Báo cáo Y học: Association of human tumor necrosis factor-related apoptosis inducing ligand with membrane upon acidification pdf

Association of human tumor necrosis factor-related apoptosis inducing ligand with membrane upon acidification Gyu Hyun Nam and Kwan Yong Choi National Research Laboratory of Protein Folding and Engineering, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, Republic of Korea Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) has been known to induce tumor-specific apoptosis and to share the structural and functional char- acteristics with the proteins of TNF family. Recently, the crystal structure of human TRAIL showed that TRAIL is a homotrimeric protein whose subunits contain mainly b-sheets. We characterized the structural changes of recombinant human TRAIL induced by acidification and the biological implication of the structural characteristics at acidic pH in the interaction with the lipid bilayer. At acidic pH below pH 4.5, TRAIL resulted in substantial structural changes to a molten globule (MG)-like state. Far-UV CD spectrum of TRAIL indicated that the acidification induced a-helices that are absent in the native state. TRAIL at acidic pH exhibited significant change of tertiary structures as reflected in the near-UV CD spectrum. Thermal transition curve indicated that there was less cooperation at acidic pH than at neutral pH in the thermal denaturation of TRAIL. Moreover, TRAIL at the MG-like state not only enhanced the binding ability to liposomes, but also increased the release rate of a fluorescent dye, calcein, encapsulated in liposomes. The binding assay with anilinonaphthalene-8- sulfonic acid revealed that the surface hydrophobicity of TRAIL was increased while tryptophan residues became more exposed to solvent as judged by blue shift of the maximum fluorescence wavelength. Taken together, our results demonstrate that the acidification of human TRAIL induces the MG-like state in vitro and makes the membrane permeable through the favorable interaction of TRAIL with the membrane, implicating that general intrinsic properties such as TRAIL, TNF-a and lymphotoxin are shared by TNF family members. Keywords: TNF-related apoptosis inducing ligand; TNF family; acidification; molten globule (MG)-like state; a-helices. Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) is a member of the TNF family and its soluble form exhibited apoptotic activity for various cancer cell lines with minimal cytotoxicity toward normal tissues both in vitro and in vivo [1–5]. TRAIL has been known to be involved in CD4 + T cell-mediated and monocyte-induced cytotoxicity [6,7]. TRAIL has drawn a great deal of attention in the field of cell death because it induces apoptosis in most transformed cells and some virally infected cells, but not in normal cells [1,2]. However, despite the ubiquitous existence of TRAIL and its ability to induce apoptosis in many different tumor cells, little is known about structural characteristics for its biological action. TNF-a and lymphotoxin (LT) are trimeric proteins whose subunits contain mainly b-sheets, and belong to the TNF family together with TRAIL. Drastic structural changes to a molten globule (MG)-like state have been observedinTNF-a and LT at acidic pH values. Upon acidification, TNF-a and LT were found not only to have non-native secondary structures [8,9], but also to be able to be inserted into lipid bilayers [9,10]. Besides the TNF family proteins, a variety of proteins at acidic pH have been demonstrated to form the MG-like state retaining their partial secondary structures but lacking complete tertiary structures. For both bovine [11] and human [12] a-lactalbumin, as well as for bovine carbonic anhydrase B [13], MG-like states also were induced at acidic pH values. It has been proposed that the biological function of the MG- like state may be implicated in membrane interaction as shown in the case of colicin A at acidic pH [14–16]. TRAIL is a protein which can self-associate into a trimer. The crystal structure of human TRAIL [17,18] and the crystal structure in complexes with death receptor-5 [19–21] revealed that the individual TRAIL subunit mostly consists of antiparallel b-sheets and can be organized to form a jellyroll b-sandwich. Very recently, a unique zinc binding site was found and a zinc ion was known to be important for the trimeric structure of TRAIL [18]. TRAIL shares with TNF-a and LT characteristics such as sequence homology, three-dimensional structure, and cytotoxicity. In case of TNF-a and LT, the tumor cell killing activities enhanced by acidification led to the discovery that TNF-a and LT share the abilities to associate with and penetrate membranes, and the structural characteristics acquired by acidification Correspondence to K. Y. Choi, National Research Laboratory of Protein Folding and Engineering, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, 790–784, Republic of Korea. Fax: + 82 54 2792199, Tel.: +82 54 2792295, E-mail: kchoi@postech.ac.kr Abbreviations: ANS, 1-anilinonaphthalene-8-sulfonic acid; Myr 2 Gro- PCho, dimyristoylglycerophosphocholine; LT, lymphotoxin; MG, molten globule; PtdSer, phosphatidylserine; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis inducing ligand. (Received 6 June 2002, revised 6 August 2002, accepted 9 September 2002) Eur. J. Biochem. 269, 5280–5287 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03242.x provided a reasonable explanation for the acid-enhanced membrane interactions [9,10,22–24]. While TRAIL has been investigated mainly on the molecular mechanism for receptor-mediated apoptosis, few studies have been carried out with TRAIL concerning the structure–function rela- tionship under environments such as acidic pH values. Thus, it would be interesting to compare the structural features of TRAIL with those of two well-characterized TNF family proteins, TNF-a and LT, in their association with membranes upon acidification. In this study, acidification of TRAIL resulted in the drastic structural changes to an MG-like state where the secondary structure was significantly changed and non- native a-helices were induced. In its MG-like state, TRAIL could make membranes permeable by enhancing its liposome binding ability. Our results demonstrate that the structural changes of TRAIL are responsible for the increased membrane permeability which is mediated by enhanced membrane binding under acidic environments, implicating that the general intrinsic properties are shared by TNF family members. EXPERIMENTAL PROCEDURES Preparation of recombinant human TRAIL The truncated recombinant TRAIL containing amino acids 114–281, referred to from now on as TRAIL, coding for the extracellular region of the full-length human TRAIL, was produced and purified as described previously [25]. The TRAIL gene, inserted into downstream of the T7 promoter in a plasmid vector, pET-3a [26], was introduced into Escherichia coli strain BL21(DE3). Bacterial cells were grown while the expression of TRAIL was induced with 1m M isopropyl- D -thiogalactoside at 37 °C for 4 h. The bacterial cells were harvested and resuspended in a buffer solution containing 20 m M sodium phosphate, pH 7.0, 100 m M NaCl, and 1 m M dithiothreitol. After sonication, soluble fractions were obtained by centrifugation and applied onto an SP Sepharose Fast Flow column (Amer- sham Pharmacia Biotech). The bound fraction was concen- trated by use of Centri-prep (Amicon) and then loaded onto Superdex 200 HR 10/30 column (Amersham Pharmacia Biotech) for gel-filtration chromatography. The purified TRAIL appeared as a single band on SDS/PAGE analysis (data not shown). CD spectroscopy CD spectroscopic analyses were performed with a spectro- polarimeter (Jasco, 715). A cuvette with a path length of 2 mm for far-UV region (200–250 nm) or a path length of 5 mm for near-UV region (250–300 nm) was used for the CD spectral measurements. The temperature of the cuvettes was adjusted to 25 °C by use of a Peltier type temperature controller (Jasco, PTC-348WI). The protein concentration was 10 l M for far-UV CD measurements and 50 l M for near-UV CD measurements, respectively. TRAIL was dissolved in a buffer containing 100 m M NaCl and 1 m M dithiothreitol with either 20 m M sodium phosphate, pH 7.0 or 20 m M sodium acetate, pH 4.5. CD spectra were obtained with a scanning speed of 10 nmÆmin )1 and a bandwidth of 2 nm. Scans were collected at 1-nm intervals with a response time of 0.25 s and were accumulated three times. Each spectrum was corrected by subtracting the spectrum of the buffer at the respective pH. Thermal denaturation was monitored by measuring molar ellipticity at 222 nm in the same spectropolarimeter upon the temperature change with the heating rate of 30 °CÆh )1 . Fluorescence measurements Fluorescence spectra were obtained by use of a spectroflu- orimeter (Shimadzu, RF-5401) equipped with a thermo- statically controlled cell holder. TRAIL samples (each 10 l M ) at neutral and acidic pH values were prepared, respectively, with the same buffers as those in the CD spectroscopic measurements. To obtain TRAIL in its unfolded state, it was dissolved in 6 M guanidine hydro- chloride. Fluorescence spectra of TRAIL were obtained between 300 and 400 nm at 25 °C with the excitation wavelength at 295 nm. Each spectrum represented the average of three sequential scans with the spectrum of the buffer being subtracted. ANS binding assay Binding experiment with anilinonaphthalene-8-sulfonic acid (ANS; Molecular Probes) was performed by incubating TRAIL with ANS at acidic or neutral pH. The final concentrations of TRAIL and ANS were both 10 l M .The mixture of TRAIL and ANS was incubated at either pH 7.0 or 4.5 for 15 min. The incubated solution was then excited at 380 nm and the fluorescence changes were subsequently monitored between 400 nm and 600 nm at 25 °C. Fluores- cence spectra were obtained by a spectrofluorimeter (Shim- adzu, RF-5401) using a cuvette with a path length of 10 mm, and subtracted from the spectrum of the ANS solution without TRAIL. Liposome preparation Dimyristoylglycerolphosphocholine (Myr 2 Gro-PCho; Sig- ma) and bovine brain phosphatidylserine (PtdSer; Sigma) were dried in a glass test tube with a stream of nitrogen. Large unilamellar vesicles of about 1000-A ˚ diameter were prepared according to the reverse-phase evaporation method as described previously [23]. Briefly, they were prepared in a buffer containing 20 m M sodium phosphate, pH 7.0, 100 m M NaCl and 1 m M dithiothreitol. The liposomes were extruded through a polycarbonate mem- brane (Nucleopore, Pleasanton, CA, USA) of 0.1 lm pore diameter and then diluted into the respective buffer; a buffer containing 20 m M sodium phosphate, 100 m M NaCl, and 1m M dithiothreitol for pH 7.0 or pH 6.0; and a buffer containing 20 m M sodium acetate, 100 m M NaCl, and 1m M dithiothreitol for pH 5.0 or 4.5. Phospholipid concentration was determined according to the phosphorus assay method as described previously [27]. Liposome binding assay The amount of liposome-bound TRAIL was determined as follows: TRAIL at 10 lgÆmL )1 was incubated at 25 °Cfor 30 min with or without liposomes containing 100 l M phospholipid in the respective buffer solution at pH 7.0, Ó FEBS 2002 Association of human TRAIL with membrane at acidic pH (Eur. J. Biochem. 269) 5281 6.0, 5.0 and 4.5, respectively, as described in the liposome preparation. The mixture was then centrifuged at 13 000 g for 5 min and subjected to 15% SDS/PAGE. Densitometric scanning of the protein bands on the gel was performed to quantify the liposome-bound TRAIL. Leakage assay Liposomes at pH 7.0, 6.0, 5.0 and 4.5 were prepared as described in the liposome preparation except the buffer containing calcein (Sigma), a fluorescent dye. Liposomes at the respective pH were prepared in the buffer containing 1m M calcein. The liposome suspension was sonicated and then applied onto a Sephadex G-50 column (Amersham Pharmacia Biotech) to separate free calcein from liposome- entrapped calcein. The reaction was initiated by adding various amounts of TRAIL to the liposome suspension containing 1 m M of calcein and 50 l M of phospholipid in the buffer of the respective pH at 25 °C. The reaction was stopped 2 min after the initiation. The leakage rate of calcein in the liposomes was determined by monitoring the fluores- cence spectraof calcein.Excitation andemission wavelengths were 470 and 520 nm, respectively. Triton X-100 was added to release calcein encapsulated in liposomes and then the fluorescence intensity of calcein was determined to assess the amount of calcein in the liposome. RESULTS Structural analyses of TRAIL by CD spectroscopy The effects of acidification on the contents of secondary structures in TRAIL were estimated by CD spectroscopy. The far-UV CD spectra of TRAIL at pH 7.0 and at pH 4.5 are shown in Fig. 1A. At pH 7.0, the spectrum of TRAIL exhibited a typical pattern reflecting the high content of b-sheet structures with a single negative maximum ellipticity around 218 nm as observed for TNF family proteins. The negative ellipticities from 200 to 220 nm were increased significantly upon lowering pH to below 4.0. To estimate the spectral transition in the far-UV region by CD spectroscopy upon the pH change, the spectra were monitored at pH 5.0, 4.5 and 4.0, respectively. The distinct spectral change was observed below pH 4.5. The negative ellipticity at 222 nm was increased from about )2200 °Æcm )2 Ædmol )1 at pH 7.0 to )3200 at pH 4.5 (Fig. 1A). This spectral change strongly indicates that a-helical structure was induced upon acidifi- cation. The near-UV CD spectrum of TRAIL at pH 7.0 exhibited strongly negative ellipticities at 275 and 285 nm. These signals were drastically weakened in an acidic environment (Fig. 1B). Thus, at pH 4.5 TRAIL possesses a structure different from that in its native state and with a significant change of tertiary structure. Meanwhile, the effect of zinc ions on the conformation of TRAIL at acidic pH was investigated by analyzing the CD spectra of TRAIL, as zinc ions were recently found to be important to the structure of TRAIL [18]. The far- and near-UV CD spectra of TRAIL at pH 4.5 were not significantly altered in metal-free solution (data not shown). Thermal denaturation at neutral and acidic pH values To investigate the effect of pH on the cooperation of TRAIL for unfolding, the thermal denaturation was monitored by measuring the changes in molar ellipticity at 222 nm at various temperatures. No significant changes in the CD ellipticity were observed at neutral pH values up to 46 °C (Fig. 2). In the range of 68–79 °C, however, a drastic transition in sigmoidal transition curve of the ellipticity was observed, with a transition midpoint at 74 °C, indicating that the thermal transition at neutral pH is cooperative. On the other hand, the negative ellipticity at pH 4.5 was gradually increased upon raising temperature, and the thermal transition was not cooperative and occurred over a much wider temperature range than at neutral pH. Fluorescence spectroscopy Fluorescence spectra were obtained to analyze the effect of pH on the structure of TRAIL. The fluorescence spectrum Fig. 1. CD spectra of TRAIL at pH 7.0 (solid line) and pH 4.5 (dashed line). (A) Far-UV and (B) near-UV CD spectra of TRAIL at pH 7.0 and 4.5, respectively, are shown. The protein concentrations were 10 l M for the far-UV CD measurements and 50 l M for the near-UV CD measurements. TRAIL was dissolved in either 20 m M sodium phosphate, pH 7.0, or 20 m M sodium acetate, pH 4.5, containing 100 m M NaCl and 1m M dithiothreitol, respectively. All the CD spectra were obtained after the incubation of the protein with the buffer at the respective pH. 5282 G. H. Nam and K. Y. Choi (Eur. J. Biochem. 269) Ó FEBS 2002 of TRAIL at acidic pH lies between that of the native state and of the unfolded state. As shown in Fig. 3, the maximum fluorescence wavelength (k max ) of TRAIL at pH 7.0 was 330 nm and shifted to 350 nm at unfolded state. The k max of TRAIL was shifted to 337 nm at pH 4.5 and its fluores- cence intensity was decreased by about 40% relative to that at pH 7.0. These results reveal that aromatic residues such as tryptophan might be partially exposed to an aqueous environment at acidic pH values [16]. When the pH was returned to 7.0, the k max was decreased reversibly to 330 nm. Interaction with ANS To compare the relative hydrophobic surface area of TRAIL between the native and acidic states, binding experiments with ANS were performed. ANS, a fluorescent probe, has been utilized to monitor conformational changes of the protein with the subsequent exposure of hydrophobic binding sites on proteins, as described previously [28,29]. Fluorescence spectra and quantum yield of ANS were shown to be sensitive to the environment around the probe [29]. Changes of the pH between 7.0 and 4.5 in the absence of TRAIL had no effect on the ANS spectrum, and ANS alone did not display any significant fluorescence intensity between 400 and 600 nm (data not shown). Compared with the free dye in the solution, TRAIL resulted in a drastic increase in the fluorescence intensity upon binding ANS. ANS was bound to TRAIL at acidic pH values more favorably than at neutral pH (Fig. 4). When ANS was bound to TRAIL at pH 7.0, the ANS fluorescence was marginally enhanced, suggesting that TRAIL at neutral pH can bind ANS weakly. When the pH was lowered to 4.5, the fluorescence intensity was increased dramatically (by about 2.5-fold), and the k max of TRAIL was blue-shifted from 514 nm to 487 nm. These spectral changes imply that, at acidic pH, TRAIL might contain hydrophobic sites which bind ANS more strongly than at neutral pH. Shifting the pH from 4.5 back to 7.0 resulted in resumption of the original spectrum at pH 7.0, indicating that the exposure of hydrophobic binding sites of TRAIL seemed to be revers- ible (data not shown). Fig. 3. Fluorescence spectra of TRAIL at pH 7.0 (solid line), pH 4.5 (dashed line), and unfolded state (dotted line), respectively. The protein samples at pH 7.0 and pH 4.5 were prepared, respectively, in the same buffers used for the CD measurements. The sample of TRAIL in its unfolded state was prepared in 6 M guanidine hydrochloride. The protein samples at 10 l M concentration were excited at 295 nm and the emission spectra were observed at between 300 and 400 nm. Fig. 4. Interaction of ANS either without (dotted line) or with TRAIL at pH 7.0 (solid line) and pH 4.5 (dashed line). The protein samples at 10 l M wereincubatedwith10l M of ANS at the indicated pH values. The protein samples were excited at 380 nm and fluorescence spectra were obtained at between 400 and 600 nm. Fig. 2. Thermal transition curves for the molar ellipticity of TRAIL at pH 7.0 (solid line) and pH 4.5 (dashed line). Cooperation of TRAIL for thermal denaturation was observed at pH 7.0 when the CD signal at 222 nm was monitored with increasing temperature. The concentra- tion of TRAIL was 10 l M and the heating rate was 30 °CÆh )1 .The protein samples were prepared in a buffer containing 100 m M NaCl and 1 m M dithiothreitol with either 20 m M sodium phosphate, pH 7.0, or 20 m M sodium acetate, pH 4.5. Ó FEBS 2002 Association of human TRAIL with membrane at acidic pH (Eur. J. Biochem. 269) 5283 Binding of TRAIL to liposome membrane To assess the extent of binding capability of TRAIL with lipid bilayer at various pH values, TRAIL was incubated at pH 7.0, 6.0, 5.0 and 4.5, respectively, with liposomes which had been prepared from the mixtures of Myr 2 Gro-PCho and PtdSer. The protein samples alone were not precipitated at the respective pH upon centrifugation, as judged by SDS/ PAGE analysis. The amount of TRAIL bound to liposomes was observed to decrease with increasing pH (Fig. 5). Only a marginal amount of TRAIL was bound to Myr 2 Gro- PCho/PtdSer vesicles at pH values above 5.0. The amount of TRAIL bound to liposomes was below 10% of the total TRAIL added at pH 5.0–7.0, whereas it was increased up to about 35% at pH 4.5, indicating that TRAIL can be bound to lipid vesicles more favorably at acidic pH than at neutral pH. Release of liposome-entrapped dye induced by TRAIL As estimated by SDS/PAGE, TRAIL was bound more favorably to lipid vesicles consisting of Myr 2 Gro-PCho and PtdSer at acidic pH values. In many cases, the binding of a protein with lipid bilayer disrupts the bilayer integrity to make the membranes permeable [30,31]. To ascertain whether TRAIL alters the permeability of the membrane, the release of a fluorescent dye, calcein, entrapped in the liposome consisting of Myr 2 Gro-PCho and PtdSer (1 : 1 molar ratio) was monitored in the presence of TRAIL at various pH values. Myr 2 Gro-PCho/PtdSer vesicles alone were not leaky at pH 4.5–7.0. Figure 6 shows the pH dependence of the release rate of calcein induced by TRAIL. Upon adding TRAIL, marginal release of calcein from liposomes was observed above pH 6.0, but the permeability was changed below pH 6.0. Particularly, the release rate of the dye was increased drastically upon lowering pH below 5.0. Therefore, our results indicate that TRAIL could induce the release of calcein from liposomes through the interaction with lipid vesicles. Figure 7 shows the depend- Fig. 5. pH dependence of binding of TRAIL to liposome. TRAIL at 10 lgÆmL )1 was incubated at 25 °C with liposomes consisting of Myr 2 Gro-PCho and PtdSer (1 : 1 molar ratio, 100 l M phospholipid) at various pH valuess (lane 1, pH 7.0; lane 2, pH 6.0; lane 3, pH 5.0; lane 4, pH 4.5). After 30 min, the liposome-bound TRAIL was sep- arated from liposome-free TRAIL by use of a Sephadex G-50 column. The amount of liposome-bound TRAIL was determined by scanning the protein bands on the SDS/PAGE gel. Lane 5 represents TRAIL standard (5 lg). Fig. 6. pH dependence of the release rate of calcein from liposomes induced by TRAIL. TRAIL at 1 lgÆmL )1 was added to liposome suspensions consisting of Myr 2 Gro-PCho and PtdSer (1 : 1 molar ratio, 50 l M phospholipid) containing calcein at pH 7.0, 6.0, 5.0 and 4.5. Increase of calcein fluorescence was monitored at 25 °Cwith excitation and emission wavelengths of 470 and 520 nm, respectively. The fluorescence intensity of calcein after adding Triton X-100 devoid of TRAIL was taken as 100%. Three independent measurements were performed and the error bars represent one standard deviation of the measurements. Fig. 7. Dependence of the release rate of calcein from liposomes on the amountofTRAILatpH4.5.Various amounts of TRAIL were added to liposomes consisting of Myr 2 Gro-PCho and PtdSer (1 : 1 molar ratio, 50 l M phospholipid) containing calcein at pH 4.5. The release rate was determined by monitoring the increase in the fluorescence intensity of calcein released from liposomes with excitation and emis- sion wavelengths of 470 and 520 nm, respectively, at 25 °C while the amount of TRAIL is increased. Three independent measurements were performed and the error bars represent one standard deviation of the measurements. 5284 G. H. Nam and K. Y. Choi (Eur. J. Biochem. 269) Ó FEBS 2002 ence of the release rate on the amount of TRAIL at pH 4.5. The leakage rate was proportional to the amount of TRAIL, indicating that the observed release from liposomes could be induced by TRAIL. DISCUSSION Acidification induced dramatic structural changes in TRAIL. The structural changes of TRAIL at acidic pH resulted in an MG-like state which retained a substantial secondary structure but lost a rigid tertiary structure. TRAIL at acidic pH was observed to contain not only increased hydrophobic surface but also a non-native a-helical structure. Moreover, TRAIL was found to be able to bind to the lipid membrane in such a way as to make the membrane permeable when pH was lowered. Thus, the structural changes of TRAIL by acidification provide an explanation for intrinsic properties of TRAIL toward the membrane, as observed in such structurally and evolutio- narily related cytokines as TNF-a and LT. TRAIL in its native state consists mostly of b-sheets. When it was partially unfolded by acidification, it induced an MG-like state with a substantial change in the secondary structure. One of the important features of TRAIL in its MG-like state is that it contains the non-native a-helices upon acidification. A similar observation was made for TNF-a, another member of the TNF family [8,32]. The far- UV CD spectrum of TRAIL at acidic pH is very similar to that in the acid-unfolded TNF-a, suggesting that the content of the secondary structure at acidic pH should be similar to and the induction of non-native a-helices seems to be shared by two proteins that belong to the TNF family. The induction of a-helices has been known to mediate the insertion of proteins into membrane [9,24,33]. Thus, the induction of a-helices might give a structural basis for membrane interaction properties of TRAIL upon acidifi- cation. Besides similar features of MG-like states between TNF-a and TRAIL, TNF family proteins also have a high sequence homology and their crystal structures consist of jellyroll b-sheets with close similarities [17,34,35]. In particular, the number and location of tryptophan residues are conserved precisely among TRAIL, TNF-a and LT in all species analyzed. There are two tryptophans per monomer. The red shift of k max as well as the decrease of tryptophan fluorescence intensity occurring when TRAIL was acidified indicates that environments surrounding the tryptophan residues of TRAIL at acidic pH values become more polar relative to those at neutral pH; such spectral changes were also observed with two other TNF family proteins, TNF-a and LT [8,9]. The structural changes that enable the positional shift of tryptophan residues to the surface of TRAIL are accompanied by the exposure of hydrophobic binding sites for the dye ANS. The ANS fluorescence alone was not affected over a wide range of pH. However, a substantial increase of fluorescence intensity was observed for TRAIL exposed to ANS at pH 4.5 compared with at pH 7.0. These changes in fluorescence properties are characteristic of ANS bound to proteins through hydro- phobic interactions. The extent of ANS binding at acidic pH is similar among TRAIL, TNF-a and LT [9,24], suggesting that both exposure of hydrophobic surface to bind ANS and the positional shift of the tryptophan residue to an aqueous environment are common phenomena among the three TNF family proteins. In case of TNF-a and LT, the hydrophobic surface exposed by acidification [9,24] and induction of non-native a-helices [8] provides a possible explanation for the acid- enhanced membrane-interaction properties of two proteins. It would be also interesting to investigate whether the structural changes of TRAIL at acidic pH, as characterized in this study, can induce membrane binding and subsequent membrane leakage. TRAIL could lead to membrane leakage induced by intrinsic membrane interaction using purified TRAIL and liposome systems. Release rate of liposome-entrapped dye induced by TRAIL as well as membrane binding ability of TRAIL were found to be dependent on the pH of the local environment. TRAIL exhibited increased membrane binding with decreasing pH. Concomitantly, it induced membrane leakage below pH 5.0, as the release rate of calcein was significantly increased. Therefore, the present results clearly show that TRAIL induced release of calcein from liposomes consisting of Myr 2 Gro-PCho and PtdSer when it was more tightly bound to them. Taken together, our results suggest that the structural changes of TRAIL at acidic pH might potentially be significant for interactions with the membrane. Even though receptor-mediated apoptosis for TRAIL was extensively investigated, little information is available for the regulation of the expression of TRAIL. TNF-a and LT of the same TNF family to which TRAIL belongs were found to be cleaved in vivo by metalloprotease and released from the activated macrophage where the local environment becomes acidic [36–38]. The soluble forms of TNF-a and LT displayed dramatic structural changes at low pH, and their membrane binding and channel forming activities were remarkably enhanced at low pH [9,10,24]. In addition, the biological cytotoxicity of TNF-a was increased at low pH [10,24]. Recent studies showed that soluble TRAIL is generated in vitro by cysteine proteases [39] and was released from the monocytes and macrophages by lipopolysaccha- ride stimulation [40], like TNF-a and LT. Alternatively, when TRAIL is located in acidic environments such as endocytic vesicles, endosomes or lysosomes by receptor- mediated endocytosis, it might perturb or disrupt their membrane structure similar to the case of TNF-a, whose cytotoxicity seems to be related to receptor-mediated endocytosis [41,42]. With these observations, structural changes of TRAIL induced by acidification reveal that TRAIL might have a potential acid-enhanced cytotoxicity and its intrinsic property be also shared by those of TNF-a and LT. In conclusion, TRAIL shows striking similarities to TNF-a and LT in terms of structural features and membrane-interaction properties in acidic environments. The acid-enhanced ability of TRAIL for membrane binding which results in the membrane leakage is closely associated with structural changes to the MG-like state which include the induction of a-helices and the exposure of hydrophobic binding sites, as judged by the movement of tryptophan residues to an aqueous environment. Even if our studies are not conceptually novel, they will contribute to the under- standing of the intrinsic properties of TRAIL for inducing membrane disruption under acidic environments, as observed in other members of the TNF family. More Ó FEBS 2002 Association of human TRAIL with membrane at acidic pH (Eur. J. Biochem. 269) 5285 detailed analyses on the biochemical characteristics of TRAIL might give valuable information for in vivo function of TRAIL. ACKNOWLEDGMENTS The recombinant TRAIL gene encoding amino acids 114–281 of the full-length human TRAIL was kindly supplied by Professor Byung-Ha Oh. We would like to thank Dr Sun-Shin Cha for his assistance in TRAIL purification and Jung Hwan Kim for his technical help in liposome preparation. This research was supported by grants from the programs of National Research Laboratory sponsored by Korean Ministry of Science and Technology and G. H. N. was supported in part by the Brain Korea 21 project. REFERENCES 1. Wiley, S.R., Schooley, K., Smolak, P.J., Din, W.S., Huang, C.P., Nicholl, J.K., Sutherland, G.R., Smith, T.D., Rauch, C., Smith, C.A. et al. (1995) Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 3, 673–682. 2. Pitti, R.M., Marster, S.A., Ruppert, S., Donahue, C.J., Moore, A. & Ashkenazi, A. (1996) Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family. J. Biol. Chem. 271, 12687–12690. 3. Griffith, T.S., Chin, W.A., Jackson, G.C., Lynch, D.H. & Kubin, M.Z. 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