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 apoptosisinducing ligand with membrane upon acidification Gyu Hyun Nam and Kwan Yong Choi National Research Laboratory of Protein Foldi

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 byacidification 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 tertiarystructures 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 onlyenhanced the binding abilityto liposomes, but also increased the release rate of a fluorescent dye, calcein, encapsulated in liposomes The binding assaywith anilinonaphthalene-8-sulfonic acid revealed that the surface hydrophobicity of TRAIL was increased while tryptophan residues became more exposed to solvent as judged byblue 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 familymembers

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 familyand its

soluble form exhibited apoptotic activityfor 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 abilityto induce

apoptosis in manydifferent 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 familytogether with TRAIL Drastic structural changes to a molten globule (MG)-like state have been observed in TNF-a and LT at acidic pH values Upon acidification, TNF-a and LT were found not onlyto have non-native secondarystructures [8,9], but also to be able to

be inserted into lipid bilayers [9,10] Besides the TNF family proteins, a varietyof proteins at acidic pH have been demonstrated to form the MG-like state retaining their partial secondarystructures 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 maybe 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 mostlyconsists

of antiparallel b-sheets and can be organized to form a jellyroll b-sandwich Veryrecently, 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 discoverythat TNF-a and LT share the abilities to associate with and penetrate membranes, and the structural characteristics acquired byacidification

Correspondence to K Y Choi, National Research Laboratoryof

Protein Folding and Engineering, Division of Molecular and Life

Sciences, Pohang Universityof 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)

provided a reasonable explanation for the acid-enhanced

membrane interactions [9,10,22–24] While TRAIL has

been investigated mainlyon 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 familyproteins, 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

secondarystructure was significantlychanged and

non-native a-helices were induced In its MG-like state, TRAIL

could make membranes permeable byenhancing its

liposome binding ability Our results demonstrate that the

structural changes of TRAIL are responsible for the

increased membrane permeabilitywhich is mediated by

enhanced membrane binding under acidic environments,

implicating that the general intrinsic properties are shared

byTNF familymembers

E X P E R I M E N T A L P R O C E D U R E S

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

1 mM isopropyl-D-thiogalactoside at 37°C for 4 h The

bacterial cells were harvested and resuspended in a buffer

solution containing 20 mM sodium phosphate, pH 7.0,

100 mMNaCl, and 1 mMdithiothreitol After sonication,

soluble fractions were obtained bycentrifugation and

applied onto an SP Sepharose Fast Flow column

(Amer-sham Pharmacia Biotech) The bound fraction was

concen-trated byuse 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 byuse of a Peltier type temperature

controller (Jasco, PTC-348WI) The protein concentration

was 10 lM for far-UV CD measurements and 50 lM for

near-UV CD measurements, respectively TRAIL was

dissolved in a buffer containing 100 mMNaCl and 1 mM

dithiothreitol with either 20 mMsodium phosphate, pH 7.0

or 20 mM 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 bysubtracting the spectrum of the buffer at the respective pH Thermal denaturation was monitored bymeasuring 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 byuse of a spectroflu-orimeter (Shimadzu, RF-5401) equipped with a thermo-staticallycontrolled cell holder TRAIL samples (each

10 lM) 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 6M 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 byincubating TRAIL with ANS at acidic or neutral pH The final concentrations of TRAIL and ANS were both 10 lM 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 bya 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 (Myr2Gro-PCho; Sig-ma) and bovine brain phosphatidylserine (PtdSer; SigSig-ma) 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, theywere prepared in a buffer containing 20 mMsodium phosphate,

pH 7.0, 100 mM NaCl and 1 mM 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 mMsodium phosphate, 100 mM NaCl, and

1 mM dithiothreitol for pH 7.0 or pH 6.0; and a buffer containing 20 mM sodium acetate, 100 mM NaCl, and

1 mM dithiothreitol for pH 5.0 or 4.5 Phospholipid concentration was determined according to the phosphorus assaymethod as described previously[27]

Liposome binding assay The amount of liposome-bound TRAIL was determined as follows: TRAIL at 10 lgÆmL)1was incubated at 25°C for

30 min with or without liposomes containing 100 lM phospholipid in the respective buffer solution at pH 7.0,

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

quantifythe 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

1 mMcalcein 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 byadding

various amounts of TRAIL to the liposome suspension

containing 1 mMof calcein and 50 lMof 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 bymonitoring the

fluores-cence spectra of calcein Excitation and emission wavelengths

were 470 and 520 nm, respectively Triton X-100 was added

to release calcein encapsulated in liposomes and then the

fluorescence intensityof calcein was determined to assess the

amount of calcein in the liposome

R E S U L T S

Structural analyses of TRAIL by CD spectroscopy

The effects of acidification on the contents of secondary

structures in TRAIL were estimated byCD 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 familyproteins The

negative ellipticities from 200 to 220 nm were increased

significantlyupon lowering pH to below 4.0 To estimate the

spectral transition in the far-UV region byCD 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 ellipticityat 222 nm was increased from about)2200 °Æcm)2Ædmol)1at 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 stronglynegative ellipticities at 275 and 285 nm These signals were drasticallyweakened 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 tertiarystructure Meanwhile, the effect of zinc ions on the conformation of TRAIL at acidic

pH was investigated byanalyzing the CD spectra of TRAIL, as zinc ions were recentlyfound to be important to the structure of TRAIL [18] The far- and near-UV CD spectra of TRAIL at pH 4.5 were not significantlyaltered 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 bymeasuring the changes in molar ellipticityat

222 nm at various temperatures No significant changes in the CD ellipticitywere 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 ellipticitywas observed, with a transition midpoint at 74°C, indicating that the thermal transition at neutral pH is cooperative On the other hand, the negative ellipticityat pH 4.5 was graduallyincreased 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

1 m dithiothreitol, respectively All the CD spectra were obtained after the incubation of the protein with the buffer at the respective pH.

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 (kmax) of TRAIL at pH 7.0 was

330 nm and shifted to 350 nm at unfolded state The kmaxof

TRAIL was shifted to 337 nm at pH 4.5 and its

fluores-cence intensitywas decreased byabout 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 kmax was decreased reversiblyto

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 displayanysignificant 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 intensityupon binding ANS

ANS was bound to TRAIL at acidic pH values more

favorablythan at neutral pH (Fig 4) When ANS was

bound to TRAIL at pH 7.0, the ANS fluorescence was

marginallyenhanced, suggesting that TRAIL at neutral pH

can bind ANS weakly When the pH was lowered to 4.5, the

fluorescence intensitywas increased dramatically(byabout

2.5-fold), and the kmax of TRAIL was blue-shifted from

514 nm to 487 nm These spectral changes implythat, at

acidic pH, TRAIL might contain hydrophobic sites which bind ANS more stronglythan 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 were incubated with 10 l 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.

Binding of TRAIL to liposome membrane

To assess the extent of binding capabilityof 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 Myr2Gro-PCho

and PtdSer The protein samples alone were not precipitated

at the respective pH upon centrifugation, as judged bySDS/

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 Myr2

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 favorablyat acidic pH than at neutral

pH

Release of liposome-entrapped dye induced by TRAIL

As estimated bySDS/PAGE, TRAIL was bound more

favorablyto lipid vesicles consisting of Myr2Gro-PCho and

PtdSer at acidic pH values In manycases, 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 permeabilityof the membrane,

the release of a fluorescent dye, calcein, entrapped in the

liposome consisting of Myr2Gro-PCho and PtdSer (1 : 1

molar ratio) was monitored in the presence of TRAIL at

various pH values Myr2Gro-PCho/PtdSer vesicles alone

were not leakyat pH 4.5–7.0 Figure 6 shows the pH

dependence of the release rate of calcein induced byTRAIL

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 byuse of a Sephadex G-50 column.

The amount of liposome-bound TRAIL was determined byscanning

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 °C with excitation and emission wavelengths of 470 and 520 nm, respectively The fluorescence intensityof 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 amount of TRAIL at pH 4.5 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 bymonitoring the increase in the fluorescence intensityof 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.

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 byTRAIL

D I S C U S S I O N

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

secondarystructure but lost a rigid tertiarystructure

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 wayas to make

the membrane permeable when pH was lowered Thus, the

structural changes of TRAIL byacidification provide an

explanation for intrinsic properties of TRAIL toward the

membrane, as observed in such structurallyand

evolutio-narilyrelated cytokines as TNF-a and LT

TRAIL in its native state consists mostlyof b-sheets

When it was partiallyunfolded byacidification, 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 verysimilar to

that in the acid-unfolded TNF-a, suggesting that the

content of the secondarystructure at acidic pH should be

similar to and the induction of non-native a-helices seems to

be shared bytwo 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 familyproteins 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

preciselyamong TRAIL, TNF-a and LT in all species

analyzed There are two tryptophans per monomer The

red shift of kmax as well as the decrease of tryptophan

fluorescence intensityoccurring 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 familyproteins, TNF-a

and LT [8,9] The structural changes that enable the

positional shift of tryptophan residues to the surface of

TRAIL are accompanied bythe 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 intensitywas 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 familyproteins

In case of TNF-a and LT, the hydrophobic surface exposed byacidification [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 byintrinsic membrane interaction using purified TRAIL and liposome systems Release rate of liposome-entrapped dye induced by TRAIL as well as membrane binding abilityof 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 significantlyincreased Therefore, the present results clearlyshow that TRAIL induced release of calcein from liposomes consisting of Myr2Gro-PCho and PtdSer when it was more tightlybound to them Taken together, our results suggest that the structural changes of TRAIL at acidic pH might potentiallybe significant for interactions with the membrane

Even though receptor-mediated apoptosis for TRAIL was extensivelyinvestigated, little information is available for the regulation of the expression of TRAIL TNF-a and

LT of the same TNF familyto which TRAIL belongs were found to be cleaved in vivo bymetalloprotease 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 remarkablyenhanced 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 bycysteine 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 byacidification reveal that TRAIL might have a potential acid-enhanced cytotoxicity and its intrinsic propertybe also shared bythose 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 abilityof TRAIL for membrane binding which results in the membrane leakage is closelyassociated with structural changes to the MG-like state which include the induction of a-helices and the exposure of hydrophobic binding sites, as judged bythe movement of tryptophan residues to an aqueous environment Even if our studies are not conceptuallynovel, theywill 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

detailed analyses on the biochemical characteristics of

TRAIL might give valuable information for in vivo function

of TRAIL

A C K N O W L E D G M E N T S

The recombinant TRAIL gene encoding amino acids 114–281 of the

full-length human TRAIL was kindlysupplied byProfessor 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 bygrants from the programs of

National Research Laboratorysponsored byKorean Ministryof

Science and Technologyand G H N was supported in part bythe

Brain Korea 21 project.

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