Acta Cardiol Sin 2016;32:604-611
Original Article
doi: 10.6515/ACS20150825A
Basic Science
Hydrogen Sulfide Modulates the
S-Nitrosoproteome and the Mitochondrial
Morphology in Endothelial Cells
Tsan-Wan Chiu,1# Ying-Lun Chen,2,3,4# Chien-Yi Wu,5,6 Pei-Ling Yu,7 Ying-Hua Shieh8 and Bin Huang7,9,10,11
Background: Hydrogen sulfide (H2S) is one of the endogenous gaseous molecules promoting the production of
nitric oxide (NO) which has cardioprotective functions. However, the role of the H2S-mediated protein Snitrosoproteome and its subsequent physiological response remains unclear.
Methods: Endothelial cells EAhy 926 were treated with 50 mM of H2S for 2 hours. The NO bound S-nitrosoproteins
were purified by a biotin-switch and then digested by trypsin. Resulting peptides from control and H2S treatment
were separately labeled by isobaric tag for relative and absolute quantitation 114/115, quantified by liquid
chromatography tandem-mass spectrometry and analyzed by ingenuity pathway analysis (IPA) software. The
microP software was applied to analyze the morphological changes of mitochondria.
Results: With the treatment of H2S, 416 S-nitrosylated proteins were identified. IPA analysis showed that these
proteins were involved in five signaling pathways. The NO-bound cysteine residues and the S-nitrosylation levels
(115/114) were shown for ten S-nitrosoproteins. Western blot further verified the S-nitrosylation of thioredoxindependant peroxide reductase, cytochrome c oxidase and cytochrome b-c1 complex that are involved in the
mitochondrial signaling pathway. H2O2-induced mitochondrial swelling can be reduced by the pretreatment of H2S.
Conclusions: The H2S-mediated endothelial S-nitrosoproteome has been confirmed. In the present study, we have
proposed the cardioprotective role of H2S via maintaining mitochondrial homeostasis.
Key Words:
Endothelial cell · Hydrogen sulfide · MicroP · Mitochondria · Mitophagy · S-nitrosoproteome
INTRODUCTION
Received: May 18, 2015
Accepted: August 25, 2015
1
Division of Cardiology, Ten Chan General Hospital, Chung-Li, Taoyuan;
2
Department of Medicine, MacKay Medical College, New Taipei City;
3
Department of Anesthesiology, MacKay Memorial Hospital; 4MacKay
Junior College of Medicine, Nursing and Management, Taipei;
5
Department of Pediatrics, E-Da Hospital; 6School of Medicine,
College of Medicine, I-Shou University; 7Department of Biomedical
Science and Environmental Biology, College of Life Science, Kaohsiung
Medical University, Kaohsiung; 8Division of Family Medicine, Wan
Fang Medical Center, Taipei Medical University, Taipei; 9Center for
Biomarkers and Biotech Drugs; 10Center for Infectious Disease and
Cancer Research, Kaohsiung Medical University; 11Department of
Biological Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan.
Address correspondence and reprint requests to: Dr. Bin Huang,
Department of Biomedical Science and Environmental Biology,
College of Life Science, Kaohsiung Medical University, No. 100,
Shihchuan 1st Rd., San Ming District, Kaohsiung 80708, Taiwan. Tel:
886-7-312-1101 ext. 2704; Fax: 886-7-322-7508; E-mail: huangpin2
@yahoo.com.tw
#
Equally contributed to the manuscript.
Acta Cardiol Sin 2016;32:604-611
Gas molecules regarded as signal transmitters in the
cardiovascular system have been studied for many years.1
Overall, the diverse physiologic actions of three cellular
gaseous molecules, carbon monoxide, nitric oxide (NO)
and hydrogen sulfide (H 2 S) are the primary focus of
these discussions.2 Recently, the interplay of H2S and NO
in the cardiovascular system has been proposed. H 2 S
can increase NO generation through the promotion of
endothelial nitric oxide synthase activity.3,4
The toxicity of H2S on cells has been recognized for
several decades. In recent years, more attention has
been directed towards H2S as the third gaseous mediator, which has been shown to exhibit vasodilatory activity both in vitro and in vivo. 5 Of the three enzymes,
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Hydrogen Sulfide Modulates the S-Nitrosoproteome
analysis, the posttranslational proteome modulated
carcinogenesis and cardiovascular physiology were reported in the earlier studies.20,21
Here, we particularly want to characterize the regulatory signaling cascade of the S-nitrosoproteome affected by H2S.
cystathionine-g-lyase, cystathionine-b-synthetase and
3-mercaptopyruvate sulfurtransferase, can utilize L-cysteine as a substrate to produce H 2 S. 6 Dysfunction of
H2S-producing enzymes results in physiological disorders
such as homocystinuria, which is characterized by mental retardation, skeletal abnormalities, increased urine
homocysteine, increased risks of thromboembolism,
and early onset of atherosclerosis.7,8 H2S was also found
to prevent vascular remodeling from endothelial damage and was shown to regulate vascular tone and angiogenesis via S-sulfhydration of the potassium channel.1,9
NO is the most studied gaseous molecule that exhibits a significant function in the regulation of the
cardiovascular integrity through a posttranslational Snitrosylation on the cysteine.10 In our previous study, a
mechanical shear flow was regarded as protective for
endothelial cells, leading to a series S-nitrosylation of
proteins. 11 The mechanisms of NO in preventing ischemia/reperfusion injury were reported through the
S-nitrosylated proteins, such as F1F0-ATPase, reduced the
generation of Ca2+ and reactive oxygen species in mitochondria.12 NO is known to prevent irreversible oxidative
stress and to protect from several diseases including cancer, diabetes and neuron degeneration.13,14 However, the
abnormal S-nitrosylation of hypoxia-inducible factor 1alpha, matrix metalloproteinase 9, and protein kinase B
(Akt) induces hypertension, stroke and diabetes.15 Therefore, it is important to consider the equilibrium of NOmediated S-nitrosylation.
NO-mediated protein S-nitrosylation can be preliminarily identified by biotin-switch methodology.16 However, several studies have focused on how to increase
the sensitivity of S-nitrosylation 17 compared to former
methodology.17,18 Isobaric tag for relative and absolute
quantitation (iTRAQ) is an isobaric labeling method that
uses stable isotope molecules to be covalent bonded to
the N-terminus and side chain amines of proteins. 19
Therefore, in the present study, the S-nitrosoproteins
were purified by the biotin-switch method, labeled by
iTRAQ reagent and identified by tandem mass spectrometry. Ingenuity pathway analysis (IPA) is a softwarebased mechanism that can generate the interplay and
the network of proteins in particular signal pathways so
that the scientists can further investigate the implications of these proteins in the indicated physiology. With
iTRAQ-labeling-based quantitative proteomics and IPA
MATERIALS AND METHODS
Cell culture
The endothelial cell (EC) line EAhy 926 was generously donated by Cora-Jean S. Edgell, from the University of North Carolina, Chapel Hill. ECs were cultured in
Dulbecco’s Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (FBS, 10%), streptomycin (100 mg/ml), and penicillin (100 U/ml). ECs were replaced by the same medium containing 2% FBS and incubated overnight prior to the NaHS (50 mM for 2 h, as a
donor of H2S) treatments.
Cell lysis and protein extraction
ECs were washed with buffer (0.14 M NaCl, 4 mM
KCl, 11 mM glucose, 10 mM HEPES pH 7.4) after treatment, and then lysed with 100 mL of lysis buffer (250
mM HEPES pH 7.7, 1 mM ethylenediaminetetraacetic
acid (EDTA), 0.1 mM neocuproine, 0.4% (w/v) CHAPS).
After centrifugation, the supernatant was collected and
protein concentrations were determined using a BCA reagent (Thermo Fisher Scientific, Waltham, MA, USA).
Biotin switch and the purification of S-nitrosoproteins
The biotin switch method was used subsequent to a
previous study.16 Briefly, cell lysates were obtained by
sonication and using a lysis buffer. The free cysteine
thiols in the proteins were blocked by S-methylthiolation with 20 mM MMTS. The MMTS-blocked proteins
were labeled with 4 mM biotin-HPDP (Thermo Fisher
Scientific) and 1 mM ascorbate. The biotinylated proteins (i.e., the former S-nitrosoproteins) can be purified
by using neutravidin-agarose beads (15 mL per mg of initial protein) in neutralization buffer (20 mM HEPES pH
7.7), 100 mM NaCl, 1 mM EDTA, 0.5% (v/v) Triton X100). The beads were washed in washing buffer (20 mM
HEPES pH 7.7, 600 mM NaCl, 1 mM EDTA, 0.5% (v/v) Triton X-100), and then incubated with elution buffer (20
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Acta Cardiol Sin 2016;32:604-611
Tsan-Wan Chiu et al.
sion-induced dissociation (CID) MS/MS of the five most
intense precursor ions in the ion trap, followed by five
higher-energy collisional dissociation (HCD) MS/MS scans
on the same precursors. Peptide precursor ions were selected with an isolation window of 2.0 Da, and a target
value of 1 ´ 106. Dynamic exclusion was implemented
with a repeat count of 1 and exclusion duration of 180 s.
The NCE was set to 35% for CID, and 60% for HCD to facilitate the reporter ion generation.
mM HEPES pH 7.7, 100 mM NaCl, 1 mM EDTA, 100 mM
2-mercaptoethanol).
In-gel protein digestion and iTRAQ labeling
The biotinylated proteins were solidified by acrylamide and then digested with trypsin (In-Gel Tryptic Digestion kit, Thermo Fisher Scientific) for 4 h at 37 °C.
The resulting tryptic peptides were labeled with the
iTRAQ 4-plex reagent as follows: iTRAQ 114 for control
and iTRAQ 115 for NaHS treatment (50 mM, 2 h) according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA), except that the labeling
time was doubled. Labeling efficiency was evaluated
with a Mascot search where the respective isobaric
modifications were set as variable modifications on the
N-terminus and lysine. The labeled digests were then
mixed and dried using a speed vacuum concentrator
(Savant SC110; Thermo Fisher Scientific).
Data analysis
Data file processing, protein identification, and relative abundance quantification were performed using
Proteome Discoverer v.1.3 (Thermo Fisher Scientific).
The CID and HCD raw spectra were extracted and searched against the forward and decoy human Swiss-Prot
database using the Proteome Discoverer with MASCOT
search algorithm (Matrix Science, Boston, MA, USA).
The search parameters used were as follows: peptide
ion mass tolerance of 100 ppm; CID and HCD fragment
ion mass tolerance of 0.8 Da and 0.1 Da, respectively; 2
missed trypsin cleavages; variable oxidation of methionine, carboxamidomethylation of cysteine, deamidation
of asparagine and glutamine, acetylation of lysine; iTRAQ
labeling of the N-termini of peptides and lysine side
chain residues; and a false discovery rate < 1.0%. In the
CID-HCD dual scan configuration, peptide identification
was obtained from the CID scan. Quantitation based on
iTRAQ reporter ions was accomplished with a mass
tolerance of 20 ppm. The iTRAQ reporter ion intensities
were extracted from the HCD scans and mapped to the
same precursor ion identified from the corresponding
CID scan. Thereafter, all peptide ratios were normalized
against the median protein ratio.
Liquid chromatography tandem-mass spectrometry
(LC-MS/MS) analysis
The peptide mixtures were fractionated using strong
cation-exchange chromatography. Briefly, samples were
reconstituted in 25% (v/v) acetonitrile and 5 mM KH2PO4
and loaded onto a PolySULFOETHYL A column (PolyLC,
Columbia, MD, USA) in a high performance liquid chromatography (HPLC) system (Agilent Technologies, Palo
Alto, CA, USA). Peptides were eluted with a linear gradient of 0-350 mM KCl (5 mM KH 2 PO 4 in 25% (v/v)
acetonitrile, pH 2.7). A split-flow configuration of HPLC
was used for online nanoLC separation and was coupled
to an Orbitrap mass spectrometer (Thermo Fisher Scientific). The peptide samples were injected into a homemade capillary trap column (2 cm ´ 100 mm i.d., packed
with Magic C18AQ reversed-phase material, 5 mm, 200
Å, Michrom BioResources, Auburn, CA, USA). Then they
were separated in a 13 cm ´ 75 mm i.d. capillary column
(10 mm electrospray tip, packed with Magic C18AQ, 5
mm, 100 Å). For iTRAQ-labeled peptides, mass spectra
were acquired in the positive ion mode at a selected
mass range of 350-1600 m/z. The mass spectrometer
was run in a top-ten configuration with one MS scan followed by ten MS/MS scans. The acquisition of MS/MS
spectra was operated in parallel mode, allowing accurate mass measurements of precursors in the Orbitrap
concurrent with the acquisition of data-dependent ColliActa Cardiol Sin 2016;32:604-611
Ingenuity pathway analysis
The 416 identified S-nitrosoproteins were evaluated
using IPA (Ingenuity Systems, Redwood City, CA, USA).
The modulated proteins were grouped by known relationships into the canonical pathway analysis of the cardiovascular signaling network. The hypothetical protein
interaction clusters between the signal components are
indicated. IPA also computes the cellular compartment
of the linked functional pathway. The proteins with direct interaction were linked by solid line, and the proteins with indirect interaction was indicated by dash
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Hydrogen Sulfide Modulates the S-Nitrosoproteome
level of NO.4 In the present study, H2S-induced S-nitrosoproteins were identified by iTRAQ-labeling-based
quantitative proteomics (Figure 1A). In total, 416 protein with posttranslational S-nitrosylation were identified. These proteins were classified into six groups according to the reported functions such as transcription
factor, heat shock protein, membrane protein, cytoskeleton protein, mitochondrial protein and signaling protein (Figure 1B).
line. The P score is calculated as -log10 (p value), which
refers to the probability of matching the input proteins
in a protein-protein linkage from the Ingenuity Knowledge Base by random chance.
Western blot analysis
Forty micrograms of biotinylated lysate before immunoprecipitation [Before immunoprecipitation (IP)],
and 500 ng of neutravidin-purified biotinylated (i.e.
S-nitrosylated) protein (After IP) were mixed with equal
volume of sample buffer [Tris-HCl (62.5 mM, pH 6.8), sodium dodecyl sulfate,sodium salt (SDS) (3%, w/v), 2mercaptoethanol (5%, v/v), glycerol (10%, v/v)] and
then separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The gel was transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) and immunoblotted with antibody PRDX3 (1:2000, Thermo Fisher Scientific), COX5A
(1:1000, Thermo Fisher Scientific) and UQCRC2 (1:1000,
Abcam, Cambridge, MA, USA). The membranes were
visualized with the SuperSignal West Femto reagent
(Thermo Fisher Scientific) on X-ray films. The images on
X-ray films were scanned using a digital scanner (Microtek
International Inc., Hsinchu, Taiwan) and the density was
calculated by the Progenesis Samespots v2.0 software
(NonLinear Dynamics, Newcastle, UK) from three repeats.
Signaling pathway predicted from S-nitrosoproteins
There were five signaling pathways including the
p53 signal, mitochondrial function, PI3/AKT, Integrinlinked kinase (ILK) and epithelial adherens junctions
were predicted to have a direct interaction in the IPA
analysis (Figure 2A). The S-nitrosylation site and the relative S-nitrosylation levels (115/ 114) of ten proteins involved in the pathway were indicated (Figure 2B).
Validation of protein S-nitrosylation
By using biotin switch coupled with western blot,
the S-nitrosylation of thioredoxin-dependent peroxide
reductase (PRDX3), cytochrome c oxidase subunit 5A
(COX5A) and cytochrome b-c1 complex subunit 2
(UQCRC2) were verified (Figure 3).
H2O2-induced mitochondrial swelling can be
attenuated by H2S
The ECs were treated with 400 mM H2O2 for 4 h to
induce oxidative stress. Under this stress, mitochondria
tend to swell up to 4-fold their original size as compared
to the control. However, the swollen shape of mitochondria can be reduced by pretreatment with H2S (Figure 4).
Observation of mitochondrial morphology
The ECs were seeding on the cover glass slide (2 cm
´ 2 cm) and then incubated with H2O2 (400 mM, 4 h) or
with a pretreatment of NaHS (50 mM, 2 h). The cells
were dehydrated by methanol for 2 min. Slides were
co-incubated with MitoTracker fluorescent dye according to the user’s guideline and observed by a laser scanning confocal imaging system (OlympusFluoview300)
consisting of an Olympus BX51 microscope and a 20
mW-output argon ion laser. The images were further
subjected to the MicroP software analysis for determining morphological changes of mitochondria.
DISCUSSION
H2S is a toxic gaseous molecule applied in endothelial research that has been studied previously.4 By using
fluorescent probes that can detect the cellular levels of
NO and H2S separately, we found that only H2S can enhance the production of NO. As a result, NO-mediated
protein S-nitrosylation under H2S treatment was further
studied here by using a biotin switch coupled with
iTRAQ-labeled quantitative proteomics. IPA software offers a significant suggestion in signaling cross interac-
RESULTS
H2S modulates the S-nitrosylation of endothelial
proteins
According to a previous study, H2S can induce the
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Tsan-Wan Chiu et al.
A
B
Figure 1. Detection of S-nitrosoproteins. (A) The S-nitrosoproteins from different treatments were purified by biotin-switch. The resulting tryptic
peptides were labeled with iTRAQ 114 (control) and 115 (H2S treatment) separately. (B) After the analysis of LC-MS/MS and IPA, S-nitrosoproteins
were classified into six groups according to their annotated function. LC-MS/MS, liquid chromatography tandem-mass spectrometry; IPA, ingenuity
pathway analysis; iTRAQ, isobaric tag for relative and absolute quantitation.
B
A
Figure 2. IPA prediction of S-nitrosoproteins. (A) Five distinct signaling pathways were obtained from IPA analysis. (B) Ten S-nitrosoproteins with
identified nitrosylated cysteine residue and the relative level of S-nitrosylation (115/114) were indicated.
Acta Cardiol Sin 2016;32:604-611
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Hydrogen Sulfide Modulates the S-Nitrosoproteome
A
B
C
D
E
F
chondrial electron transport chain. NO can be produced
in the mitochondria by either Complex III or IV, and the
produced NO can compete with O2 to the binding site of
cytochrome c oxidase and results in the reduced respiration rate.23 In spite of this, the possible cytoprotective
role of cytochrome-mediated NO production was also
proposed.24 In the reported article of protein S-nitrosylation during preconditioning and ischemia/reperfusion injury, the S-nitrosylated site of cytochrome c
oxidase subunit 5B (COX5B, Cys115) and of the cytochrome b-c1 complex subunit 1 (UQCRC1, Cys268) were
verified.25 Therefore, it remains doubtful whether the
inhibition of cytochrome complexes at NO is the consequence of S-nitrosylation on this protein.
With the concern that H 2 S profoundly modulates
the S-nitrosylation of mitochondrial proteins, the integrity of mitochondria is evaluated in the present study.
Mitochondrial morphology that correlated to mitochondrial autophagy (mitophagy) was discussed and software that can distinguish the status of mitophagy according to the ratio of fusion/fission was developed.26,27
H2O2 is a strong oxidant that has always been applied in
mimicking oxidative stress within mitochondria.28 With
a treatment of H 2 S, the ATP synthesis, mitochondrial
membrane potential (DYm) and cytochrome c release
from mitochondria indicated that mitochondrial function had recovered.29 Similar to this previous study, we
further used the MitoTracker, a fluorescent dye showing
similar effects as JC-1 reagent to observe the morphological changes of mitochondria under different treatments. The mitochondrial morphology, particularly the
swollen shape, indicating a process of mitophagy, can be
identified by microP software. As a result, we found that
H2O2-induced swollen mitochondria can be remediated
by H2S treatment.
In addition to the effect of S-nitrosylation on mitochondrial proteins, the acetylation also shows an important role in modulating the function of proteins and
thereby affects the integrity of mitochondria.30 H2S is recently confirmed as an enhancer for sirtuin 3 (SIRT 3)
protein activation and increasing cell longevity.31 Due to
its apparent capacity to detect cellular acetylome,32 the
protein acetylation that was separately modulated by
H2S can be further investigated. This would provide improved insight into the mechanism of how H2S increases
mitochondrial function through protein acetylation in
Figure 3. The verification of S-nitrosoprotein. (A, C, E) Forty micrograms of biotinylated lysate before immunoprecipitation (Before IP),
and 500 ng of neutravidin-purified biotinylated protein (After IP) were
separated by SDS-PAGE. The membranes were immunoblotted with antibody PRDX3 (1:2000), COX5A (1:1000) and UQCRC2 (1:1000). (B, D, F).
The relative level of S-nitrosoprotein was shown as means ± SE from
three repeats. Statistical significance (* p < 0.05, ** p < 0.01) was
analyzed by using Fisher’s Least significant difference procedure.
tions. In the present study, we found that these NOmodified proteins were involved in p53 signal, mitochondrial function, PI3/AKT, ILK and epithelial adherens
junction. Within these signal pathways, mitochondrial
function attracts more attention because mitochondrial integrity was profoundly affected by S-nitrosylation. 10 This was also consistent with the study that
mitochondria possess an alternative pathway for NO
synthesis.22
Within the three validated mitochondrial S-nitrosylated proteins, PDRX3, UQCRC2 and COX5A were further confirmed by western blot (Figure 3). Other proteins, such as PRDX1, COX5B and HSP90AA1 could not
be confirmed, which might be explained by the specificity of the used antibodies. PRDX3 is an enzyme showing
antioxidative function in mitochondria. However, the effect of S-nitrosylation on PRDX3 function remains unclear. The cytochrome b-c1 complex, also known as Complex III, was confirmed to contain the subunit 2 core
protein (UQCRC2). It is followed by cytochrome c oxidase (Complex IV) to complete the last step of the mito609
Acta Cardiol Sin 2016;32:604-611
Tsan-Wan Chiu et al.
A
B
C
Figure 4. H2S decreased H2O2-induced swelling of mitochondria. (A) The endothelial cell incubated with H2O2 or H2O2 pretreated with H2S were
stained by MitoTracker fluorescent dye and observed by confocal microscopy. (B, C) The percentage of different mitochondrial shape including small
globule, swollen globule, and tubule and loop were identified by MicroP software. The relative fold of each shape of mitochondria from different
treatment were shown as compared to the control. Data are shown as means ± SE from three repeats. Statistical significance (** p < 0.01) was analyzed by using Fisher’s least significant difference procedure.
ACKNOWLEDGEMENTS
addition to S-nitrosylation.
Taken together, this study provides valuable insight
of H2S-modulated S-nitrosoproteome in regulating mitochondrial morphology under oxidative stress. The correlation of proteins involved with mitophagy should further be investigated.
This work was supported by the Kaohsiung Medical
University “Aim for the Top Universities Grant”, grant
No. KMU-TP104E13, KMU-TP105E11, KMU-TP105G00,
KMU-TP105G01, KMU-TP105G02, 105-P032, and also
(MOST 104-2632-M-037-001). We are grateful to the
core facility laboratory of the Institute of Biomedical
Sciences, Academia Sinica, Mithra Solution Biotechnology Inc. and Center for Resources, Research & Development of Kaohsiung Medical University for their
mass spectrometric analyses. We are also grateful to
Dr. Hans-Uwe Dahms for critical reading of the manuscript.
CONCLUSIONS
Through the technique of shotgun proteomics and
the algorithm of mitochondria morphology, we provided
a preliminary evidence of H2S in preventing mitochondria from oxidative damage.
Acta Cardiol Sin 2016;32:604-611
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Hydrogen Sulfide Modulates the S-Nitrosoproteome
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CONFLICT OF INTEREST
The authors declare no conflict of interest.
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