ORIGINAL PAPER

Characterisation of the Bax-nucleophosmin interaction:the importance of the Bax C-terminus

Jane Thompson Æ Keith Finlayson Æ Eliane Salvo-Chirnside Æ David MacDonald ÆJames McCulloch Æ Lorraine Kerr Æ John Sharkey

Published online: 24 January 2008

� Springer Science+Business Media, LLC 2008

Abstract The molecular chaperone nucleophosmin has

been identified as a novel Bax binding protein with this

interaction proposed to be a key event in the activation and

translocation of Bax in mitochondrial dysfunction and

apoptotic cell death. Using a proximity assay, we have

quantitatively defined the high affinity and saturable inter-

action between Bax and nucleophosmin indicative of a

competitive and specific mechanism. Binding of full length

Bax to nucleophosmin was only observed after conforma-

tional change was induced using non-ionic detergents (e.g.,

NP-40). The Bax-nucleophosmin interaction was inhibited

by a Bax C-terminal antibody (IC50 = 1 nM) but minimally

affected by antibodies directed against either the N-terminus

or a-helices 4 and 5. Bcl-2 and p53 inhibited the interaction

between full length activated Bax and nucleophosmin. The

proximity assay based on the Bax-nucleophosmin interac-

tion was robust and reproducible (Z0 = 0.50) facilitating its

use for screening a small chemical library. A low molecular

weight non-peptide compound, 2-(5-methyl-2-phenyl-1,

3-thiazol-4-yl)ethanohydrazide, partially inhibited the Bax-

nucleophosmin interaction (IC50 = 100 nM) and also

attenuated UV-induced cell death of HEK293 cells. The

present investigations demonstrate the importance of

exposure of the C-terminus of Bax for its interaction with

nucleophosmin. These protein–protein interaction assays

provide a technical approach both for the study of Bax-

interacting proteins and for the discovery of novel anti-

apoptotic agents.

Keywords Bcl-2 family � Apoptosis � Cell death �Proximity assay

Introduction

Bax, a pro-apoptotic member of the Bcl-2 family, is pri-

marily found in the cytosol of healthy cells in a soluble

inactive state while anti-apoptotic members such as Bcl-2

and Bcl-xL act to protect the outer mitochondrial mem-

brane (OMM) integrity [1, 2]. Bcl-2 and Bcl-xL both

contain domains BH1-4 and protect the OMM from dis-

ruption by Bax [3, 4]. Evidence has shown that insertion of

the BH3 domain of Bax into a hydrophobic cleft on the

surface of Bcl-2 and Bcl-xL appear necessary for their

hetero-dimerization with Bax for the suppression of

apoptosis [5]. Following an apoptotic stimulus, BH3 only

proteins can activate Bax [6], leading to a conformational

change and exposure of its N- and C-terminal domains. The

C-terminus of Bax is then targeted to the OMM enabling

membrane insertion and pore formation [7]. The resultant

loss of cytochrome c and apoptogenic factors through

mitochondrial pores leads to the formation of apoptosomes,

activation of effector caspses and cell death ensues [8].

L. Kerr and J. Sharkey have contributed equally to this work.

J. Thompson (&) � K. Finlayson � E. Salvo-Chirnside �D. MacDonald � J. McCulloch � L. Kerr � J. Sharkey

Astellas CNS Research in Edinburgh, The University of

Edinburgh, The Chancellors Building, 49 Little France Crescent,

Edinburgh EH16 4SB, UK

e-mail: jthompson@cyclacel.com

Present Address:J. Thompson

Cyclacel Pharmaceutical Inc, Dundee Technopole,

James Lindsay Place, Dundee DD1 5JJ, UK

Present Address:E. Salvo-Chirnside

Institute of Stem Cell Research, The University of Edinburgh,

Kings Buildings, West Mains Road, Edinburgh EH9 3JQ, UK

123

Apoptosis (2008) 13:394–403

DOI 10.1007/s10495-007-0177-2

Several proteins in the cytosol have been identified as

molecular chaperones or retention factors which regulate

monomeric Bax, preventing the conformational changes

associated with apoptosis and protecting against activation

by BH3 only proteins. Nucleophosmin has recently been

identified as a Bax interacting protein [9]. It is a nucleolar

phosphoprotein and molecular chaperone which is abun-

dantly expressed in the nucleolus and continuously shuttles

between the nucleus and cytoplasm to prevent protein

aggregation, promote histone assembly and acetylation

dependent transcription [10–12]. Nucleophosmin has been

shown to be localized in the cytosol prior to Bax mito-

chondrial translocation following staurospaurine induced

apoptosis in SHSY5Y cells in vitro and focal cerebral

ischemia in vivo [9].

We have developed an in vitro based binding assay

(amplified luminescent proximity homogenous assay) to

study this novel protein–protein interaction at the molecular

level. We established conditions to examine the binding of a

C-terminal peptide and full length Bax protein to nucleo-

phosmin. Various antibodies or peptides known to interact

with Bax or nucleophosmin were used in the assay to allow

a systematic characterisation of the interaction between

nucleophosmin and full length Bax or a C-terminal Bax

peptide. We have demonstrated that Bax binds to nucleo-

phosmin by its C-terminal tail and that small molecule

inhibitors could be identified. Recent studies indicating that

nucleophosmin may be a chaperone involved in transloca-

ting activated Bax to the mitochondria at the critical point of

apoptotic cell death, highlights the therapeutic potential of

modulating the Bax-nucleophosmin interaction which

should aide the identification of drugs for the treatment of

certain neurodegenerative disorders or neoplasias.

Materials and methods

All chemicals were obtained from Sigma (Dorset, UK)

unless otherwise indicated.

Expression and purification of recombinant Bax

and nucleophosmin

The cDNAs encoding Bax and nucleophosmin (Invitrogen,

Paisley, UK) were subcloned from Gateway� pENTRTM221

UltimateTM human ORF clones. Using forward and reverse

primers respectively for Bax (50-ATG GAC GGG TCC GGG

GAG CA-30 and 50-CTA GCC CAT CTT CTT CCA GAT

GG-30) and nucleophosmin (50-ATG GAA GAT TCG ATG

GAG ATG G-30 and 50-GCT GGG TTC TAA AGA GAC

TTC C-30), PCR was used to obtain the complete coding

regions from the pENTRTM221 clones. Amplified products

for Bax and nucleophosmin were TOPO-TA cloned into the

N-terminal His-tag fusion vector pCRT7-NT-TOPO (Invit-

rogen) and sequenced on both strands by automated

sequencing (MWG, London, UK). The nucleophosmin

construct was transformed into BL21 cells (Invitrogen) and

protein expression induced by 1 mM IPTG for 4 h. Cells

were lysed (Fast-break lysis buffer, 1/10 dilution; Promega,

Southampton, UK) and His-tagged nucleophosmin captured

with MagneHisTM beads (for 2 min, Promega). Beads were

captured using a magnetic stand and washed 4 times in

MagneHisTM binding/wash buffer. Bound protein was eluted

in 500 mM imidazole and dialysed against Tris buffer

(50 mM Tris-HCl pH7.4, 150 mM NaCl, 1 mM EDTA,

5 mM DTT, 0.5% NP-40, 0.5% Triton-X100, 10% glycerol)

using Slide-A-Lyzer cassettes (10 KDa MWCO; Pierce,

Northumberland, UK). Full length His-tagged Bax was

produced using ExpresswayTM in vitro expression system

(Invitrogen) according to the manufacturer’s instructions

using 1 lg pCRT7-TOPO-Bax DNA. Recombinant Bax was

purified using MagneHisTM purification kit (Promega) as

described for nucleophosmin except final dialysis was

against 150 mM NaCl, 10 mM HEPES-KOH pH7.4.

Immunoprecipitation and Western blotting

Immunoprecipitation was performed using an antibody

against active Bax (Bax 6A7, 5/200 mg protein; BD Bio-

sciences, Oxford, UK). Complexes were isolated with protein

G-conjugated paramagnetic dynabeads (Dynal Biotech,

Bromborough, UK) and analysed by Western blotting using

BaxNT antibody (1/2000; Upstate, Chandlers Ford, UK).

Biotinylation of Bax protein

To enable conjugation to streptavidin-coated donor beads

(PerkinElmer, Beaconsfield, UK) full length Bax was

biotinylated using the ProtOnTM Biotin labelling Kit

(Vector Laboratories, Peterborough, UK) as described by

the manufacturer. Briefly, 1 mg of recombinant Bax in

100 ll of buffer (150 mM NaCl, 10 mM HEPES-KOH

pH7.4) was biotinylated by adding 2 ll of labelling

reagent. After 45 min incubation at room temperature, the

reaction was terminated by the addition of 2 ll stop solu-

tion. To remove excess biotin from the labelled Bax,

dialysis was carried out against 2 l of buffer (150 mM

NaCl, 10 mM HEPES-KOH pH7.4) overnight at 4�C.

Proximity assay

Protein–protein interactions were analysed using an

amplified luminescence proximity homogeneous assay

Apoptosis (2008) 13:394–403 395

123

(AlphaScreenTM technology; PerkinElmer) in 384-well

white polystyrene proxiplates (PerkinElmer). Assays com-

prised nucleophosmin monoclonal antibody-conjugated

acceptor beads and streptavidin-coated donor beads (20 lg/

ml of each), Bax (biotinylated peptide or full length protein)

and nucleophosmin, with putative inhibitors as indicated, in

a total assay volume of 25 ll unless otherwise stated.

A monoclonal antibody directed against the C-terminus

of nucleophosmin (Sigma) was conjugated with Alpha-

ScreenTM acceptor beads (PerkinElmer) using a standard

amination procedure. The nucleophosmin antibody

(0.01 mg) was added to 0.3 mg of unconjugated acceptor

beads in 5% sodium cyanoborohydride, 0.02 M 2-[N-

morpholino] ethanesulfonic acid (MES), pH6.0 and incu-

bated for 48 h at 37�C in the dark. A block was performed

with 0.1 M carboxymethoxylamine-hemihydrochloride

(CMO; 1 h) and the beads washed 3 times in 0.1 M Tris

buffer pH8.0. Conjugated beads were finally prepared as a

5 mg/ml solution in 25 mM HEPES, 0.1 M NaCl, pH 7.4

(PerkinElmer).

The left and right arms of the proximity assay were

verified to determine if the assay was functional. To

ascertain if biotinylated Bax C-terminal peptide bound to

the streptavidin-coated donor beads, biotinylated acceptor

beads (20 ug/ml) were incubated for 30 min at 30�C with

biotinylated Bax C-terminal peptide (biotin-TVTIF-

VAGVLTASLTIWKKMG; CSS-Albachem, Gladsmuir,

UK) prepared in 150 mM NaCl, 10 mM HEPES-KOH

pH7.4, 0.1% Tween 20) at a concentration range (0.03–

3,000 nM). Streptavidin-coated donor beads were added

and incubation performed for a further 60 min prior to

analysis using a multi-label plate reader (Envision Exci-

teTM; PerkinElmer). Similarly to determine successful

conjugation of nucleophosmin monoclonal antibody to

acceptor beads, the putatively conjugated acceptor beads

were incubated for 30 min with biotinylated secondary

antibody at a concentration range (0.003–3 nM). Strepta-

vidin-coated donor beads were added and incubation

performed for a further 60 min prior to analysis.

The optimal concentrations of Bax C-terminal peptide

and nucleophosmin required to produce a maximal signal

in the assay were established by incubating constant con-

centrations of nucleophosmin (5, 15, 150 and 350 nM) with

Bax C-terminal peptide at a concentration range (0.3–

1,000 nM). The binding of the two molecules at the opti-

mal concentrations (100 nM Bax C-terminal peptide and

15 nM nucleophosmin) was also measured over time (0–

4 h). An optimal incubation for the proteins following

donor bead addition was established at 90 min and was

used thereafter.

To investigate the interaction between full length Bax

and nucleophosmin, serial dilutions of each protein were

performed in assay buffer (150 mM NaCl, 10 mM HEPES-

KOH pH7.4, 0.01% BSA) containing either 0.1% CHAPS,

0.1% Nonidet P-40 (NP-40) or 0.1% Tween 20. Due to the

instability of the full length Bax protein in long-term

storage, production was limited to small quantities. The

concentration of full length Bax protein used in subsequent

competition assays (38 nM) was determined from dilution

of stock preparation producing the maximal signal in a

serial dilution study.

Competition studies were performed using Bax biotin-

ylated C-terminal peptide (100 nM) or full length protein

(38 nM), nucleophosmin (15 nM) and putative inhibitors (if

present) at concentrations ranging from (0.03–1,000 nM)

preincubated at room temperature for 45 min. Nucleo-

phosmin antibody-conjugated acceptor beads were added

and a further incubation of 30 min at 30�C performed. After

the addition of streptavidin-coated donor beads, the plate

was incubated at 30�C for a further 90 min prior to analysis.

Putative peptide inhibitors used were Bcl-2 (His-tagged,

C-terminal truncated; Merck, Nottingham, UK), p53 pep-

tide (Abcam, Cambridge, UK), antibodies raised against

the Bax N-terminus (Bax-NT; Upstate), a-helices 4 and 5

(Ab4; Merck) and C-terminus (1D3; Abcam). C-terminal

Bax antibody at concentration range (0.03–1,000 nM) was

used as a positive inhibition control of the Bax-nucleo-

phosmin interaction while buffer and the individual

proteins alone served as negative controls.

Identification of non-peptide inhibitors

of the Bax-nucleophosmin interaction

A small library of 800 chemically diverse compounds

(HitfinderTM, Maybridge, Cornwall, UK) was assessed for

the ability to block the Bax-nucleophosmin interaction.

Compounds, supplied as a dry film (1 lmole) in 96 well

plates, were diluted to a 50 mM master stock in 100%

DMSO. The final concentration of compounds used in the

assay was 1 lM in 0.02% DMSO. C-terminal Bax peptide

and nucleophosmin were prepared in assay buffer contain-

ing 0.1% Tween 20 to a final assay concentration of 100 nM

and 15 nM respectively and the assay performed as

described above. C-terminal Bax antibody (3 nM final

concentration) was used as a positive inhibition control of

the Bax peptide-nucleophosmin interaction while buffer and

the individual proteins alone served as negative controls.

Cell culture and UV-induced apoptosis

HEK293 cells were seeded into 4 well plates at a density of

0.5 9 105 cells/well and maintained in Dulbecco’s Modi-

fied Eagles Medium (DMEM) supplemented with 2 mM

Glutamine and 1% non essential amino acids at 37�C in a

396 Apoptosis (2008) 13:394–403

123

humidified 5% CO2 atmosphere. Following a 24 h incu-

bation period media was removed from wells and treated

with UV-C (30 J/m2, 254 nm) for 5 s using a GS gene

linker UV chamber (BioRad, Hemel Hempstead, UK) with

the lids removed. After irradiation, the cells were supplied

with complete medium containing either vehicle alone

(0.01% DMSO) or vehicle plus 10 lM inhibitor (2-(5-

methyl-2-phenyl-1,3-thiazol-4-yl) ethanohydrazide). Con-

trol cells which did not receive UV irradiation were

replaced with the same medium as experimental plates.

Cell viability was then measured by MTS assay (Promega)

following a further incubation period of 24 h.

Results

Development of a Bax-nucleophosmin proximity assay

Nucleophosmin was identified as a Bax-binding protein by

affinity chromatography [9]. We have subsequently devel-

oped a proximity assay to characterise this novel protein–

protein interaction in detail. The principle behind the

amplified luminescent proximity homogenous assay (Al-

phaScreenTM) is that upon excitation, a chemiluminescent

signal is produced when donor and acceptor beads are sep-

arated by a distance of less than 200 nm. Thus when two

proteins of interest (separately conjugated to donor and

acceptor beads) interact, the beads are brought into close

proximity and a luminescent signal is produced [13]. We

have used this technology to characterise the interaction

between Bax and nucleophosmin and a schematic diagram of

the assay design is depicted in Fig. 1a. A biotinylated peptide

corresponding to the last 21 C-terminal amino acids of Bax

was attached to streptavidin-coated donor beads. Attachment

was verified by the ability of this peptide to disrupt the

interaction between streptavidin-coated donor beads and

biotin-coated acceptor beads (Fig. 1b, c). A monoclonal

antibody raised against nucleophosmin was directly conju-

gated to acceptor beads and to confirm this, a biotinylated

secondary antibody was used to link streptavidin-coated

donor beads thus generating a signal (Fig. 1d, e). Having

verified both halves of the proximity assay (biotinylated Bax

peptide attachment to streptavidin-coated donor beads and

acceptor beads conjugated with nucleophosmin monoclonal

antibody) optimal assay conditions were determined.

C-terminal Bax peptide (0.3–100 nM) produced a con-

centration-dependent increase in binding signal when

incubated with constant concentrations of nucleophosmin.

The maximal signal was observed when the nucleophosmin

concentration was 15 nM (Fig. 2a). Using this concentra-

tion of nucleophosmin and varying the Bax concentration,

a maximal signal was observed with 100 nM C-terminal

Bax peptide (Fig. 2b). Using these optimal concentrations,

the luminescent signal increased with incubation time

reaching equilibrium at 90 min (Fig. 2c). Therefore all

subsequent experiments utilizing Bax peptide were per-

formed with 100 nM C-terminal Bax peptide and 15 nM

nucleophosmin with incubation for at least 90 min.

Nucleophosmin only binds to active Bax

To investigate whether nucleophosmin interacts with full

length Bax, recombinant protein was expressed in vitro,

biotinylated and attached to streptavidin-coated donor

beads. However, no binding between nucleophosmin and

full length Bax was observed unless a conformational

change was induced by the non-ionic detergents Nonidet

P-40 (NP-40) and Tween 20 (Fig. 3a). This is consistent

with the view that the C-terminus of inactive Bax is

sequestered within a hydrophobic pocket of the BH3

domain and therefore not available to interact with other

proteins unless conformationally activated [14]. Our initial

experiments were performed in 0.1% CHAPS-containing

buffer which does not alter the conformation of Bax, unlike

non-ionic detergents [15]. Verification that NP-40 induced

a Bax conformational change was obtained by immuno-

precipitation using the conformation-sensitive Bax 6A7

antibody (Fig. 3b). Moreover, in the presence of both non-

ionic detergents nucleophosmin-Bax interactions were

detected (Fig. 3a), the greatest being observed with NP-40.

In all further experiments using full length Bax, NP-40 was

included in the reaction buffer.

Molecular analysis of Bax-nucleophosmin interaction

To elucidate further which regions of Bax are involved in

binding nucleophosmin, competition studies were per-

formed with antibodies raised against specific domains of

Bax. An antibody targeted to the C-terminus of Bax (C-

term Ab) inhibited the Bax peptide-nucleophosmin inter-

action with an apparent IC50 of 1.66 ± 0.54 nM

(mean ± SEM; Fig. 4a). While no inhibition was observed

using an N-terminally directed antibody (N-term Ab), an

antibody raised against a-helices 4 and 5 (Ab4) partially

inhibited binding (*40%), although only at the highest

concentration tested (10 nM; Fig. 4a). Similarly the inter-

action between full length Bax and nucleophosmin was

inhibited by the C-terminal antibody (IC50 = 1.43 ± 0.65

nM) but not by either of the other two antibodies (Fig. 4b).

Data obtained from the competition studies gave Hillslope

values = 1 for Bax and nucleophosmin suggesting a

stoichiometric binding of 1:1.

Competition studies were performed using reagents

known to interact with nucleophosmin and Bax. p53, which

Apoptosis (2008) 13:394–403 397

123

binds to nucleophosmin [16], partially inhibited (by 60%)

the Bax peptide-nucleophosmin interaction (IC50 = 65 ±

24 nM; Fig. 5a). In contrast Bcl-2, a known Bax inter-

acting protein [3], had little effect on Bax peptide-

nucleophosmin binding (Fig. 5a). Similar competition

studies were performed using full length Bax protein. In

contrast to the data obtained with the C-terminal Bax

peptide, the interaction between the full-length proteins

was disrupted by both reagents, generating IC50 values of

100 nM and 200 ± 70 nM for Bcl-2 and p53 respectively

(Fig. 5b).

Identification of non-peptide inhibitors of the

Bax-nucleophosmin interaction

In addition to investigating protein–protein interactions at

the molecular level, the proximity assay can also be used as

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Fig. 1 Validation of proximity assay to analyse Bax-nucleophosmin

interaction. (a) Diagrammatic representation of the proximity assay

design to analyse the Bax-nucleophosmin interaction. Streptavidin-

coated donor beads (D) attached to a biotinylated Bax C-terminal

peptide and acceptor beads (A) conjugated with a nucleophosmin

monoclonal antibody attached to nucleophosmin. (b) and (c) Com-

petition of the interaction between streptavidin-coated donor beads

and biotin-coated acceptor beads with increasing concentration of

biotinylated Bax C-terminal peptide (0.03–3,000 nM) verified attach-

ment of the peptide to the donor beads. (d) and (e) In the presence of

streptavidin-coated donor beads and acceptor beads conjugated with a

nucleophosmin monoclonal antibody, a concentration dependent

increase in signal was observed with a biotinylated secondary

antibody (0.003–3 nM) verifying successful conjugation of acceptor

beads to the nucleophosmin monoclonal antibody

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Fig. 2 Interaction of Bax C-terminal peptide with nucleophosmin.

(a) Binding of Bax C-terminal peptide (0.3–100 nM) and constant

concentrations of nucleophosmin (5 nM, open triangles; 15 nM, filled

triangles; 150 nM, open circles and 350 nM, filled circles) demon-

strated a concentration dependent increase in signal with a maximal

response using 15 nM nucleophosmin. (b) Bax C-terminal peptide

and nucleophosmin (15 nM) interaction demonstrated a maximal

signal at 100 nM Bax. At higher concentrations of protein the

characteristic hook effect was seen due to quenching of the signal. (c)

Time course of C-terminal Bax peptide (100 nM) and nucleophosmin

(15 nM) binding measured over 4 h demonstrated equilibrium

conditions had been obtained by 90 min. Negative control reactions

contained donor and acceptor beads plus either protein alone. Error

bars represent sem; separate experiments (n = 3)

398 Apoptosis (2008) 13:394–403

123

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Fig. 3 Nucleophosmin binds to conformationally active Bax. (a)

Measurement of the interaction between full length Bax and

nucleophosmin in the presence of various detergents: 0.1% CHAPS

(filled triangles), 0.1% Tween 20 (filled circles) or 0.1% NP-40 (open

circles). Binding was only evident in the presence of the non-ionic

detergents NP-40 and Tween 20. Error bars represent sem; separate

experiments (n = 3). (b) Bax in the absence or presence of NP-40

was immunoprecipitated with the conformation specific antibody Bax

6A7 and precipitated samples Western blotted with a pan reactive

antibody BaxNT. Conformationally activated full length Bax was

only detected following treatment with NP-40

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Fig. 4 Competition of Bax-nucleophosmin interaction with Bax

antibodies. Competition studies of the interactions between (a) C-

terminal Bax peptide (100 nM) or (b) full length Bax (38 nM) and

nucleophosmin (15 nM) were performed using a N-terminally

directed Bax antibody (N-term Bax Ab; filled circles, solid line), a

C-terminally directed Bax antibody (C-term Bax Ab; filled circles

dashed line) or an antibody raised against Bax a helices 4 and 5 (Bax

Ab4; filled triangles, dotted line) over the concentration range 0.03–

10 nM. Error bars represent sem; separate experiments (n = 3)

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Fig. 5 Competition of Bax-nucleophosmin interaction with p53 and

Bcl-2. Competition studies of the interactions between (a) C-terminal

Bax peptide (100 nM) or (b) full length Bax (38 nM) and nucleo-

phosmin (15 nM) were performed using a p53 peptide (0.3–

1,000 nM, open circles) or Bcl-2 (0.3–300 nM, filled triangles). C-

terminally directed Bax antibody (C-term Bax Ab; 0.03–10 nM; filled

circles, dashed line) was included as a positive control. Error bars

represent sem; separate experiments (n = 3)

Apoptosis (2008) 13:394–403 399

123

a robust pharmacological screen with a signal to noise ratio

of 135 and a Z0 value of 0.50 ± 0.01 over 3 separate

experiments (Fig. 6a).

To identify modulators of the Bax-nucleophosmin

interaction, we evaluated a small, chemically diverse

library of 800 compounds. This screen yielded 17 com-

pounds that were either potential inhibitors or enhancers of

the Bax peptide-nucleophosmin interaction (defined as

inhibiting or enhancing the signal by [50%). The results

from a representative experiment are shown in Fig. 6b.

Putative positive compounds were further analysed over a

concentration range of 1 pM to 10 lM. One compound,

2-(5-methyl-2-phenyl-1,3-thiazol-4-yl)ethanohydrazide with

a chemical composition of C12H13N3OS and a molecular

weight of 247.3 Daltons (Da), showed inhibition similar to

that observed in the first round of screening with an IC50 of

around 100 nM although the maximal inhibition pro-

duced by this agent was approximately 50% (Fig. 6c).

Specific inhibition of the Bax-nucleophosmin interaction by

2-(5-methyl-2-phenyl-1,3-thiazol-4-yl)ethanohydrazide was

confirmed by titration of the compound over the same

concentration range 1 pM-10 uM in a streptavidin-biotin

detection assay. Inhibition was not observed in this assay

indicating there was no quench of the chemiluminescent

signal and action of the compound was specific to the Bax-

nucleophosmin interction (Fig. 6d).

Bax-nucleophosmin inhibitor blocks UV

irradiation-induced cell death

The utility of 2-(5-methyl-2-phenyl-1,3-thiazol-4-yl)etha-

nohydrazide in modulating cell death was assessed using

an in vitro model in which apoptotic cell death was

induced in HEK293 cells by exposure to UV irradiation

[17]. Twenty-four hours after exposure to 5 s of UV, cell

viability was reduced to 53.85 ± 3.29%. Treatment with

2-(5-methyl-2-phenyl-1,3-thiazol-4-yl)ethanohydrazide

0

20

40

60

80

100

120

140

160

180

TotL

NPML B2 E2 H2 C3 F3 A4 D4 G4 B5 E5 H5 C6 F6 A7 D7 G7 B8 E8 H8 C9 F9A10 D10 G10 B11 E11 H11

Buff R

Compound number

% S

ign

al

B)2-(5-methyl-2-phenyl-1,3-thiazol-4-yl)ethanohydrazide

C12H13N3OS (247.3Da)

Well Number0 10 15 20

RF

U (

x100

0)

0

20

40

60

80

100 C-terminal Bax/NPM signalBackground signal

A)

% In

hib

itio

n o

f to

tal s

ign

al

0

20

40

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80

100

120

0

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2-(5-methyl-2-phenyl-1,3-thiazol-4-yl)ethanohydrazidestreptavidin/ biotin

[Protein] Log M

-13 -12 -11 -10 -9 -8 -7 -6 -5 -4

[Protein] Log M

-13 -12 -11 -10 -9 -8 -7 -6 -5 -4

% In

hib

itio

n o

f to

tal s

ign

al

C-terminal Bax /NPM 2-(5-methyl-2-phenyl-1,3-thiazol-4-yl)

D)C)

5

Fig. 6 Identification of a partial inhibitor of the Bax-nucleophosmin

interaction. (a) Assay performance was confirmed by a Z0 value of

0.5 ± 0.01 for the proximity assay and a signal to noise ratio of 135.

Data from a representative plate are shown of experiments performed

in triplicate. (b) Bar chart showing proximity assay results from a

representative plate in the screen of a small chemical library from

which the hit compound was identified. The chemical structure,

composition and molecular weight (247.3 Da) of 2-(5-methyl-2-

phenyl-1,3-thiazol-4-yl)ethanohydrazide is shown. (c) The interaction

between Bax C-terminal peptide (100 nM) and nucleophosmin

(15 nM) was partially inhibited by 2-(5-methyl-2-phenyl-1,3-thia-

zol-4-yl)ethanohydrazide over the concentration range 1 pM-10 lM

(filled triangles, solid line) as compared to vehicle treatment (filled

circles, dashed line). (d) The interaction between streptavidin coated

donor beads and biotin coated acceptor beads was not inhibited by

2-(5-methyl-2-phenyl-1,3-thiazol-4-yl)ethanohydrazide over the con-

centration range 1 pM-10 lM (filled triangles, solid line)

400 Apoptosis (2008) 13:394–403

123

(10 lM) showed a partial but significant (P-value =

0.002, t-test) rescue of cells (by 17.85%) from the UV-

induced reduction in cell viability (Fig. 7).

Discussion

We have recently identified the nuclear chaperone nucle-

ophosmin as a Bax-binding protein and demonstrated that

in animal models of stroke, nucleophosmin moves from the

nucleolus to the cytosol at a time when Bax is conforma-

tionally active, before it translocates to the mitochondria to

induce apoptotic cell death [9]. We have now developed a

proximity assay which has enabled us to characterise this

novel protein–protein interaction at the molecular level.

The proximity assay demonstrated that nucleophosmin

bound to a peptide corresponding to the Bax C-terminal

domain of the native protein. This region is normally

sequestered within the structure of cytosolic Bax and

therefore not available for binding [14]. In addition, no

interaction between full length Bax and nucleophosmin

was observed unless the detergents NP-40 or Tween 20

were included in the reaction buffer. The non-ionic deter-

gent NP-40 has previously been shown to induce a

conformational change in Bax [15] and we confirmed this

by immunoprecipitation with the conformation-sensitive

Bax antibody 6A7. The 6A7 antibody recognises an epi-

tope at the N-terminus of Bax (amino acids 12–24) which

is exposed when the conformation of Bax changes

following treatment with certain detergents or after the

induction of apoptosis [14]. The full length Bax-nucleo-

phosmin interaction was not disrupted by an antibody

raised against the N-terminus of Bax although it was dis-

rupted by an antibody raised against the Bax C-terminus. It

is less clear why very high concentrations of an antibody

recognising a-helices 4 and 5 caused a modest reduction;

steric hindrance is unlikely as antibodies of a similar size

did not produce a similar effect at this concentration.

However recent data have also demonstrated a role for Bax

a-helices 5 and 6 in the interaction of Bax with mito-

chondrial membranes [18]. Only the C-terminal Bax

antibody markedly inhibited the Bax-nucleophosmin

interaction. Taken together our data suggest that nucleo-

phosmin only binds to conformationally altered (activated)

Bax via its C-terminal domain which is normally inacces-

sible in cytosolic Bax.

The importance of helix a9, the C-terminal domain in

the targeting of Bax to the mitochondria following acti-

vation remains controversial. Following an apoptotic

stimulus, Bax undergoes conformational change [19] and it

has been proposed that exposure of the N- and C-terminal

domains of Bax occurs before targeting to the mitochondria

is mediated by the C-terminal domain alone [20]. Several

studies support this theory; GFP-tagged C-terminal trun-

cated Bax expressed in COS cells does not translocate to

mitochondria following apoptosis [19] whereas a Bax

polypeptide devoid of the N-terminal ART domain (Bax-

DART) is constitutively targeted to the mitochondria [21].

Furthermore mutation of Ser184 to Val (S184V), deletion

of the Ser184 (DSer184) altogether in the C-terminal

domain of full length Bax or a 21 amino acid C-terminal

peptide are also sufficient to induce targeting to the mito-

chondria [22]. The interaction of nucleophosmin with

mutated Bax peptide (S184V) in a similar proximity assay

[9] is consistent with the importance of amino acid residues

in the C-terminus region for biological activity.

Recombinant human Bcl-2, a known Bax-binding pro-

tein [23, 24], blocked the interaction between full length

Bax and nucleophosmin but did not affect the binding

between the Bax C-terminal peptide and nucleophosmin.

These data suggest that Bcl-2 binds to a domain which is

absent from the C-terminal Bax peptide but present in the

full length protein such as the BH3 domain or N-terminal

region and are consistent with data in the literature as the

BH3 domain of Bax has been shown to be essential for

binding to Bcl-2 [25]. The role of the Bax BH3 domain (a-

helix 2) in binding to nucleophosmin is still unclear as we

were unable to source a suitable antibody against this

region to perform inhibition studies and it is possible that

the block observed using Bcl-2 was due to steric hindrance

of another binding site. In contrast to the data obtained with

Bcl-2, a peptide fragment of p53, a known nucleophosmin-

rotibihnIelciheVrotibihnIelciheV

Cel

l Su

rviv

al (

%)

0

02

04

06

08

001

021

VUoN VUsceS5

*

* p 200.0=

Fig. 7 Attenuation of UV-induced cell death in HEK293 cells by

2-(5-methyl-2-phenyl-1,3-thiazol-4-yl)ethanohydrazide. HEK293

cells were treated with vehicle or inhibitor (2-(5-methyl-2-phenyl-

1,3-thiazol-4-yl)ethanohydrazide). Cell death was induced using UV

irradiation (30 J/m2, 5 s) and cell viability was measured by MTS

assay 24 h later. Following UV treatment, cell viability was reduced

to 53.85 ± 3.29% in vehicle treated cells. However a 17.85%

increase in survival was observed in those cells treated with inhibitor

compared to vehicle following UV irradiation (P-value = 0.002,

t-test). Survival was expressed as a percentage relative to untreated

cells. Error bars represent sem; separate experiments (n = 3)

Apoptosis (2008) 13:394–403 401

123

binding protein [16], disrupted the interaction between

nucleophosmin and both forms of Bax. The p53 binding

region is located in the C-terminal half of nucleophosmin

[16, 26] and recent data produced using chemical cross-

linking, proteolytic digestion and mass spectrometry-based

peptide mapping have narrowed this down to residues 249–

262 [27]. This p53-binding region of nucleophosmin

overlaps with the immunizing peptide (residues 226–294)

used to raise the monoclonal antibody [28] which we used

to attach nucleophosmin to the acceptor beads in our

proximity assay. Therefore it is likely that the p53 peptide

competed for nucleophosmin binding at the C-terminal

domain attached to the acceptor beads rather than the Bax-

nucleophosmin protein–protein interaction. As a proximity

assay signal was generated with Bax when nucleophosmin

was attached to the acceptor beads by its C-terminal

domain, our data also implicate the N-terminal half of

nucleophosmin as important for Bax binding. This region

contains the signals required for nucleophosmin localiza-

tion to the nucleolus and sequences essential for its

chaperone activity [29, 30] consistent with our identifica-

tion of nucleophosmin as a Bax chaperone which

translocates from the nucleolus to the cytoplasm following

apoptotic stimuli [9, 31].

Recently identified chaperones that regulate the target-

ing of Bax to the mitochondria bind to either the N- or C-

terminus and these include humanin, 14-3-3, Ku70 and

clusterin [32–35]. As with nucleophosmin, clusterin inter-

acts with a conformation-altered Bax, but to prevent

apoptotic activity [35]. Data published from this lab sug-

gests nucleophosmin is a regulator of Bax mediated cell

death both in vitro and in vivo [9]. Following stauro-

sporine treatment of SH-SY5Y cells, nucleophosmin was

localised in the cytosol prior to Bax mitochondrial trans-

location. In vivo, focal cerebral ischaemia led to

nucleophosmin translocation and Bax conformational

change [9]. Nucleophosmin acts as a chaperone to regulate

the stability and transcriptional activation of ARF and p53

in another apoptotic pathway [16, 36–38]. Binding of ARF

by nucleophosmin activates mdm2 causing p53 to be down

regulated resulting in cell proliferation [38]. However in

the absence of nucleophosmin, ARF binds to mdm2 caus-

ing p53 accumulation and cell cycle arrest via downstream

targets such as BH3 only proteins and Bax [39].

The proximity assay technology used to characterise the

molecular interplay between Bax and nucleophosmin, can

also be used as a high-throughput pharmacological screen for

inhibitors/modulators of this interaction. The reliability of this

384-well assay is reflected by a signal to noise ratio of 135, an

inter-plate variance of 14.8% (across days) and a Z0 value of

0.50 which puts this assay at the cusp between marginal and

excellent as a high-throughput screen [40]. A manual screen of

a small chemical library (800 compounds) identified 17

potential compounds that modulated Bax peptide-nucleo-

phosmin binding either by inhibiting or enhancing the signal

by 50%. One hit compound, 2-(5-methyl-2-phenyl-

1,3-thiazol-4-yl)ethanohydrazide, produced a concentration-

dependent partial inhibition of the Bax-nucleophosmin

interaction with an IC50 value of 100 nM. The success rate is

supportive of extending the analysis to the major chemical

libraries of the pharmaceutical industry which contain more

than 250,000 diverse compounds. Moreover, this compound

partially reversed the cell death induced by UV treatment of

HEK293 cells. We previously demonstrated that RNAi

knockdown of nucleophosmin expression protected against

staurosporine-induced apoptosis and that the level of protec-

tion was proportionate to the reduction in expression levels

[9]. Together these data suggest that the Bax-nucleophosmin

interaction may be involved in the response to different

apoptotic stimuli. Thus the Bax-nucleophosmin protein–pro-

tein interaction may represent a novel therapeutic target for the

numerous disease states associated with dysregulated

apoptosis.

In conclusion, the present report provides compelling

new data in three aspects of the interaction between Bax

and nucleophosmin:

(1) The kinetics of the binding of Bax and nucleophos-

min are indicative of a competitive and specific

biological mechanism.

(2) The binding of full length Bax to nucleophosmin

requires the conformational change which Bax under-

goes early in the apoptotic cascade, exposing the

C-terminal region.

(3) The proximity assay described provides a drug

discovery platform for the identification of low

molecular weight modulators of apoptosis.

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