Proteomics-based Target Identification
2003; Elsevier BV; Volume: 278; Issue: 52 Linguagem: Inglês
10.1074/jbc.m309039200
ISSN1083-351X
AutoresHarry Towbin, Kenneth W. Bair, James A. DeCaprio, Michael J. Eck, Sunkyu Kim, Frederick R. Kinder, Anthony A. Morollo, Dieter Mueller, Patrick Schindler, Hyun Kyu Song, Jan van Oostrum, Richard Versace, Hans Voshol, Jeanette M. Wood, Sonya Zabludoff, Penny E. Phillips,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoLAF389 is a synthetic analogue of bengamides, a class of marine natural products that produce inhibitory effects on tumor growth in vitro and in vivo. A proteomics-based approach has been used to identify signaling pathways affected by bengamides. LAF389 treatment of cells resulted in altered mobility of a subset of proteins on two-dimensional gel electrophoresis. Detailed analysis of one of the proteins, 14-3-3γ, showed that bengamide treatment resulted in retention of the amino-terminal methionine, suggesting that bengamides directly or indirectly inhibited methionine aminopeptidases (MetAps). Both known MetAps are inhibited by LAF389. Short interfering RNA suppression of MetAp2 also altered amino-terminal processing of 14-3-3γ. A high resolution structure of human MetAp2 co-crystallized with a bengamide shows that the compound binds in a manner that mimics peptide substrates. Additionally, the structure reveals that three key hydroxyl groups on the inhibitor coordinate the di-cobalt center in the enzyme active site. LAF389 is a synthetic analogue of bengamides, a class of marine natural products that produce inhibitory effects on tumor growth in vitro and in vivo. A proteomics-based approach has been used to identify signaling pathways affected by bengamides. LAF389 treatment of cells resulted in altered mobility of a subset of proteins on two-dimensional gel electrophoresis. Detailed analysis of one of the proteins, 14-3-3γ, showed that bengamide treatment resulted in retention of the amino-terminal methionine, suggesting that bengamides directly or indirectly inhibited methionine aminopeptidases (MetAps). Both known MetAps are inhibited by LAF389. Short interfering RNA suppression of MetAp2 also altered amino-terminal processing of 14-3-3γ. A high resolution structure of human MetAp2 co-crystallized with a bengamide shows that the compound binds in a manner that mimics peptide substrates. Additionally, the structure reveals that three key hydroxyl groups on the inhibitor coordinate the di-cobalt center in the enzyme active site. Bengamides are natural products originally isolated from marine sponges (1Quiñoà E. Adamczeski M. Crews P. Bakus G.J. J. Org. Chem. 1986; 51: 4494-4497Crossref Scopus (104) Google Scholar). Bengamide B is one of the most potent members of the family; it causes growth inhibition in vitro at low nm concentrations on all human tumor cell lines tested (2Thale Z. Kinder F.R. Bair K.W. Bontempo J. Czuchta A.M. Versace R.W. Phillips P.E. Sanders M.L. Wattanasin S. Crews P. J. Org. Chem. 2001; 66: 1733-1741Crossref PubMed Scopus (87) Google Scholar). In vivo, it significantly inhibits growth of MDA-MB435 human breast cancer xenografts at well tolerated doses (3Kinder F.R. Versace R.W. Bair K.B. Bontempo J.M. Cesarz D. Chen S. Crews P. Czuchta A.M. Jagoe C.T. Mou Y. Nemzek R. Phillips P.E. Tran L.D. Wang R.M. Weltchek S. Zabludoff S. J. Med. Chem. 2001; 44: 3692-3699Crossref PubMed Scopus (62) Google Scholar). In vitro studies of bengamide B suggested its activity might be due to inhibition of a novel target, because its pattern of activity in the NCI 60 cell line screening panel was unique compared with that of other chemotherapeutic agents (2Thale Z. Kinder F.R. Bair K.W. Bontempo J. Czuchta A.M. Versace R.W. Phillips P.E. Sanders M.L. Wattanasin S. Crews P. J. Org. Chem. 2001; 66: 1733-1741Crossref PubMed Scopus (87) Google Scholar). Although bengamide B was a potent lead structure, its limited availability from natural sources, the complexity of its synthesis, and its relatively poor solubility prevented further development of the compound as a therapeutic agent. Therefore, a synthetic chemistry program was initiated to develop analogues of bengamide B with enhanced solubility and ease of synthesis. LAF389 was selected from a number of analogues because of its equivalent activity in vitro against a broad panel of human tumor cell lines and its improved activity in human tumor xenograft models (3Kinder F.R. Versace R.W. Bair K.B. Bontempo J.M. Cesarz D. Chen S. Crews P. Czuchta A.M. Jagoe C.T. Mou Y. Nemzek R. Phillips P.E. Tran L.D. Wang R.M. Weltchek S. Zabludoff S. J. Med. Chem. 2001; 44: 3692-3699Crossref PubMed Scopus (62) Google Scholar). As was the case with bengamide B, the profile of LAF389 in standard cytotoxicity assays and tests of its effects on conventional cytotoxic targets did not suggest the molecular target of the compound. Because such knowledge can help to develop more potent or selective inhibitors and is also helpful in selecting the most responsive tumor types for clinical use, a number of approaches were used to determine a molecular target. LAF389 was not active in assays for DNA binding and damage, topoisomerase binding, polymerization of microtubules and actin, or proteasome function (data not shown). Similarly, no changes in mRNA transcription were detected in studies on transcriptional effects where cells were challenged for 8 h with LAF389. The relatively short exposure time was chosen to narrow the responses to initial effects on or close to the affected target. Here, we report on the identification of a molecular target for bengamides based on proteomic studies performed on cells treated in vitro with LAF389. Proteomics approaches have the potential to discover effects at the level of posttranslational modifications of proteins, an area where other powerful techniques such as RNA profiling experiments are not directly informative. In this case, proteomic analysis has proven pivotal to the discovery of methionine aminopeptidases as a molecular target for bengamides. Cloning, Expression, and Assay of Human Methionine Aminopeptidases—Human MetAp 1 and 2 were PCR-amplified based on published sequences (4Li X. Chang Y.-H. Biochim. Biophys. Acta. 1995; 1260: 333-336Crossref PubMed Scopus (35) Google Scholar, 5Li X. Chang Y.-H. Biochem. Biophys. Res. Commun. 1996; 227: 152-159Crossref PubMed Scopus (54) Google Scholar, 6Nagase T. Miyajima N. Tanaka A. Sazuka T. Seki N. Sato S. Tabata S. Ishikawa K. Kawarabayasi Y. Kotani H. Nomura N. DNA Res. 1995; 2: 37-43Crossref PubMed Scopus (114) Google Scholar), expressed as N-terminal glutathione S-transferase-tagged proteins in insect cells, and purified over GSH columns. Enzyme assays were run using MetAp enzyme (5.2 μg/ml, 100 nm) incubated with Met-Ala-Ser tripeptide substrate (Bachem; 1 mm) and inhibitor compounds in 66 μl of assay buffer for 1 h at room temperature. Assay buffer was 50 mm KH2PO4, pH 7.5, 0.4 mm CoCl2, 0.02% bovine serum albumin. Reactions were terminated, and released methionine was visualized by the addition of 80 μl of ninhydrin solution (30 mm ninhydrin, 30 mm CdCl2, 12.2% acetic acid). Plates were incubated 1 h at room temperature and read at 490 nm. Purification and Synthesis of Bengamide Analogues—The syntheses of bengamide E, LAF153, and LAF389 have been reported previously (3Kinder F.R. Versace R.W. Bair K.B. Bontempo J.M. Cesarz D. Chen S. Crews P. Czuchta A.M. Jagoe C.T. Mou Y. Nemzek R. Phillips P.E. Tran L.D. Wang R.M. Weltchek S. Zabludoff S. J. Med. Chem. 2001; 44: 3692-3699Crossref PubMed Scopus (62) Google Scholar, 7Kinder Jr., F.R. Wattanasin S. Versace R.W. Bair K.W. Bontempo J. Green M.A. Lu Y.J. Marepalli H.R. Phillips P.E. Roche D. Tran L.D. Wang R.M. Waykole L. Xu D.D. Zabludoff S. J. Org. Chem. 2001; 66: 2118-2122Crossref PubMed Scopus (36) Google Scholar). Structures of the compounds are given in Fig. 1. Cell Culture and Proliferation Assays—H1299 human small cell lung carcinoma and A549 human non-small cell lung carcinoma cells were cultivated in RPMI 1640 medium supplemented with 10% fetal calf serum and glutamine. Human umbilical vein endothelial cells (HUVEC) 1The abbreviations used are: HUVEChuman umbilical vein endothelial cellsCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidsiRNAshort interfering RNAIEFimmuno-electrophoresisMALDI-MSmatrix-assisted laser desorption ionization mass spectrometryESMS/MSelectrospray tandem mass spectrometryMetApsmethionine aminopeptidases. were purchased from Clonetics and cultured according to the manufacturer's protocol. All maintenance media contained 100 units/ml penicillin and 100 μg/ml streptomycin. Antiproliferative effects of LAF389 in vitro were measured as described previously (3Kinder F.R. Versace R.W. Bair K.B. Bontempo J.M. Cesarz D. Chen S. Crews P. Czuchta A.M. Jagoe C.T. Mou Y. Nemzek R. Phillips P.E. Tran L.D. Wang R.M. Weltchek S. Zabludoff S. J. Med. Chem. 2001; 44: 3692-3699Crossref PubMed Scopus (62) Google Scholar). human umbilical vein endothelial cells 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid short interfering RNA immuno-electrophoresis matrix-assisted laser desorption ionization mass spectrometry electrospray tandem mass spectrometry methionine aminopeptidases. IEF Sample Preparation and Immunoblotting—Cell samples were washed with phosphate-buffered saline and the cell pellet was dissolved in a buffer containing 4% CHAPS, 7 m urea, 2 m thiourea, 10 mg/ml dithiothreitol, and 1% pharmalytes (pH 3-10) (8Rabilloud T. Adessi C. Giraudel A. Lunardi J. Electrophoresis. 1997; 18: 307-316Crossref PubMed Scopus (403) Google Scholar). Isoelectric focusing of samples and transfer to membranes was performed as described (9Towbin H. Özbey Ö. Zingel O. Electrophoresis. 2001; 22: 1887-1893Crossref PubMed Scopus (40) Google Scholar). Two-dimensional Electrophoresis and Proteomics Techniques—The methodology described earlier (10Mueller D.R. Schindler P. Coulot M. Voshol H. van Oostrum J. J. Mass Spectrom. 1999; 34: 336-345Crossref PubMed Scopus (48) Google Scholar) was used. For resolving the 14-3-3 isoforms, ultrazoom pH gradient strips in the range of pH 4.5-5.5 (Amersham Pharmacia Biotech, Uppsala, Sweden) were used. Western Blots—Rabbit polyclonal antibodies against 14-3-3 proteins (pan-reactive and isotype-specific) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). A monoclonal antibody specific to the amino terminus of 14-3-3γ was raised against a peptide corresponding to the N-terminal 15 amino acid residues, beginning with methionine, coupled to keyhole limpet hemocyanin. Specificity of the antibody was confirmed by testing against both the immunizing peptide as well as a 15-mer peptide missing the initial methionine and beginning with an acetylated valine (data not shown). siRNA—MetAp2 suppression was performed using an siRNA sequence designed as described (11Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8104) Google Scholar). The targeting sequence was AAUGCCGGUGACACAACAUGA (Dharmacon Research). The control mismatch sequence was AAUGCCGGCGCUACAACAUGA. Duplex RNA was introduced into H1299 cells using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's protocol. Duplex RNA was introduced into HUVECs by electroporation. Structure Determination—The full-length human methionine aminopeptidase-2 protein was expressed by using a baculovirus/insect cell system and was purified as described previously (12Liu S. Widom J. Kemp C.W. Crews C.M. Clardy J. Science. 1998; 282: 1324-1327Crossref PubMed Scopus (390) Google Scholar). Crystals isomorphous to the previously described unliganded and fumagillin-bound MetAp2 (13Otwinowski Z. Minor W. Methods Enzymol. 1997; 276 (Part A): 307-326Crossref Scopus (38436) Google Scholar) were obtained by vapor diffusion at 4 °C by combining 2 μl of MetAp2 protein in storage buffer (10 mm Hepes, pH 7.0, 150 mm NaCl, 1 mm CoCl2, and 10% glycerol) with 2 μl of a reservoir solution containing 30% t-butyl alcohol and 50 mm sodium citrate, pH 5.5. For co-crystallization with LAF153, an approximately 4-fold molar excess of the inhibitor was added to the protein solution (in ethanol). Diffraction data were recorded at 100 K on the A1 station at the Cornell High Energy Synchrotron Source and processed using DENZO and SCALEPACK (13Otwinowski Z. Minor W. Methods Enzymol. 1997; 276 (Part A): 307-326Crossref Scopus (38436) Google Scholar). The structure was determined by rigid body refinement of the unliganded MetAp2 structure (Protein Bata Bank code 1BN5) against the LAF153 data set. The structure and inhibitor was fit to the electron density with the program O (14Jones T.A. Kjeldgaard M. Methods Enzymol. 1997; 277: 173-208Crossref PubMed Scopus (504) Google Scholar). Crystallographic refinement with the program CNS (15Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16946) Google Scholar) yielded a final R value of 20.4% (Rfree = 21.1%) using all data to 1.6 Å resolution. Crystallographic data collection and refinement statistics are presented in Table II. Coordinates for the x-ray structure of the MetAp2/LAF153 complex have been deposited with the Protein Data Bank under code 1QZY.Table IIData collection and refinement statisticsData collection statisticsSpace groupC2221Cell parameters (Å)a = 90.61, b = 99.04, c = 101.38Resolution rangeaValues in the parentheses are for the reflections in the highest resolution shell (1.66 − 1.6 Å). (Å)50.0 − 1.6 (1.66 − 1.60)Unique/total reflections59,626/679,600Redundancy11.4CompletenessaValues in the parentheses are for the reflections in the highest resolution shell (1.66 − 1.6 Å). (%)98.8 (89.8)RmergeaValues in the parentheses are for the reflections in the highest resolution shell (1.66 − 1.6 Å).bRmerge=∑h∑i|I(h,i)-〈I(h)〉|/∑h∑II(h,i), where I(h,i) is the intensity of the ith measurement of reflection h and 〈I(h)〉 is the average value over multiple measurements. (%)6.0 (45.0)Refinement and model statisticsR-factor/RfreecR=∑||Fo|-|Fc||/∑|Fo|, where Rfree is calculated for 5% test set of reflections. (%)20.4/21.1Resolution range (Å)50.0 − 1.6Number of protein atoms2,787Number of inhibitor atoms32 (LAF153: 27, t-BuOH: 5)Number of water molecules/Co2+ ions272/2RMSDdRMSD, root-mean-square deviation. bond lengths (Å)0.009RMSD bond angles (°)1.40Average B-value (Å2)Main-chain/side-chain20.5/23.1LAF153/t-BuOH/water/Co2+21.8/34.7/32.7/18.3a Values in the parentheses are for the reflections in the highest resolution shell (1.66 − 1.6 Å).b Rmerge=∑h∑i|I(h,i)-〈I(h)〉|/∑h∑II(h,i), where I(h,i) is the intensity of the ith measurement of reflection h and 〈I(h)〉 is the average value over multiple measurements.c R=∑||Fo|-|Fc||/∑|Fo|, where Rfree is calculated for 5% test set of reflections.d RMSD, root-mean-square deviation. Open table in a new tab A Proteomics Approach Identifies an Alteration in a 14-3-3 Protein after LAF389 Treatment—A pilot study was performed using two-dimensional electrophoresis to analyze differences in protein expression of H1299 cells after treatment with a natural bengamide, bengamide E. In the initial experiments, roughly 1500 protein spots were visualized, and a limited number of differences were observed after bengamide treatment. Protein spots that were reproducibly altered in intensity were identified using MALDI-MS. Determination of tryptic peptide masses permitted identification of the parent protein in most cases (Table I). Examination of the differentially expressed proteins did not immediately suggest a common pathway affected by bengamide treatment, because with the exception of MetAp2 and eIF2α, the proteins are not known to be associated or co-regulated. One feature of the identified proteins was that a subset had treatment-dependent changes in charge, rather than changes in mass or abundance. Such changes are likely to arise from post-translational modifications, including phosphorylation and acetylation. A notable protein included in this set was a member of the 14-3-3 protein family. 14-3-3 proteins are ubiquitous cytosolic adaptor proteins that bind to specific phosphoserine-containing motifs and modulate intracellular signaling, cell cycle control, transcriptional control, and apoptosis (16Tzivion G. Shen Y.H. Zhu J. Oncogene. 2001; 20: 6331-6338Crossref PubMed Scopus (258) Google Scholar). In humans, seven isoforms are encoded by a multigene family. Several isoforms are post-translationally modified by phosphorylation (17Megidish T. Cooper J. Zhang L. Fu H. Hakamori S. J. Biol. Chem. 1998; 273: 21834-21845Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 18Dubois T. Rommel C. Howell S. Steinhussen U. Soneji Y. Morrice N. Moelling K. Aitken A. J. Biol. Chem. 1997; 272: 28882-28888Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) and N-terminal acetylation (19Martin H. Patel Y. Jones D. Howell S. Robinson K. Aitken A. FEBS Lett. 1993; 331: 296-303Crossref PubMed Scopus (71) Google Scholar).Table IProteins with altered mobility in two-dimensional gel electrophoresis after treatment of H 1299 cells with 25 μM bengamide E for 8 hProtein nameChangePP2A inhibitor42 kDa, pI 4.0 in control, pI 4.4 in treatedNucleophosmin B2339 kDa, pI 4.8 in control, pI 5.3 in treatedMethionine aminopeptidase 280 kDa, pI 5.7 in control, pI 5.5 in treatedL protein75 kDa, pI 7.8 in control, pI 7.7 in treated67 kDa laminin receptor36 kDa, pI 4.4 intensity up in controlTriosephosphate isomerase30 kDa, pI 7.2 intensity up in treatedeIF2α40 kDa, pI 4.8 in control, pI 5.0 in treatedEB135 kDa, pI 5.0 intensity up in treated14-3-3 isoforms30 kDa, pI 4.5 in control, pI 4.6 in treated Open table in a new tab Further analysis of the altered 14-3-3 proteins was pursued by using cells treated with the synthetic bengamide LAF389. Initial experiments with two-dimensional gel electrophoresis confirmed that LAF389 treatment of H1299 cells shifted the isoelectric point of a 14-3-3 isoform, similar to the effects seen in pilot experiments using bengamide E (Fig. 2A). To broaden the observation of bengamide effects on changes in 14-3-3 isoforms and characterize which of the closely related 14-3-3 isoforms were altered by bengamide treatment, an analytical blotting method was used that resolved 14-3-3 isoforms by isoelectric focusing of cell lysates, followed by Western blotting using antibodies to 14-3-3 proteins (9Towbin H. Özbey Ö. Zingel O. Electrophoresis. 2001; 22: 1887-1893Crossref PubMed Scopus (40) Google Scholar). In experiments not shown, we studied the kinetics and LAF389 concentration dependence of the appearance of the base-shifted 14-3-3 protein. The band became detectable after about 8 h and accumulated for 48 h. After 24 h of incubation, effects in H1299 cells were observed at 3 nm LAF389, with gradual increases up to 300 nm, the highest concentration tested. The bengamide-responsive 14-3-3 isoform was then identified by testing cell lysates from LAF389-treated and control H1299 cells with the IEF immunoblotting method. The immunoblots were probed in parallel samples with isoform-specific polyclonal antibodies (Fig. 2B). Only the 14-3-3γ specific antibody recognized the inducible and more basic band, consistent with the shift seen in the original two-dimensional gels. The observed pH shift of 0.07 pH units was compatible with the theoretically calculated shift of 0.06 for a loss of one negative charge. For the generality of the response, three cell lines (U2OS, MDA-MB-435, and A549) were analyzed by this method, now using the γ-specific antibody. In all cell lines, LAF389 induced the base-shifted form of 14-3-3γ (data not shown). To corroborate these findings, extracts of LAF389-treated H1299 cells as well as control cells were separated by two-dimensional electrophoresis, where the first dimension was on a zoom gel (pH 4.5-5.5 over a distance of 18 cm). The region of the 14-3-3 proteins was identified from a contact blot of the two-dimensional gel that was probed with a pan-isoform-reactive anti-14-3-3 antibody. A series of proteins in the antibody-reactive region, including the additional isoform, was cut from a silver-stained two-dimensional gel for mass spectrometric analyses. The spots were digested with trypsin and analyzed by MALDI mass spectrometry. Data base searching resulted in the identification of several 14-3-3 family proteins. The LAF389-induced spot gave the best fit to human 14-3-3γ. The LAF389-inducible Form of 14-3-3γ Lacks N-terminal Processing—Upon close inspection of the MALDI mass spectra of tryptic digests of the induced and constitutive forms of 14-3-3γ, a unique peak at m/z 1156.57 could be seen in the spectrum of the constitutive form that was absent in the spectrum of the induced form (Fig. 3A). This mass matched that calculated for [M+H]+ of the acetylated N-terminal peptide VDREQLVQK (amino acids 2-10). In the spectrum of the induced form, no mass was detected that fit this sequence with or without N-terminal acetylation. Instead, in both spectra, peaks at m/z 1245.61 and 1261.59 were observed. Three tryptic peptides of 14-3-3γ had masses matching m/z 1245.61: peptides (111-120) ([M+H]+ = 1245.51 Da), (163-172) ([M+H]+ = 1245.62 Da), and (1-10) ([M+H]+ = 1245.66 Da). Therefore, assignment based solely on mass was not possible. The peak at m/z 1261.59 matched perfectly the mass of the N-terminal tryptic peptide (1-10) (M(oxi)VDREQLVQK, [M+H]+ = 1261.65 Da) if its methionine was considered as oxidized (as revealed by the metastable loss of CH3SOH). Coincidentally, a second peptide which also contained an oxidized methionine (163-172, EHM(oxi)QPTHPIR, [M+H]+ = 1261.61 Da) perfectly matched that molecular mass as well. Interestingly, relative to a reference peak at m/z 1197.63, the intensity of the peak at m/z 1261.59 was lower in the spectrum of the constitutive isoform versus that of the induced isoform. This result suggested that the peak at m/z 1261.59 possibly corresponded to a mixture of two peptides in the induced form, whereas that peak would have been generated by a single peptide (163-172) in the constitutive form. This possibility was confirmed by MALDI-MS analysis of endoproteinase Lys-C digests of the inducible and constitutive isoforms of 14-3-3γ (Fig. 3B). The MALDI spectrum showed that the peak at m/z 1261.57 (matching the unprocessed N-terminal peptide M(oxi)VDREQLVQK) was found only in the spectrum of the induced 14-3-3γ form, whereas the peak at m/z 1156.57 (matching the acetylated N-terminal peptide VDREQLVQK) was found only in the constitutive form (Fig. 3B). The combined data from both MS methods (Lys-C and trypsin digest) resulted in sequence coverage of 94 and 82% for the constitutive and induced forms of 14-3-3γ, respectively. Confirmation of the assignment of the N-terminal sequences was performed by nano-ESMS/MS sequencing. Direct selection of the triply charged parent ion at m/z 421.22 from the Lys-C digest of the induced 14-3-3γ isoform and subsequent fragmentation produced sufficient y and b type fragment ions to identify unambiguously the sequence of the peptide as M(oxi)VDREQLVQK (1-10). A similar analysis of the amino-terminal peptide of the tryptic digest of the constitutive 14-3-3γ confirmed the complete sequence as acetyl-VDREQLVQK (2-10) (data not shown). The lack of acetylation in the inducible isoform readily explained the focusing position at a higher pH relative to the constitutive isoform. LAF389 Inhibits Methionine Aminopeptidases Directly—The retention of the initiator methionine in the inducible 14-3-3γ isoform suggested LAF389 treatment of cells reduced MetAp activity, but the data could not distinguish between a direct effect of LAF389 on MetAp activity versus LAF389 effects on proteins that regulated MetAp activity. To address this question, both human MetAp isoforms were expressed in recombinant form and tested in an enzymatic assay using a methionine-containing peptide as substrate (Fig. 4A). Both MetAp1 and 2 were inhibited by LAF389, thus establishing MetAps as direct targets of LAF389. Fumagillin, a well known covalently reacting inhibitor specific for MetAp2 (20Sin N. Meng L. Wang M.Q.W. Wen J.J. Bornmann W.G. Crews C.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6099-6103Crossref PubMed Scopus (589) Google Scholar, 21Griffith E.C. Su Z. Turk B.E. Chen S. Chang Y.-H. Wu Z. Biemann K. Liu J.O. Chem. Biol. 1997; 4: 461-471Abstract Full Text PDF PubMed Scopus (402) Google Scholar), was tested in parallel in these assays. For MetAp2, the IC50 of LAF389 was 800 nm in this assay format, compared with 30 nm for fumagillin. In contrast to LAF389, however, fumagillin caused at most 20% inhibition of MetAp1 even at 300 nm. LAF389 Has Pronounced Activity on Endothelial Cells— Fumagillin selectively inhibits endothelial cell proliferation, and structure-activity relation studies of fumagillin analogues have shown that its anti-proliferative effect correlates well with MetAp2 enzyme inhibition (21Griffith E.C. Su Z. Turk B.E. Chen S. Chang Y.-H. Wu Z. Biemann K. Liu J.O. Chem. Biol. 1997; 4: 461-471Abstract Full Text PDF PubMed Scopus (402) Google Scholar, 22Griffith E.C. Su Z. Niwayama S. Ramsay C.A. Chang Y.-H. Liu J.O. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15183-15188Crossref PubMed Scopus (233) Google Scholar). Therefore, it was of interest to test whether LAF389 could inhibit endothelial cell proliferation. In monolayer growth assays, LAF389 was a potent inhibitor of HUVEC proliferation, with a 20 nm IC50, roughly 10-fold more potent than on A549 tumor epithelial cells (Fig 4B). The endothelial specificity of LAF389 was less pronounced than that seen with fumagillin, which had ∼1000-fold selectivity for endothelial growth inhibition. LAF389 also differed from fumagillin in that substantial cell death of A549 cells was seen at micromolar concentrations of LAF389, whereas even at 10 μm, fumagillin was only cytostatic for this carcinoma cell line. MetAp processing of 14-3-3γ was inhibited in endothelial cells treated with LAF389, as detected by IEF-Western blotting using a polyclonal antibody to the γ isoform (Fig. 5A, left). The unprocessed form of 14-3-3γ could also be detected with a monoclonal antibody raised against a 15-amino acid peptide from the amino terminus of 14-3-3γ (Fig. 5A, right). This monoclonal antibody permitted detection of the unprocessed form of 14-3-3γ in Western blots of lysates resolved on conventional SDS-PAGE gels. Treatment of endothelial cells with either LAF389 or fumagillin caused the appearance of the unprocessed form of 14-3-3γ, with the maximal response to fumagillin at 1 nm and maximal response to LAF389 at 1 μm (Fig. 5B). Unprocessed 14-3-3γ could also be detected in cells treated with siRNA to lower the level of MetAp2 (Fig. 5C). The level of MetAp2 was reduced ∼75% by this treatment. Both endothelial cells and tumor epithelial cells had detectable levels of the unprocessed form of 14-3-3γ upon treatment with MetAp2 siRNA, suggesting that in both cell types, 14-3-3γ may be processed at least in part by the MetAp2 isoform. Crystal Structure of a MetAp2/bengamide Complex—To understand the mechanism of inhibition of methionine aminopeptidases by bengamides, we co-crystallized human MetAp2 with LAF153. LAF153 is the primary metabolite of LAF389 both in cells and in vivo and is an equally potent inhibitor of human MetAp2 in vitro (data not shown). The structure, refined at 1.6 Å resolution (Table II), reveals that LAF153 binds in a manner that closely mimics that expected for a polypeptide substrate. The MetAp2 active site is a deep invagination in the surface of the enzyme, with a dinuclear metal center that is critical for enzymatic activity (23Lowther W.T. Matthews B.W. Biochim. Biophys. Acta. 2000; 1477: 157-167Crossref PubMed Scopus (253) Google Scholar). The innermost portion of the active-site cleft forms the P1 pocket, which recognizes the N-terminal methionine of a substrate polypeptide. The rather hydrophobic P1′ pocket, which accommodates the penultimate residue, lies in the central portion of the cleft, whereas the P2′ pocket is formed at its solvent-exposed surface. In the LAF153 inhibitor complex, the hydrophobic alkenyl linkage with attached tert-butyl alcohol substituent mimics the methionine side chain and occupies the deeply buried P1 pocket, the hydroxymethyl moiety extends into the P1′ pocket, and the caprolactam ring is coordinated in the solvent-exposed P2′ region (Fig. 6A). The 3, 4, and 5 hydroxyl groups of the inhibitor coordinate the two active-site cobalt ions. As discussed below, the geometry of the interactions of the hydroxyl groups with the di-cobalt center is remarkably similar to that observed for the main chain amide and carbonyl groups of peptidic inhibitors of aminopeptidases (24Lowther W.T. Orville A.M. Madden D.T. Lim S. Rich D.H. Matthews B.W. Biochemistry. 1999; 38: 7678-7688Crossref PubMed Scopus (137) Google Scholar). The P1 pocket is formed largely by residues Phe-219, His-382, and Ala-414. The t-butyl group of LAF153 is in hydrophobic contact with each of these residues. The double bond through which the t-butyl group is attached is likely required for its proper positioning within this pocket. Interestingly, in the unliganded MetAp2 protein, which was crystallized in a buffer containing butanol (12Liu S. Widom J. Kemp C.W. Crews C.M. Clardy J. Science. 1998; 282: 1324-1327Crossref PubMed Scopus (390) Google Scholar), a butanol molecule is bound in the same position as that of the t-butyl group of LAF153. The smaller P1′ pocket is formed by Leu-328, Phe-366, and His-231. The side chain imidazole of His-231 hydrogen bonds with the carbonyl group that is adjacent to the methoxy side chain. We expect that this portion of the inhibitor mimics the penultimate residue in a peptide substrate (Fig. 6E). The size and character of the P1′ pocket is consistent with the requirement for small, uncharged amino acids in this position in protein substrates (23Lowt
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