Arsenic trioxide targets Hsp60, triggering degradation of p53 and survivin

Article abstract of DOI:10.1039/d1sc03119h

The mechanisms of action of arsenic trioxide (ATO), a clinically used drug for the treatment of acute promyelocytic leukemia (APL), have been actively studied mainly through characterization of individual putative protein targets. There appear to be no studies at a system level. Herein, we integrate metalloproteomics through a newly developed organoarsenic probe, As-AC (C20H17AsN4O3S2) with quantitative proteomics, allowing 37 arsenic binding and 250 arsenic regulated proteins to be identified in NB4, a human APL cell line. Bioinformatics analysis reveals that ATO disrupts multiple physiological processes, in particular, chaperone-related protein folding and cellular response to stress. Furthermore, we discover heat shock protein 60 (Hsp60) as a vital target of ATO. Through biophysical and cell-based assays, we demonstrate that ATO binds to Hsp60, leading to abolishment of Hsp60 refolding capability. Significantly, the binding of ATO to Hsp60 disrupts the formation of Hsp60-p53 and Hsp60-survivin complexes, resulting in degradation of p53 and survivin. This study provides significant insights into the mechanism of action of ATO at a systemic perspective, and serves as guidance for the rational design of metal-based anticancer drugs. This journal is

Full text of DOI:10.1039/d1sc03119h

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Arsenic trioxide targets Hsp60, triggering
degradation of p53 and survivin†
Cite this: DOI: 10.1039/d1sc03119h
Xuqiao Hu,‡a Hongyan Li,‡a Tiffany Ka-Yan Ip,a Yam Fung Cheung,a Mohamad Koohi-
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
Moghadam,ad Haibo Wang,a Xinming Yang,a Daniel N. Tritton,a Yuchuan Wang,a
a
ae
b
Yi Wang,
Eric Wai-Choi Tse
Runming Wang,a Kwan-Ming Ng,
Hua Naranmandura,
c
a
*
and Hongzhe Sun
The mechanisms of action of arsenic trioxide (ATO), a clinically used drug for the treatment of acute
promyelocytic leukemia (APL), have been actively studied mainly through characterization of individual
putative protein targets. There appear to be no studies at a system level. Herein, we integrate
metalloproteomics through a newly developed organoarsenic probe, As-AC (C20H17AsN4O3S2) with
quantitative proteomics, allowing 37 arsenic binding and 250 arsenic regulated proteins to be identified
in NB4, a human APL cell line. Bioinformatics analysis reveals that ATO disrupts multiple physiological
processes, in particular, chaperone-related protein folding and cellular response to stress. Furthermore,
we discover heat shock protein 60 (Hsp60) as a vital target of ATO. Through biophysical and cell-based
assays, we demonstrate that ATO binds to Hsp60, leading to abolishment of Hsp60 refolding capability.
Significantly, the binding of ATO to Hsp60 disrupts the formation of Hsp60-p53 and Hsp60-survivin
complexes, resulting in degradation of p53 and survivin. This study provides significant insights into the
mechanism of action of ATO at a systemic perspective, and serves as guidance for the rational design of
metal-based anticancer drugs.
Received 9th June 2021
Accepted 15th July 2021
DOI: 10.1039/d1sc03119h
rsc.li/chemical-science
acting on the two fusion partner.7 However, cytotoxicity of ATO
was also found for the system that lacks the PML-RARa fusion
protein,4 suggesting other biological targets may also be
Introduction
Arsenic trioxide (ATO) is an effective chemotherapeutic drug
either used alone or in combination therapy for the treatment of
acute promyelocytic leukemia (APL).1–3 It also shows efficacy
against other hematologic malignancies and other solid
tumors.4,5 It has been demonstrated that arsenic facilitates
profound cellular alteration including induction of apoptosis,
inhibition of proliferation, stimulation of differentiation and
inhibition of angiogenesis.6 The molecular mechanism of
action of ATO (i.e., arsenite) for APL has long been attributed to
degradation of the disease-causing oncoprotein, PML-RARa by
accountable for its efficacies. Given the thiophilic nature of
arsenic, ATO binds large numbers of proteins,8 and differenti-
ating key targets from these proteins remains to be challenging.
Although enormous efforts have been made towards validation
of potential targets of ATO on individual proteins, including
hexokinase-2 (HK2),9 peptidyl-prolyl cis–trans isomerase NIMA-
interacting 1 (PIN1),10 MTHFD1 (ref. 11) and mutated NPM1,12
such studies are unlikely to provide a systemic view on the
mechanism of action of ATO.
Proteomics has been well adapted to track proteins regulated
by metallodrugs.13 Previously, global proling of arsenic-
binding proteins has been performed using a human pro-
teome microarray, allowing 360 arsenic-binding proteins to be
identied.9 However, these proteins may not be authentic
arsenic binding proteins in live cells. Owing to the complexity of
arsenic–protein interactions in cells, it is a considerable chal-
lenge to track arsenic binding proteins particularly in live cells.
Various approaches have been employed to mine arsenic-
binding proteins, among which small-molecule-based uores-
cent probes have been used in the detection and visualization of
proteins owing to their less-obvious steric effects and fast
labeling properties.14 For example, biarsenical uorescent
probes e.g. FlAsH-EDT2, which was initially designed to image
aDepartment of Chemistry and CAS-HKU Joint Laboratory of Metallomics on Health
and Environment, The University of Hong Kong, Hong Kong SAR, P. R. China.
E-mail: hsun@hku.hk
bDepartment of Toxicology, School of Medicine and Public Health, Zhejiang University,
Hangzhou, P.R. China
cDepartment of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong
Kong, Queen Mary Hospital, Hong Kong, P. R. China
dDivision of Applied Oral Sciences and Community Dental Care, Faculty of Dentistry,
University of Hong Kong, Hong Kong SAR, P. R. China
eDepartment of Chemistry, Shantou University, Shantou, Guangdong, 515063, P. R.
China
† Electronic supplementary information (ESI) available: Experimental procedures,
supplementary gures, identied proteomes and bioinformatics data. See DOI:
10.1039/d1sc03119h
‡ These authors contribute equally to this work.
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proteins with genetically fused tetracysteine motifs (CCXXCC)
in live cells,15 were also immobilized to beads or resin with some
modication for specic capture arsenic-binding proteins.16
However, this type of probe is unable to identify proteins with
spatially cysteine-rich regions or with even one critical
cysteine.17 For phenylarseneoxide (PAO) based uorescent
probes,18 such as FAsH, although they could image vicinal
dithiol rich proteins, downstream identication of these
proteins might result in potential dissociation of the probe from
proteins of interest. Affinity chromatography-based approaches
have also been employed to site-specically label large libraries
of arsenic binding proteins.19
Herein, we design and synthesize a new uorescent probe
AsIII-AC, via conjugation of an arsenic moiety with a uorophore
and an arylazide. The probe can rapidly enter cells to label
intracellular arsenic binding proteins. An arylazide was incor-
porated into the probe, enabling the labelled proteins to be
anchored upon photoactivation and subsequently identied
through high-throughput proteomics. By integration with
quantitative proteomics, we comprehensively identify 37 As-
binding and 250 As-regulated proteins in live NB4 cells.
Subsequent bioinformatics analysis reveals 10 hub proteins as
potential key targets of ATO. We further validate that ATO binds
to Hsp60 (HSPD1), abolishing its folding/refolding activity.
Signicantly, such a binding disrupts the complexes of Hsp60-
p53 and Hsp60-survivin, leading to degradation of p53 and
survivin, which contributes to ATO-induced apoptosis of
leukemia cells. Hsp60 therefore serves as an important target of
the anti-leukemia drug.
Fig. 1 (a) Synthesis of As-AC. (b) Excitation (lex ¼ 355 nm) and emis-
sion (lem ¼ 448 nm) spectra of As-AC.
We then examined the feasibility of selective labelling and
identication of arsenic-binding proteins by As-AC in vitro.
Incubation of 10 molar equivalents of cysteine-containing
proteins e.g. BIR3 or SlyD (Fig. S5 and Table S1†) with As-AC
(1 mM) led to a time-dependent increase in uorescence inten-
sity upon photo-activation, with a plateau reached at around
10 min, where ca. 8-fold increase in uorescence intensity was
observed (Fig. 2a and S6†), possibly attributable to the change of
the conjugated environment.25 In contrast, nonspecic binding
of As-AC to proteins that have no cysteines e.g. SlyDDC
(Fig. S7a†) and ubiquitin (Fig. S7b†) resulted in only 3-fold
uorescence changes. Other thiol-containing small molecules
e.g. glutathione (GSH), 1,4-dithiothreitol (DTT), 2-mercaptoe-
thanol (b-ME) or cysteine (Fig. S7c†) also exhibited negligible
effects on protein labelling by the probe. The mixtures of As-AC
with BIR3 or ubiquitin were irradiated under UV light (365 nm)
for ca. 20 min to achieve the maximum uorescence intensity
then subjected to SDS-PAGE analysis and uorescence imaging.
We observed an intense blue uorescence band at a molecular
weight of 10 kDa, corresponding to BIR3 but not ubiquitin in
the uorescence image (Fig. 2b). Binding of BIR3 to AsIII (as ATO
or As-AC) was further conrmed by MALDI-TOF mass spec-
trometry. As shown in Fig. 2c, peaks at m/z of 10 987, 11 058 and
11 365 correspond to the intact BIR3 protein, the protein with
one arsenic bound (calcd 11 059) and the protein with one
probe bound (calcd 11 365). Collectively, we demonstrate that
the probe selectively targets arsenic binding-proteins, and the
arylazide is critical for signicant uorescence turn-on upon UV
activation (Fig. S8†).
Results and discussion
Synthesis of an organoarsenic uorescent probe and systemic
identication of AsIII-binding and -regulated proteins in NB4
cells
We design an organoarsenic probe As-AC (1) consisting of an
arsenic moiety, coumarin as a uorophore and arylazide. By
taking the advantage of arylazide, the labelled arsenic binding
proteins could be subsequently identied via conventional pro-
teomics.20,21 We rst synthesized 2-p-aminophenyl-1,3,2-
dithiarsenolane (3) from p-arsanilic acid (6) according to a previ-
ously reported method.22 The azido compound (2) was synthe-
sized from 7-amino-4-methyl-3-coumarinylacetic acid using the
procedure developed previously.21 Finally, (2) and (3) were
conjugated together by EDCI (1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide) coupling to afford the coumarin azide-based
probe As-AC (1) (Fig. 1a and Schemes S1–S3†). The purity of As-
AC was conrmed by 1H and 13C NMR (Fig. S1 and S2†) and
electrospray ionization mass spectrometry, which gave rise to
a peak at m/z of 522.60 assignable to [M + Na]+ (calcd 523.00)
(Fig. S3†). As-AC exhibits a maximum absorption at 355 nm and
emits at 448 nm (Fig. 1b) with a quantum yield (FF) of 0.050
under physiologically relevant conditions. The low quantum yield
of As-AC is attributed to the presence of an azide at the seventh
position of the coumarin moiety, which quenches the uores-
cence of coumarin.23,24 Negligible toxicity of the probe in
mammalian cells was noted (Fig. S4†).
We then evaluated whether the probe could selectively label
arsenic binding proteins in live cells. The probe (1 mM) was rst
incubated with E. coli cells overexpressing either SlyD, SlyDDC
or SlyD CmutA, where ve cysteine residues were mutated into
alanines, then subjected to UV irradiation prior to SDS-PAGE
analysis. An intense blue band was observed on the uores-
cence image only for cells overexpressing SlyD, but not SlyDDC
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irradiation at 365 nm for 20 min prior to lysis, subsequently the
labelled proteins were separated by 2D gel electrophoresis and
then identied through peptide mass ngerprinting (Fig. S10†).
In total, 37 and 31 putative arsenic binding proteins were
identied by the As-AC probe from NB4 and HL60 respectively
(Tables S2 and S3†), among which 21 mutual proteins were
found in both cells. The Gene Ontology (GO) enrichment anal-
ysis illustrated different functional and component of cate-
gories of the non-mutual proteins (Fig. S11†), demonstrating
the utility of the probe in proling arsenic binding proteins in
cells.
Amongst 37 identied As-binding proteins in NB4 cell, ten
proteins have also been identied previously by other tech-
niques (Table S2,† highlighted in red).19,26 Further analysis of
the structures of newly identied arsenic binding proteins
revealed that majority proteins contain Cys–Cys pairs, which
likely coordinate to arsenic, including TPI (pdb: 4POC); CLIC1
(pdb: 1K0O); VCP (pdb: 3HU2) etc. However, some proteins e.g.
NPM1 (pdb: 5EHD); HSPA9 (pdb: 6P2U) contain no Cys–Cys
pairs. Despite of its thiophilic nature, arsenic was also shown
previously to coordinate to histidine and serine in the crystal
structures.27,28 This suggests conventional “so-hard’’ theory
might not be applicable to proteins.
To verify the identied proteins are indeed authentic As-
binding proteins, we overexpressed and puried two proteins
i.e., GSTP1 and PPA1 (ESI†) and examined their interaction with
ATO by MALDI-TOF mass spectrometry. As shown in Fig. S12,†
one and up to six As(III) ions were found to bind GSTP1 and
PPA1 respectively. To further validate that these proteins bind to
ATO in cellulo, we selected 8 putative AsIII binding proteins, i.e.,
TPT1, EEF1B2, ENO1, NPM1, VCP, IMPDH2, LDHB and NME1
for verication by cellular thermal shi assay (CETSA),
a method that is widely used to investigate target engagement of
drug candidates in a cellular context.29 Supplementation of
1 mM ATO to the cell culture medium for 24 h caused thermal
melting curves of these proteins shied evidently (Fig. S13†),
indicative of the binding of ATO to these proteins in cellulo.
To prole the intact proteome of leukemia in response to
arsenic trioxide, ATO-treated and non-treated NB4 cell extracts
were compared in a TMT quantitative experiment. The differ-
ences of protein expression between the control and ATO-
treated groups were quantied by LC-MS/MS. 870 distinct
proteins were identied from biological triplicate runs by at
least two high-condence peptides per protein with a con-
dence level of 95% and global false discovery rate (FDR) of less
than 1%. In total, 250 proteins including 141 up-regulated and
109 down-regulated proteins were identied as signicant
differentially expressed proteins (p <0.05) with >1.3-fold or
<0.77-fold change in abundance compared to the control group
(ESI Data 1†).
Fig. 2 Feasibility of labelling As-binding proteins by As-AC. (a) Fluo-
rescence spectra of As-AC (1 mM) at different time intervals after
addition of BIR3 (10 mM). Inset: time-dependent fluorescence changes
(lem ¼ 448 nm) of As-AC upon binding to BIR3 protein. (b) Selectivity
of As-AC towards BIR3 and ubiquitin (Ub) examined by SDS-PAGE after
UV activation. The fluorescence image and SDS-PAGE are also shown.
(c) MALDI-TOF-MS spectra of BIR3 protein (30 mM) in the absence and
presence of 4 molar equivalents of AsIII (as ATO) and As-AC. (d) and (e)
Fluorescence image and SDS-PAGE showing that As-AC selectively
labels overexpressed SlyD but not SlyDDC and SlyD CmutA in E. coli.
KML603 cells without (control) and with overexpressed SlyD, SlyDDC
or SlyD CmutA were incubated with As-AC (10 mM) under dark for
30 min at 37 ꢀC, followed by UV activation, lysed, then subjected to
13% SDS-PAGE gel analysis. (f) MALDI-TOF-MS spectra of SlyD (30 mM)
in the absence and presence of 4 molar equivalents of AsIII (as ATO)
and As-AC. (g) Confocal images of As-AC labelled HeLa cells pre-
incubated with different concentrations of AsIII (as ATO). Scale bar: 20
mm. (h) Relative fluorescent intensity plotted against different
concentrations of ATO pre-incubated with the cells.
or SlyD CmutA (Fig. 2d and e), conrming that the probe
entered bacterial cells and labeled SlyD. Binding of AsIII (as ATO
or As-AC) was further conrmed by MALDI-TOF mass spec-
trometry. As shown in Fig. 2f, the peaks at m/z of 21 237, 21 312
and 21 616, correspond to the intact protein, the protein with
one arsenic bound (calcd 21 309) and the protein with one
probe bound (calcd 21 615) respectively. In contrast, negligible
uorescence intensities were observed for the control groups
(Fig. 2d and e). Similar experiments were performed with HeLa
cells and we observed intense and faint blue uorescence
signals in the cytoplasm and nuclear region of HeLa cells
respectively (Fig. S9†). Pretreatment of the cells with increasing
amounts of ATO for 1 hour prior to As-AC probe led to dose-
dependent decrease in the uorescence intensity (Fig. 2h),
suggesting that ATO competes with As-AC in protein binding.
We nally applied the probe to NB4 cells and another human
leukemia cell line HL60 for comparison, to track arsenic
binding proteins at a proteome-wide scale. The cells were
treated with 10 mM As-AC for 30 min, then subjected to UV
Bioinformatics analysis of arsenic-associated proteins
We used ClueGO to build gene ontology (GO) analysis network
for the 37 AsIII-binding proteins. From the network, we found
As-binding proteins were functionally arranged into 6 clusters
and the most signicantly enriched clusters were proteins
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including HSPD1, HSPA5, HSPA8, HSPA9, LAMP1, HNRNPA1,
CASP3, RPS6, ACTB and CCT5 (Fig. 3c), which are likely served
as key targets of ATO. Amongst these hub proteins, four of them
are referred to heat shock proteins (HSP) superfamily, which
were highly expressed in acute myeloid leukemia and have been
utilized as targets for the development of therapeutics against
AML.31 Ribosomal protein (RPS) is responsible for protein
synthesis in living cells.32 Mutations of this ribosomal protein
family genes are of high risk in developing leukemia.33 Caspase
3, activated by ATO, plays a central role in the execution-phase
of cell apoptosis as well as progression of acute myeloid
leukemia.34 Other hub proteins, e.g. ACTB,35 LAMP1,36
HNRNPA1 have been reported to be involved in cancer cell
development, invasion, progression and chemoresistance.37
Amongst the 10 hub proteins identied in the AsIII-associated
PPI, HSPD1 (Hsp60) appears to be the most up-regulated
arsenic binding protein (Fig. S14†). Hsp60 has been previ-
ously exploited as a potential target for cancer therapies.38,39
Further analysis of the protein–protein interaction network of
Hsp60 (Fig. 3d) revealed that Hsp60 has direct interaction with
two important apoptosis related proteins i.e., BIRC5 (survivin)
and TP53. We therefore selected Hsp60 for further investigation
to see what relevant biological pathways are disrupted by ATO.
Fig. 3 Bioinformatics analysis of arsenic associated proteins. (a) GO
enrichment generated by ClueGO for 37 As-binding proteins detected
by As-AC probe. Colors represent different GO clusters. Each node
represents a GO biological process and the edges show the connec-
tivity between each node based on the connection of the proteins in
their biological pathways. The size of the nodes depends on the
number of proteins grouped. (b) GO enrichment analysis of proteins
differentially regulated by ATO. Genes with p-value <0.05 were
selected. ꢂlog 10 of the enrichment p-values for selected GO cate-
gories (biological process, molecular function) are plotted relative to
z-scores of differential expression ratios in each category. Those
nodes with ꢂlog(p-value) >10 are labelled. Circle size denotes the
number of genes in each group. (c) As-associated protein–protein
interactions (AsPI) network in NB4 cells. Proteins are colored and
shaped according to their different properties in the network. The
central nodes are represented by those of larger size with high degree
and betweenness centrality (BC) values within the top 10% of the total
nodes. (d) PPI networks projecting the proteins functionally associated
with Hsp60 (HSPD1).
ATO binds to Hsp60 and inhibits its activity
Hsp60 protein was overexpressed in E. coli (DE3) and puried
with high purity being conrmed by SDS-PAGE (ESI and
Fig. S15a†) and size exclusion chromatography (Fig. S15b†).
Hsp60 was incubated with six molar equivalents (eq.) of ATO
and then subjected to a home-made GE-ICP-MS for simulta-
neous analysis of metals/metalloids and its bound protein.40
127I-labeled proteins were used as an internal standard to cali-
brate the molecular weight (MW) and Hsp60 without treatment
of ATO was used as a control. Incubation of ATO to Hsp60
resulted in appearance of a single 75As peak at the MW of ca. 60
kDa corresponding to a monomer of AsIII-bound Hsp60
(Fig. 4a), suggesting that AsIII binds to Hsp60 in vitro. The
stoichiometry of AsIII to Hsp60 was determined to be 1.90 ꢃ 0.11
by ICP-MS through quantication of 34S for protein concentra-
tion and 75As in ATO-treated Hsp60, aer removal of excess non-
bound arsenic ions by size exclusion chromatography
(Fig. S16†). Given that arsenic is highly thiophilic, we then
examined whether the three Cys residues (Cys213, Cys418 and
Cys423) are involved in arsenic binding by the measurement of
free thiols of Hsp60 pre-incubated with different molar ratios of
ATO using Ellman's assay. As shown in Fig. 4b, about 3 eq. of
ATO could completely deplete free thiols, indicating that
cysteine residues are involved in arsenic binding. Collectively,
we demonstrate that arsenic binds to Hsp60 mainly via cysteine
residues. We then examined whether ATO binds to Hsp60 in
cells by cellular thermal shi assay (CETSA).29 NB4 cells was
exposed to 1 mM ATO (IC50 ¼ 0.9 mM, 24 h) for 24 h, and then
subjected to CETSA according to a previous protocol.41 Treat-
ment of ATO led to a shi of melting point from 60.6 ꢀC to
62.6 ꢀC (Fig. 4c), indicative of cellular engagement of AsIII to
Hsp60 in NB4 cells.
related to chaperone cofactor-dependent protein refolding (GO:
005108, p ¼ 6.75 ꢁ 10ꢂ7) and acute phase response (GO:
000695, p ¼ 1.05 ꢁ 10ꢂ3) (Fig. 3a and ESI Data 2†). In particular,
chaperone proteins, including HSPA5, HSPA8, HSPA9, HSPD1,
ST13 that signicantly enriched in the former GO term were
largely related to the response to stress.30 Then we performed
GO enrichment analysis for 250 arsenic-regulated proteins
identied by quantitative proteomics. GOPlot was used to
perform analysis and those GO terms with p-value <0.05 were
selected for further analysis. As shown in Fig. 3b, the proteins
that were most enriched in nine GO terms with the best z-scores
and most signicant p-values are involved in translational
initiation, viral transcription, protein folding, ER translocation,
nonsense-mediated mRNA decay, rRNA processing, RNA
binding, protein binding and unfolded protein binding, indic-
ative of the general functional disruption in NB4 under ATO
stress (Tables S4–S6†).
To further functionally categorize the identied arsenic-
associated proteins i.e., 37 arsenic-binding and 250 arsenic-
regulated proteins,
a
protein–protein interaction (PPI)
network was generated. The top 10 proteins with the highest
betweenness centrality scores were selected as hub proteins
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apoptosis by formation of Hsp60-survivin and Hsp60-p53
complexes, which will stabilize survivin levels and restrain the
function of p53.44 The wild-type p53 is a tumor suppressor,45
involving in different types of leukemia diagnosis and
therapy.46,47 Survivin, belonging to the family of inhibitor of
apoptosis protein (IAP), has been considered as an attractive
candidate for cancer therapy.48
We then asked whether binding of ATO to Hsp60 disrupts
the Hsp60-survivin or Hsp60-p53 complexes, resulting in the
release of p53 and survivin, which could be degraded rapidly by
a protease. We performed co-immunoprecipitation assay for
endogenous proteins in NB4 cell lysates. In ATO un-treated
control, immunoprecipitated survivin from NB4 cell lysates
contained co-associated Hsp60, indicative of formation of
a complex between Hsp60 and survivin (Fig. 5a and S17†).
Similarly, Hsp60-p53 complex was also detected by co-
immunoprecipitation in the cell lysates. However, upon treat-
ment of the cell with increasing amounts of ATO, the co-
associated Hsp60 in these protein complexes decreased
(Fig. 5b and S17†), conrming that ATO disrupts the ability of
Hsp60 to recruit target proteins, i.e., p53 and survivin.
Fig. 4 Arsenic binds and functionally disrupts Hsp60. (a) GE-ICP-MS
profiles of apo-Hsp60, ATO treated Hsp60, and 127I-labeled internal
standards. (b) Free thiol contents in Hsp60 after treatment with
different concentrations of ATO measured by Ellman's assay. Inset
shows the absorbance at 412 nm vs. amounts of ATO. (c) Thermal
melting curves of Hsp60 protein in intact leukemia cells, treated with
or without arsenic trioxide. Data are presented as the mean SEM from
at least three independent experiments. (d) Effect of ATO on ATPase
activity of Hsp60. ATP content was determined by Malachite Green
Phosphate Assay Kit (POMG-25H) after incubation of Hsp60 (2 mM)
with or without ATO for 30 min at 37 ꢀC. (e) ATO inhibits Hsp60/Hsp10
refolding activity. Time-dependent refolding activity of Hsp60/Hsp10
in the presence of gradient concentrations of ATO.
We further examined whether ATO affected the co-
localization of Hsp60 with survivin or p53 in NB4 cells by
Hsp60 is an ATP-dependent protein that facilitates the folding
of a wide range of substrates to the native state and prevents their
aggregation.42 ATPase is essential for Hsp60 to conduct its activity
either by recruiting its co-chaperonin Hsp10 or releasing protein
aer nal folding and assembly.43 We rst examined whether
binding of arsenic inhibited the ATPase activity of Hsp60 by a well-
established in vitro assay. As shown in Fig. 4d, ATPase activity of
Hsp60 was suppressed by 59.0% at 8 mM ATO. We then examined
the refolding activity of Hsp60 using a typical chaperone assay, in
which the substrate protein was rst denatured chemically. The
refolding activity of Hsp60/Hsp10 was then monitored both in the
absence and presence of gradient amounts of ATO. In the absence
of ATO, the denatured substrate was refolded effectively by Hsp60/
Hsp10 in a time-dependent manner, and ca. 80% of the substrate
protein was fully-folded in around 60 min. The presence of ATO
signicantly reduced refolding activity of Hsp60/Hsp10. As shown
in Fig. 4e, Hsp60/Hsp10 could only recover 34.3%, 25.8% and
12.3% of the substrate activity in the presence of 1.6, 4.0 and 8.0
mM ATO respectively. Our combined data conrm that binding of
ATO to Hsp60 attenuates ATPase activity and abolishes its refold-
ing activity.
Fig. 5 Binding of arsenic to Hsp60 disrupts complexation of Hsp60
with p53 and survivin, leading to degradation of p53 and survivin. (a)
and (b) IP-western blots of endogenous Hsp60 associated with sur-
vivin or p53 in NB4 cells treated with ATO at different concentrations.
(c) and (d) Immunolocalization of endogenous Hsp60 with p53 and
survivin. Co-staining was performed using antibodies specific for
Hsp60 (green), survivin (red), p53 (red) and a fluorochrome conjugated
secondary antibodies against rabbit or mouse IgG. Cells treated with
ATO (1.2 mM) or without (as control, upper panel) were analyzed using
confocal microscopy (ꢁ63 oil lens). The images were visualized with
a Zeiss microscope. Scale bars: 4 mm. (e) and (f) Arsenic mediates
proteasome degradation of p53 and survivin. Treatment of NB4 cells
with MG132, a 26S proteasome inhibitor prevents arsenic induced
protein degradation in cells. Western blots were prepared with the
extracts of NB4 cells untreated or pretreated with 25 mM MG132 for 4 h
in the absence and presence of 0.8 mM As2O3, b-actin was used as
control. Protein contents were quantified by densitometric analysis.
Means ꢃ SEM; n ¼ 3; *p <0.05, **p <0.01 versus vehicle control. (g)
Proposed the mechanism of ATO induced apoptosis through inhibi-
Binding of ATO to Hsp60 disrupts the complexes of p53-
Hsp60 and survivin-Hsp60, leading to degradation of p53 and
survivin
The protein–protein interaction network shows that Hsp60
(HSPD1) functionally associated with BIRC5 (SVV family) and
TP53 (P53 family) (Fig. 3d). Hsp60 regulates tumour cell tion of key target Hsp60 and degradation of survivin and p53.
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immunouorescence. In the control group, both Hsp60 and proteomics, As-binding and As-regulated proteins were tracked
p53, which were immobilized with anti-Hsp60 antibody (green) at a proteome-wide scale.
and anti-p53 antibody (red), were evenly distributed in the
Bioinformatics analysis of these arsenic-associated proteins
cellular compartment with certain co-localization (yellow) of (and proteome) and their PPI networks allowed us to uncover
Hsp60 and p53 (Fig. 5c). Upon exposure to ATO, Hsp60 tends to ten central nodes in the AsIII–protein-interaction (API) network
be non-dispersed and concentrated in certain part of the cell, with seven of them being As-binding proteins in leukemia cells.
whereas p53 concentrated in different part of the cell and co- We show that Hsp60 binds to ATO both in vitro and in cellulo.
localization of the two proteins was hardly observed. Simi- Such a binding disrupts its folding/refolding activity together
larly, survivin is evenly distributed inside cells in the control with Hsp10 owing to abolished ATPase activity of Hsp60 by ATO.
group, whereas co-localization of Hsp60 and survivin was only By using immunoprecipitation and immunouorescence, we
observed in the control but not ATO-treated cells (Fig. 5d). demonstrate that treatment of ATO to NB4 cells resulted in
These data demonstrate that binding of ATO to Hsp60 results in dissociation or disrupting the formation of Hsp60-p53 and
dissociation or disruption of formation of Hsp60-p53 and Hsp60-survivin complexes, leading to degradation of p53 and
Hsp60-survivin complexes in cells.
survivin. Owing to its high expression levels in tumors, Hsp60 is
We next examined whether the p53 and survivin are an ideal target for anticancer therapy due to less toxicity to
degraded via the proteasomal pathway. NB4 cells were pre- normal cells/tissues. Moreover, targeting complexes such as
treated with or without 25 mM MG132, an inhibitor of 26S survivin-Hsp90,51 Hsp60-p53 and Hsp70/90-p53 have been
proteasome, for 4 h and protein levels of both survivin and p53 demonstrated to be effective in anticancer therapies.44,52 Our
in NB4 cells were measured by western blot. As shown in Fig. 5e combined data have demonstrated clearly that Hsp60 serves as
and f, time-dependent decreases in the levels of both p53 and an important target of ATO. Other hub proteins identied in
survivin were observed in ATO-treated NB4 cells in the absence this study may warrant further studies in future. The integrative
of MG132. However, upon addition of MG132, the protein levels approach we report here also provides a general platform for
remained almost unchanged. These results indicate that both system-wide understanding therapeutic and toxicology of
p53 and survivin undergo proteasomal degradation, in consis- arsenic (and other metallodrugs).
tence with previous reports.49,50 Taken together, we demonstrate
that ATO binds and functionally abolishes Hsp60, leading to
dissociation of the complexes of Hsp60-survivin and Hsp60-
Author contributions
p53. Both p53 and survivin are subsequently degraded, which H. S. and H. L. conceived the project and H. S., H. L., X. H., E. T.
may partially contribute to ATO-induced apoptosis (Fig. 5g).
and N. H. designed the experiments; X. H., T. K.-Y. I., D. T.,
H. W. and X. Y. performed the experiments; Y. F. C., M. K. and X.
H. performed the MS-based quantitative experiment; K. M., X.
H., R. W., and Y. W. performed the bioinformatics; and H. L., X.
Conclusions
In spite of extensive studies for decades, the mechanism of H. and H. S. principally wrote the manuscript with input from
action of ATO is still not fully understood. Arsenic trioxide was all.
demonstrated to bind multiple proteins or enzymes.8 However,
it remains unknown what the primary or key targets are.
Conventionally, the putative protein targets of ATO were
Conflicts of interest
selected and characterized individually, an approach that does There are no conicts to declare.
not provide a holistic view on the biological or pathological
mechanisms of action of ATO. Given the complexity of arsenic–
protein interaction in cells, integration of different approaches
Acknowledgements
is necessary to comprehensively uncover arsenic-associated This work is supported by the Research Grants Council of Hong
proteins, which provides rich resources for downstream inves- Kong (1711618M, 17304614P, 17333616P and 17307017P), the
tigation of biological pathways disrupted by ATO at a system University of Hong Kong (URC), and Norman & Cecilia Yip
level.
foundation. We acknowledge Faculty Core Facility of LKS
We design and synthesize an organo-arsenic uorescent Faculty of Medicine (the University of Hong Kong) for the
probe, which could rapidly enter live cells to selectively label support on confocal imaging and thank the Center for Genomic
AsIII-binding proteins. These proteins were subsequently iden- Sciences, Li Ka Shing Faculty of Medicine for the mass spec-
tied via conventional proteomics approach upon photo- trometry facilities.
activation of arylazide of the probe. The arylazide not only
enabled signicant uorescence turn-on, but also strengthened
the binding between the probe and the labelled proteins. We
Notes and references
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proteins to be mined at a proteome-wide scale. Moreover, our
method enables the weakly, even transiently arsenic bound
proteins to be identied. Together with quantitative
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