59
Enzyme Mediated S- glutathionylation Danyelle M. Townsend Director, Analytical Redox Biochemistry Nebraska, June 11, 2019
Enzyme Mediated S-
glutathionylation
Danyelle M. Townsend Director,
Analytical Redox Biochemistry
Nebraska, June 11, 2019
www.creative-proteomics.com
Post-translational modifications:
Highly Regulated Complexity of Life
# cysteinesin genome
Biological complexity 200,000 cysteines in human proteome
Possible Cysteine Post translational modifications:
• thiol/disulfide, thiolate, sulfenate, sulfinate, sulfonate
• S-glutathionylation
• S-nitrosylation
• S-cysteinylation
• S-sulfydration (persulfide)
• S-thiohemiacetylation
• S-succinylation
• S-isoprenylation
• S-farnesylation
• S-geranylgeranylation
• S-palmitoylation
Cysteine: Post-translational Modifications
From: Tew et al, 2016
# cysteines in genome
Biological complexity
200,000 cysteines in human proteome
https://www.researchgate.net/publication/232226611_Oxidative_Modification_of_Proteins_An_Emerging_Mechanism_of_Cell_Signaling
H2N
CH2
COOH
SH
H2N
CH2
COOH
S
S
HN
O
COOHNH
O
COOH
NH2
Low pK
cysteine
residue
S-glutathionylation
Deglutathionylation
H2N
CH2
COOH
S
OH
O O
H2N
CH2
COOH
S
OH
O
H2N
CH2
COOH
S
OH
H2N
CH2
COOH
S.
Cysteinyl radical Sulfinic acid Sulfonic acidSulfenic acid
[O]
[N]
S-Glutathionylation
Protected
Glutaredoxin
GSTO
Sulfiredoxin
GST
OxidationReduction
degrade
Gstp1/p2-/- mice
HPC : hematopoietic progenitor cells
`✓
GSTP knockout mice
0 15 30 60 120 240 min0 5 10 15 20 25 uM250
75
50
30
250
75
50
30
GST-WT
GST-KO
PABA/NO PABA/NO 15 uM
Enhanced S-glutathionylation in GST WT- MEFs
GST-WT
0 15 30 60 120 240 (min)
IB: Actin
0 50 100 200 300 500 uM
IB: PSSG GST-KO
NOV-002 NOV-002 (100 uM)
IB: PSSG
Townsend et al., 2009, JBC
Enhanced S-glutathionylation in GST-WT MEFs
S-glutathionylation of actin alters focal contacts
Increased S-glutathionylation in GST WT mice
GSTP: cys 47 & 101 are
S-glutathionylated
From: Townsend et al, JBC: 284, 436-445, 2009
APOPTOSIS PROLIFERATION
Enzyme mediated S-glutathionylation
P
JNK
c-jun
GSTπ
P
S-glutathionylated
oligomer
c-jun
JNK
c-jun
GSTπ
GSTπ
Townsend, D.M. 2007 Molecular Intervention
Townsend and Tew. 2012 ARS
s
Energy MetabolismSignaling Pathways
Calcium
homeostasis
GSTP is auto-regulated by
(trans) S-glutathionylation
GSTP S-glutathionylates ER resident proteins
Mass Spectrometry Identification of S-glutathionylated ER proteins
Protein ID MW (kDa)
Calnexin 97
Calreticulin 60
Endoplasmin 94
SERCA2A 114
GRP78, Bip 78
Protein disulfide isomerase (A5) 59
GSTP interacts with ER protein
and enhances S-glutathionylation
GSTP
BIP
PDI
GSTP
Calnexin
GSTP
Calreticulin
GSTP
IP: Calnexin
IP: BIP
IP: Calreticulin
IP: PDI
Total IP IP IgG
Disulfiram
(10µM)
- - + -
Gstp1/p2+/+
Gstp1/p2-/-
How does cytosolic GSTP
catalyze S-glutathionylation of
Endoplasmic Reticulum resident
proteins?
GSTP
Calnexin
GAPDH
Gstp1/p2 + - + - + -
ER Cyto Nuc
Histone H3
SERCA2
GSTP is a resident ER Protein
GSTP is a resident Endoplasmic
Reticulum (ER) protein
BIP GSTP Merge - TRIODAPI - nuclear
CalnexinDAPI - nuclear GSTP Merge - TRIO
GSTP regulates ER Calcium homeostasis
0%
20%
40%
60%
80%
100%
120%
0 500 1000 1500
ER
Calc
ium
ho
meo
sta
sis
(% o
f co
ntr
ol)
Time (second)
Gstp1/p2+/+Gstp1/p2-/-
Thapsigargin Gstp1/p2+/+
Gstp1/p2-/-
Thapsigargin
(nM)
Velcade
(nM)
Gstp1/p2+/+ 121.7 23.6
Gstp1/p2-/- 16.5 3.0
ratio 7.4 7.9
Thapsigargin and Velcade are more toxic to
Gstp1/p2-/- BMDDCs
Viability (IC50 value)
Endoplasmic Reticulum (ER)Albert Claude 1945
Lace work structure”
Rough ER
• Protein synthesis: Secreted, membrane bound, digestive enzymes, hormones and antibodies
• Translocon docking sites for ribosome
• GSH/ GSSG ~10:1
Smooth ER:
• Lipid / phospholipid and steroid synthesis
• Calcium ion storage
Sarcoplasmic ER:
Calcium storage: major role in excitation / contraction coupling
Demand exceeds capacity ……..
Conditions Probable effects
temperature shifts
(e.g., heat-shock)
protein denaturation
protein aggregation
heavy metals, arsenite,
iodoacetamide
reaction with sulfhydryl goups,
conformational changes in protein
anoxia, hydrogen peroxide,
superoxide ions, free radicals
oxygen toxicity, free radical
fragmentation of proteins
proteasome inhibitors inhibition of proteolysis
amytal, azide, dinitrophenol,
ionophores
antimycin
inhibition of oxidative phosphorylation,
changes in redox state,
covalent modification of proteins
hydroxylamine cleavage of asparagine-glycine bonds
ethanol translation errors
amino acid analogs, puromycin abnormal proteins
Agents/Treatments that induce ER stress response
Grek and Townsend (2013): ER stress and Cancer Biology:
Gstp1/p2-/- mice
HPC : hematopoietic progenitor cells
`✓
GSTP knockout mice
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3375108/
ER dysfunction in liver disease
Toxicology of Acetaminophen
Journal of Clinical and Translational Hepatology 2016;4(2):131-142
Increased resistance to acetaminophen hepatotoxicity
in mice lacking glutathione S-transferase PiColin J. Henderson*, C. Roland Wolf* Neil Kitteringham,Helen Powell §, Diana Otto*, and B. Kevin Park
NAPQI + GSH Detox (NOT related to GSTP)
NAPQI + Protein adduct equivalent
GSTP +/+ decreased GSH/GSSG
** Toxic role of GSTP downstream of protein conjugation of NADPQI
J Pharmacol Exp Ther. 2013 Jan;344(1):286-94. doi: 10.1124/jpet.112.199067. Epub 2012 Oct 23.
Mouse liver protein sulfhydryl depletion after acetaminophen exposure.Yang X, Greenhaw J, Shi Q, Roberts DW, Hinson JA, Muskhelishvili L, Davis K, Salminen WF.
Source
Division of Systems Biology, National Center for Toxicological Research, Jefferson, Arkansas, USA.
Protein – SH depleted regions correlated with areas exhibiting histopathologic injury
NOT GLOBAL DECREASE IN LIVER
http://www.ncbi.nlm.nih.gov/pubmed/23093024http://www.ncbi.nlm.nih.gov/pubmed?term=Yang X[Author]&cauthor=true&cauthor_uid=23093024http://www.ncbi.nlm.nih.gov/pubmed?term=Greenhaw J[Author]&cauthor=true&cauthor_uid=23093024http://www.ncbi.nlm.nih.gov/pubmed?term=Shi Q[Author]&cauthor=true&cauthor_uid=23093024http://www.ncbi.nlm.nih.gov/pubmed?term=Roberts DW[Author]&cauthor=true&cauthor_uid=23093024http://www.ncbi.nlm.nih.gov/pubmed?term=Hinson JA[Author]&cauthor=true&cauthor_uid=23093024http://www.ncbi.nlm.nih.gov/pubmed?term=Muskhelishvili L[Author]&cauthor=true&cauthor_uid=23093024http://www.ncbi.nlm.nih.gov/pubmed?term=Davis K[Author]&cauthor=true&cauthor_uid=23093024http://www.ncbi.nlm.nih.gov/pubmed?term=Salminen WF[Author]&cauthor=true&cauthor_uid=23093024
J Pharmacol Exp Ther. 2012 Feb;340(2):360-8. doi: 10.1124/jpet.111.187948. Epub 2011 Nov 1.
Changes in mouse liver protein glutathionylation after
acetaminophen exposure.Yang X, Greenhaw J, Ali A, Shi Q, Roberts DW, Hinson JA, Muskhelishvili L, Beger
R, Pence LM, Ando Y, Sun J, Davis K, Salminen WF.
1h = Increased P-SSG
24h = Decreased P-SSG at site of necrosis
PROTEIN OXIDATION 10x > APAP-Conjugation
Zhang, K. et al. J. Biol. Chem. 2004
Three UPR transducers and one master regulator
GRP78 / BiP: Glucose related protein / Binding Immunoglobin Protein
Decreased S-glutathionylation of
BiP in GSTP1P2 knockout mouse
liver
GSTp
GAPDH
BiP
112213_SCW_II_141_Glut_BIP_FirstZT #16877 RT: 94.18 AV: 1 NL: 1.91E4F: ITMS + c NSI d Full ms2 [email protected] [220.00-1710.00]
300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
x5 x5
34DLGTTYSC(glut)VGVFK46MH+ = 1694.74 Da
M+2H+ = 847.87 Da
y2294.16
y4450.23
b5-H2O
470.22
b4387.23
y5549.34
(y11-129)2+
669.46
y112+
733.98
y3393.26
(M+2H-129-H2O)2+
774.56
(M+2H-129)2+
783.53
y6-129-H2O
810.65
(M+2H-H2O-NH3)2+
829.60
y7-129
915.44
y6957.50
y71044.46
y8-129
1078.50
y81207.56
y9-129
1179.58
y91308.57
b91245.51
b11-129
1272.50
y11-129
1337.61
b111401.63
y111466.72
b121548.49
b9-129
1116.51
BiP is s-glutathionylated on C41 and C420
BiP
PSS
G
Merge
HEPG2 Cells pre-treated with NAC (and
treated with 15mM APAP for 16hrs) have
28% increase in GSH
RESULTS:
0
20
40
60
80
100
120
140
160
Relative GSH concentration
**
BiP-SSG is concurrent with APAP
induced liver toxicity
• BiP: GSTpi dissociates when under ER stress
• GSTpi KO mice have 42% less SSG-BiP than GSTpi WT mice
• NAC treatment increases GSH / decreases BiP-SSG and reduces apoptosis
S-glutathionylation of BiP
attributes to multiple
myeloma resistance
Anti GSHCell lysate IP: IgG IP: BiP
MS MR US URMS MR US URMS MR US UR
MM resistant cells have higher S-glutationylated levels of BiP.
112213_SCW_II_141_Glut_BIP_FirstZT #16877 RT: 94.18 AV: 1 NL: 1.91E4F: ITMS + c NSI d Full ms2 [email protected] [220.00-1710.00]
300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e Ab
unda
nce
x5 x5
34DLGTTYSC(glut)VGVFK46
MH+ = 1694.74 Da
M+2H+ = 847.87 Da
y2294.16
y4450.23
b5-H2O
470.22
b4387.23
y5549.34
(y11-129)2+
669.46
y112+
733.98
y3393.26
(M+2H-129-H2O)2+
774.56
(M+2H-129)2+
783.53
y6-129-H2O
810.65
(M+2H-H2O-NH3)2+
829.60
y7-129
915.44
y6957.50
y71044.46
y8-129
1078.50
y81207.56
y9-129
1179.58
y91308.57
b91245.51
b11-129
1272.50
y11-129
1337.61
b111401.63
y111466.72 b12
1548.49
b9-129
1116.51
112213_SCW_II_141_Glut_BIP_FirstZT #24932 RT: 130.02 AV: 1 NL: 5.13E4F: ITMS + c NSI d Full ms2 [email protected] [185.00-2000.00]
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relat
ive A
bund
ance
x5
418DVC(glut)PLTLGIETVGGVMTK435
MH+ = 2138.02 Da
M+3H+ = 713.34 Da
y2248.25
y3379.35
y5535.35
y6592.33
(M+3H-129-H2O)3+
664.50
or
b102+-H2O
b92+
609.62
b3623.34
y4478.46
b32+
312.31
b3-129
494.34
b111447.93
b91217.47
b81104.47
b71047.59
b172+
996.
74
b162+
945.
94
b152+
880.
65
b122+
774.
14
or
y8-
H2O
y9921.58
(M+3H-H2O-NH3)3+
701.65 b142+
831.
15
y8792.
49b13
2+
802.
39
y7691.55
Wavelength, nm
180 200 220 240 260 280
Mo
lecu
lar E
llip
ticit
y,
-40000
-30000
-20000
-10000
0
10000
20000
BIP
BIP-SSG
CD Analysis
Wavelength, nm
300 320 340 360 380 400 420 440 460
Em
issio
n,
co
un
ts/s
ec
0.0
2.0e+4
4.0e+4
6.0e+4
8.0e+4
1.0e+5
1.2e+5
1.4e+5
BIP
GSS-BIP
Tryptophanyl Fluorescence
0
50
100
150
200
BiP BiP-SSG
AT
Pas
e ac
tivit
y (
%)
0
50
100
150
200
BiP BiP-SSGH
old
ase
acti
vit
y (
%)
BiP
GSH
Merge
Disulfiram - +
S
S+TLK1
99 R
R+TLK1
99
U266 5.4 3.2 27.5 10.1
MM1.s 5.3 4.1 26.5 9.8
S: BTZ sensitive MM cells; R: BTZ resistant MM cells
Table 1. Cross resistance
IC50 (fold resistance)MM1s.S MM1s.R U266 U266R
BTZ (nM) 5.4 27.5 (~5) 5.3 26.5 (~5)
ThG (nM) 8 18 (~2) 6 35 (~6)
TuM
(ng/mL)
13 43 (~3) 12 43 (~3.5)
Table 2. IC50 values of BTZ (nM)
Proteosome Resistance in Multiple Myeloma Confers
Collateral Resistance to ER Stress Inducers
SO
D2
GS
TP
Grx
1
Prx
6
Trx
1
Trx
2
Srx
GPx
1/2
Prx5
Prx2
GPx
4
PDI
S R S R
MM1s U266
S R S R
MM1s U266
UPR proteins Redox enzyme
IRE1
BiP
GRP94
GAPDH
S R S R
MM1s U266
GR
MM resistant cells have different expression of UPR proteins and
redox enzymes
0
0.5
1
1.5
2
2.5
3
3.5
Pro
tein
ex
pre
ssio
n
(rel
ativ
e to
sen
siti
ve
cell
s)
UPR and Redox enzyme profile
MM1s S MM1s R U266 S U 266 R
CHOP
ATF4
Actin
0 6 18 24 0 6 18 24
hrs
Btz (5 nM)
Control Telintra
MM1.S
1 1.1 2.4 3.3 0.9 1.1 2.3
3.4
1 1.0 1.4 1.1 0.7 1.3 3.6
2.1
Telintra reverses resistance to Velcade (Btz) in
multiple myeloma.
NH
ONH
SO
Telintra
COOC2H5
COOC2H5H2N
0
0.2
0.4
0.6
0.8
1
1.2
0.001 0.01 0.1 1 10 100 1000
Cellviab
ility(relativetoControl)
Btz (nM)
Multiplemyeloma Sensitive
Sensitive+TLK
BtzResistant
BtzResistant+TLK
Unfolded and misfolded proteins magically appear?
125
100
75
50
25
0
0 20 40 60
Perc
en
tS
urv
ival
µM PABA/NO µM PABA/NO0 5 15 25 40
NO
Gen
era
tio
n
F
old
I
ncrease
50
40
30
20
10
PABA/NO induced NO generation is
concurrent with P-SSG and apoptosis
Townsend et al., Cancer Research, 2009
• Β-lactate
dehydrogenase• Rho GDP
Dissociation inhibitor
• Nucleophosmin
• Protein Disulfide
Isomerase
• ATP synthase
• Chaperonin / HS65
• Glucosidase
• Elongation factor II
Untreated 30μM PABA/NO
Coomassie: topα-Glutathionylation: bottom
Townsend et al., Mol. Pharm. 2006
2D – SDSMALDI-TOFIdentification of S-glutathionylated
proteins
Protein Disulfide Isomerase
• 55 kDa Homodimer
• 14 PDI isoforms in ER
• Two – UPRE in promotor– (Kox = 1mM)
• Widely distributed across eukaryoticTtiainsest aul., 2e00s6:Cell: 14(1): 61-73– mM Concentrations
– 1% total pr. Content of cells
– Decreased PDI leads to protein aggregation (ie Anti-chaperone)
Disulfide Formatio
nSH SH
Sulfydryl
S-SSH SH
PDI
PDI
Disulfide
Disulfide
Isomerizations
SH SH
PDI
PABA/NO
IP:α-PDI
IP:IgG
DTT
- - + + +
+ - - + +
- + + - -
- - - - +
250
105
75
50
WB:PSSG
WB:PDI
PDI-SSG is concurrent with UPR activation
PDI
PABA/NO
GSH
+ + +
+
-
-
+
+
+
Mr (kDa)
75
50
WB: PSSG
WB: P-SNO
WB: PDI
75
50
75
50
Active Site Cysteine Residues Are
Molecular Targets of PABA/NO
PDI-SSG Alters Structure / Function
120
100
80
60
40
20
0
Per
cen
tAct
ivit
y
0 25 50 100
μM PABA/NO
250 300 350 400
Wavelength, nm
450 500 550
-20
0
20
40
60
100
80 GSTSG
GST Intact
200 220 240
Wavelength,nm
260 280
-40
180
-20
0
20
40
60
80 120
GSTINTACT
GSTSG
Circular Dichroism Intrinsic Fluorescence
C47
W38
Generation of S-glutathionylation
refractory PDI (conversion of basic a.a.
adjacent to catalytic cysteines to neutral)WCGHCK
a domain
WCGHCK
a’domain
PDI-WT
WCGFCL WCGFCLPDI-FLFL
Histidine (H)
Lysine (K)
Phenylalanine
(F)
Leucine
(L)
Alanine
(A)
PDIFLFL is refractory to S-glutathionylation
WB: P-SSG
WB: PDI
In Vitro PDIWT
0 25 50
PABA/NO
PDIFLFL
100 0 25 50 100
PDIWT PDIFLFL
- + - +
Rotenone
FLAG-
PDIWT
- +
FLAG-PDIFLFL
- + PABA/NO+
In Cells
Ctr
IP: FLAG
WB: PDI-
PSSGWB: PDI
WB: P-SSG
WB: PDI
500 1000 Rotenone(µM)0 100 250
Rotenone (uM)
Mean
O.D
.
0
2
4
6
0
5
10
0
2
4
PD
I
mR
NA
fold
incre
ase
- + -
+PDIWT PDIFLFL
- + -
+PDIWT PDIFLFL
- + - +PDIWT PDIFLFL
BIP
mR
NA
fold
incre
ase
CH
OP
mR
NA
fold
incre
ase
S-glutathionylation
refractory PDI1) Blunts the toxic effects of rotenone
2) Diminishes activation of the UPR in
PC12 neuronal cells
PDI is a chaperone for ER
What are the consequences of S-
glutahtionylation of PDI on ER stability and
transcriptional activation?
PDI: Chaperone for Estrogen Receptor alpha
Xiong, Y et al, Int J Cell Biol. 2012
PSSG
PDI
PABA/NO (h) 0 0.5 2
75
50
75
50
IP: PDI
75
50
0 0.5PABA/NO (h) 2
IP: ER
PDI
ER
75
50
PDI-SSG blunts chaperone activity with
ERα
Xiong et al., 2011. IJCB
A
PDI FLAG
BPDI OEVector
ER
PABA/NO (h)0 4 8
PDI
Actin
0 4 8
Xiong, Y et al, Int J Cell Biol. 2012
120
100
80
60
40
20
0
PDI level affects PABA/NO-stimulated ER degradation
E2 (20 nM)
0 2 4 8 (h)
ER
ER
c-Myccyclin D1
p21WAF1/CIP1
Actin
PABA/NO (20 M)
0 2 4 8 (h)
ER
ERcyclin D1
p21WAF1/CIP1
Actin
Cyclin D1 mRNA and protein were down-regulated by anti-estrogen ICI182780
(Faslodex). ICI182780 also increased the expression of the CDK inhibitor p21WAF1/
CIP1 (Watts CK, et al. 1995)
PDI-SSG has anti-estrogen effects
Grek, C and Townsend, D.M. (2013)
ER stress is worth stressing over…
Parkinson’s
A
G
E
Huntington’s
Alzheimer's
Heart Disease
ER stress has been linked to the following medical conditions:
Conclusions
GSTP catalyzes the forward reaction of S-
glutathionylation toward a large number of
proteins
GSTP is an ER resident protein (high levels).
GSTP forms protein interactions with ER resident
proteins important in regulating the UPR
GSTP protects from ER stress agents, including
Velcade, and has a role in cancer drug resistance.
