Identification of HSP90 as a direct target of artemisinin for its anti-inflammatory activity: Via quantitative chemical proteomics

Article abstract of DOI:10.1039/c9ob01264h

The anti-malarial drug artemisinin (ART) possesses potent anti-inflammatory activity, yet its underlying mechanism of action has remained elusive. Here we employed quantitative chemical proteomics to in situ profile the cellular targets of ART and identified heat shock protein 90 (HSP90) as a direct target. Further study revealed that ART suppressed the production of nitric oxide (NO) in macrophages via inhibiting the interaction between HSP90 and inducible NO synthase (iNOS).

Full text of DOI:10.1039/c9ob01264h

Organic &
Biomolecular Chemistry
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PAPER
Identification of HSP90 as a direct target of
artemisinin for its anti-inflammatory activity via
quantitative chemical proteomics†
Cite this: DOI: 10.1039/c9ob01264h
Guolin Wu, a Bao Cheng, a Hui Qian, a Shengming Ma a,b and
a
Qin Chen
*
The anti-malarial drug artemisinin (ART) possesses potent anti-inflammatory activity, yet its underlying
mechanism of action has remained elusive. Here we employed quantitative chemical proteomics to in situ
profile the cellular targets of ART and identified heat shock protein 90 (HSP90) as a direct target. Further
study revealed that ART suppressed the production of nitric oxide (NO) in macrophages via inhibiting the
interaction between HSP90 and inducible NO synthase (iNOS).
Received 31st May 2019,
Accepted 26th June 2019
DOI: 10.1039/c9ob01264h
rsc.li/obc
hensively and unbiasedly via ABPP combined with quantitative
chemical proteomics. Non-specific binding is one of the most
Introduction
In addition to anti-malarial, anti-cancer, anti-viral and anti- serious problems for ABPP, which can be minimized by quan-
bacterial effects,1–3 the potential application of artemisinin titative proteomics.18
(ART) and its derivatives in the treatment of immune diseases
has also been drawing increasing attention, due to their sup-
pressive effects on both innate and adaptive immune cells.4,5
Macrophages, representing a key component of the innate
Results and discussion
immune system, can produce pro-inflammatory cytokines,
In order to avoid the bulky size of the reporter tag which can
such as TNFα and nitric oxide (NO), which are vital for macro-
limit the proper uptake of the probes by the target proteins, an
phage-mediated inflammatory and immune reactions. The
ART probe with a clickable alkyne (2) was synthesized follow-
members of the ART family have been reported to downregu-
ing previously reported procedure with some modifications
late the release of these cytokines from macrophages via mul-
(Fig. 1A).14,15,19–22 To verify its inhibition on NO production in
tiple signaling pathways, such as NF-κB,6,7 STAT-1,8 etc.9,10
macrophages, RAW264.7 macrophage cells were pre-treated
However, the direct targets of ART accounting for its anti-
with different concentrations of parent ART (1) or 2 for 1 h
inflammatory function are not well understood.
before stimulation with 10 ng mL−1 Lipopolysaccharide (LPS)
Activity-based protein profiling (ABPP) is a well-established
for 24 h, and the Griess reaction was employed to measure the
strategy that has been widely used to identify the targets of
level of NO in the cultural medium. As shown in Fig. 1B, 2 is
small molecules.11–13 Recently, the targets of ART for its anti-
as potent as 1 in inhibiting NO production, suggesting that
malarial14,15 and anti-cancer activities16,17 have been identified
the introduction of a terminal alkyne handle did not affect its
by using ABPP. However, the cellular targets of ART to execute
anti-inflammatory activity. Therefore, 2 should be suitable for
its anti-inflammatory activity are likely different from those in
proteomic profiling as an ART probe.
malaria parasites or cancer cells. To better understand the
To visualize the target proteins covalently bound to ART
mode of action of ART for its anti-inflammatory activity, we
probe, cell lysates were prepared from 2-treated RAW264.7
sought to screen its molecular targets in macrophages compre-
cells, clicked with a fluorescent dye tetramethyrhodamine
(TAMRA) via well-established copper-catalyzed alkyne–azide
cycloaddition (CuAAC) methodology,23 and resolved by
SDS-PAGE. Only the proteins covalently bound to 2 can be
labeled and visualized by fluorescent scanning. As shown in
Fig. 2A, compared with the negative control, multiple proteins
were labeled by TAMRA after RAW264.7 cells were treated with
2 for 1 h, while Coomassie blue staining indicated an equal
amount of cell lysate was loaded in each lane. The level of
aResearch Center for Molecular Recognition and Synthesis, Department of Chemistry,
Fudan University, 220 Handan Lu, Shanghai 200433, P. R. China.
E-mail: chenq@fudan.edu.cn
bState Key Laboratory of Organometallic Chemistry, Shanghai Institute of Organic
Chemistry, Chinese Academy of Sciences, 345 Lingling Lu, Shanghai 200032,
P. R. China
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c9ob01264h
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target labeling increased along with the extension of treatment
time (Fig. 2A), which was also enhanced by increasing concen-
tration of 2 (Fig. 2B). To determine if 2 shares the same targets
as 1, competitive assay was performed whereby cells were pre-
incubated with 100 μM of 1 before addition of 50 μM of 2. As
shown in Fig. 2C, significant reduction in the intensity of the
majority of bands was observed after pretreatment with 1, indi-
cating that 1 and 2 share the same protein targets. Based on
these data, we carried out intracellular labeling experiments to
visualize the cellular localization of proteins potentially bound
to ART. The cells were treated with 2 or DMSO for 8 h, and sub-
jected to CuAAC reaction with TAMRA-azide. The cell nucleus
was stained with 4,6-diamidino-2-phenylindole (DAPI). As
shown in Fig. 2D, TAMRA fluorescence could barely be
observed in the cells treated with vehicle control (DMSO),
while high level of TAMRA fluorescence distributed universally
in the cells treated with 2 for 8 h. This observation demon-
strated that 2 was cell-permeable and the target proteins were
widely distributed throughout the cells.
We next performed large-scale pull down and isobaric
tandem mass tagging (TMT)-based quantitative mass spec-
trometry to enrich and identify the target proteins, as illus-
trated in Fig. 3. After incubation with 2 for 8 h with or without
pretreatment with 1 for 1 h, the cells were harvested and sub-
jected to protein extraction. The target proteins were labeled
by biotin, pulled down by streptavidin beads and subjected to
on-bead trypsin digestion. The resulting peptides were labeled
with TMT reagents 126 and 127 respectively, followed by
LC-MS/MS analysis. The proteins were quantified by the inten-
sities of their corresponding TMT reporter ions (126 and 127)
and the competitive ratio (126/127) of each protein was calcu-
lated. The targets were defined as those giving a statistically
significant (t-test, p < 0.05) reduction of abundance in the
competition group versus the control group, with a competitive
ratio of 1.3 as the cut-off threshold.
Fig. 1 (A) ART (1) and the clickable ART probe with an alkyne (2) (B) 1
and
2
dose-dependently
inhibited
NO
production
in
RAW264.7 macrophage cells. The level of NO was normalized to the
vehicle control. The data represented the mean values S.D. from three
independent experiments. *p < 0.05.
A total of 199 proteins were identified as potential targets
of ART (ESI Table†). Gene ontology (GO) analysis revealed that
the targets of ART were involved in multiple pathways includ-
ing protein synthesis, carbon metabolism, with influences on
a number of biological processes, such as translation, protein
folding and stabilization, oxidation–reduction process (Fig. 4 &
Fig. 2 (A) Time-dependent in situ fluorescence labeling of RAW264.7
cells with 2. Fluo (fluorescent scanning); COO (Coomassie blue
staining). (B) Dose-dependent in situ fluorescence labeling of
RAW264.7 cells with 2. (C) Competitive assay: cells were pretreated
with 1 (100 μM) for 1 h before 2 (50 μM) was added. Arrow: the bands
that were markerly reduced by pretreatment with 1. (D) Images of
cellular localization of ART targets by fluorescent microscopy.
Images in A–D are representative of triplicate experiments,
respectively.
Fig. 3 Workflow for affinity purification and quantitative mass spec-
trometry-based proteome profiling of ART binding targets.
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Fig. 4 Gene ontology (GO) analysis of ART target proteins. BP (biologi-
cal process); CC (cellular component); MF (molecular function).
S1†). In consistency with the result of cellular fluorescent
labeling, GO analysis also showed that the target proteins of
ART widely distributed throughout the cells (Fig. 4).
Among these putative target proteins (Fig. S2†), some pro-
teins have been identified and validated in malaria parasites
and cancer cells,14,17 such as pyruvate kinase (PKM), L-lactate
dehydrogenase (LDH), glyceraldehyde-3-phosphate dehydro-
genase (GAPDH), peroxiredoxin-1 (PRDX1) and transferrin
receptor protein 1 (TFRC). Artesunate (a derivative of artemisi-
nin) has been reported to protect the lung from sepsis-induced
injury via hemo oxygenase-1 (HMOX1) modulation.24 Heme
oxygenase 2 (HMOX2), which plays a critical role in inflamma-
tory and reparative response,25 was also found among the
targets with top competitive ratio.
Fig. 5 (A) Validation of the interaction between HSP90 and
2 by
western blot. (B) Effect of 1 on the expression of iNOS. RAW264.7 cells
were stimulated with 10 ng mL−1 LPS for 8 h after pretreated with 50 μM
of 1 or 5 μM of geldanamycin (GM) for 1 h. (C) Effect of 1 on the inter-
action between HSP90 and iNOS. All the experiments in A, B and C have
been repeated for three times and representative blots were shown. (D)
Covalent docking model of ART bound to the human HSP90β (PDB
code: 3NMQ) N-terminus. The artemisinin is colored in cyan and the
proteins are colored in green for HSP90β. (E) A model for the mecha-
nism underlying the anti-inflammatory activity of ART.
Heat shock protein 90 (HSP90), which is among the targets
with top statistical significance, caught our attention.
HSP90 has been reported to play an important role in the
induction and the stability of inducible NO synthase significantly inhibited the LPS-induced expression of iNOS in
(iNOS),26,27 which is a major source of NO during inflam- primary rat microglia30 and astrocytoma.31 The different
mation. Furthermore, HSP90 also enhanced the activity of effects of ART on the expression of iNOS may be due to the
iNOS via direct binding.28,29 To examine if HSP90 was difference in cellular context and species,32 which deserves
accounted for ART-regulated NO production, we firstly vali- further study.
dated the interaction between 2 and HSP90. After RAW264.7
To examine the effect of ART on the interaction of iNOS
cells were treated with 2 for 8 h with or without pre-incubation and HSP90, RAW264.7 cells were pre-incubated with or
with 1, cell lysates were prepared and subjected to click chem- without 50 μM of 1 for 1 h followed by stimulating with 10 ng
istry with biotin-azide. Then target proteins were enriched mL−1 LPS for 8 h. Cell lysates were collected and subjected to
using streptavidin beads followed by western blot with HSP90 co-immunoprecipitations with anti-iNOS antibody. As shown
antibody. As shown in Fig. 5A, HSP90 was pulled down from in Fig. 5C, HSP90 was immunoprecipitated by antibody
the cell lysates treated with 2, whereas the band intensity was against iNOS in DMSO-treated cells, indicating the association
drastically decreased in the presence of 1, indicating a specific between HSP90 and iNOS. However, the level of HSP90 pulled
interaction between HSP90 and ART.
down by antibody against iNOS markedly decreased in the
We next investigated whether the interaction between ART presence of 1 (Fig. 5C), suggesting that ART interfered the
and HSP90 could inhibit NO production via reducing HSP90- interaction between HSP90 and iNOS, which in turn inhibited
regulated iNOS expression. Little iNOS was detected in cells NO production.
without LPS treatment, while LPS stimulated the expression of
To further investigate the binding mode between HSP90
iNOS in a time-dependent manner (Fig. S3†). Geldanamycin and ART, we carried out covalent docking studies using
(GM), a specific HSP90 inhibitor, significantly inhibited the Schrödinger software. As shown in Fig. 5D, the amino of Lys58
induction of iNOS by LPS for 8 h, whereas 1 had little effect on was assigned as the reaction site. According to the covalent
this process (Fig. 5B). However, it has been reported that ART docking model of ART with HSP90β, the carbonyl group of
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ART can form covalent bond with Lys58. Additionally, the resi- 15 min at 4 °C). The protein pellet was washed twice with pre-
dues Ala55, Ile96, Met98, Leu107 and Phe138 in HSP90β can chilled methanol and air dried. The proteins were dissolved in
form hydrophobic interactions with ART. It has been reported electrophoresis sample buffer and separated by SDS-PAGE fol-
that ATP binding drives tertiary and quaternary structural lowed by in-gel fluorescence scanning with a FUJIFILM FLA
rearrangements of HSP90, which potentiate the association 9000 plus DAGE fluorescence scanner. The total protein levels
between HSP90 and client protein.33,34 The covalent binding were determined by Coomassie staining.
between ART and Lys58 of the HSP90 ATP binding site may
Fluorescent microscopy
block the binding of ATP to HSP90 and thereby, decrease the
association between HSP90 and iNOS. Further characterization RAW264.7 cells were seeded on coverslips in a 24-well plate
of the binding mode between HSP90 and ART and the influ- and cultured overnight. After treatment with 100 μM of ART
ence of ART on the interaction between HSP90 and iNOS are probe for 8 h, the cells were washed twice with PBS and fixed
in progress in our laboratory.
with 4% paraformaldehyde for 15 min at room temperature.
Fixed cells were permeabilized with 0.1% Triton X-100 for
10 min followed by blocking with 3% BSA for 1 h. Then the
cells were treated with 500 μL of freshly prepared reaction
mixture (10 μM TAMRA-azide, 100 μM TBTA, 1 mM TCEP and
1 mM CuSO4) for 1 h at room temperature. After being washed
with 0.05% Tween-20 in PBS for three times, the cells were
stained with DAPI (2 μg mL−1) for 5 min. Cells were observed
and photographed with a Leica TSC SP8 STED 3X fluorescent
microscope.
Experimental
Synthesis of the ART probe (2)
Compound 2 was synthesized as detailed in ESI.†
Cell culture
Mouse macrophage RAW264.7 cells were purchased from
ATCC (Manassas, VA) and cultured in Dulbecco’s Modified
Eagle Medium (DMEM) supplemented with 10% FBS, 100
U mL−1 of penicillin, and 100 μg mL−1 of streptomycin at
37 °C in humidified air containing 5% CO2.
Affinity enrichment and LC-MS/MS
Cell lysates were prepared at a concentration of 2 mg mL−1 and
each of 500 μL of the sample was used for click chemistry. For
each click reaction, biotin-azide (100 μM), TBTA ligand
(100 μM), TCEP (1 mM), and CuSO4 (1 mM) were sequentially
added to the samples. After incubation for 1 h at room temp-
erature in the dark, the samples were precipitated with pre-
chilled acetone, washed with methanol, and air dried. The pro-
teins were dissolved in 1 mL of PBS containing 0.1% SDS by
brief sonication. 50 μL of streptavidin beads (GE Healthcare,
Chicago, IL) were added to each sample. After gently rotated at
room temperature for 2 h, the beads were washed with 1% SDS
in PBS and triethylammonium bicarbonate (TEAB, 100 mM)
containing 4 M Urea, respectively, for three times in sequential
order. The proteins on-bead were then reduced by incubation
with 200 μL of dithiothreitol (DTT, 10 mM) for 30 min at 50 °C
followed by treatment with 200 μL of iodoacetamide (55 mM)
for 30 min at 37 °C. After washing with 50 mM of TEAB for five
times, the proteins were digested with 1 μg of trypsin (Thermo
Scientific, Waltham, MA) for 16 h at 37 °C and labeled with
Measurement of NO production
RAW264.7 cells were seeded in 96-well plates at a density of
40 000 cells per well. After cultured overnight, the cells were
pre-treated with ART 1 or ART probe 2 at different concen-
trations for 1 h before stimulation with LPS (10 ng mL−1). The
level of NO in cultural medium was quantified by using the
Griess reaction 24 h later. Briefly, 50 μL of the culture super-
natant was incubated with 100 μL of Griess reagent (1% sulfa-
nilamide and 0.1% N-1-naphthylethylenediamine dihy-
drochloride in 2.5% H3PO4) at room temperature for 10 min.
The absorbance at 540 nm was measured with a microplate
reader (SpectraMax M2, Molecular Devices). The concentration
of NO was calculated from the nitrite standard (sodium nitrite)
reference curve.
In situ proteome labeling
Cells were grown to 80–90% confluence in 6 cm dishes and TMTduplex Isobaric Label Reagent Set (Thermo Scientific,
treated with 100 μM ART probe for indicated times. The cells Waltham, MA) according to the manufacturer’s instructions.
were washed twice with PBS to remove the excessive probe and The labeled peptides were combined, cleaned up by using
lysed with lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, Pierce C-18 Spin Columns (Thermo Scientific, Waltham, MA),
0.1% Triton X-100 and protease inhibitor cocktail) followed by dried on a SpeedVac, and re-dissolved with 20 μL of formic
sonication. Equal amounts of samples (50 μg) were used for acid/acetonitrile/H2O (v : v : v = 0.5%/2%/97.5%).
fluorescent labeling. For each click reaction, tetramethyrhoda-
LC-MS/MS analysis was performed on a Thermo C18
mine (TAMRA)–azide (100 μM), tris[(1-benzyl-1H-1,2,3-triazol-4- PepMap100 precolumn (300 μm × 5 mm) and eluted on a
yl)methyl]amine (TBTA, 100 μM), tris(2-carboxyethyl)phos- Thermo Acclaim PepMap RSLC analytical column (75 μm ×
phine (TCEP, 1 mM), and CuSO4 (1 mM) were sequentially 15 cm). Mobile phase A (0.1% formic acid in H2O) and mobile
added to the samples. After incubation for 1 h at room temp- phase B (0.1% formic acid in acetonitrile) were used to estab-
erature in the dark, the click reactions were terminated by lish the 120-minute gradient comprised of 85 min of 4–30% B,
adding pre-chilled acetone. The proteins were precipitated at 15 min of 30–50% B, and 5 min of 90% B, followed by re-equi-
−20 °C for 1 h, followed by centrifugation (13 000 rpm for librating at 4% B for 15 min. The flow rate was 0.3 μL min−1
.
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Peptides were then analyzed on Thermo Orbitrap Fusion Jiangsu, China) or iNOS and then secondary antibodies
Lumos mass spectrometer in a data-dependent manner, with conjugated with HRP. Protein bands were visualized by
automatic switching between MS and MS/MS scans using a enhanced chemiluminescence (ECL kit, Pierce Biotechnology,
cycle time 3 s. MS spectra were acquired at a resolution of Rockford, IL).
120 000 with AGC target value of 4 × 105 ions or a maximum
integration time of 50 ms. The scan range was limited from
375 to 1500 (m/z). Peptide fragmentation was performed via
high energy collision dissociation (HCD) with the energy set at
Conclusions
In summary, we performed the global profiling of the target
38 NCE. The MS/MS spectra were acquired at a resolution of
50 000 with AGC target value of 1 × 105 ions or a maximum
proteins of ART for its anti-inflammatory activity by using
ABPP combined with quantitative chemical proteomics.
integration time of 105 ms. The fixed first m/z was 120, and
the isolation window was 0.7 (m/z).
Furthermore, we elucidated a novel mechanism that ART
inhibited NO production in macrophage via direct interaction
Proteomics data analysis
with HSP90, which blocked the association between HSP90
and iNOS (Fig. 5E). Our findings shed new light on the mecha-
nism of action of ART in its anti-inflammatory activity, which
provided new clues to discover ART derivatives as novel anti-
inflammatory agents.
Protein identification and quantification were performed
using Proteome Discoverer 2.1 software (Thermo Scientific).
Data were matched to annotation data downloaded from
Uniprot (http://www.uniprot.org/) in October, 2018 (proteome
ID: UP000000589, total sequence 16 997). Peptide sequences
(and hence protein identity) were determined by matching
Uniprot protein databases with the acquired fragmentation
pattern by SEQUEST HT algorithm. The precursor mass toler-
ance was set to 10 ppm and fragment ion mass tolerance to
0.02 Da. One missed cleavage site of trypsin was allowed.
Carbamidomethyl (C) and TMT-duplex (K and N-terminal)
were used as a fixed modification. Oxidation (M) was used as
variable modifications. All spectra were searched against
protein database using a target false discovery rate (FDR) of
1%. Protein ratios were calculated as the median of all peptide
hits belonging to a protein. Since our data that were normally
distributed met the criteria for Student’s t-test, the p-values of
each category of TMT ratios were subjected to the Student’s
t-test, and only proteins identified have p-values of compe-
tition ratio less than 0.05 were considered statistical significant
targets. The standard deviation (S.D.) was calculated to be
0.15, so with the use of “1 + 2 × S.D.” formula, we use competi-
tive ratio of 1.3 as the cut-off threshold. The proteomics experi-
ments were carried out in biological duplicates.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This study was supported by the National Natural Science
Foundation of China (21690063, 81572695). We would like to
thank Dr Weichao Li and Dr Youli Xiao for LC-MS/MS analysis.
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RAW264.7 cells were pre-treated with ART (50 μM) or DMSO
for 1 h before stimulation with 10 ng mL−1 of LPS for 8 h.
After being washed with PBS for three times, the cells were
lysed in RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM
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