Pharmacia Corporation, Kalamazoo, Michigan 49001
Submitted 21 September 2003 ; accepted in final form 9 October 2003
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ABSTRACT |
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insulin sensitizers; thiazolidinediones; photoaffinity probes; novel site of action; mitoNEET
Although there have been considerable attempts to exploit this hypothesis (36), and structural information regarding the putative target is available (46), there are no superior drugs on the horizon. Furthermore, there are some inconsistencies in the hypothesis that direct activation of PPAR might explain all of the pharmacology. Thus not all PPAR
activators have antidiabetic actions (30), and some potent antidiabetic analogs are not good PPAR
activators (5, 37, 42). Partial reduction in PPAR
expression results in increased insulin sensitivity (33), and naturally occurring mutations also do not offer a clear picture (e.g., 17, 19, 44). Finally, in humans, pioglitazone (Actos), a weaker activator of PPAR
(31), generally produces a greater reduction in circulating lipids (reduction in triglycerides and total cholesterol/HDL cholesterol) than does the more potent activator rosiglitazone (Avandia) (2, 15, 24, 29).
Given the lack of certainty about the mechanism of action of this important class of drugs (28, 35), we have explored the possibility of an alternate site of binding of the thiazolidinediones. We have approached this problem by using high specific activity tritiated pioglitazone and a structurally related iodinated photoaffinity probe.
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MATERIALS AND METHODS |
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Membrane isolation. Crude rat liver, skeletal muscle, and brain mitochondrial-enriched fractions were prepared as follows. Briefly, Sprague-Dawley rats were anesthetized, and hindleg muscle, liver, and whole brain were removed to a cold buffer, MLB (in mM: 225 sucrose, 6 K2HPO4, 5 MgCl2, 20 KCl, and 2 EGTA, pH 7.4). Tissues were chopped, rinsed, and homogenized with a polytron (setting 7; 3x, 15 s) in 5 volumes of MLB. After removal of the unbroken cells and nuclei (750 g), the mitochondrial-enriched fraction was collected at 15,800 g for 5 min. The loose pellet was discarded, and the dense central pellet was resuspended in MLB and recollected at 11,800 g for 10 min. The final pellets were resuspended in 50 mM Tris (pH 8) at 58 mg/ml total protein and frozen at -80°C.
Bovine brain mitochondria were harvested from steer brains. The rinsed brains were homogenized in fractionation buffer (250 mM sucrose, 50 mM Tris, pH 8.0, containing 1 mg/ml pepstatin A, 5 mg/ml leupeptin, 10 mg/ml bacitracin, and 0.1 mM PMSF). After removal of nuclei at 2,250 rpm in a Sorval SS-34 rotor, the mitochondrial pellet was harvested at 20,000 g (13,000 rpm in a Sorval SS-34 rotor) and further enriched by sucrose density centrifugation. Membrane fractions were collected from the top of the 1.18 and 1.20 density bands, resuspended in 50 mM Tris, and collected by centrifugation. The fractions ("B3/B4") were stored at -80°C until use. Lower-density bands were collected as controls. Succinate cytochrome c reductase was used as the mitochondrial marker (12), and protein was measured by the bicinchoninic acid method (Pierce).
3T3-L1 preadipocytes were grown and differentiated as previously described (41). Preadipocytes or fully differentiated adipocytes (11 days) were lifted off the plate with trypsin, and the cells were washed with Dulbecco's PBS and homogenized with 15 strokes with a motor-driven Teflon homogenizer. For the differentiated adipocytes, both floating and pelleted cells were included. After removal of the low-speed (750 g) pellet, the second pellet was collected at 18,000 g for 10 min. The pellets were resuspended in 50 mM Tris (pH 8), equalized for protein content, and used for cross-linking and Western blots as we will describe.
Binding and cross-linking assays. [3H]pioglitazone binding was conducted using intact mitochondrial fractions and also fractions solubilized in 20 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Intact membrane solutions were thawed and diluted to a concentration of 1 mg/ml in 50 mM Tris, pH 8.0. The reactions were conducted in 1.5-ml polypropylene microfuge tubes. The assay volume was 400 µl, containing 100 µl of membranes, 100 µl of 0.4% gamma globulin, 100 µl of 4% DMSO with or without the indicated concentration of competitor, and 100 µl of [3H]pioglitazone (0.2 µCi). The tubes were mixed and incubated at ambient temperature on a rocker for 60 min. Bound counts were detected after sedimentation of membranes (18,000 g, 5 min). For study of the solubilized binding, membrane stocks were solubilized in 20 mM CHAPS, and bound counts were separated from free ones with dextran-coated charcoal (5% charcoal/0.5% dextran in 50 mM Tris, pH 8.0). All binding assays were conducted in triplicate.
Cross-linking reactions were carried out in a final volume of 200 µl, containing 100 µl of membranes (adjusted for total protein), 50 ml of 4% DMSO with or without competing thiazolidinedione (TZD; e.g., 100 µM PNU-91325, 25 µM final concentration), and 50 µl of carrier-free 125I-PNU-101074 (0.10.2 µCi/tube). Specific cross-linking was defined as labeling that was prevented in the presence of competing active TZD (29). An appropriate amount of 125I-PNU-101074 in acetonitrile was dried under vacuum in the dark immediately before use. The reactions were incubated for 15 min at room temperature and stopped by exposure to UV light in open tubes (180,000 µJ in a Stratalinker). The cross-linked samples were then rinsed with 50 mM Tris (pH 8.0) after centrifugation at 18,000 g for 5 min. The rinsed pellets were resuspended in 100 µl of 50 mM Tris.
Purification and identification of cross-linked protein. Optimal selective solubilization of the specifically cross-linked protein (m17) was obtained by bringing the resuspended pellet to 1% Triton X-114. After rocking at room temperature for 5 min, the bulk of the cross-linked m17 was recovered in the supernatant after centrifugation at 18,000 g for 15 min. The cross-linked m17 also remained in the supernatant after centrifugation at 450,000 g in a Beckman TLA 100 ultracentrifuge for 30 min. This removed many of the contaminating proteins.
Triton X-114 was removed from the sample by precipitation and flotation of the detergent with ammonium sulfate. The addition of equal volumes of 1.5 M ammonium sulfate to the Triton X-114 solution precipitated the proteins, leaving the detergent in solution. The precipitation procedure was repeated 3 times to maximize the yield of precipitated protein. Special care was taken to ensure that the protein precipitate did not float on the Triton-containing supernatant. The precipitated protein was concentrated for direct separation on SDS-PAGE gels (1020% or 18% Tris-glycine) or for HPLC.
HPLC was conducted by using both UV scanning diode array detector and an in-line gamma-C flow cell (Packard). Purification of the m17 protein was achieved with a Phenomenex Synergi MAX-RP C12 column (4.6 x 250 mm, 4 µm). The guard column was a 4 x 2.0-mm RP-1 SecurityGuard Cartridge (Phenomenex). The selections of the column and guard columns were made after considerable examination of standard protein columns, which gave no appreciable yield of the target protein. Samples were eluted with a programmed gradient elution starting with 70% solution A [water-0.05% trifluoracetic acid (TFA), vol/vol] and 30% solution B (Can-0.05% TFA, vol/vol). The gradient was held at 30% B for the first 15 min; B was then increased from 30 to 55% over 30 min and then increased to 80% over 15 min. Flow rates were fixed at 1 ml/min.
The specifically cross-linked proteins were excised from electrophoretic gels and were reduced, alkylated, and digested in situ with modified porcine trypsin (Promega) by use of a DigestPro robot (ABIMED). Briefly, protein gel spots were placed in reaction vials and secured in a Peltier heating/reaction block. Digested peptides were extracted with 60% acetonitrile-5% formic acid. Peptide extracts were placed in a Speed-Vac centrifuge until dry and were reconstituted in 10 µl of 5% formic acid in water.
NanoLC tandem mass spectrometry analysis (nanoLC-MS/MS) was performed on a Micromass Qtof ultima instrument coupled to a Micromass CapLC. Typically, 5 µl from a total sample amount of 5.5 µl were injected and preconcentrated using column switching. An auxiliary pump was used to preconcentrate and desalt samples on a C18 Pepmap precolumn (0.3 x 5 mm) by delivering 0.1% formic acid at 20 µl/min. After desalting, the precolumn was switched in-line with the analytical column (75-mm-ID C18 Pepmap, LC Packings) and eluted at 300 nl/min with a gradient of 0.1% formic acid in water and 0.1% formic acid containing 90% acetonitrile directly into the Qtof. Tandem MS data were acquired and processed by Micromass MassLyxn software. Nanospray MS/MS data were used to identify proteins by comparing the experimental data with predicted data derived from protein and DNA databases. Tandem MS data were searched against the NCBInr protein database with MASCOT (Matrix Science) programs maintained on the SAM Chemistry MS lab NT server.
A procedure was developed to elute the m17 after SDS-PAGE with a series of rehydration and drying steps. The individual lane bands were cut out, and groups of 10 gel slices were placed in microfuge tubes, rehydrated in 0.5 ml of water, and nutated for 1 h at room temperature. The eluted material was retained, and the gel slices were then dried in a Speed-Vac centrifuge; this procedure was repeated two additional times. The pooled, eluted m17 was concentrated and resolved on SDS-PAGE before MS/MS identification or used for generation of CNBr fragments in 70% formic acid (overnight at room temperature). CNBr fragments were released by water elution. No further recovery occurred by electroelution of these gels. The samples were finally concentrated, run on 18% Tris-glycine gels (Invitrogen), and blotted to Immobilon-Psq (Millipore). The blots were stained with 0.1% Coomassie R-250, destained, air dried, and exposed to Biomax MS film at -80°C. Amino-terminal sequencing was performed by automated Edman degradation on an Applied Biosystems model 492 Procise cLC protein sequencer.
Generation of antibodies and Western blotting. The protein identified from cross-linking with PNU-101074 was evaluated for potential antigenic peptides with a set of computer programs developed in-house by F. J. Kezdy and R. A. Poorman. Three peptides were chosen and synthesized on an Applied Biosystems 433A peptide synthesizer by use of a HBTU/NMP protocol with the Fmoc (9-fluorenylmethoxycarbonyl) group as the NH2- or amino-protecting group. The crude peptides were precipitated, purified by reverse-phase HPLC (Vydac C18, 22 x 250 mm, 10 µm), and characterized by open access electrospray mass spectrometry.
Peptides A (CGGKAMVNLQIQKDDPKVV-OH), B (KVVHAFDMGDLGDKAVWC-OH), and C (CGGNEETGDNVGPLIIKKKET) (compare with sequence in Fig. 5) were sent to Covance Research Products (Denver, PA) for conjugation to keyhole limpet hemocyanin and immunization of rabbits. Serum was tested against a spotted concentration-dose curve (0.0110 mg) against all peptides. Positive reactions were obtained from the first bleed onward for peptides A and B. Peptide C did not elicit an immune response. Antisera to A or B did not cross-react to any of the other peptides. Western analysis was conducted by running protein samples on reducing 18% Tris-glycine gels and blotting to polyvinyldifluoride membranes. The blotted membranes were incubated with a 1:30,000 dilution of anti-mitoNEET peptide B. Detection of the immunoreactive bands was determined by incubating with a 1:50,000 dilution of alkaline phosphatase-conjugated monoclonal anti-rabbit IgG (Sigma no. A-2556) followed by development with BCIP/NBT Blue Liquid Substrate (Sigma no. B-3804). Anti-prohibitin (1:400 dilution; Research Diagnostics) was used as a mitochondrial protein marker (23). The developed blots were dried and exposed to Biomax MS film.
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Association of mitochondrial proteins with the synthetic target. The 108-amino acid protein identified as the putative target of TZD cross-linking was synthesized by solid phase to contain an NH2-terminal biotin for attachment of streptavidin agarose (4%) beads (Sigma, S-1638). Peptide with or without an NH2-terminal biotin extension (25 µg/50 µl bead suspension) was incubated in a total volume of 250 µl for 1 h at 4°C, followed by addition of 50 µl of 1 mM biotin and 200 µl of the solubilized mitochondrial preparation and incubation for an additional 2 h. The beads were then washed 6 times with 1 ml of 10 mM Tris, 150 mM NaCl, and 0.4 mM CHAPS. Proteins were eluted with a minimal volume (60 µl) of 0.1 M glycine and 150 mM NaCl, pH 2.3. There was no binding of solubilized mitochondrial proteins to beads alone or beads plus the peptide not containing the NH2-terminal biotin.
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RESULTS |
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Attempts to isolate the soluble [3H]pioglitazone-binding fraction met with little success. The activity migrated as a large complex in the void volume of size-exclusion columns and as a single peak on ion exchange, but the activity was rapidly lost, especially when the samples were not kept at a pH close to 8 (not shown). A photoaffinity probe was made to mark the site of attachment of the TZD for further characterization.
As shown in Fig. 3A, a single specifically cross-linked band (competed by the active TZD, PNU-91325) marked by the arrow (m17), was found in the crude mitochondrial fraction obtained from a variety of tissue sources. Competition for cross-linking of this band by both pioglitazone and ciglitazone was similar to the competition for [3H]pioglitazone binding (Fig. 3B). Furthermore, both [3H]pioglitazone binding (Fig. 3C) and cross-linking of the m17 protein (Fig. 3D) correlated with the mitochondrial marker succinate cytochrome c reductase in subcellular fractions. Similar results were obtained for rat skeletal muscle and liver (not shown).
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The cross-linked band could not be resolved by two-dimensional gel electrophoresis (not shown), a technique that has proven successful for other proteins identified by this technique (9). The specifically cross-linked band was purified from bovine brain and rat liver mitochondria by two approaches. Cross-linked mitochondria were solubilized with 1% Triton X-114, also resulting in a partial enrichment with respect to total protein. Further enrichment and concentration of m17 were accomplished by precipitating the solubilized cross-linked protein with 0.75 M ammonium sulfate (AS). This was the optimal concentration of AS that allowed precipitation of the protein while keeping the Triton in solution. Concentration and removal of the Triton X-114 were essential for optimal separation by HPLC (see below).
The concentrated m17 was separated by HPLC, as described in MATERIALS AND METHODS. Identical results were obtained from either fresh rat liver mitochondrial samples or bovine brain mitochondrial fractions, suggesting that a similar target protein was involved. A representative pattern of the separation by HPLC is shown in Fig. 4A. Identification of the radioactive peak was simplified by the in-line radiometric detector (Fig. 4A, bottom right). The m17 peak eluted at 30 min under these conditions at
55% acetonitrile. Parallel runs with samples from cross-linking incubations that contained the competitor PNU-91325 lacked this peak (not shown). SDS-PAGE together with autoradiography demonstrated that this method provides a successful purification of the specifically PNU-101074-cross-linked protein (Fig. 4).
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The m17 cross-linked protein was also concentrated in high yield by a water elution procedure from unfixed, unstained gels (Fig. 4B). For this approach, 80 individual tubes were cross-linked with or without PNU-91325, solubilized with Triton X-114, concentrated by AS precipitation, and then subjected to SDS-PAGE on 18% Tris-glycine gels that were not fixed or stained. The bands of interest were marked and cut out as described in MATERIALS AND METHODS. Figure 4B shows an autoradiogram of a representative gel before and after the band of interest was cut out for water elution of the cross-linked m17 band. Reexposure of these gels confirmed that the center of the specific band had been excised. This procedure produced the highest yield of m17 cross-linked protein.
Purified m17 bands from unfixed, unstained 18% Trisglycine gels were rinsed and processed for proteomic identification, as described in MATERIALS AND METHODS. Preparations from both rat liver mitochondria and bovine mitochondrial fractions identified the same tryptic peptides shown in bold in Fig. 5A. These sequences are annotated "similar to hematopoietic stem/progenitor cells protein MDS029" ID gi 15488774: (BC013522 [GenBank] ). The mouse protein sequence is shown. The predicted human and mouse proteins are virtually identical.
We attempted to confirm the identification by NH2-terminal sequencing. Sequencing of the intact protein was unsuccessful, suggesting that the NH2 terminus might be blocked. Gel digestion with CNBr generated a <6-kDa cross-linked fragment. The blot showing the intact and CNBr-digested fragment is shown in Fig. 5B. Partial sequence data were obtained from this fragment supporting the MS/MS identification of the labeled protein (Fig. 5B).
We next sought to generate antibodies against this protein. Three peptides from the predicted non-membrane-spanning region were selected and synthesized. Antisera generated against both peptide A and peptide B recognized the m17 on Western blots; however, the greatest reactivity was with the serum generated from rabbits immunized with peptide B. Figure 6 demonstrates a representative Western blot of cross-linking reactions with sucrose density fractions obtained from bovine brain. As predicted from previous results, the cross-linked band was enriched in the higher-density protein bands that are enriched in mitochondrial marker (in agreement with results shown in Fig. 3, C and D). After SDS-PAGE, representative gels for these samples were transferred to membranes for Western blots using preimmune (Fig. 6, top left), anti-peptide B (Fig. 6, top center), or anti-prohibitin, a known mitochondrial protein (23) (Fig. 6, top right). Prohibitin and m17 immunoreactivity were in the same fractions, and the m17 staining was overlaid by the TZD-specific cross-linking (Fig. 6, bottom) representative panels. The antibody also recognized a protein band of the same size as the specifically cross-linked band in rat liver and skeletal muscle mitochondrial fractions; other cell fractions were negative (not shown).
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A further correlation of the m17 protein by Western blot analysis and cross-linking by the TZD probe is shown in Fig. 7. As can be seen in the autoradiograms depicting the TZD-specific cross-linking, there is little cross-linking (Fig. 7, B and D, lanes 14) and no measurable immunostaining in the Western blots (Fig. 7C) in the crude membranes from preadipocytes (lanes 14), whereas both end points are found in membranes from the differentiated cells (lanes 58). Overexposure of the autoradiograms did demonstrate very low levels of cross-linking in the preadipocytes (not shown).
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Although antibodies against the m17 peptides recognized the intact protein, they were not useful for immunoprecipitation purposes (not shown). Because the tritiated pioglitazone-binding activity in solubilized membranes migrated as a large complex (see RESULTS), we assumed that the m17 might associate with other mitochondrial proteins and that this might provide some hint as to its function. The 108-amino acid protein was synthesized by a solid-phase approach with an NH2-terminal biotin and fastened to Streptavidin beads, as described in MATERIALS AND METHODS. Solubilized mitochondrial preparations were then incubated with the beads with or without the attached synthetic protein. Figure 8 shows an example of a silver-stained gel containing the contents released from these beads. In the absence of peptide (-, beads only), no bound proteins were eluted when the pH of the washing solution was reduced. In the presence of the peptide (+), a subset of proteins was eluted from beads incubated with solubilized mitochondria from brain, skeletal muscle, or liver. The protein bands were cut from the gel and identified by nanoLC-MS/MS, as described in MATERIALS AND METHODS. A representative data set obtained from a solubilized rat liver mitochondria preparation is shown in Table 1. The subset of proteins collected by m17 from mitochondria from various sources included complex 3, ATP synthase, and enzymes involved in -oxidation of fatty acids.
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DISCUSSION |
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We found that high specific activity [3H]pioglitazone bound to a saturable site with relatively high affinity in the mitochondria. Active, but not inactive, analogs competed for the binding. We could not show specific [3H]pioglitazone binding in any cytosolic or nuclear fractions. Because isolation of the [3H]pioglitazone-binding protein(s) proved difficult, we designed a photoaffinity ligand that could be iodinated to allow purification of the protein responsible for drug binding. We have previously had success with photoaffinity ligands to locate the binding of drugs with uncharacterized binding sites (9). A single protein was cross-linked in a specific fashion in the same tissue fractions that bound [3H]pioglitazone. This protein (m17) was enriched in the same subcellular fractions as [3H]pioglitazone binding and demonstrated the same specificity with respect to competition by unlabeled analogs (Fig. 3).
Two approaches were taken to purify the protein from two tissue sources, bovine brain and rat liver. One involved selective solubilization with Triton X-114, followed by reversephase HPLC and SDS-PAGE electrophoresis. The second approach involved preparative scale electrophoresis followed by elution of the cross-linked protein from the unfixed gels, CNBr cleavage, and then electrophoresis and sequencing of the labeled fragment. The partially purified fractions submitted to trypsin digestion and MS/MS analysis, as described in MATERIALS AND METHODS, identified two common tryptic peptides from both tissue sources on three different occasions (Fig. 5). The identification was confirmed by NH2-terminal sequencing of the 6-kDa CNBr fragment produced after preparative scale electrophoresis (Fig. 5B).
To provide more evidence for support of the identification, antibodies were generated to allow measurement of the protein by Western blot. In support of the findings, the antibodies recognized a protein of the same size in the same tissue fractions. Importantly, both the protein and cross-linking were undetectable under standard conditions in cultured preadipoctyes, and both were measurable upon differentiation of the cells into adipocytes. The common detection under these conditions supports the conclusion that we have identified the mitochondrial protein that is cross-linked by the thiazolidinedione probe. The increased expression upon differentiation of adipocytes suggests that the protein may be involved in lipid metabolism.
Because we were unable to immunoprecipitate the cross-linked protein with the anti-peptide antibodies, we asked whether the synthetic protein would bind to solubilized mitochondrial proteins. Chromatography of soluble binding studies with tritiated pioglitazone had indicated that the mitochondrial target was associated with a large number of proteins. The identification of these proteins might provide insight as to function. The synthetic target protein bound a set of mitochondrial proteins, including components of complex 3, ATP synthase, and pyruvate dehydrogenase. Of particular interest was the association of key enzymes involved in fatty acid oxidation, especially the trifunctional enzyme complex (11). On the basis of its unique sequence and mitochondrial location, we suggest the name mitoNEET for this previously unidentified target for the thiazolidinediones. MitoNEET may serve as a supporting framework upon which mitochondrial metabolism can be channeled and controlled.
It is well known that insulin resistance is associated with elevated tissue levels of long-chain acyl-CoA esters (10). Furthermore, treatment of animals with pioglitazone can lower the tissue levels of long-chain acyl-CoA esters and even protect against their buildup during lipid infusions (49). The buildup of long-chain acyl-CoA esters could result in insulin resistance by a number of mechanisms, including activation of specific protein kinase C isoforms (50) or generation of ceramide (7). Thiazolidinediones could improve insulin sensitivity secondary to lowering the long-chain CoAs after a direct interaction with this novel mitochondrial target. Such an effect could include more efficient oxidation (11), perhaps even including a feedforward effect by keeping malonyl-CoA levels in check (38, 39).
In conclusion, a novel mitochondrial target protein has been identified for the insulin-sensitizing thiazolidinediones. This protein may play a role in regulating mitochondrial oxidation of fatty acids, and modulation of this target may be involved in the mechanism of action of these drugs. The groundwork is now laid to test this hypothesis. Because PPAR is known to be centrally involved in the production of new fat stores (32) and induction of glycerol kinase (16), promoting the storage of fat, it is possible that elimination of direct modulation of PPAR
while maintaining the mitochondrial interaction may produce an improved therapeutic profile.
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ACKNOWLEDGMENTS |
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Current addresses of J. R. Colca, W. G. McDonald, J. W. Leone, and W. R. Mathews: Pfizer St. Louis, 700 Chesterfield Parkway North, Chesterfield, MO 63198; of D. J. Waldon: Guilford Pharmaceuticals, 6611 Tributary St., Baltimore, MD 21224; of J. M. Lull and C. A. Bannow: Proteos, 4717 Campus Drive Innovations Center, Kalamazoo, MI 49008; and of E. T. Lund, Pfizer, 2800 Plymouth Rd., Ann Arbor, MI 48105.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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