Identifying Glyceraldehyde 3-Phosphate Dehydrogenase as a Cyclic Adenosine Diphosphoribose Binding Protein by Photoaffinity Protein-Ligand Labeling Approach

Article abstract of DOI:10.1021/jacs.6b08088

Cyclic adenosine diphosphoribose (cADPR), an endogenous nucleotide derived from nicotinamide adenine dinucleotide (NAD⁺), mobilizes Ca²⁺ release from endoplasmic reticulum (ER) via ryanodine receptors (RyRs), yet the bridging protein(s) between cADPR and RyRs remain(s) unknown. Here we synthesized a novel photoaffinity labeling (PAL) cADPR agonist, PAL-cIDPRE, and subsequently applied it to purify its binding proteins in human Jurkat T cells. We identified glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as one of the cADPR binding protein(s), characterized the binding affinity between cADPR and GAPDH in vitro by surface plasmon resonance (SPR) assay, and mapped cADPR's binding sites in GAPDH. We further demonstrated that cADPR induces the transient interaction between GAPDH and RyRs in vivo and that GAPDH knockdown abolished cADPR-induced Ca²⁺ release. However, GAPDH did not catalyze cADPR into any other known or novel compound(s). In summary, our data clearly indicate that GAPDH is the long-sought-after cADPR binding protein and is required for cADPR-mediated Ca²⁺ mobilization from ER via RyRs.

Full text of DOI:10.1021/jacs.6b08088

Subscriber access provided by Fudan University
Article
Identifying GAPDH as a Cyclic Adenosine Diphosphoribose (cADPR)
Binding Protein by Pho-toaffinity Protein-Ligand Labeling Approach
Kehui Zhang, Wei Sun, Lihong Huang, Kaiyuan Zhu, Longchao Zhu, Qian Wang, Yingying
Lu, Hongmin Zhang, Hongwei Jin, Li-He Zhang, Liangren Zhang, and Jianbo Yue
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b08088 • Publication Date (Web): 12 Dec 2016
Downloaded from http://pubs.acs.org on December 13, 2016
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of the American Chemical Society is published by the American Chemical
Society. 1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 32
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Graphic abstract
118x96mm (300 x 300 DPI)
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 2 of 32
Identifying GAPDH as a Cyclic Adenosine Diphosphoribose (cADPR) Binding Protein by
Photoaffinity Protein-Ligand Labeling Approach
1
2
3
4
5
6
7
Kehui Zhang1, 2, Wei Sun2, 3, Lihong Huang2, Kaiyuan Zhu2, Longchao Zhu2, Qian Wang2, Yingying
Lu2, Hongmin Zhang3, Hongwei Jin1, Li-He Zhang1*, Liangren Zhang1*, Jianbo Yue2*
8
9
1State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking
University, Beijing 100191, China
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2Department of Biomedical Sciences, City University of Hong Kong, Hong Kong, China
3 Department of Biology and Shenzhen Key Laboratory of Cell Microenvironment, South University of
Science and Technology of China, Shenzhen 518052, China
Key words: cADPR, GAPDH, Ryanodine receptor, Ca2+, photoaffinity labeling
ABSTRACT: Cyclic adenosine diphosphoribose (cADPR), an endogenous nucleotide derived from NAD,
mobilizes Ca2+ release from ER via ryanodine receptors (RyRs), yet the bridging protein(s) between
cADPR and RyRs remain(s) unknown. Here we synthesized a novel photoaffinity labeling cADPR
agonist, PAL-cIDPRE, and subsequently applied it to purify its binding proteins in human Jurkat T cells.
We identified glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as one of cADPR binding protein(s),
characterized the binding affinity between cADPR and GAPDH in vitro by SPR assay, and mapped the
cADPR’s binding sites in GAPDH. We further demonstrated that cADPR induces the transient interaction
between GAPDH and RyRs in vivo, and GAPDH knockdown abolished cADPR-induced Ca2+ release.
On the other hand, GAPDH did not catalyze cADPR into any other known or novel compound(s). In
summary, our data clearly indicate that GAPDH is the long-sought-after cADPR binding protein and is
required for cADPR-mediated Ca2+ mobilization from ER via RyRs.
cADPR production that leads to calcium release or
influx, establishing cADPR as a second messenger
INTRODUCTION
Cyclic adenosine diphosphoribose (cADPR)-
mediated Ca2+ signaling pathway is involved in a
1b,
6
.
CD38 is the dominant enzyme for
synthesizing cADPR in mammalian systems and
CD38 knockout mice show a number of
physiological defects, including metabolic
disorder, impaired immune responses, and social
1
wide variety of cellular processes , e.g. abscisic
acid-signaling 2, calorie restriction in gut stem cell
3, circadian clock in plants , and long-term
4
5
synaptic depression in hippocampus . Many
1a,
7
behavioral changes
.
Although the
1
extracellular stimuli have been shown to induce
ACS Paragon Plus Environment

Page 3 of 32
Journal of the American Chemical Society
physiological importance of cADPR has been well
with P2O5 (1.0 g) in chloroform (4 mL) in ice
1
2
3
4
5
6
7
8
3-4,
documented
8, the molecular mechanism
water bath for 2 h, followed by column
chromatography (PE/EA) to purify compound 3.
Compound 3 (6.721 g, 30.38 mmol) and acetic
anhydride (11mL) were incubated with boron
trifluoride-diethyl etherate complex (3 mL) at 0 °C
for 2 h to generate compound 4, which was then
incubated with TMSBr in DCM to produce
compound 5. NHS ester of compound 9
(compound 10) was synthesized by adding NHS
(1.2eq) and EDCI•HCl (1.2 eq) and stirring at ice
water bath for 16 h. Compound 12 was
synthesized using similar condition as described
previously13, and the deprotection of which
produced compound 13. Compound 14 was
synthesized by bis-phosphorylation of compound
13. The intra-molecular cyclization of compound
mediating the cADPR signaling remains elusive 9.
It has been shown that cADPR targets
ryanodine receptors (RyRs) on the endoplasmic
9
10
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
reticulum (ER) in many cell types , yet cADPR
does not directly act on the receptor 11. It is
possible that cADPR binds to an accessory protein
in the channel complex. Some candidates have
been suggested, including calmodulin (CALM)
and FK506-binding protein 12 (FKBP12) 12. Thus
far, this elusive cADPR binding protein remains to
be identified, let alone other regulators involved in
cADPR signaling.
Here, we synthesized a photoaffinity labeling
cADPR analogue, PAL-cIDPRE, applied it to
purify its novel binding proteins in human Jurkat
T cells, and identified GAPDH as one of the
cADPR binding proteins.
1
14 produced N3-cIDPRE (compound 15). H
NMR (400 MHz, MeOD) δ 8.58 (d, J = 23.6 Hz,
1H), 8.16 (d, J = 2.3 Hz, 1H), 6.23 (s, 1H), 6.15
(dd, J = 10.8, 5.7 Hz, 1H), 5.85 (dd, J = 22.3, 6.1
Hz, 1H), 5.39 – 5.33 (m, 1H), 5.23 (dd, J = 45.4,
10.9 Hz, 1H), 4.35 (t, J = 6.4 Hz, 1H), 3.87 – 3.74
(m, 3H), 3.58 – 3.31 (m, 4H), 1.53 (s, 3H), 1.37 (s,
3H). 31P NMR (162 MHz, decoupled with 1H,
MeOD) δ -10.44(d), -11.30(d). HRMS(ESI-TOF-
EXPERIMENTAL SECTION
Chemistry- Compound 9 and 11 were synthesized
as described previously13. Briefly, sodium
benzoate (17.6 g, 122 mmol, 1.2 eq) was added to
a solution of epichlorohydrin (9.25 g, 100 mmol)
in toluene (90 ml) along with tetra-n-
butylammonium bromide as a catalyst. The
mixture was stirred at 110 °C for 2 h followed by
column chromatography (PE/EA) to purify
compound 2. Then compound 2 (2.025 g, 9.15
mmol) in diemthyloxymethane (4 ml) was mixed
-
): calcd for C17H22N7O12P2 [(M-H)-], 578.08072;
found, 578.08088. NH2-cIDPRE (compound 16)
was synthesized through reduction of compound
15 via Staudinger reaction. PAL-cIDPRE
(compound 17) was finally produced by
31
incubating compound 10 with NH2-cIDPRE. P
2
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 32
NMR (162 MHz, decoupled with 1H, MeOD) δ -
10.39(d), -11.61(d). HRMS(ESI-TOF-): calcd for
concentration of 10 μM and incubated at 4 °C for
1
2
3
4
5
6
7
8
2 h. The samples were subsequently exposed to
UV 365 nm at 4 °C for 30 min. Azide biotin was
then added at a final concentration of 20 µM
followed by the addition of catalyst. The catalyst
was composed of CuSO4 (20 μM), TECP (Tris(2-
carboxyethyl)phosphine hydrochloride) (25 μM)
was applied to reduce Cu(II) to Cu(I), and THPTA
(Tris(3-hydroxypropyltriazolylmethyl)amine) (60
μM) was added to stabilize Cu(I). After rotating at
room temperature for 18 h, cold acetone was
added to the reaction to precipitate proteins. Then
the pellet was washed with cold acetone for three
times and dissolved in 1 % (w/v) SDS in PBS. The
PAL-cIDPRE-bound proteins purified by
streptavidin beads (Invitrogen) were eluted by
boiling for 10 min in sample loading buffer and
subjected to electrophoresis on 4-20% SDS
polyacrylamide gradient gels. The gel was then
visualized using silver staining or SYPRO Ruby
staining. The protein bands which disappeared or
became less abundant in the samples which were
preincubated with cIDPRE were excised, in-gel
digested with trypsin, and analyzed by LC-
MS/MS on a ProteomeX-LTQ mass spectrometer
(Institute of Biophysics, CAS, Beijing, China) to
identify the interacting proteins. Database
searches were performed by using MASCOT.
C26H34N7O13P2[(M-H)-],
714.1695;
found,
714.1704.
Cell culture- Jurkat cells (from ATCC) were
maintained in RPMI 1640 medium (RPMI 1640,
powder, Invitrogen) with 10% fetal bovine serum
(FBS, Invitrogen) and 1% penicillin-streptomycin
(P/S, Invitrogen). HEK293 cells (from ATCC)
were maintained in Dulbecco’s Modified Eagle
Medium (DMEM, powder, Invitrogen) with 10%
fetal bovine serum (FBS, Invitrogen) and 1%
penicillin-streptomycin (P/S, Invitrogen). Human
coronary artery smooth muscle cells (HCASMC),
kindly provided by Dr Guirong Li of the
University of Hong Kong, were maintained in α-
medium/F-12 medium (1:1) with 20% fetal bovine
serum (FBS, Invitrogen), epidermal growth factor
(EGF, recombinant human, 0.5ng/ml) and
fibroblast growth factor (FGF, recombinant
human, 2ng/ml). All cells were maintained at
37°C with 5% CO2 and 95% humidity and they
were passaged every 2 or 3 days.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Photoaffinity purification- cIDPRE was used as a
competitor of photoaffinity labeling probe PAL-
cIDPRE. Briefly, for the control experiment,
cIDPRE was incubated with cell lysate in a final
concentration of 1 mM at 4 °C for 2 h, while for
the experimental group, the same volume of MQ
water was added instead of cIDPRE. Then PAL-
cIDPRE was added to the cell lysate in a final
Western blot analyses- Cells were lyzed in an ice-
cold lysis buffer (50 mM HEPES at pH 7.5, 0.15
M NaCl, 1 mM EDTA, 1% Nonidet P-40, 150 μM
PMSF, 10 mM NaF, 10 ng/ml leupeptin, 1 mM
3
ACS Paragon Plus Environment

Page 5 of 32
Journal of the American Chemical Society
DTT, and 1 mM sodium vanadate) and passed
balanced salt solution (HBSS) for 30 min in the
1
2
3
4
5
6
7
8
9
through a 21-gauge needle several times to
disperse any large aggregates. Protein
concentrations of the cell lysates were determined
by Bradford protein assay. 30 μg of protein per
lane was diluted in the standard SDS-sample
buffer and subjected to electrophoresis on 10%
SDS-polyacrylamide gels or gradient gels (4-20%,
purchased from Bio-rad). Proteins were then
transferred to an Immobilon PVDF membrane
(Millipore, Billerica, MA), blocked with 5% milk
in TBST (20 mM Tris, 150 mM NaCl, pH 7.6),
and incubated with the primary antibodies
overnight. After washed with TBST, the blots
were probed with a secondary antibody (1:5000
dilution) for detection by chemiluminescence. The
antibodies used in the western blot analyses were:
anti-GAPDH (1:1000 dilution), Sigma-Aldrich;
anti-RyRs (1:500) and anti-VCP (1:1000), Santa
Cruz Biotechnology, Inc.
dark at 37 °C. The cells were then washed with
HBSS twice and incubated in 200 μl of HBSS with
or without calcium. Thereafter, the cells were put
on the stage of an Olympus inverted
epifluorescence microscope for measuring
fluorescence intensity at 340 nm and 380 nm.
Images were collected by a CCD camera and
analyzed by the Cell^R software.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
shRNA and lentivirus production and infection-
Three shRNA oligoes against human GAPDH
(Table S1) were cloned into the pLKO.1 vector
for expressing shRNA. The lentivirus production
and infection were performed as described
previously15. The shRNA knockdown efficiencies
were assessed by western blot analyses.
Immunocytochemistry-
Immunocytochemistry
was performed as described previously 16. Briefly,
Jurkat cells (1×106 cells) were incubated with
saponin (50 μg/ml in PBS) at room temperature for
25 min, followed by cADPR, ADPR, or NAD+
(0.5 mM) treatment for indicated times. Cells were
then fixed by paraformaldehyde (4% w/v) at room
temperature for 30 min, spread on a gelatin coated
cover glass, and air dried for 20 min. Thereafter,
the cover glasses were blocked with 1% normal
donkey serum, 1% BSA, 0.1% Triton X-100 in
PBS for 1 h, and incubated with primary
antibodies (anti-GAPDH, G8795, Sigma-Aldrich,
1:200 dilution; anti-RyRs, sc-13942, Santa Cruz,
1:100 dilution) for 2 h, followed by secondary
Calcium measurement- Calcium measurement
14
was performed as described previously . Jurkat
cells (2 ×105 cells/well) or HEK293 cells (6 ×104
cells/well) were plated in 24-well plates coated
with 100 or 10 μg/ml poly-L-lysine (purchased
from Sigma), respectively. Human coronary artery
smooth muscle cells were plated in 24-well plates
without coating poly-L-lysine. Jurkat cells were
incubated in serum free medium overnight for
adherence while HEK293 and HCASMCs were
incubated in regular medium. The adherent cells
were incubated with 2 μM Fluo-2 AM in Hanks’
4
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 32
antibody (Alexa Fluor® 488 Goat Anti-Mouse
relatively low laser excitation power, typically 60
1
W/cm2 for 656.5 nm laser in Alexa 647 channel
and 80 W/cm2 for 750 nm laser in Alexa 750 nm
channel. During the STORM acquisition, the laser
power was raised to 4k W/cm2 and 4.5k W/cm2,
respectively. Each super-resolution image was
reconstructed from a movie containing 20,000-
30,000 frames recorded. The data was used to
calculate a two-dimensional (2D) Gaussian
distribution that was assumed to center on the
location of a single dye molecule. The final
resolution was determined to be ~20 nm in both
channels based on average fitting error.
2
3
4
5
6
7
8
9
IgG, A11008, 1:500 dilution; Alexa Fluor® 555
Donkey Anti-Rabbit IgG, A31572, Life
Technologies, 1:500 dilution) incubation for 1 h.
DAPI was used to stain the nuclei. Cells were
imaged using a Zeiss LSM 880 Laser Scanning
Microscope.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Super resolution imaging- Super resolution
imaging was performed as described previously 17.
Briefly, Jurkat cells were treated and immobilized
on gelatin coated cover glass as described in
immunocytochemistry. After incubated with
primary antibodies, the cover slips were incubated
with secondary antibodies (Alexa Fluor 647 anti-
rabbit for RYR primary antibody and Alexa Fluor
750 anti-mouse for GAPDH primary antibody) at
room temperature for 2 h. Cells were then fixed
with 3% PFA-0.05% glutaraldehyde at room
temperature for 20 min, and immersed in STORM
imaging buffer (50 mM TCEP (phosphine tris(2-
Immunoprecipitation- Immunoprecipitation was
performed as described previously18. Briefly, anti-
Flag antibody beads (Invitrogen) and Protein A
beads (GE Healthcare) were used to pull down
Flag-GAPDH
and
ryanodine
receptors,
respectively. Flag-GAPDH overexpressing Jurkat
cells were lyzed and 50 μl of anti-Flag beads were
added to the lysate. The mixture was incubated
with gentle rocking at 4 °C for 2 h, and centrifuged
for 30 s at 4°C to discard the supernatant. The
pellet was washed for three times with PBST (PBS
with 0.1% Tween 20), and 5×SDS sample buffer
was added to the beads. The sample was heated in
boiling water bath for 10 min, loaded on SDS-
PAGE gel, and subjected to western blot analysis.
Similarly, RyRs primary antibody was incubated
with Protein A beads with gentle rocking at 4 °C
for 2 h. The beads were then washed and added to
the lysate of Jurkat cells. The mixture was
5
carboxyethyl)phosphine),
2
mM
COT
(cyclooctatetraene), 5 U/ml pyranose oxidase,
10%(w/v) glucose, 57 μg/ml catalase, 1 mM
ascorbic acid, and 1mM methyl viologen in 200
mM Tris-HCL, pH9.0). Stochastic optical
reconstruction microscopy (STORM) images
were acquired by the STORM system
(NanoBioImaging Ltd, Hong Kong, China) with
the dual-channel imaging of Alexa Fluor647- and
Alexa Fluor750-immunolabeled samples. Prior to
STORM imaging, the desired position was located
using conventional fluorescence image with
ACS Paragon Plus Environment

Page 7 of 32
Journal of the American Chemical Society
incubated at 4 °C for another 2 h, followed by
To evaluate the effects of cADPR on the
1
2
3
4
5
6
7
8
9
cADPR (0.5 mM) treatment for the indicated
times. Thereafter, the sample was immediately
washed and heated with 5×SDS sample buffer in
boiling water bath, loaded on SDS-PAGE gel, and
subjected to western blot analysis.
enzymatic activity of GAPDH, cADPR at varied
concentration was added into the aforementioned
reaction mixture before the start of the recording
the A340.
HPLC analysis of cADPR or NAD+ in GAPDH
enzymatic reaction- HPLC analysis of cADPR or
NAD+ in GAPDH enzymatic reaction was
performed as described previously20. Briefly, a
column of AG MP-1 resin (10×120 mm) was used
to analyze cADPR, NAD+, or other adenine
nucleotides. The elution was performed using a
gradient of water/trifluoroacetic acid (TFA, 0.15
M in water) at a flow rate of 1 ml/min. The
gradient was 0 - 10 min 0-15% TFA, and 10 - 15
min 15-100% TFA. The UV detector of HPLC
was set at 260 nm. Samples of GAPDH enzymatic
reaction with or without cADPR were diluted with
TFA and injected into HPLC for analysis.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
GAPDH enzymatic activity assay- GAPDH
enzymatic activity assay was performed as
described previously19. Briefly, 0.1 ml of NAD+
(7.5 mM in water), 0.03 ml of DTT (0.1 M in
water) and 0.03 ml of GAPDH (0.083 mg/ml in
PBS) were mixed with 0.87 ml of the sodium
pyrophosphate buffer (0.015 M, pH 8.5,
containing 0.03 M sodium arsenate). The mixture
was then transferred to a cuvette and incubated in
o
spectrophotometer at 25 C for 3-5 minutes to
determine a blank absorbance at 340nm (A340) in
the absence of glyceraldehydes-3-phosphate
(G3P). Afterwards (time zero), 0.03 ml of 0.015M
G3P was added to the reaction and the A340 at 1
min interval were recoded for 4 or 5 minutes.
ΔA340/minute of the initial linear portion of the
curve was determined. The extinction coefficient
of NADH at 340 nm is 6.22 absorbance
units/mmol when the path length is 10 mm, and
one unit of enzymatic activity is defined as
reduction of 1 μM NAD+/min. Thus ，het GAPDH
enzymatic activity was calculated via the
following equation:
Molecular cloning- GAPDH cDNA was amplified
from HeLa cell cDNA pools and subcloned into
pRHSUL2 to engineer a His6-tagged and a Sumo
tag at the N-terminal of GAPDH, or into pENTR-
His6-Flag-C1 (Table S1). The His6-Flag-GAPDH
sequence was then recombined into the pLenti-
CMV-puro-DEST vector using the LR reaction,
according to the manufacturer’s instructions. The
different segments of RyR2 were amplified from
pCDNA-RyR2, a gift from Dr. King-Ho Cheung
of University of Hong Kong, and subcloned into
ΔA340/min
Units/mg=
6.22 × mg GAPDH/ml reaction mixture
6
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 32
PGEX-4T1 to engineer a GST-tag at the N-
terminal of RyR segments.
were dialyzed and concentrated by a 10 KD
1
2
3
4
5
6
7
8
concentrator to about 5-10 µg/ml. All purified
proteins were quantified by SDS-PAGE gel by
using BSA as the standard.
Mutagenesis-
GAPDHArg234Ala were made by site-directed
mutagenesis (Stratagene) as described
GAPDHHis179Ala
and
Surface Plasmon Resonance (SPR) assay-
Interactions between GAPDH or its mutants and
compounds were analyzed using the Biacore T200
system (GE Healthcare, Uppsala, Sweden) at 25
°C. Briefly, recombinant human GAPDH or its
mutant proteins were immobilized on a sensor
chip (CM5) using an amine coupling kit (GE
Healthcare, Buckinghamshire, UK). Final
immobilized GAPDH levels were typically
∼15000 RU. Subsequently, compounds were
injected as analytes at various concentrations and
20 mM Tris-HCl (pH 7.5 with 0.05% surfactant
P20) was used as running buffer. For binding
affinity studies, analytes were applied at indicated
concentrations in running buffer at a flow rate of
30 μl/min with a contact time of 60 second and a
dissociation time of 60 second. Chip platforms
were washed with running buffer.
9
previously18. The primers used are listed in Table
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
S1.
Recombinant protein purification- The His6-
Sumo-GAPDH construct was transformed into
BL21 (DE3) E.coli cells. Protein expression was
induced by isopropyl-d-thiogalactopyranoside
(IPTG) for overnight. The E.coli cells were then
lyzed and sonicated in an ice-chilled container.
The resulting cellular debris was removed by
centrifugation at 17000 rpm for 30 min, and the
supernatant was loaded into a 5 ml HisTrap HP
column (GE Healthcare). After extensive
washing, His6-Sumo-GAPDH protein was eluted
and dialyzed overnight with SUMO protease
buffer (20 mM Tris, pH7.5, 0.1 M Nacl, SUMO
protease) to cleave the His6-Sumo tag from
GAPDH. The reaction mixtures were again loaded
to a HisTrap HP column and the flowthrough was
collected to obtain GAPDH protein. The GAPDH
in the flow through was further purified by a
HiTrap Q column (GE Healthcare), and
concentrated by a 10 KD concentrator to about 20
µg/ml.
Molecule docking and molecular dynamic
simulations- The crystal structure of GAPDH was
obtained from RCSB Protein Data Bank
(http://www.pdb.org, PDB ID: 1U8F). The protein
was prepared with the Protein Clean tools in
Discovery Studio 2.5 (Accelrys, San Diego,
USA). In the preparation step, the CHARMm
force field was applied, the protein was
protonated, hydrogen atoms were added, and all
Similarly, the GST tagged RyR segments
were expressed and purified by a 5 ml GSTrap
column (GE Healthcare). The purified proteins
7
ACS Paragon Plus Environment

Page 9 of 32
Journal of the American Chemical Society
water molecules and the original ligand NAD+
were employed using Gaussian 09 program20. The
1
2
3
4
5
6
7
8
9
were all removed by using the protein preparation
protocols. The structure of cADPR was sketched
geometry was fully optimized and then the
electrostatic potentials around them were
determined at the HF/6-31G* level of theory. The
RESP strategy21 was used to obtain the partial
atomic charges.
in
ChemBioDraw
Ultra
(http://www.
cambridgesoft.com). Then the preparation and
1000 steps of steepest descent followed by 1000
steps of conjugate gradient minimization were
carried out for cADPR using Sybyl X 1.1.2
Molecular Modeling Suite (Version 1.1.2;
BioPharmic, LLC: San Mateo, CA, 2009). The
molecular docking simulation was carried out with
GOLD docking program (Gold version 5.2.2,
CCDC Software Ltd.: Cambridge, 2013). GOLD
is the abbreviation of genetic optimization for
ligand docking, which is a genetic algorithm for
docking flexible ligands into protein binding sites
containing flexible side chains of the target
protein. In the GOLD docking program, the initial
position of NAD+ was used to define the center of
the binding site, and the pocket was defined as all
residues within a radius of 8.0 Å. Other parameters
were remained as default. The docking results
were indicated by GoldScore fitness calculated
from contributions of hydrogen bonds and van der
Waals interactions between GAPDH and cADPR.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The starting structure of cADPR-GAPDH
complex obtained by docking was solvated in
TIP3P water using a octahedral box, which was
extended 8 Å away from any solute atom. To
neutralize the negative charges of simulated
molecules, Na+ counter-ion was placed next to
each phosphate group. MD simulation was carried
out by using SANDER module of AMBER 11.
The calculations began with 500 steps of steepest
descent followed by 500 steps of conjugate
gradient minimization with a large constraint of
500 kcal mol–1 Å-2 on the complexes atoms. Then
1000 steps of steepest descent followed by 1500
steps of conjugate gradient minimization with no
restraint on the complex atoms were performed.
Subsequently, after 20 ps of MD, during which the
temperature was slowly raised from 0 to 300 K
with weak (10 kcal mol–1 Å-2) restraint on the
complex, the final unrestrained production
simulations of 10.0 ns was carried out at constant
pressure (1 atm) and temperature (300 K). In the
entire simulation, SHAKE was applied to all
hydrogen atoms. Periodic boundary conditions
with minimum image conventions were applied to
calculate the nonbonded interactions. A cutoff of
10 Å was used for the Lennard-Jones interactions.
8
The best binding mode obtained from
molecule docking was chosen as initial structure
for molecular dynamics simulation. Molecular
dynamics (MD) simulation was performed with
AMBER 11 molecular simulation package19. To
obtain molecular mechanical parameters for
cADPR, ab initio quantum chemical methods
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 10 of 32
The final conformations of the complexes were
mM Tris-HCl, pH 8.8. The cathode buffer was 100
1
2
3
4
5
6
7
8
9
produced from the 1,000 steps of minimized
averaged structure of the last 5.0 ns of MD.
mM histidine with 0.002 % Coomassie Brilliant
Blue G-250, pH 8.0 (adjusted by Tris base). After
electrophoresis, the gel was either directly scanned
or transferred to PVDF membrane for western blot
analysis.
GST in vitro pull down assay- Binding between
GAPDH to RyR segments was assessed by the
GST fusion protein pulldown assay as described
previously 21. Briefly, GST-tag RyR segment
protein (10 µg) was incubated with 10 µl
Glutathione Sepharose 4 Fast Flow for 30 min on
ice followed by extensive washing. Thereafter,
BSA (25 µg) with or without GAPDH (10 µg) was
added to the beads followed by extensive washing.
The RyR segment protein and its associated
proteins were then eluted by glutathione and
analyzed by SDS-PAGE, coomassie brilliant blue
staining (for RyR detection), and immunoblot (for
GAPDH detection).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
RESULTS
Design and synthesis of a novel photoaffinity
labeling cADPR analogue, PAL-cIDPRE. Several
23
cADPR analogues have been synthesized
,
among which cIDPRE, a structural simplified
analogue, is a membrane-permeable cADPR
agonist in Jurkat T cells (Fig. 1)24. Based on the
structure-activity relationship of these cADPR
analogues, we found that the configuration of the
N1-glycosyl moiety on the northern ribose of
cADPR is not critical for its Ca2+ mobilization
ability. Thus, we designed and synthesized a
photoaffinity labeling cADPR analogue based on
cIDPRE, and referred it as PAL-cIDPRE (Fig. 1
and Scheme 1-3). PAL-cIDPRE is composed of
Native PAGE- A discontinuous native gel was
applied to analyze the oligomerization of
GAPDH. The recipe for a 5 ml native PAGE
stacking gel was: 0.375 M Tris-HCl (pH8.8),
4.275 ml; acrylamide/bis-acrylamide (30%/0.8%),
0.67 ml; 10% ammonium persulfate, 0.05 ml;
TEMED, 5 μl. For a 10 ml 8% separating gel, the
recipe was: 0.375 M Tris-HCl (pH8.8), 7.29 ml;
acrylamide/bis-acrylamide (30%/0.8%), 2.6 ml;
10% ammonium persulfate, 0.1 ml; TEMED, 10
μl. The 2x sample buffer was 0.625M Tris-HCl
(pH8.8) containing 50% glycerol and 1%
Coomassie Brilliant Blue G-250. As previously
reported 22, the anode buffer for native gel was 100
an amino-cIDPRE ligand moiety and
a
photoreactive group carrying a clickable terminal.
Notably, the ligand moiety has the northern ribose
replaced by a branched ether chains. Its
photoreactive group, diazirine, could bind the
related protein under UV irradiation and its
terminal alkynyl group could connect with a biotin
moiety by click reaction. Biotin was altered to
carry a corresponding clickable terminal of an
azide group and was separately synthesized (Fig.
9
ACS Paragon Plus Environment

Page 11 of 32
Journal of the American Chemical Society
S1A). The structure of PAL-cIDPRE was
antagonist, 8-Br-cADPR (Fig. 2C), or RyR2 and
1
characterized by 1H, 13C, 31P NMR, and
highresolution mass spectrometry (Fig. S1B-S1F).
In principle, after PAL-cIDPRE interacts with its
receptor(s), applied UV irradiation could generate
a reactive species that covalently binds the probe
to its receptor, and the binding protein(s) could
then be purified and identified.
RyR3 double knockdown26 (Fig. 2D) significantly
inhibited PAL-cIDPRE-induced Ca2+ increases in
Jurkat cells. Collectively, it is clear that PAL-
cIDPRE is a cell permeant cADPR agonist and can
trigger Ca2+ releases via RyRs.
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Identification of GAPDH as a cADPR binding
protein. Subsequently, we incubated PAL-
cIDPRE with Jurkat cell crude extracts in the
presence or absence of cIDPRE, followed by UV
irradiation to generate the reactive species that
crosslinks the cIDPRE to its target receptors. CuI
and azide-biotin were then added to the mixture to
form a triazole ring between biotin and the
protein-cIDPRE complexes via click reaction.
Afterwards, the cIDPRE-bound proteins were
purified by streptavidin-coupled Dynabeads® and
analyzed by mass spectrometry analyses (Fig.
3A). As shown in Figure 3B (right panel), S2A and
S2B, several protein bands appeared in samples
treated with PAL-cIDPRE alone, and two of these
bands were significantly competed off by cIDPRE
pre-incubation, suggesting that these two PAL-
cIDPRE labeled proteins are specific. Surprisingly,
one of the proteins (#1 in the gels in Fig. 3B and
S2A) turned out to be glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) by mass spec analyses
(Fig. S2C and S2D), and western blot analyses
confirmed that GAPDH in Jurkat cell crude extract
was specifically labeled with PAL-cIDPRE,
which was competed off by cIDPRE pre-
incubation (left panel in Fig. 3B). We then
10
Pharmacological characterization of PAL-
cIDPRE. We next examined the ability of PAL-
cIDPRE to induce Ca2+ release in human Jurkat T
cells. The PAL-cIDPRE markedly increased
cytosolic Ca2+ in a concentration-dependent
manner either in the presence or absence of
extracellular Ca2+, and the cytosolic Ca2+ increase
observed in the presence of extracellular Ca2+ was
significantly higher and sustained compared to
that in the absence of extracellular Ca2+ (Fig. 2A),
suggesting that Ca2+ influx also contributes.
Pretreating cells with thapsigargin, a specific
SERCA inhibitor, abolished PAL-cIDPRE-
induced Ca2+ increase in the absence of
extracellular Ca2+, consistent with the fact that
cADPR triggers Ca2+ release from the ER pools
(Fig. 2B). These data also indicate that PAL-
cIDPRE induces Ca2+ release from ER pools,
accompanied with extracellular Ca2+ influx.
Ample evidence indicates that cADPR targets
ryanodine receptors (RyRs) on the ER membrane
for Ca2+ mobilization in many cell types 25. Indeed,
pretreatment with a RyR antagonist, high
concentrations of ryanodine, or a cADPR
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 12 of 32
characterized the affinity between wild type
(Fig. S5). As expected, the SPR assay showed that
1
the KD value of cADPR to GAPDHHis179Ala mutant
protein was markedly increased to 82 μM (Fig.
4B) and cADPR even did not bind specifically to
GAPDHArg234Ala mutant protein (Fig. 4C). These
data indicate that both His179 and Arg234 in
GAPDH are indeed two key residues for the
interaction between cADPR and GAPDH. We
also used NAD+, a known binding partner of
GAPDH, in the SPR assay as a control, and found
that His179Ala or Arg234Ala mutation did not
significantly affect the binding affinity between
NAD+ and GAPDH (Fig. 5A-5C). Thus, these
data further indicate that His179 and Arg234 in
GAPDH are specific for its binding with cADPR,
not NAD+.
2
3
4
5
6
7
8
9
GAPDH protein and cADPR by a surface plasmon
resonance (SPR) assay, and found that cADPR
specifically bound to the recombinant GAPDH
proteins (Fig. S3) immobilized on the CM5 chip.
The calculated KD value of cADPR from the SPR
assay was around 8.59 μM (Fig. 3C). As a control,
ADPR, a nucleotide derived from NAD or
cADPR, showed no specific binding to GAPDH
immobilized on the CM5 chip by SPR assay (Fig.
S4). These data suggest that GAPDH is a specific
cADPR binding protein.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Mapping cADPR’s binding residues in GAPDH.
We also performed the molecule docking and
molecular dynamic simulations to predict the
binding sites in GAPDH for cADPR based on the
crystal structures of GAPDH27 and cADPR28. As
shown in Figure 4A, cADPR binding to GAPDH
could induce a conformational change of GAPDH,
and potential salt-bridges exist between two H
atoms of N2, N3 from guanidine group of Arg234
in GAPDH and the O4 from cADPR, and
hydrogen bonds between the H atom of N1 from
iminazole of His179 in GAPDH and the O5 from
cADPR, with distances of 1.8 Å, 2.0 Å, 1.9 Å,
respectively (Fig. 4A). This analysis suggests that
Arg234 and His179 in GAPDH might be the
potential binding sites for cADPR. We, therefore,
purified recombinant GAPDHArg234Ala and
GAPDHHis179Ala proteins (Fig. S3), and both
mutant proteins existed in the form of tetramers in
vitro, suggesting that both are properly folded
Effects of cADPR on GAPDH’s catalytic activity.
GAPDH, a traditional “housekeeping gene”, plays
essential role in glycolysis and gluconeogenesis.
GAPDH catalyzes oxidative phosphorylation of
D-glyceraldehyde 3-phosphate (G3P) to 1,3-
bisphospho-D-glycerate with inorganic phosphate
as the co-substrate and NAD+ as co-enzyme, in
which NAD+ can receive a hydride ion to be
29
reduced to NADH
(Fig. S6A). We, thus,
performed a standard in vitro GAPDH assay:
GAPDH + G3P ± NAD, in the presence or absence
of cADPR, and assessed GAPDH enzymatic
activity by measuring rate of NAD+ conversion to
NADH. We found that cADPR at higher
concentration
only
marginally
inhibited
GAPDH’s oxidative phosphorylation activity
11
ACS Paragon Plus Environment

Page 13 of 32
Journal of the American Chemical Society
(Fig. 6A and S6B). In addition, we analyzed the
affected by GAPDH depletion. In addition,
1
2
3
4
5
6
7
8
9
aforementioned reactions by HPLC to detect the
reaction end products. Interestingly, GAPDH +
G3P + NAD reaction not only produced the
NADH peak but also ADPR and nicotinamide
peak (Fig. S6C), indicating that GAPDH can
hydrolyze NAD to nicotinamide and ADPR. Yet,
in reaction of GAPDH + G3P + cADPR ± NAD,
GAPDH failed to catalyze or hydrolyze cADPR
into ADPR or any other compounds (Fig. 6B).
These data suggest that cADPR is not the catalytic
substrate of GADPH in glycolysis at least in vitro.
GAPDH knockdown had little effects on store-
operated Ca2+ entry (SOCE) (Fig. S7E). Notably,
ATP level in GAPDH knockdown cells was much
lower than that in control cells (Fig. S7F), and
these cells started to die after 3 passages. Taken
together, these data suggest that GAPDH is
specifically involved in cADPR-mediated
cytosolic Ca2+ mobilization.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
cADPR transiently increases the interaction
between GAPDH and RyRs. Since cADPR incites
Ca2+ release from ER via RyRs but it does not
directly bind to RyRs, we speculate that GAPDH
might be the bridging protein between cADPR and
RyRs. Not surprisingly, we found that there is a
basal interaction between GAPDH and RyRs in
Jurkat cells (Fig. S8A), and cADPR treatment
transiently but markedly increased the interaction
between RyRs and GAPDH as shown by the Co-
IP experiments (Fig. 8A and S8B). Furthermore,
we treated Jurkat cells with saponin to partially
permeabilize cell membrane, which enables the
cytosolic GAPDH to leak out of the cells but spare
the organelle or membrane associated one.
Strikingly, we found that addition of cADPR to the
permeabilized cells transiently induced the co-
localization of GAPDH with RyRs by
immunostaining analyses with antibodies against
GAPDH and anti-RyRs, respectively (Fig. 8B and
S8C). Pre-incubating the antibodies with
respective peptides abolished the positive staining
pattern in Jurkat cells (Fig. S8D), supporting the
GAPDH is required for cADPR-mediated Ca2+
release. GAPDH is actually more than just a
catalyzing enzyme in glycolysis but functions
across the major cellular compartments to be
involved in many important cellular events 30. We
reason that cADPR might bind to GAPDH to
trigger Ca2+ release from ER. As expected,
GAPDH knockdown markedly inhibited NPE-
cADPR or PAL-cIDPRE induced cytosolic Ca2+
increase in Jurkat cells (Fig. 7A and 7B), RyR3-
expressing HKE293 cells (Fig. 7C), or human
coronary artery smooth muscle (HCVSM) cells
(Fig. 7D). On the other hand, GAPDH knockdown
did not affect ATP or histamine induced Ca2+
increases in Jurkat cells (Fig. S7A and S7B),
suggesting that GAPDH is not involved in IP3-
mediated Ca2+ mobilization. Likewise, GAPDH
knockdown did not change ionomycin or
thapsigargin induced Ca2+ increases (Fig. S7C and
S7D), suggesting that the ER Ca2+ pool is not
12
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 14 of 32
specificity of both antibodies. Surprisingly,
Moreover, addition of cADPR to the binding
1
2
3
4
5
6
7
8
9
washing saponin-treated cells with PBS abolished
cADPR-induced co-localization of GAPDH with
RyRs (Fig. S8E). These data suggest that cADPR
forms complex with the leaked GAPDH
extracellularly and the cADPR-GAPDH complex
then transiently flows back into cells to interact
with RyRs. As controls, ADPR, a metabolite of
cADPR or NAD+, or NAD+ itself, failed to induce
the localization of GAPDH with RyRs in saponin-
treated Jurkat cells (Fig. S8F and S8G).
Furthermore, super-resolution imaging analyses
confirmed that GAPDH was transiently co-
localized with RyRs in saponin-treated Jurkat cells
upon cADPR treatment (Fig. 8C). Collectively,
these data indicate that cADPR treatment
transiently increases the interaction between
GAPDH and RyRs in vivo.
complex increased the binding affinity between
region 7 (residues 3512-4560), not region 6
(residues 2210-2643), and GAPDH (Fig. 9B).
Subsequently, we assessed which domain(s) in
GAPDH is(are) involved in the interaction with
the region 7 (residues 3512-4560) in RyR2.
Surprisingly, the in vitro GST pull-down
experiments showed that the catalytic domain, not
NAD binding domain, of GAPDH bound with the
region 7 (residues 3512-4560) in RyR2 (Fig. 9C).
Notably, the region 7 is actually the central
domain of RyRs. Most recently, the structures of
RyRs in both the open and closed states were
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
determined
by
single-particle
electron
cryomicroscopy, and it has shown that the rotation
of central domain of RyRs leads to the dilation of
31
the cytoplasmic gate through coupled motions .
Mapping the regions in RyRs for GAPDH
interaction. To assess which region(s) in RyRs
is(are) involved in the interaction with GAPDH,
Nevertheless, these data suggest that the
interaction between GAPDH and RyRs is specific,
and cADPR treatment increases the interaction
between GAPDH and RyRs in vitro.
we
purified
several
GST-fusion
recombinantproteins covering different RyR2
regions located at its N-terminal cytosolic domain
except regions from amino acid residues 2644-
3511, which appeared to be in the inclusion body
(Fig. 9A). The in vitro GST pull-down
experiments were subsequently performed to
determine the region in RyRs where GAPDH
binds. As shown in Figure 10A, two regions, 6
(residues 2210-2643) and 7 (residues 3512-4560),
in RyR2 exhibited weak interaction with GAPDH.
DISCUSSION
Here we identified GAPDH as one of
cADPR’s binding protein(s) by photoaffinity
protein-ligand labeling approach (Fig. 3 and S2),
and demonstrated that GAPDH is required for
cADPR-induced Ca2+ release from ER in several
cell lines (Fig. 7). Notably, GAPDH did not
catalyze cADPR into any other known or novel
compound(s) (Fig. 6B). Moreover, we found that
13
ACS Paragon Plus Environment

Page 15 of 32
Journal of the American Chemical Society
cADPR transiently increased the interaction
Thus, the binding affinity between cADPR and
1
2
3
4
5
6
7
8
9
between GAPDH and RyRs both in vitro and in
vivo (Fig. 8 and 9). Taken together, our data
indicate that cADPR targets GAPDH to RyRs, and
thus triggers Ca2+ release from ER.
GAPDH might be underestimated in SPR assay
compared to that in vivo.
RyRs form a class of intracellular calcium
channels in various cells and tissues such as
muscles and neurons. It is the major cellular
mediator of calcium-induced calcium release in
cells, a key event for triggering muscle contraction
35. It is thought that the final functional output of
RyRs is determined by a complex interplay of
fluctuating Ca2+ levels, tonic and short-lived
cytosolic modulators, and protein–protein
interactions. For example, FKBP12 binds to a
closed state of RyRs and thus decreases RyRs’
sensitivity to Ca2+ 36. Along this line, the structure
of a closed state RyR1 in complex with FKBP12
was recently determined by single-particle
Interestingly, another protein (band 2 in Fig.
3B and S2A) might also be a specific PAL-
cIDPRE’s binding protein since it was competed
off by cIDPRE pre-incubation, yet its identity
remained to be determined due to its low
abundancy after the final purification step. Efforts
will be continued to identify this protein in the
near future. Upon its identification, its role in
cADPR-mediated Ca2+ mobilization and its
relationship with GAPDH and RyRs upon cADPR
treatment will be studied accordingly.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
It has been shown that cADPR specifically
binds to microsomes from sea urchin eggs with KD
around 17 nM 32. In human Jurkat cells, Guse et al.
found that stimulation of the T-cell receptor
markedly increased intracellular cADPR levels
from 0.5 µM to 2 µM, which is responsible for
subsequent Ca2+ signaling and T cell activation 33.
Here, we found that the binding affinity between
cADPR and GAPDH is around KD 9 µM (Fig. 4C).
Obviously, the aforementioned mysterious
protein(s) (band 2 in Fig. 3B and S2A) might be
involved in the interaction between cADPR and
GAPDH as well. In addition, it is well recognized
that it is difficult not to change the biological
functionality of molecules during the process of
37
electron cryomicroscopy , and this closed state
RyR1 structure shed light on high ion conductance
by RyRs and the long-range allosteric regulation
of channel activities. It has been suggested that
cADPR treatment induces the disassociation of
FKBP12 from RyR2 and thus activates RyRs for
Ca2+ releasing, and phosphorylation of RyR2 at
Ser2808 or Ser2815 might be required for the
release of FKBP12 from RyR2 as well 38. Thus, it
is of interest to assess the effects of the binding
between cADPR and GAPDH on FKBP12’s
association with RyRs and the phosphorylation
state of RyRs.
cADPR binding to GAPDH might induce a
conformation change on GAPDH as revealed by
14
34
immobilizing the molecules to the sensor chip .
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 16 of 32
the molecule docking and molecular dynamic
between GAPDH and RyRs in the absence
1
2
3
4
5
6
7
8
9
simulations (Fig. 4A), and obviously this awaits
to be confirmed by the crystal structure of
GAPDH-cADPR complex. We also found that
two regions, residues 2210-2643 and residues
3512-4560, in RyR2 exhibited weak interaction
with GAPDH in vitro (Fig. 9A). Surprisingly,
cADPR addition only increased the binding
affinity between region (residues 3512-4560),
not region (residues 2210-2643), of RyR2 and
GAPDH in vitro (Fig. 9B). One possibility is
that the RyR2 region (residues 2210-2643) fails
to fold correctly in vitro resulting in the inability
of the cADPR-GAPDH complex to efficiently
interact with it. Another possibility is that
cADPR binds to GAPDH thus changes
GAPDH’s binding pattern with RyRs, showing
higher affinity with one region in RyRs than
another one. Additional in vivo assays are
needed to assess these possibilities, and the
ultimate answer might lie on solving the cryo-
EM structure of cADPR-GAPDH-RyRs
complex.
cADPR (Fig. S8A) and cADPR treatment
transiently increased the interaction between
GAPDH and RyRs (Fig. 8A-8C and S8B). Not
surprisingly, GAPDH knockdown markedly
inhibited the ability of caffeine, a known RyRs
agonist, to induce intracellular Ca2+ release in
both Jurkat cells and RyR3-expressing HEK293
cells (Fig. S9), suggesting that GAPDH is
important for RyRs-mediated Ca2+ release in
general.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Accumulating evidence indicate that
GAPDH is more than just a catalyzing enzyme
but functions across the major cellular
compartments to play essential roles in many
important cellular events, such as DNA repair,
tRNA export, membrane fusion and transport,
cytoskeletal dynamics, and cell death30. Indeed,
dysregulation of GAPDH has been associated
with numerous human diseases, e.g.
neurodegenerative disorders and cancers 41. It
would be of interest to study whether the
cADPR-GAPDH-RyR-Ca2+ cascade contributes
to these functions and disorders. The pleiotropic
functions of GAPDH are regulated by
posttranslational modification and subcellular
Glucose
or
glycolytic
pathway
intermediaries has long been found to modulate
intracellular Ca2+ oscillation39. For example,
intracellular glucose inhibited cADPR-mediated
42
localization of GAPDH . Yet, the mechanism
Ca2+ spiking, but potentiated IP3-induced Ca2+
underlying the regulation of GAPDH in these
non-glycolytic cellular events remains elusive.
The presumably conformational changes of
GAPDH (Fig. 4A), and its trans-localization to
ER upon cADPR binding (Fig. 8B and 8C)
46
spiking
.
Several glycolytic enzymes,
including GAPDH, has also been shown to form
40
a ternary complex in skeletal muscle triads .
Here we found that there is a weak interaction
15
ACS Paragon Plus Environment

Page 17 of 32
Journal of the American Chemical Society
should add another layer of regulation on
Figure S4. ADPR failed to specifically bind to
1
2
3
GAPDH.
GAPDH in the SPR assay.
4
5
Figure S5. Characterization of recombinant
ASSOCIATED CONTENT
6
7
GAPDH
wildtype,
GAPDHHis179Ala
,
and
Supplemental Table S1. Primer sequences used
GAPDHArg234Ala proteins by blue native (BN)-
PAGE electrophoresis. Upper panel: Coomassie
blue G250 staining; bottom panel: GAPDH
immunoblot.
8
9
in the study.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure S1. Synthesis scheme of azide-biotin and
characterization of PAL-cIDPRE. (A) Azide-
biotin synthesis scheme. (B) to (D) 1H (B), 13C (C)
, and 31P NMR (D) of compound 15. (E) and (F)
The 31P NMR (E) and high resolution mass
spectrometry (F) analyses of PAL-cIDPRE.
Figure S6. The effects of cADPR on the catalytic
activity of GAPDH in vitro. (A) Schematic of
GAPDH assay. (B) GAPDH enzymatic activity
was assessed by measuring rate of NAD+
conversion to NADH (A340nm). cADPR at higher
concentration marginally inhibited GAPDH’s
oxidative phosphorylation activity. (C) HPLC
analysis of the GAPDH + G3P ± NAD+ reaction.
Quantification of nicotinamide, NADH, and
ADPR peaks in the reactions (NAD + GAPDH +
G3P ± cADPR) of Fig. S6C and Fig. 6B are
expressed as area under curve (AUC) ± S.D., n =
3.
Figure S2. Purification of the cADPR binding
proteins by a photoaffinity purification approach.
(A) and (B) The final elution of PAL-cIDPRE
binding proteins from Jurkat cell extract in the
presence or absence of cIDPRE was subjected to
electrophoresis on a SDS-PAGE gel, and
visualized by silver staining (A) or detected by
streptavidine-HRP
immunoblot
(B).
(C)
Schematic of GAPDH peptides recovered by mass
spec analyses. (D) Peptides recovered by mass
spec analyses (heighted by color, over 72%
peptide recovery rate) and MS/MS spectra of two
GAPDH peptides.
Figure S7. GAPDH knockdown in Jurkat cells did
not have any significant effects on ATP (100 µM)
(A), histamine (100 µM) (B), ionomycin (1 µM)
(C), or thapsigargin (1 µM) (D) induced cytosolic
Ca2+ increases, nor did it affected SOCE (E) in
Jurkat cells. Quantification of intracellular Ca2+
peak values are expressed as mean ± S.D., n = 3
(15-30 cells in each independent experiment). (F)
GAPDH knockdown markedly decreased ATP
Figure S3. Purification and quantification of
recombinant GAPDH wildtype, GAPDHHis179Ala
and GAPDHArg234Ala proteins. BSA of indicated
concentrations was used as standard to quantify
GAPDH concentration.
,
16
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 18 of 32
production in HEK293, RyR3-HEK293, and
AUTHOR INFORMATION
Corresponding Author
1
2
3
Jurkat cells.
4
5
6
7
8
9
*Correspondence and requests for materials
should be addressed to J.Y, L.H.Z, and L.Z
Figure S8. cADPR transiently increases the
interaction between GAPDH and RyRs in human
Jurkat cells. (A) Basal interaction between
GAPDH and RyRs as shown by co-IP experiments
in Jurkat cells. (B) cADPR at different
Author Contributions
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The manuscript was written through contributions
of all authors. / All authors have given approval to
the final version of the manuscript.
concentrations
transiently
increased
the
interaction between GAPDH and RyRs in Jurkat
cell extract. (C) Treatment of saponin-
permebilized Jurkat cells with cADPR (0.5 mM)
transiently induced the co-localization of GAPDH
with RyR as shown by confocal image analyses of
GAPDH and RyR. Co-localization scan of
immune staining of GAPDH and RyRs in Jurkat
cells treated with cADPR for the indicated time
were performed. (D) Pre-incubation of GAPDH or
RyR antibody with respective blocking peptide
abolished the positive signaling. (E) In saponin-
treated cells followed by PBS washing, cADPR
failed to induce the co-localization of GAPDH
with RyRs. (F) and (G) ADPR (0.5 mM) (F) or
NAD+ (0.5 mM) (G) failed to induce the co-
localization of GAPDH with RyRs in saponin-
treated Jurkat cells.
Funding Sources
This work was supported by Hong Kong Research
Grant Council (RGC) grants (785213 and
17126614), ITS/261/14, CAS-Croucher Funding
Scheme, and Guangdong-Hong Kong joint
innovation
Research
Scheme
(#2016A050503010) to JY, research grants from
Shenzhen government and from SUSTC
(ZDSYS20140509142721429
and
FRG-
SUSTC1501A-24) to HZ, and National Natural
Science Foundation of China (NSFC): 91213302,
81673279 (to L. Zhang) and 81573273 (to L-H.
Zhang).
ACKNOWLEDGMENT
We thank members of Yue lab for their advice on
the preparation of this manuscript.
Figure S9. GAPDH was required for caffeine (20
mM)-induced Ca2+ increases in mammalian cells.
(A) and (B) GAPDH knockdown significantly
inhibited caffeine-induced intracellular Ca2+
increases in Jurkat cells (A) or RyR3-expressing
HEK293 cells (B).
ABBREVIATIONS
RyR, ryanodine receptor; cADPR, cyclic ADP-
ribose; NAD+, nicotinamide adenine dinucleotide;
SOCE, store operated calcium entry; IP3, inositol
trisphosphate; SPR, surface plasmon resonance;
17
ACS Paragon Plus Environment

Page 19 of 32
Journal of the American Chemical Society
G3P, D-glyceraldehyde 3-phosphate; GAPDH,
CB 2005, 15, 874-8; (b) Kannan, M. S.; Fenton, A.
1
2
3
4
5
6
7
8
9
glyceraldehyde 3-phosphate dehydrogenase; PAL-
cIDPRE, photoaffinity labeling cIDPRE.
M.; Prakash, Y. S.; Sieck, G. C. Am J Physiol
1996, 270, H801-6; (c) Prakash, Y. S.; Kannan, M.
S.; Walseth, T. F.; Sieck, G. C. Am J Physiol 1998,
274, C1653-60.
REFERENCES
7.
(a) Lee, H. C. Curr Mol Med 2004, 4, 227-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
37; (b) Malavasi, F.; Deaglio, S.; Funaro, A.;
Ferrero, E.; Horenstein, A. L.; Ortolan, E.;
Vaisitti, T.; Aydin, S. Physiol Rev 2008, 88, 841-
86; (c) Deterre, P.; Berthelier, V.; Bauvois, B.;
Dalloul, A.; Schuber, F.; Lund, F. Chem Immunol
2000, 75, 146-68.
1.
(a) Guse, A. H. Curr Med Chem 2004, 11,
847-55; (b) Galione, A.; Churchill, G. C. Sci STKE
2000, 2000, PE1.
2.
Bruzzone, S.; Moreschi, I.; Usai, C.;
Guida, L.; Damonte, G.; Salis, A.; Scarfi, S.;
Millo, E.; De Flora, A.; Zocchi, E. Proc Natl Acad
Sci U S A 2007, 104, 5759-64.
8.
(a) Jin, D.; Liu, H. X.; Hirai, H.;
Torashima, T.; Nagai, T.; Lopatina, O.; Shnayder,
N. A.; Yamada, K.; Noda, M.; Seike, T.; Fujita,
K.; Takasawa, S.; Yokoyama, S.; Koizumi, K.;
Shiraishi, Y.; Tanaka, S.; Hashii, M.; Yoshihara,
T.; Higashida, K.; Islam, M. S.; Yamada, N.;
Hayashi, K.; Noguchi, N.; Kato, I.; Okamoto, H.;
Matsushima, A.; Salmina, A.; Munesue, T.;
Shimizu, N.; Mochida, S.; Asano, M.; Higashida,
H. Nature 2007, 446, 41-5; (b) Nagamune, K.;
Hicks, L. M.; Fux, B.; Brossier, F.; Chini, E. N.;
Sibley, L. D. Nature 2008, 451, 207-10.
3.
(a) Yilmaz, O. H.; Katajisto, P.; Lamming,
D. W.; Gultekin, Y.; Bauer-Rowe, K. E.;
Sengupta, S.; Birsoy, K.; Dursun, A.; Yilmaz, V.
O.; Selig, M.; Nielsen, G. P.; Mino-Kenudson, M.;
Zukerberg, L. R.; Bhan, A. K.; Deshpande, V.;
Sabatini, D. M. Nature 2012, 486, 490-5; (b)
Igarashi, M., Guarente, L. Cell 2016, 166, 436-
450.
4.
Dodd, A. N.; Gardner, M. J.; Hotta, C. T.;
Hubbard, K. E.; Dalchau, N.; Love, J.; Assie, J.
M.; Robertson, F. C.; Jakobsen, M. K.; Goncalves,
J.; Sanders, D.; Webb, A. A. Science 2007, 318,
1789-92.
9.
Lund, F.; Ziegler, M. Sci Signal 2009, 2, mr1.
10. (a) Galione, A.; Lee, H. C.; Busa, W. B.
Koch-Nolte, F.; Haag, F.; Guse, A. H.;
5.
Reyes-Harde, M.; Empson, R.; Potter, B.
Science 1991, 253, 1143-6; (b) Galione, A.;
White, A.; Willmott, N.; Turner, M.; Potter, B. V.;
Watson, S. P. Nature 1993, 365, 456-9; (c) Hua,
S. Y.; Tokimasa, T.; Takasawa, S.; Furuya, Y.;
Nohmi, M.; Okamoto, H.; Kuba, K. Neuron 1994,
V.; Galione, A.; Stanton, P. K. Proc Natl Acad Sci
U S A 1999, 96, 4061-6.
6.
(a) Yamasaki, M.; Thomas, J. M.;
Churchill, G. C.; Garnham, C.; Lewis, A. M.;
Cancela, J. M.; Patel, S.; Galione, A. Curr Biol :
18
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 20 of 32
12, 1073-9; (d) Barry, V. A.; Cheek, T. R.
Wu, W. T.; Markus, H.; Yue, J. Autophagy 2014,
10, 1895-905.
17. Zhao, T.; Wang, Y.; Zhai, Y.; Qu, X.;
1
2
3
4
5
6
7
8
Biochem J 1994, 300, 589-97; (e) Lee, H. C.;
Aarhus, R.; Graeff, R. M. J Biol Chem 1995, 270,
9060-6; (f) Noguchi, N.; Takasawa, S.; Nata, K.;
Tohgo, A.; Kato, I.; Ikehata, F.; Yonekura, H.;
Okamoto, H. J Biol Chem 1997, 272, 3133-6; (g)
Kunerth, S.; Langhorst, M. F.; Schwarzmann, N.;
Gu, X.; Huang, L.; Yang, Z.; Zhang, L.; Mills, S.
J.; Zhang, L. H.; Potter, B. V.; Guse, A. H. J Cell
Sci 2004, 117, 2141-9; (h) Fulceri, R.; Rossi, R.;
Bottinelli, R.; Conti, A.; Intravaia, E.; Galione, A.;
Benedetti, A.; Sorrentino, V.; Reggiani, C.
Biochem Biophys Res Commun 2001, 288, 697-
702.
Cheng, A.; Du, S.; Loy, M. M. Opt Express 2015,
23, 1879-87.
9
18.
Yue, J.; Ferrell, J. E., Jr. Mol Cell Biol
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2006, 26, 5300-9.
19.
Zhang, J.; Snyder, S. H. Proc Natl Acad
Sci U S A 1992, 89, 9382-5.
20.
Graeff, R.; Liu, Q.; Kriksunov, I. A.;
Kotaka, M.; Oppenheimer, N.; Hao, Q.; Lee, H. C.
J Biol Chem 2009, 284, 27629-36.
21.
Sambrook, J.; Russell, D. W. CSH Protoc
2006, 2006, 3757.
11.
C.; Galione, A. Biochem J 2001, 359, 451-7.
12. (a) Walseth, T. F.; Aarhus, R.; Kerr, J. A.;
Thomas, J. M.; Masgrau, R.; Churchill, G.
22.
Niepmann, M.; Zheng, J. Electrophoresis
2006, 27, 3949-51.
23.
(a) Tsuzuki, T.; Sakaguchi, N.; Kudoh, T.;
Lee, H. C. J Biol Chem 1993, 268, 26686-91; (b)
Tang, W. X.; Chen, Y. F.; Zou, A. P.; Campbell,
W. B.; Li, P. L. Am J Physiol Heart Circ Physiol
2002, 282, H1304-10.
Takano, S.; Uehara, M.; Murayama, T.; Sakurai,
T.; Hashii, M.; Higashida, H.; Weber, K.; Guse, A.
H.; Kameda, T.; Hirokawa, T.; Kumaki, Y.; Potter,
B. V.; Fukuda, H.; Arisawa, M.; Shuto, S. Angew
Chem Int Ed Engl 2013, 52, 6633-7; (b)
Swarbrick, J. M.; Graeff, R.; Garnham, C.;
Thomas, M. P.; Galione, A.; Potter, B. V. Chem
Commun 2014, 50, 2458-61.
13.
Gu, X.; Yang, Z.; Zhang, L.; Kunerth, S.;
Fliegert, R.; Weber, K.; Guse, A. H.; Zhang, L. J
Med Chem 2004, 47, 5674-82.
14.
Wei, W.; Lu, Y.; Hao, B.; Zhang, K.;
Wang, Q.; Miller, A. L.; Zhang, L. R.; Zhang, L.
24.
Zhang, L.; Yue, J.; Zhang, L. H. Chem Rec
H.; Yue, J. Stem cells 2015, 33, 2664-73.
2015, 15, 511-23.
15.
Lu, Y.; Hao, B. X.; Graeff, R.; Wong, C.
25.
(a) Higashida, H.; Salmina, A. B.;
W.; Wu, W. T.; Yue, J. J Biol Chem 2013, 288,
Olovyannikova, R. Y.; Hashii, M.; Yokoyama, S.;
Koizumi, K.; Jin, D.; Liu, H. X.; Lopatina, O.;
Amina, S.; Islam, M. S.; Huang, J. J.; Noda, M.
Neurochem Int 2007, 51, 192-9; (b) Lee, H. C.;
24247-63.
16.
Lu, Y.; Dong, S.; Hao, B.; Li, C.; Zhu, K.;
Guo, W.; Wang, Q.; Cheung, K. H.; Wong, C. W.;
19
ACS Paragon Plus Environment

Page 21 of 32
Journal of the American Chemical Society
Aarhus, R.; Graeff, R.; Gurnack, M. E.; Walseth,
T. F. Nature 1994, 370, 307-9.
26. Yu, P. L.; Zhang, Z. H.; Hao, B. X.; Zhao,
32.
81.
33.
Lee, H. C. J Biol Chem 1991, 266, 2276-
1
2
3
4
5
6
7
8
9
Guse, A. H.; da Silva, C. P.; Berg, I.;
Y. J.; Zhang, L. H.; Lee, H. C.; Zhang, L.; Yue, J.
Skapenko, A. L.; Weber, K.; Heyer, P.;
Hohenegger, M.; Ashamu, G. A.; Schulze-Koops,
H.; Potter, B. V.; Mayr, G. W. Nature 1999, 398,
70-3.
J Biol Chem 2012, 287, 24774-83.
27.
(a) Cowan-Jacob, S. W.; Kaufmann, M.;
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Anselmo, A. N.; Stark, W.; Grutter, M. G. Acta
Crystallogr D 2003, 59, 2218-27; (b) Charron, C.;
Talfournier, F.; Isupov, M. N.; Littlechild, J. A.;
Branlant, G.; Vitoux, B.; Aubry, A. J Mol Biol
2000, 297, 481-500.
34.
Pattnaik, P. Appl Biochem Biotech 2005,
126, 79-92.
35.
(a) Franzini-Armstrong, C.; Protasi, F.
Physiol Rev 1997, 77, 699-729; (b) Protasi, F.;
Shtifman, A.; Julian, F. J.; Allen, P. D. Am J
Physiol Cell Physiol 2004, 286, C662-70; (c)
Yano, M.; Yamamoto, T.; Ikeda, Y.; Matsuzaki,
M. Nat Clin Pract Card 2006, 3, 43-52.
28.
Lee, H. C.; Aarhus, R.; Levitt, D. Nat
Struct Biol 1994, 1, 143-4.
29.
Seidler, N. W. Adv Exp Med Biol 2013,
985, 1-36.
30.
(a) Chang, C.; Su, H.; Zhang, D.; Wang,
36.
Marx, S. O.; Reiken, S.; Hisamatsu, Y.;
Y.; Shen, Q.; Liu, B.; Huang, R.; Zhou, T.; Peng,
C.; Wong, C. C.; Shen, H. M.; Lippincott-
Schwartz, J.; Liu, W. Mol Cell 2015, 60, 930-40;
(b) Yun, J.; Mullarky, E.; Lu, C.; Bosch, K. N.;
Kavalier, A.; Rivera, K.; Roper, J.; Chio, II;
Giannopoulou, E. G.; Rago, C.; Muley, A.; Asara,
J. M.; Paik, J.; Elemento, O.; Chen, Z.; Pappin, D.
J.; Dow, L. E.; Papadopoulos, N.; Gross, S. S.;
Cantley, L. C. Science 2015, 350, 1391-6.
Jayaraman, T.; Burkhoff, D.; Rosemblit, N.;
Marks, A. R. Cell 2000, 101, 365-76.
37.
(a) Zalk, R.; Clarke, O. B.; des Georges,
A.; Grassucci, R. A.; Reiken, S.; Mancia, F.;
Hendrickson, W. A.; Frank, J.; Marks, A. R.
Nature 2015, 517, 44-9; (b) Efremov, R. G.;
Leitner, A.; Aebersold, R.; Raunser, S. Nature
2015, 517, 39-43; (c) Yan, Z.; Bai, X. C.; Yan, C.;
Wu, J.; Li, Z.; Xie, T.; Peng, W.; Yin, C. C.; Li,
X.; Scheres, S. H.; Shi, Y.; Yan, N. Nature 2015,
517, 50-5.
31.
(a) des Georges, A.; Clarke, O. B.; Zalk,
R.; Yuan, Q.; Condon, K. J.; Grassucci, R. A.;
Hendrickson, W. A.; Marks, A. R.; Frank, J. Cell
2016, 167, 145-157; (b) Peng, W.; Shen, H.; Wu,
J.; Guo, W.; Pan, X.; Wang, R.; Chen, S. R.; Yan,
N. Science 2016, 354, aah5324.
38.
(a) Zheng, J.; Wenzhi, B.; Miao, L.; Hao,
Y.; Zhang, X.; Yin, W.; Pan, J.; Yuan, Z.; Song,
B.; Ji, G. Cell Calcium 2010, 47, 449-57; (b)
Zhang, X.; Tallini, Y. N.; Chen, Z.; Gan, L.; Wei,
B.; Doran, R.; Miao, L.; Xin, H. B.; Kotlikoff, M.
20
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 22 of 32
I.; Ji, G. Cardiovasc Res 2009, 84, 253-62; (c)
A 2015, 112, E2217-24; (b) Sen, N.; Hara, M. R.;
1
2
3
4
5
6
7
8
Wang, Y. X.; Zheng, Y. M.; Mei, Q. B.; Wang, Q.
S.; Collier, M. L.; Fleischer, S.; Xin, H. B.;
Kotlikoff, M. I. Am J Physiol-Cell Ph 2004, 286,
C538-46.
Kornberg, M. D.; Cascio, M. B.; Bae, B. I.;
Shahani, N.; Thomas, B.; Dawson, T. M.;
Dawson, V. L.; Snyder, S. H.; Sawa, A. Nat Cell
Biol 2008, 10, 866-73.
9
39.
(a) Chini, E. N.; Dousa, T. P. Arch
42.
(a) Sawa, A.; Khan, A. A.; Hester, L. D.;
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Biochem Biophys 1999, 370, 294-9; (b) Cancela,
J. M.; Mogami, H.; Tepikin, A. V.; Petersen, O. H.
Curr Biol : CB 1998, 8, 865-8.
Snyder, S. H. Proc Natl Acad Sci U S A 1997, 94,
11669-74; (b) Tristan, C.; Shahani, N.; Sedlak, T.
W.; Sawa, A. Cell Signal 2011, 23, 317-23; (c)
Hara, M. R.; Agrawal, N.; Kim, S. F.; Cascio, M.
B.; Fujimuro, M.; Ozeki, Y.; Takahashi, M.;
Cheah, J. H.; Tankou, S. K.; Hester, L. D.; Ferris,
C. D.; Hayward, S. D.; Snyder, S. H.; Sawa, A.
Nat Cell Biol 2005, 7, 665-74.
40.
Brandt, N. R.; Caswell, A. H.; Wen, S. R.;
Talvenheimo, J. A. J Membrane Biol 1990, 113,
237-51.
41.
(a) Suzuki, M.; Sasabe, J.; Miyoshi, Y.;
Kuwasako, K.; Muto, Y.; Hamase, K.; Matsuoka,
M.; Imanishi, N.; Aiso, S. Proc Natl Acad Sci U S
21
ACS Paragon Plus Environment

Page 23 of 32
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
22
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 24 of 32
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
23
ACS Paragon Plus Environment

Page 25 of 32
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
24
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 26 of 32
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
25
ACS Paragon Plus Environment

Page 27 of 32
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
26
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 28 of 32
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
27
ACS Paragon Plus Environment

Page 29 of 32
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
28
ACS Paragon Plus Environment