 fmol of DHHC2 was preincubated with inhibitor for 8 min at 25°C. Prewarmed RHM was added to give a final concentration of 1.1 μM [3H]palmCoA in 25 μl and the reaction proceeded for 2 min before stopping with 5× gel loading buffer. Reactions were processed similar to those inFig. 2.Fig. 5Inhibitor effect on enzyme autoacylation. DHHC2 (250 fmol) was preincubated with inhibitor and assayed as described in Materials and Methods. The film was exposed for 8 days.View Large
Год издания: 2008
Авторы: Benjamin C. Jennings, Marissa J. Nadolski, Yiping Ling, Meredith Beckham Baker, Marietta L. Harrison, Robert J. Deschenes, Maurine E. Linder
Издательство: Elsevier BV
Источник: Journal of Lipid Research
Ключевые слова: Protein Kinase Regulation and GTPase Signaling, Drug Transport and Resistance Mechanisms, Metabolism, Diabetes, and Cancer
Другие ссылки: Journal of Lipid Research (PDF)
Journal of Lipid Research (HTML)
DOAJ (DOAJ: Directory of Open Access Journals) (HTML)
Europe PMC (PubMed Central) (PDF)
Europe PMC (PubMed Central) (HTML)
PubMed Central (HTML)
PubMed (HTML)
Journal of Lipid Research (HTML)
DOAJ (DOAJ: Directory of Open Access Journals) (HTML)
Europe PMC (PubMed Central) (PDF)
Europe PMC (PubMed Central) (HTML)
PubMed Central (HTML)
PubMed (HTML)
Открытый доступ: hybrid
Том: 50
Выпуск: 2
Страницы: 233–242