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Monitoring Interactions of Bcl-2 Family Proteins in 96-Well Plate Assays
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[24] M o n i t o r i n g I n t e r a c t i o n s o f B c l - 2 F a m i l y P r o t e i n s i n 96-Well Plate Assays B y J o s l ~ - L u I s D I A Z , TILMAN
OLTERSDORF,a n d
LAWRENCE C. FRITZ
Introduction The bcl-2 gene family encodes a series of proteins involved in the regulation of programmed cell death. Members of the family can be grouped into two distinct sets with antagonistic functions. 1-5 For example, Bcl-2, Bcl-XL, and Bcl-w have antiapoptotic, protective functions, and prevent the activation of downstream death-effector caspase proteases. In contrast, proteins such as Bax, Bak, Bad, and Bid have proapoptotic roles and can antagonize the cell-protective functions of Bcl-2. 6-9 A large number of studies have indicated that Bcl-2 family members can dimerize, forming homodimers and/or heterodimers, and that these interactions are important for their function. 1° Yang and Korsmeyern have proposed a "life-death rheostat" model, in which the response of a cell to an apoptotic signal is determined by the ratio of antiapoptotic to proapoptotic Bcl-2 family proteins. In this model, apoptosis is regulated by the competitive dimerization between various pairs of family members. Bcl-2 family members share stretches of sequence homology known as BH1, BH2, BH3, and BH4 domains. 12'13The antiapoptotic family members 1 y. Tsujimoto, J, Cossman, E. Jaffe, and C. Croce, Science 228, 1440 (1985). 2 D. M. Hockenbery, G. Nunez, C. Milliman, R. D. Screiber, and S. J. Korsmeyer, Nature (London) 348, 334 (1990). 3 D. L. Vaux, I. L. Weissman, and S. K. Kim, Science 258, 1955 (1992). 4 T. Miyashita and J. C. Reed, Blood 81, 151 (1993). 5 L. H. Boise, M. Gonzalez-Garcia, C. E. Postema, L. Ding, T. Lindsten, L. Turka, X. Mao, G. Nunez, and C. B. Thompson, Cell 74, 597 (1993). 6 Z. Oltvai, C. Milliman, and S. J. Korsmeyer, Cell 74, 609 (1993). 7 S. N. Farrow, J. H. M. White, I. Martinou, T. Raven, K.-T. Pun, C. J. Grinham, J,-C. Martinou, and R. Brown, Nature (London) 374, 731 (1995). s T. Chittenden, E. A. Harrington, R. O'Connor, C. Flemington, R. J. Lutz, G. I. Evan, and B. C. Guild, Nature (London) 374, 733 (1995). 9 M. C. Kiefer, M. J. Brauer, V. C. Powers, J. J. Wu, S. R. Umansky, L. D. Tomei, and P. J. Barr, Nature (London) 374, 736 (1995). to X. M. Yin, Z. N. Oltvai, and S. J. Korsmeyer, Nature (London) 369, 321 (1994). 11 E. Yang and S. J. Korsmeyer, Blood 88, 386 (1996). t2 T. Chittenden, C. Flemington, A. B. Houghton, R. G. Ebb, G. J. Gallo, B. Elangovan, G. Chinnadurai, and R. J, Lutz, EMBO J. 14, 5589 (1995). 13H. Zha, C. Aime-Sempe, T. Sato, and J. C. Reed, Z Biol. Chem. 271, 7440 (1996).
METHODS IN ENZYMOLOGY, VOL. 322
Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. 0076-6879/09 $30.00
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generally possess all four homology domains. The three-dimensional structure of Bcl-XL has been solved and demonstrates that the homology domains fold into a compact structure, forming a prominent hydrophobic groove on the surface of the protein) 4 In contrast, the proapoptotic family members contain only a subset of the homology domains; some contain domains BH1-312'13'15 but others possess only the BH3 domain) 6,17 Although the proapoptotic family members vary in structure, mutagenesis experiments suggest that the BH3 domain is both necessary and sufficient for these proteins to heterodimerize with Bcl-2 and Bcl-XL and to induce apoptosis. 12'~3'18 Structural studies have now shown that the BH3 domain of Bak forms an a helix that binds into the hydrophobic groove of BclXL.29Indeed, the formation of both homodimers and heterodimers is dependent on BH3 domain binding, because BH3-derived peptides can fully inhibit the interactions between the full-length proteins. 2° The mechanism by which the antiapoptotic Bcl-2 family members inhibit cell death remains unknown. However, mutagenesis studies suggest that these antiapoptotic proteins have intrinsic cell survival activity, 21 implying that this activity is inhibited when proapoptotic, BH3-containing proteins bind into the hydrophobic groove. Thus, the groove can be viewed as a pharmacological binding site for modulating apoptosis in cells. For example, compounds that bind the groove of Bcl-2 might mimic the proapoptotic proteins and inhibit cell survival function. Alternatively, compounds might bind into the groove in such a manner as to block the binding of proapoptotic proteins, but not inhibit the intrinsic activity of Bcl-2. Such a compound would be predicted to disinhibit Bcl-2, promoting survival. In 14S. W. Muchmore, M. Sattler, H. Liang, R. P. Meadows, J. E. Harlan, H. S. Yoon, D. Nettesheim, B. S. Changs, C. B. Thompson, S. Wong, S. Ng, and S. W. Fesik, Nature (London) 381, 335 (1996). is S. Ottilie, J.-L. Diaz, W. Home, J. Chang, Y. Wang, G. Wilson, S. Chang, S. Weeks, L. C. Fritz, and T. Oltersdorf, J. Biol. Chem. 272, 30866 (1997). 16j. M. Boyd, G. J. Gallo, B. Elangovan, A. B, Houghton, S. Malstrom, B. J. Avery, R. G. Ebb, T. Subramanian, T. Chittenden, R. J. Lutz, and G. Chinnadurai, Oncogene 11, 1921 (1995). 17K. Wang, X.-M. Yin, D. T. Chao, C. L. Milliman, and S. J. Korsmeyer, Genes Dev. IO, 2859 (1996). 18S, Ottilie, J.-L. Diaz, J. Chang, G. Wilson, K. M. Tuffo, S. Weeks, M. McConnell, Y. Wang, T. Oltersdorf, and L. C. Fritz, J. Biol. Chem. 272, 16955 (1997). 19M. Sattler, H. Liang, D. Nettesheim, R. P. Meadows, J. E. Harlan, M. Eberstadt, H. S. Yoon, S. B. Shuker, B. S. Chang, A. J. Minn, C. B. Thompson, and S. W. Fesik, Science 275, 983 (1997). 20T. W. Sedlak, Z. N. Oltvai, E. Yang, K. Wang, L. H. Boise, C. B. Thompson, and S. J. Korsmeyer, Proc. Natl. Acad. Sci. U.S.A. 92, 7834 (1995). 21 E. H.-Y. Cheng, B. Levine, L. H. Boise, C. B. Thompson, and J. M. Hardwick, Nature (London) 379, 554 (1996).
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either case, such compounds would be expected to block the binding of a BH3-containing protein to the groove-containing protein. Therefore, binding assays that measure the interaction of Bcl-2 family proteins may be useful in identifying compounds that promote or inhibit apoptosis. Principle of Assay Interactions between Bcl-2 family proteins have generally been studied by immunoprecipitation analyses and yeast two-hybrid systems. 1°'12,13'1s However, these methods are not optimal for establishing the quantitative high-throughput assays necessary for the screening of large chemical libraries. The method described below is a quantitative binding assay that can be carried out in a 96-well format, using any combination of recombinant Bcl-2 family protein pairs. The assay is a solid-phase binding assay with an enzyme-linked immunosorbent assay (ELISA) readout, where one member of the protein pair is attached to the surface of a polystyrene 96-well plate in a nonspecific manner, The second protein is incubated and allowed to bind to the first protein, and then is specifically detected by a mouse monoclonal antibody. The readout for the assay is the colored product of a substrate converted by alkaline phosphatase enzyme, covalently conjugated to a second antibody. Assay Methods Bacterial Expression Plasmids Human Bcl-2, Bcl-XL, Bax, and Bad proteins are expressed in bacteria. Because soluble binding partners are desired, the C-terminal membranespanning domains of Bcl-2, Bcl-xe, and Bax are deleted. Bcl-2 and Bcl-XL are cloned into the pGEX-4T-1 (Pharmacia, Piscataway, N J) vector, and are expressed as fusion proteins with N-terminal glutathione S-transferase (GST) tags. The construct for expression of GST-Bcl-2, encoding a fragment containing amino acids 1-218 of human Bcl-2, is a generous gift of J. Reed (Burnham Institute, La Jolla, CA). The construct for expression of GSTBcl-xL, encoding a fragment including amino acids 1-211, is obtained from a full-length Bcl-xL cDNA template by polymerase chain reaction (PCR) with Pfu polymerase (Stratagene, La Jolla, CA). The primer sequences are 5' A G T ATC G A A T-I?C A T G TCT CAG A G C A A C CGG 3' and 5' TAC A G T CTC G A G CTA GTI" G A A GCG T r c CTG GCC CT 3' with EcoRI as a 5' cloning site and XhoI as a 3' cloning site. Proteins with N-terminal six-histidine (6H) tags are expressed using the pET-15b (Novagen, Madison, WI) vector. The construct for expression of
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6-histidine-Bax, encoding amino acids 1-170, is obtained by PCR with the following primers: 5' ACG TAC CAT ATG GAC GGG TCC GGG GAG 3' and 5' TAC AGT CTC GAG CTA CCA CGT GGG CGT CCC A A A 3'. The fragment is cloned into the 5' NdeI and 3' XhoI cloning sites of pET-15b. Constructs encoding 6H-Bcl-XL (amino acids 1-211), 6H-Bcl-2 (amino acids 1-218), and 6H-Bad (amino acids 1-168) are obtained in an analogous fashion. Protein Expression
For expression, pGEX-4-based plasmids are transformed into Escherichia coli XL-1 blue (Stratagene). Bacteria are grown in 3-liter batch cultures in shaker flasks to an absorbance of 0.85 at 600 nm at 37 °. Cultures are cooled to 25 or 30° and isopropyl-/3-D-thiopyranoside is added to induce protein expression. Exact conditions vary and are optimized for each of the various proteins. Cultures are grown until they reach an optical density of 3 units at 600 nm, or, for a maximum of 3 hr. Bacteria are harvested by pelleting at 4000g for 10 min. Bacterial pellets are resuspended in 50 ml of the appropriate binding buffer for further purification. Bacteria are lysed by sonication followed by processing through a microfluidizer (Microfluidics International, Newton, MA). Lysates are pelleted at 20,000g for 20 min. For GST fusion proteins only the soluble fraction is processed further. In the case of Bcl-XL and Bax the bulk of the expressed protein is contained in this soluble fraction, whereas for Bcl-2, a significant amount is contained in the insoluble pellet. GST fusion proteins are purified by one-step affinity purification, using glutathione-Sepharose 4B (Pharmacia Biotech, Uppsala, Sweden) on a fast protein liquid chromatography (FPLC) system, essentially following the manufacturer instructions. Proteins with N-terminal 6H tags are expressed in the E. coli host strain BL-21 or BL-21-plysS and processed essentially like GST fusion proteins. Affinity chromatography is performed with NiNTA Superflow (Qiagen, Chatsworth, CA), following the manufacturer recommendations. When the bulk of the expressed protein is sequestered in inclusion bodies, the insoluble fraction of the bacterial extracts is solubilized in the appropriate loading buffer with 7 M urea. Ni-NTA chromatography is performed in the presence of urea. Chromatography is followed by a buffer exchange step using disposable PD-10 gel-filtration columns (Pharmacia Biotech). In some cases the final storage buffer and conditions must be optimized to avoid solubility problems. The purity of proteins obtained by these methods is assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentrations are determined routinely by a bicinchoninic acid method
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(Pierce, Rockford, IL) with a bovine serum albumin (BSA) standard. Expressed proteins are considered sufficiently pure when no contaminating bands can be detected in Coomassie-stained gels loaded with approximately 1 txg of protein per lane.
In Vitro Protein-Protein Binding Assay Procedure 1. Coating of plate: Dilute purified 6H-Bax to 4 txg/ml in phosphatebuffered saline (PBS) and coat onto 96-well microtiter plates (50 txl/well) (Immunosorb; Nunc, Roskilde, Denmark) for 18 hr at 4 °. 2. Blocking of plate: Wash the plates two times with PBS containing 0.05% (v/v) Tween 20 (PBS-T) and then block with 150/zl of 2% (w/v) BSA in PBS for 2 hr at room temperature. 3. Second protein: Wash the plates two times with PBS-T and incubate with a range of concentrations (20 to 0.0001/xM) of GST-Bcl-2 in PBS-T containing 0.5% (w/v) BSA (50 tzl/well). Allow the interaction to proceed for 2 hr at room temperature before washing the plates five times with PBS-T. 4. Primary antibody: The wells are then incubated for i hr at room temperature with 50/zl of the primary antibody, a mouse anti-GST monoclonal antibody used at I ng/ml in PBS-T plus BSA, before being washed five times with PBS-T. In our case, we use antibody 7E5A6 (a kind gift of J. Reed), but an anti-GST monoclonal antibody is commercially available from Santa Cruz Biotechnology (Santa Cruz, CA). 5. Secondary conjugated antibody: Add alkaline phosphatase-conjugated goat anti-mouse antibody (50 t~l/well; Jackson ImmunoResearch Laboratories, West Grove, PA) at a concentration of 1 ~g/ml and incubate for 1 hr at room temperature, and then wash the plates five times. 6. Substrate: Add 50 tzl of enzyme substrate, p-nitrophenyl phosphate (Kirkegaard & Perry, Gaithersburg, MD) at 4 mg/ml in 10 mM diethanolamine (pH 9.5) containing 0.5 mM MgC12, allow the reaction to progress for 15 min at room temperature, and then stop the reaction by the addition of 0.4 M N a O H (50 ~l/well). Read the optical density of the wells at 405 nm in a spectrophotometer (Molecular Devices, Palo Alto, CA). Use of Assay to Map Binding Site The solid-phase protein-protein binding assay may be used to determine protein sequences important for dimer formation. This may be done by running competition assays to test the ability of whole proteins and peptides to inhibit the dimerization of the protein pairs. For competition assays, the liquid-phase GST-tagged protein is used at a constant concentration. In
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Bcl-2 FAMILYPROTEINS
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the case of GST-Bcl-2, we use a concentration of 80 nM throughout. This concentration was determined in preliminary experiments to be on the rate-limiting part of the binding curve. The coating and blocking steps are carried out as described above, after which the appropriate peptides (50 /zl/well) are added to the wells in increasing concentrations [0.156-80 tzM in PBS-T plus 0.5% (w/v) BSA]. The plates are incubated for 1 hr at room temperature and then the second protein is added to the wells without removing the test compounds. The subsequent steps are exactly as described in the procedure above from step 3 onward. The controls usually run on each plate include a vehicle control and a negative control peptide containing a single amino acid change.
Method for Screening Compounds An adaptation of the procedure described above may be used to screen for compounds that inhibit the dimerization of the protein pairs. For screening, the liquid-phase GST-tagged protein is also used at a constant concentration throughout. The coating and blocking steps are carried out as described above, after which the compounds are added to the wells in duplicate at a concentration of 20/xM (50/zl/well) in PBS-T plus 0.5% (w/v) BSA. The plates are incubated for 1 hr at room temperature and then the second protein is added to the wells still containing the test compounds. The subsequent steps are exactly as described in the procedure above. The controls usually run on each plate include a vehicle control and a known inhibitor at a concentration titrated to give a constant 50% inhibition.
Site of Action of lnhibitors The binding assay can be modified to identify to which member of a dimer pair an inhibitory molecule is binding. After the usual coating and blocking steps using 6H-Bax, increasing concentrations of inhibitory compound (0.01 to 100/zM) are added to the wells and allowed to incubate for 2 hr at room temperature. The plates are then washed five times with PBS-T before addition of the second protein, Bcl-xL, in liquid phase. An alternative assay is then set up, in which the plates are coated with Bcl-xL and incubated with increasing concentrations of inhibitory compound. The compounds are washed off and then GST-Bax is added as the second protein in liquid phase. In this manner, only the protein on the solid phase is exposed to the inhibitory compound. Comparing the median inhibitory concentration (IC50) values for the compound incubated singly with either the Bax or the Bcl-xL protein gives an indication as to which of the dimeriz-
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INTERACTION OF B c l - 2 FAMILY PROTEINS
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ing proteins is the target for the compound. In Fig. 1, for example, Bcl-XL heterodimer formation is blocked by a peptide derived from the BH3 domain of Bax when preincubated with the Bcl-XL (ICs0 of 3.5/~M), but not when the peptide is preincubated with Bax (ICs0 > 100 ~M). Reversible and Irreversible Inhibitors
A modification of the binding assay can also be used to help decipher the mode of action of the inhibitor molecule. A constant concentration of the GST-tagged protein in liquid phase is preincubated with increasing concentrations of inhibitory compound (0.01 to 100/.~M) before addition to the 96-well plates coated with the appropriate proteins. Multiple dilution series of the compound are set up, and the preincubation time of the compound with the liquid-phase protein is varied from 0 to 24 hr. The results are plotted, and when the resulting ICs0 values are compared, we see that there is a category of compounds whose ICs0 values do not vary with time once they have reached equilibrium; these are reversible inhibitors. An
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so
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oo
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FIG. 1. A peptide derived from the BH3 domain of Bax blocks Bcl-xL heterodimer formation by binding to the Bcl-XL and not the Bax protein. Wells were coated with 6H-Bax (solid squares) or 6H-Bcl-XL (open squares) and pretreated with increasing concentrations of the peptide Bax 52-72. The unbound peptide was washed off and the wells were incubated with a constant concentration of GST-Bcl-XL (solid squares) or GST-Bax (open squares). Bound protein was quantitated in all cases by reaction with anti-GST antibody and alkaline phosphatase-coupled secondary antibody.
262
Bcl-2 FAMILYPROTEINS
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example of this is illustrated in Fig. 2, where a peptide derived from the BH3 domain of Bax (amino acids 52-72) inhibits Bcl-XL heterodimer formation with similar IC50 values irrespective of time of preincubation. In contrast, IC50 values for irreversible inhibitors vary as a function of time of incubation. Comments and Considerations In the procedures described above, we have constantly referred to the Bax/Bcl-2 or Bax/Bcl-XL dimerization as concrete examples, but this assay format may be used to investigate many of the different binding pairs in the Bcl-2 family of proteins. Some preliminary experimentation is needed to adapt this method to other binding pairs. In each case it is important to optimize the concentration of the coating protein, because the amount of protein adhering to the polystyrene surface will depend, among other things, on the size of the protein. For the screening and competition assays, it is also important to create preliminary binding curves for each pair of dimerizing proteins to be used,
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FIG. 2. A peptide derived from the BH3 domain of Bax is a reversible inhibitor of Bcl-XL heterodimer formation. Wells were coated with 6H-Bax and incubated with a constant concentration of GST-Bcl-XL that had been pretreated with increasing concentrations of competing peptide Bax 52-72 for 0 In- (open circles), 1 hr (closed circles), 3 hr (open squares), or 24 hr (closed squares). Bound protein was quantitated as in Fig. 1.
[241
INTERACTIONOF Bcl-2 FAMILYPROTEINS
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and to choose a concentration of the protein in liquid phase that lies on the steep logarithmic part of the binding curve, z2 The binding assays can be carried out with purified proteins missing the transmembrane domain. Furthermore, in our hands the presence of tags, whether located on the N terminus or C terminus, does not generally interfere with dimerization. Thus, the tags do not need to be cleaved off. On the contrary, careful selection of the tags can be used to aid the assay. The formation of Bcl-2 homodimers, for example, can be detected by using 6H and GST tags on the solid-phase and liquid-phase partners, respectively. By expressing all potential liquid-phase partners with a uniform tag, such as GST or Glu-Glu-Phe (EEF), a single anti-GST or anti-EEF antibody can be used to monitor a wide range of dimerization pairs, without having to generate a specific antibody for each protein. An antibody directed against a terminal tag is also less likely to interfere with the dimerization of the proteins, or be affected by the action of inhibitory compounds, than an antibody directed against an epitope on the main sequence of the protein itself. The assays can be run in various configurations with proteins tagged in various ways. However, in general, the assays work best when (1) the proteins to be coated onto the polystyrene plates are tagged with 6H, (2) the protein acting as a BH3 donor is used as the solid-phase partner, and (3) the partner with the hydrophobic binding cleft is used as the protein in liquid phase. For each binding pair studied, preliminary experiments should be carried out to ensure the specificity of the binding observed. In our case, for each specific interaction, saturable binding was detected, whereas only low levels of background binding were seen when the control protein BSA was used as the solid-phase binding partner (Fig. 3). To confirm that these in vitro interactions reflect the interactions observed in cells, we assessed the ability of the G145A and G138A point mutants of Bcl-2 and Bcl-XL, respectively, to bind to Bax; it has previously been shown by immunoprecipitation that these mutants fail to heterodimerize with Bax in cells. Recapitulating the cellular result, the mutants demonstrated a much-reduced binding to Bax in the plate-binding assay (Fig. 3). 22
Advantages This is an easy, reliable assay that can be used to investigate the binding properties of Bcl-2 family members with regard to homodimer and heterodimer formation. The assay can be performed with recombinant Bcl-2, Bcl-XL, and Bax proteins and is highly reproducible (Table I). The format 22 J.-L. Diaz, T. Oltersdorf, W. Horne, M. McConnell, G. Wilson, S. Weeks, T. Garcia, and L. C. Fritz, J. Biol. Chem. 272, 11350 (1997).
264
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FIG. 3. Formation of Bcl-2 family heterodimers in solid-phase binding assays. Wells were coated with 6H-Bax (solid lines) or the control protein BSA (dashed lines) and incubated with increasing concentrations of GST-Bcl-2 (filled circles), GST-Bcl-XL(filled squares), GSTBcl-2 (G145A) (open circles), or GST-Bcl-XL (G138A) (open squares). Bound protein was quantitated in all cases by reaction with anti-GST antibody and alkaline phosphatase-coupled secondary antibody. [Taken from Diaz et al. 22] o f t h e a s s a y m a k e s it a m e n a b l e to h i g h - t h r o u g h p u t screening, a n d t h e f o r m a t has c e r t a i n a d v a n t a g e s c o m p a r e d w i t h o t h e r a s s a y f o r m a t s d e s i g n e d to m e a s u r e p r o t e i n - p r o t e i n i n t e r a c t i o n s . F o r e x a m p l e , y e a s t t w o - h y b r i d assays c a n b e u s e d to m o n i t o r such i n t e r a c t i o n s , b u t with t h e a d d e d v a r i a b l e o f c o m p o u n d p e n e t r a t i o n t h r o u g h t h e y e a s t cell wall. TABLE I EC5o
VALUES
FOR
HETERODIMER
AND
HOMODIMER
FORMATION
a
Solid phase
Liquid phase
ECs0 (nM)
n
6H-Bax 6H-Bax 6H-Bcl-2 6H-Bcl-xL
GST-Bcl-2 GST-Bel-XL GST-Bcl-2 GST-Bcl-XL
26.1 _+ 10.4 14.7 + 5.3 66.2 -+ 36.0 109.7 _+ 26.0
19 17 9 1l
a 6H-Bax, 6H-Bcl-2, and 6H-Bcl-XL were coated on microtiter planes and binding curves were established with increasing concentrations of GST-Bcl-2 or GST-Bcl-XL. ECs0 values listed are the mean concentration of liquid-phase protein that yielded half-maximal binding _+standard deviation for n independent measurements. (Taken from Diaz et ai.22)
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INTERACTIONOF Bcl-2 FAMILYPROTEINS
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The assay can also be used in a quantitative manner. Indeed, we used these solid-phase binding assays to generate quantitative binding data that provided the first estimate for the strength of these protein-protein interactions. 22 The concentration of liquid-phase protein necessary for halfmaximal binding for each reaction is presented in Table I. Assumptions and Limitations Although the protein-protein binding assay described above has been used to compare the affinity of the interactions between different Bcl-2 family proteins, the binding data generated are not true Kd values. There are two main reasons why we cannot generate Kd values in these assays: first, protein binding in this method does not occur in a homogeneous liquid phase; second, this procedure involves a separation step, and therefore the assay does not measure equilibrium binding. The binding assay involves extensive washing, and therefore one would expect that binding would be detected by this procedure only for interactions where dissociation rates were comparatively low. However, the assay yields little information on the kinetics of dissociation of the dimers. There may also be an avidity effect due to the local concentration of protein bound to the solid phase. Experiments have been carried out in collaboration with Igen (Gaithersburg, MD), using their O R I G E N assay system. This is basically a method to study protein-protein interactions by electrochemiluminescence (ECL). Protein binding by this method occurs in a homogeneous liquid phase, using equilibrium binding procedures. Using ECL, we determined the Kd for the Bax/Bcl-2 interaction to be 8.4 nM (data not shown), which is reasonably close to the apparent Kd of the interaction in our plate assay (Table I). Although dimerization data generated by these in vitro assays appear consistent with observations made by other methods such as immunoprecipitation, there are two major points of variance. It has been reported that Bax can form homodimers, yet we have been unable to observe Bax homodimer formation in solid-phase binding a s s a y s . 6'13'2° This could be highlighting a problem in our assays: for example, it is possible that Bax adopts a distinct and nonphysiological conformation when binding to the solid phase. However, published studies indicate that human Bax also fails to homodimerize in a yeast two-hybrid assay. 23 Furthermore, one study reported that Bax homodimerization, as monitored by immunoprecipitation, was found to be
23 H. Zhang, B. Saeed, and S.-C. Ng, Biochem. Biophys. Res. Commun. 208, 950 (1995).
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Bcl-2 FAMILYPROTEINS
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dependent on the presence of detergent [1% (v/v) Triton X-100, as opposed to 0.05% (v/v) Tween 20 in our assays], but was not observed under more physiological conditions. 2a In addition, Bax has been shown to exist in the cytoplasm, but in that compartment it fails to form homodimers, z4 Interestingly, Bcl-XL readily formed homodimers in our assay, whereas attempts by others to observe this interaction in cells or with isolated protein have failed, a4'25 While we do not understand the basis for these differences, it is possible that a specific conformation of Bcl-XL is favored when bound to plastic, and that this conformation facilitates homodimerization. For example, this conformation may alter the positioning of the az helix (encompassing the BH3 domain), facilitating its interaction with a second molecule of Bcl-XL. Acknowledgments We thank Dr. John Reed (Burnham Institute) for the GST-Bcl-2 expression construct and anti-GST antibody, Suzanne Weeks and Gary Wilson (Idun Pharmaceuticals) for the expression and purification of the Bcl-2 family proteins, and Steven Chang for refining and implementing the methods outlined above.
24 Y.-T. Hsu and R. J. Youle, J. BioL Chem. 272, 13829 (1997). 25 A. J. Minn, L. H. Boise, and C. B. Thompson, J. BioL Chem. 271, 6306 (1996).
[25] A n a l y s i s o f D i m e r i z a t i o n o f B c l - 2 F a m i l y P r o t e i n s b y Surface Plasmon Resonance B y Z H I H U A X I E a n d JOHN C. R E E D
Introduction and Background Bcl-2 family proteins are important regulators of programmed cell death and apoptosis. 1,z These proteins either inhibit or induce cell death, with the ratios of antiapoptotic relative to proapoptotic members of the Bcl-2 family representing a critical determinant of the ultimate sensitivity or resistance of mammalian cells to various apoptotic stimuli. Many Bcl-2 family proteins can physically interact with themselves and each other, 1 j. Reed, Oncogene 17, 3225 (1998). g A. Gross, J. McDonnell, and S. Korsmeyer, Genes Dev. 13, 1899 (1999).
METHODS IN ENZYMOLOGY,VOL. 322
Copyright © 2000by AcademicPress All rightsof reproductionin any formreserved. 0076-6879/00$30.00
INTERACTIONOF Bcl-2 FAMILYPROTEINS
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[24] M o n i t o r i n g I n t e r a c t i o n s o f B c l - 2 F a m i l y P r o t e i n s i n 96-Well Plate Assays B y J o s l ~ - L u I s D I A Z , TILMAN
OLTERSDORF,a n d
LAWRENCE C. FRITZ
Introduction The bcl-2 gene family encodes a series of proteins involved in the regulation of programmed cell death. Members of the family can be grouped into two distinct sets with antagonistic functions. 1-5 For example, Bcl-2, Bcl-XL, and Bcl-w have antiapoptotic, protective functions, and prevent the activation of downstream death-effector caspase proteases. In contrast, proteins such as Bax, Bak, Bad, and Bid have proapoptotic roles and can antagonize the cell-protective functions of Bcl-2. 6-9 A large number of studies have indicated that Bcl-2 family members can dimerize, forming homodimers and/or heterodimers, and that these interactions are important for their function. 1° Yang and Korsmeyern have proposed a "life-death rheostat" model, in which the response of a cell to an apoptotic signal is determined by the ratio of antiapoptotic to proapoptotic Bcl-2 family proteins. In this model, apoptosis is regulated by the competitive dimerization between various pairs of family members. Bcl-2 family members share stretches of sequence homology known as BH1, BH2, BH3, and BH4 domains. 12'13The antiapoptotic family members 1 y. Tsujimoto, J, Cossman, E. Jaffe, and C. Croce, Science 228, 1440 (1985). 2 D. M. Hockenbery, G. Nunez, C. Milliman, R. D. Screiber, and S. J. Korsmeyer, Nature (London) 348, 334 (1990). 3 D. L. Vaux, I. L. Weissman, and S. K. Kim, Science 258, 1955 (1992). 4 T. Miyashita and J. C. Reed, Blood 81, 151 (1993). 5 L. H. Boise, M. Gonzalez-Garcia, C. E. Postema, L. Ding, T. Lindsten, L. Turka, X. Mao, G. Nunez, and C. B. Thompson, Cell 74, 597 (1993). 6 Z. Oltvai, C. Milliman, and S. J. Korsmeyer, Cell 74, 609 (1993). 7 S. N. Farrow, J. H. M. White, I. Martinou, T. Raven, K.-T. Pun, C. J. Grinham, J,-C. Martinou, and R. Brown, Nature (London) 374, 731 (1995). s T. Chittenden, E. A. Harrington, R. O'Connor, C. Flemington, R. J. Lutz, G. I. Evan, and B. C. Guild, Nature (London) 374, 733 (1995). 9 M. C. Kiefer, M. J. Brauer, V. C. Powers, J. J. Wu, S. R. Umansky, L. D. Tomei, and P. J. Barr, Nature (London) 374, 736 (1995). to X. M. Yin, Z. N. Oltvai, and S. J. Korsmeyer, Nature (London) 369, 321 (1994). 11 E. Yang and S. J. Korsmeyer, Blood 88, 386 (1996). t2 T. Chittenden, C. Flemington, A. B. Houghton, R. G. Ebb, G. J. Gallo, B. Elangovan, G. Chinnadurai, and R. J, Lutz, EMBO J. 14, 5589 (1995). 13H. Zha, C. Aime-Sempe, T. Sato, and J. C. Reed, Z Biol. Chem. 271, 7440 (1996).
METHODS IN ENZYMOLOGY, VOL. 322
Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. 0076-6879/09 $30.00
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generally possess all four homology domains. The three-dimensional structure of Bcl-XL has been solved and demonstrates that the homology domains fold into a compact structure, forming a prominent hydrophobic groove on the surface of the protein) 4 In contrast, the proapoptotic family members contain only a subset of the homology domains; some contain domains BH1-312'13'15 but others possess only the BH3 domain) 6,17 Although the proapoptotic family members vary in structure, mutagenesis experiments suggest that the BH3 domain is both necessary and sufficient for these proteins to heterodimerize with Bcl-2 and Bcl-XL and to induce apoptosis. 12'~3'18 Structural studies have now shown that the BH3 domain of Bak forms an a helix that binds into the hydrophobic groove of BclXL.29Indeed, the formation of both homodimers and heterodimers is dependent on BH3 domain binding, because BH3-derived peptides can fully inhibit the interactions between the full-length proteins. 2° The mechanism by which the antiapoptotic Bcl-2 family members inhibit cell death remains unknown. However, mutagenesis studies suggest that these antiapoptotic proteins have intrinsic cell survival activity, 21 implying that this activity is inhibited when proapoptotic, BH3-containing proteins bind into the hydrophobic groove. Thus, the groove can be viewed as a pharmacological binding site for modulating apoptosis in cells. For example, compounds that bind the groove of Bcl-2 might mimic the proapoptotic proteins and inhibit cell survival function. Alternatively, compounds might bind into the groove in such a manner as to block the binding of proapoptotic proteins, but not inhibit the intrinsic activity of Bcl-2. Such a compound would be predicted to disinhibit Bcl-2, promoting survival. In 14S. W. Muchmore, M. Sattler, H. Liang, R. P. Meadows, J. E. Harlan, H. S. Yoon, D. Nettesheim, B. S. Changs, C. B. Thompson, S. Wong, S. Ng, and S. W. Fesik, Nature (London) 381, 335 (1996). is S. Ottilie, J.-L. Diaz, W. Home, J. Chang, Y. Wang, G. Wilson, S. Chang, S. Weeks, L. C. Fritz, and T. Oltersdorf, J. Biol. Chem. 272, 30866 (1997). 16j. M. Boyd, G. J. Gallo, B. Elangovan, A. B, Houghton, S. Malstrom, B. J. Avery, R. G. Ebb, T. Subramanian, T. Chittenden, R. J. Lutz, and G. Chinnadurai, Oncogene 11, 1921 (1995). 17K. Wang, X.-M. Yin, D. T. Chao, C. L. Milliman, and S. J. Korsmeyer, Genes Dev. IO, 2859 (1996). 18S, Ottilie, J.-L. Diaz, J. Chang, G. Wilson, K. M. Tuffo, S. Weeks, M. McConnell, Y. Wang, T. Oltersdorf, and L. C. Fritz, J. Biol. Chem. 272, 16955 (1997). 19M. Sattler, H. Liang, D. Nettesheim, R. P. Meadows, J. E. Harlan, M. Eberstadt, H. S. Yoon, S. B. Shuker, B. S. Chang, A. J. Minn, C. B. Thompson, and S. W. Fesik, Science 275, 983 (1997). 20T. W. Sedlak, Z. N. Oltvai, E. Yang, K. Wang, L. H. Boise, C. B. Thompson, and S. J. Korsmeyer, Proc. Natl. Acad. Sci. U.S.A. 92, 7834 (1995). 21 E. H.-Y. Cheng, B. Levine, L. H. Boise, C. B. Thompson, and J. M. Hardwick, Nature (London) 379, 554 (1996).
[24]
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either case, such compounds would be expected to block the binding of a BH3-containing protein to the groove-containing protein. Therefore, binding assays that measure the interaction of Bcl-2 family proteins may be useful in identifying compounds that promote or inhibit apoptosis. Principle of Assay Interactions between Bcl-2 family proteins have generally been studied by immunoprecipitation analyses and yeast two-hybrid systems. 1°'12,13'1s However, these methods are not optimal for establishing the quantitative high-throughput assays necessary for the screening of large chemical libraries. The method described below is a quantitative binding assay that can be carried out in a 96-well format, using any combination of recombinant Bcl-2 family protein pairs. The assay is a solid-phase binding assay with an enzyme-linked immunosorbent assay (ELISA) readout, where one member of the protein pair is attached to the surface of a polystyrene 96-well plate in a nonspecific manner, The second protein is incubated and allowed to bind to the first protein, and then is specifically detected by a mouse monoclonal antibody. The readout for the assay is the colored product of a substrate converted by alkaline phosphatase enzyme, covalently conjugated to a second antibody. Assay Methods Bacterial Expression Plasmids Human Bcl-2, Bcl-XL, Bax, and Bad proteins are expressed in bacteria. Because soluble binding partners are desired, the C-terminal membranespanning domains of Bcl-2, Bcl-xe, and Bax are deleted. Bcl-2 and Bcl-XL are cloned into the pGEX-4T-1 (Pharmacia, Piscataway, N J) vector, and are expressed as fusion proteins with N-terminal glutathione S-transferase (GST) tags. The construct for expression of GST-Bcl-2, encoding a fragment containing amino acids 1-218 of human Bcl-2, is a generous gift of J. Reed (Burnham Institute, La Jolla, CA). The construct for expression of GSTBcl-xL, encoding a fragment including amino acids 1-211, is obtained from a full-length Bcl-xL cDNA template by polymerase chain reaction (PCR) with Pfu polymerase (Stratagene, La Jolla, CA). The primer sequences are 5' A G T ATC G A A T-I?C A T G TCT CAG A G C A A C CGG 3' and 5' TAC A G T CTC G A G CTA GTI" G A A GCG T r c CTG GCC CT 3' with EcoRI as a 5' cloning site and XhoI as a 3' cloning site. Proteins with N-terminal six-histidine (6H) tags are expressed using the pET-15b (Novagen, Madison, WI) vector. The construct for expression of
258
Bcl-2 FAMILYPROTEINS
[24]
6-histidine-Bax, encoding amino acids 1-170, is obtained by PCR with the following primers: 5' ACG TAC CAT ATG GAC GGG TCC GGG GAG 3' and 5' TAC AGT CTC GAG CTA CCA CGT GGG CGT CCC A A A 3'. The fragment is cloned into the 5' NdeI and 3' XhoI cloning sites of pET-15b. Constructs encoding 6H-Bcl-XL (amino acids 1-211), 6H-Bcl-2 (amino acids 1-218), and 6H-Bad (amino acids 1-168) are obtained in an analogous fashion. Protein Expression
For expression, pGEX-4-based plasmids are transformed into Escherichia coli XL-1 blue (Stratagene). Bacteria are grown in 3-liter batch cultures in shaker flasks to an absorbance of 0.85 at 600 nm at 37 °. Cultures are cooled to 25 or 30° and isopropyl-/3-D-thiopyranoside is added to induce protein expression. Exact conditions vary and are optimized for each of the various proteins. Cultures are grown until they reach an optical density of 3 units at 600 nm, or, for a maximum of 3 hr. Bacteria are harvested by pelleting at 4000g for 10 min. Bacterial pellets are resuspended in 50 ml of the appropriate binding buffer for further purification. Bacteria are lysed by sonication followed by processing through a microfluidizer (Microfluidics International, Newton, MA). Lysates are pelleted at 20,000g for 20 min. For GST fusion proteins only the soluble fraction is processed further. In the case of Bcl-XL and Bax the bulk of the expressed protein is contained in this soluble fraction, whereas for Bcl-2, a significant amount is contained in the insoluble pellet. GST fusion proteins are purified by one-step affinity purification, using glutathione-Sepharose 4B (Pharmacia Biotech, Uppsala, Sweden) on a fast protein liquid chromatography (FPLC) system, essentially following the manufacturer instructions. Proteins with N-terminal 6H tags are expressed in the E. coli host strain BL-21 or BL-21-plysS and processed essentially like GST fusion proteins. Affinity chromatography is performed with NiNTA Superflow (Qiagen, Chatsworth, CA), following the manufacturer recommendations. When the bulk of the expressed protein is sequestered in inclusion bodies, the insoluble fraction of the bacterial extracts is solubilized in the appropriate loading buffer with 7 M urea. Ni-NTA chromatography is performed in the presence of urea. Chromatography is followed by a buffer exchange step using disposable PD-10 gel-filtration columns (Pharmacia Biotech). In some cases the final storage buffer and conditions must be optimized to avoid solubility problems. The purity of proteins obtained by these methods is assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentrations are determined routinely by a bicinchoninic acid method
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(Pierce, Rockford, IL) with a bovine serum albumin (BSA) standard. Expressed proteins are considered sufficiently pure when no contaminating bands can be detected in Coomassie-stained gels loaded with approximately 1 txg of protein per lane.
In Vitro Protein-Protein Binding Assay Procedure 1. Coating of plate: Dilute purified 6H-Bax to 4 txg/ml in phosphatebuffered saline (PBS) and coat onto 96-well microtiter plates (50 txl/well) (Immunosorb; Nunc, Roskilde, Denmark) for 18 hr at 4 °. 2. Blocking of plate: Wash the plates two times with PBS containing 0.05% (v/v) Tween 20 (PBS-T) and then block with 150/zl of 2% (w/v) BSA in PBS for 2 hr at room temperature. 3. Second protein: Wash the plates two times with PBS-T and incubate with a range of concentrations (20 to 0.0001/xM) of GST-Bcl-2 in PBS-T containing 0.5% (w/v) BSA (50 tzl/well). Allow the interaction to proceed for 2 hr at room temperature before washing the plates five times with PBS-T. 4. Primary antibody: The wells are then incubated for i hr at room temperature with 50/zl of the primary antibody, a mouse anti-GST monoclonal antibody used at I ng/ml in PBS-T plus BSA, before being washed five times with PBS-T. In our case, we use antibody 7E5A6 (a kind gift of J. Reed), but an anti-GST monoclonal antibody is commercially available from Santa Cruz Biotechnology (Santa Cruz, CA). 5. Secondary conjugated antibody: Add alkaline phosphatase-conjugated goat anti-mouse antibody (50 t~l/well; Jackson ImmunoResearch Laboratories, West Grove, PA) at a concentration of 1 ~g/ml and incubate for 1 hr at room temperature, and then wash the plates five times. 6. Substrate: Add 50 tzl of enzyme substrate, p-nitrophenyl phosphate (Kirkegaard & Perry, Gaithersburg, MD) at 4 mg/ml in 10 mM diethanolamine (pH 9.5) containing 0.5 mM MgC12, allow the reaction to progress for 15 min at room temperature, and then stop the reaction by the addition of 0.4 M N a O H (50 ~l/well). Read the optical density of the wells at 405 nm in a spectrophotometer (Molecular Devices, Palo Alto, CA). Use of Assay to Map Binding Site The solid-phase protein-protein binding assay may be used to determine protein sequences important for dimer formation. This may be done by running competition assays to test the ability of whole proteins and peptides to inhibit the dimerization of the protein pairs. For competition assays, the liquid-phase GST-tagged protein is used at a constant concentration. In
260
Bcl-2 FAMILYPROTEINS
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the case of GST-Bcl-2, we use a concentration of 80 nM throughout. This concentration was determined in preliminary experiments to be on the rate-limiting part of the binding curve. The coating and blocking steps are carried out as described above, after which the appropriate peptides (50 /zl/well) are added to the wells in increasing concentrations [0.156-80 tzM in PBS-T plus 0.5% (w/v) BSA]. The plates are incubated for 1 hr at room temperature and then the second protein is added to the wells without removing the test compounds. The subsequent steps are exactly as described in the procedure above from step 3 onward. The controls usually run on each plate include a vehicle control and a negative control peptide containing a single amino acid change.
Method for Screening Compounds An adaptation of the procedure described above may be used to screen for compounds that inhibit the dimerization of the protein pairs. For screening, the liquid-phase GST-tagged protein is also used at a constant concentration throughout. The coating and blocking steps are carried out as described above, after which the compounds are added to the wells in duplicate at a concentration of 20/xM (50/zl/well) in PBS-T plus 0.5% (w/v) BSA. The plates are incubated for 1 hr at room temperature and then the second protein is added to the wells still containing the test compounds. The subsequent steps are exactly as described in the procedure above. The controls usually run on each plate include a vehicle control and a known inhibitor at a concentration titrated to give a constant 50% inhibition.
Site of Action of lnhibitors The binding assay can be modified to identify to which member of a dimer pair an inhibitory molecule is binding. After the usual coating and blocking steps using 6H-Bax, increasing concentrations of inhibitory compound (0.01 to 100/zM) are added to the wells and allowed to incubate for 2 hr at room temperature. The plates are then washed five times with PBS-T before addition of the second protein, Bcl-xL, in liquid phase. An alternative assay is then set up, in which the plates are coated with Bcl-xL and incubated with increasing concentrations of inhibitory compound. The compounds are washed off and then GST-Bax is added as the second protein in liquid phase. In this manner, only the protein on the solid phase is exposed to the inhibitory compound. Comparing the median inhibitory concentration (IC50) values for the compound incubated singly with either the Bax or the Bcl-xL protein gives an indication as to which of the dimeriz-
[24]
INTERACTION OF B c l - 2 FAMILY PROTEINS
261
ing proteins is the target for the compound. In Fig. 1, for example, Bcl-XL heterodimer formation is blocked by a peptide derived from the BH3 domain of Bax when preincubated with the Bcl-XL (ICs0 of 3.5/~M), but not when the peptide is preincubated with Bax (ICs0 > 100 ~M). Reversible and Irreversible Inhibitors
A modification of the binding assay can also be used to help decipher the mode of action of the inhibitor molecule. A constant concentration of the GST-tagged protein in liquid phase is preincubated with increasing concentrations of inhibitory compound (0.01 to 100/.~M) before addition to the 96-well plates coated with the appropriate proteins. Multiple dilution series of the compound are set up, and the preincubation time of the compound with the liquid-phase protein is varied from 0 to 24 hr. The results are plotted, and when the resulting ICs0 values are compared, we see that there is a category of compounds whose ICs0 values do not vary with time once they have reached equilibrium; these are reversible inhibitors. An
lOO
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so
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oo
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FIG. 1. A peptide derived from the BH3 domain of Bax blocks Bcl-xL heterodimer formation by binding to the Bcl-XL and not the Bax protein. Wells were coated with 6H-Bax (solid squares) or 6H-Bcl-XL (open squares) and pretreated with increasing concentrations of the peptide Bax 52-72. The unbound peptide was washed off and the wells were incubated with a constant concentration of GST-Bcl-XL (solid squares) or GST-Bax (open squares). Bound protein was quantitated in all cases by reaction with anti-GST antibody and alkaline phosphatase-coupled secondary antibody.
262
Bcl-2 FAMILYPROTEINS
[24]
example of this is illustrated in Fig. 2, where a peptide derived from the BH3 domain of Bax (amino acids 52-72) inhibits Bcl-XL heterodimer formation with similar IC50 values irrespective of time of preincubation. In contrast, IC50 values for irreversible inhibitors vary as a function of time of incubation. Comments and Considerations In the procedures described above, we have constantly referred to the Bax/Bcl-2 or Bax/Bcl-XL dimerization as concrete examples, but this assay format may be used to investigate many of the different binding pairs in the Bcl-2 family of proteins. Some preliminary experimentation is needed to adapt this method to other binding pairs. In each case it is important to optimize the concentration of the coating protein, because the amount of protein adhering to the polystyrene surface will depend, among other things, on the size of the protein. For the screening and competition assays, it is also important to create preliminary binding curves for each pair of dimerizing proteins to be used,
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FIG. 2. A peptide derived from the BH3 domain of Bax is a reversible inhibitor of Bcl-XL heterodimer formation. Wells were coated with 6H-Bax and incubated with a constant concentration of GST-Bcl-XL that had been pretreated with increasing concentrations of competing peptide Bax 52-72 for 0 In- (open circles), 1 hr (closed circles), 3 hr (open squares), or 24 hr (closed squares). Bound protein was quantitated as in Fig. 1.
[241
INTERACTIONOF Bcl-2 FAMILYPROTEINS
263
and to choose a concentration of the protein in liquid phase that lies on the steep logarithmic part of the binding curve, z2 The binding assays can be carried out with purified proteins missing the transmembrane domain. Furthermore, in our hands the presence of tags, whether located on the N terminus or C terminus, does not generally interfere with dimerization. Thus, the tags do not need to be cleaved off. On the contrary, careful selection of the tags can be used to aid the assay. The formation of Bcl-2 homodimers, for example, can be detected by using 6H and GST tags on the solid-phase and liquid-phase partners, respectively. By expressing all potential liquid-phase partners with a uniform tag, such as GST or Glu-Glu-Phe (EEF), a single anti-GST or anti-EEF antibody can be used to monitor a wide range of dimerization pairs, without having to generate a specific antibody for each protein. An antibody directed against a terminal tag is also less likely to interfere with the dimerization of the proteins, or be affected by the action of inhibitory compounds, than an antibody directed against an epitope on the main sequence of the protein itself. The assays can be run in various configurations with proteins tagged in various ways. However, in general, the assays work best when (1) the proteins to be coated onto the polystyrene plates are tagged with 6H, (2) the protein acting as a BH3 donor is used as the solid-phase partner, and (3) the partner with the hydrophobic binding cleft is used as the protein in liquid phase. For each binding pair studied, preliminary experiments should be carried out to ensure the specificity of the binding observed. In our case, for each specific interaction, saturable binding was detected, whereas only low levels of background binding were seen when the control protein BSA was used as the solid-phase binding partner (Fig. 3). To confirm that these in vitro interactions reflect the interactions observed in cells, we assessed the ability of the G145A and G138A point mutants of Bcl-2 and Bcl-XL, respectively, to bind to Bax; it has previously been shown by immunoprecipitation that these mutants fail to heterodimerize with Bax in cells. Recapitulating the cellular result, the mutants demonstrated a much-reduced binding to Bax in the plate-binding assay (Fig. 3). 22
Advantages This is an easy, reliable assay that can be used to investigate the binding properties of Bcl-2 family members with regard to homodimer and heterodimer formation. The assay can be performed with recombinant Bcl-2, Bcl-XL, and Bax proteins and is highly reproducible (Table I). The format 22 J.-L. Diaz, T. Oltersdorf, W. Horne, M. McConnell, G. Wilson, S. Weeks, T. Garcia, and L. C. Fritz, J. Biol. Chem. 272, 11350 (1997).
264
Bcl-2 FAMILY PROTEINS
[24]
2.00 1.75 1.50
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FIG. 3. Formation of Bcl-2 family heterodimers in solid-phase binding assays. Wells were coated with 6H-Bax (solid lines) or the control protein BSA (dashed lines) and incubated with increasing concentrations of GST-Bcl-2 (filled circles), GST-Bcl-XL(filled squares), GSTBcl-2 (G145A) (open circles), or GST-Bcl-XL (G138A) (open squares). Bound protein was quantitated in all cases by reaction with anti-GST antibody and alkaline phosphatase-coupled secondary antibody. [Taken from Diaz et al. 22] o f t h e a s s a y m a k e s it a m e n a b l e to h i g h - t h r o u g h p u t screening, a n d t h e f o r m a t has c e r t a i n a d v a n t a g e s c o m p a r e d w i t h o t h e r a s s a y f o r m a t s d e s i g n e d to m e a s u r e p r o t e i n - p r o t e i n i n t e r a c t i o n s . F o r e x a m p l e , y e a s t t w o - h y b r i d assays c a n b e u s e d to m o n i t o r such i n t e r a c t i o n s , b u t with t h e a d d e d v a r i a b l e o f c o m p o u n d p e n e t r a t i o n t h r o u g h t h e y e a s t cell wall. TABLE I EC5o
VALUES
FOR
HETERODIMER
AND
HOMODIMER
FORMATION
a
Solid phase
Liquid phase
ECs0 (nM)
n
6H-Bax 6H-Bax 6H-Bcl-2 6H-Bcl-xL
GST-Bcl-2 GST-Bel-XL GST-Bcl-2 GST-Bcl-XL
26.1 _+ 10.4 14.7 + 5.3 66.2 -+ 36.0 109.7 _+ 26.0
19 17 9 1l
a 6H-Bax, 6H-Bcl-2, and 6H-Bcl-XL were coated on microtiter planes and binding curves were established with increasing concentrations of GST-Bcl-2 or GST-Bcl-XL. ECs0 values listed are the mean concentration of liquid-phase protein that yielded half-maximal binding _+standard deviation for n independent measurements. (Taken from Diaz et ai.22)
[24]
INTERACTIONOF Bcl-2 FAMILYPROTEINS
265
The assay can also be used in a quantitative manner. Indeed, we used these solid-phase binding assays to generate quantitative binding data that provided the first estimate for the strength of these protein-protein interactions. 22 The concentration of liquid-phase protein necessary for halfmaximal binding for each reaction is presented in Table I. Assumptions and Limitations Although the protein-protein binding assay described above has been used to compare the affinity of the interactions between different Bcl-2 family proteins, the binding data generated are not true Kd values. There are two main reasons why we cannot generate Kd values in these assays: first, protein binding in this method does not occur in a homogeneous liquid phase; second, this procedure involves a separation step, and therefore the assay does not measure equilibrium binding. The binding assay involves extensive washing, and therefore one would expect that binding would be detected by this procedure only for interactions where dissociation rates were comparatively low. However, the assay yields little information on the kinetics of dissociation of the dimers. There may also be an avidity effect due to the local concentration of protein bound to the solid phase. Experiments have been carried out in collaboration with Igen (Gaithersburg, MD), using their O R I G E N assay system. This is basically a method to study protein-protein interactions by electrochemiluminescence (ECL). Protein binding by this method occurs in a homogeneous liquid phase, using equilibrium binding procedures. Using ECL, we determined the Kd for the Bax/Bcl-2 interaction to be 8.4 nM (data not shown), which is reasonably close to the apparent Kd of the interaction in our plate assay (Table I). Although dimerization data generated by these in vitro assays appear consistent with observations made by other methods such as immunoprecipitation, there are two major points of variance. It has been reported that Bax can form homodimers, yet we have been unable to observe Bax homodimer formation in solid-phase binding a s s a y s . 6'13'2° This could be highlighting a problem in our assays: for example, it is possible that Bax adopts a distinct and nonphysiological conformation when binding to the solid phase. However, published studies indicate that human Bax also fails to homodimerize in a yeast two-hybrid assay. 23 Furthermore, one study reported that Bax homodimerization, as monitored by immunoprecipitation, was found to be
23 H. Zhang, B. Saeed, and S.-C. Ng, Biochem. Biophys. Res. Commun. 208, 950 (1995).
266
Bcl-2 FAMILYPROTEINS
[251
dependent on the presence of detergent [1% (v/v) Triton X-100, as opposed to 0.05% (v/v) Tween 20 in our assays], but was not observed under more physiological conditions. 2a In addition, Bax has been shown to exist in the cytoplasm, but in that compartment it fails to form homodimers, z4 Interestingly, Bcl-XL readily formed homodimers in our assay, whereas attempts by others to observe this interaction in cells or with isolated protein have failed, a4'25 While we do not understand the basis for these differences, it is possible that a specific conformation of Bcl-XL is favored when bound to plastic, and that this conformation facilitates homodimerization. For example, this conformation may alter the positioning of the az helix (encompassing the BH3 domain), facilitating its interaction with a second molecule of Bcl-XL. Acknowledgments We thank Dr. John Reed (Burnham Institute) for the GST-Bcl-2 expression construct and anti-GST antibody, Suzanne Weeks and Gary Wilson (Idun Pharmaceuticals) for the expression and purification of the Bcl-2 family proteins, and Steven Chang for refining and implementing the methods outlined above.
24 Y.-T. Hsu and R. J. Youle, J. BioL Chem. 272, 13829 (1997). 25 A. J. Minn, L. H. Boise, and C. B. Thompson, J. BioL Chem. 271, 6306 (1996).
[25] A n a l y s i s o f D i m e r i z a t i o n o f B c l - 2 F a m i l y P r o t e i n s b y Surface Plasmon Resonance B y Z H I H U A X I E a n d JOHN C. R E E D
Introduction and Background Bcl-2 family proteins are important regulators of programmed cell death and apoptosis. 1,z These proteins either inhibit or induce cell death, with the ratios of antiapoptotic relative to proapoptotic members of the Bcl-2 family representing a critical determinant of the ultimate sensitivity or resistance of mammalian cells to various apoptotic stimuli. Many Bcl-2 family proteins can physically interact with themselves and each other, 1 j. Reed, Oncogene 17, 3225 (1998). g A. Gross, J. McDonnell, and S. Korsmeyer, Genes Dev. 13, 1899 (1999).
METHODS IN ENZYMOLOGY,VOL. 322
Copyright © 2000by AcademicPress All rightsof reproductionin any formreserved. 0076-6879/00$30.00