Established PKA's dimersation and Serca's post translational modification (PTM) in ... Prediction of Pka of all cysteine for pp1 and impact of oxidizing.
Redox regulation of protein phosphatase-1 in the heart Singh S.1, Saadatmand A. R. 3, Richter F2, Wittig I2, Vettel C. 4, Susanne Lutz1, El-Armouche A. 1,3 1Institute
of Pharmacology, University Medical Center, Georg-August-University, Göttingen, Germany 2Functional proteomics, faculty of Medicine, Goethe University, Frankfurt, Germany 3 Department of Pharmacology, University Hospital Carl Gustav Carus, Technical University Dresden, Germany 4 Institute of Clinical and Experimental Pharmacology and toxicology, Medical Faculty Mannheim, University Heidelberg, Germany
2. Effect of total phospastase (PP) and protein phosphatase -1 (PP-1) activity exposed to oxidative stress inducing agent
B
25%
C
40% 35%
20%
**
15%
** 10%
% PP-activity
% PP-activity
30% 25%
**
20%
**
15% 10%
5% 5% 0%
PP1
1
10 102 103 104
PLB-pSer16
*
100%
H2O2 (µM, 3 min.)
C
1
10 102 103 104
cMyBP-pSer282
CSQ
*
H2O2 (µM, 3 min.)
C
1
10 102 103 104
Phospho I-1
CSQ
CSQ
80% 60% 40% 20% 0%
0%
PP2A
H2O2 (µM, 3 min.) C
A
120%
PP-activity (% of control)
A
3. Impact of oxidizing agent on ß–adregenic signaling pathway
PP2A
Heart tissue
Control 100 H2O2 (µM)
PP1
NRCM
Fig. 3: (A) Percentage of PP-2A and PP-1 activity in whole heart tissue of mouse; (B) in neo-natal rat cardiomyocytes (NRCM), n=3; (C) total phosphatase activity in NRCM incubated with H2O2 (100µM) for 3 min, n=5. The values compared with t-test,*P < 0.05.
B
B
A
Fig. 5: (A) H2O2 induces oxidation of catalytic cysteine residue of PP-1 (adapted from Meng et al.,2006)
H2O2 (min, 100 µM)
0
3
6
10
15
PLB-pSer16
30
H2O2 (min, 100 µM)
0
3
6
10
15
30
C
cMyBP-pSer282 CSQ
CSQ
1.Introduction Introduction
C
Fig. 7: Representative immunoblot and quantification of (A) concentration dependency and (B) time course of H2O2 mediated crosstalk between protein kinase A and PP-1 in neonatal rat cardiomyocyte (NRCM) n=3; (C) Putative mechanism of PKA activation and PP-1 inactivation by H2O2. The values compared by one-way ANOVA, followed by Bonferroni’s multiple comparison test, *P < 0.05 vs Control
Fig. 4: (A) PP-1 activity in recombinant PP-1(rPP-1) incubated with H2O2 for 10 min, n=3; (B) PP-1 activity in rPP-1 incubated with H2O2 (200 µM) for indicated time period, n=3; (C) the reversibility of PP-1 activity in rPP-1, when incubated with H2O2 (200 µM) for 15 min and TCEP (100mM) for 5 min, n=2. The values compared by oneway ANOVA, followed by Bonferroni’s multiple comparison test, *P < 0.05 vs Control.
4. Prediction of Pka of all cysteine for pp1 and impact of oxidizing and reducing agent on PP-1
A
B Cysteine position
Pka
SEQUENCE POSITION
SEQUENCE
OXIDATION PREDICTION STATUS
CYS 39 A
18.69
39
QLTENEIRGLCLKSREIFLSQ
RED
CYS 62 A
12.64
62
LLELEAPLKICGDIHGQYYDL
CYS 105 A
12.94
105
CYS 127 A
10.12
CYS 140 A
ASA
pKa
SOD
0
13.15
RED
NOS
0.07
12.33
DRGKQSLETICLLLAYKIKYP
RED
NOS
1.65
12
127
NFFLLRGNHECASINRIYGFY
RED
SEP
4.87
5.96
13.30
140
INRIYGFYDECKRRYNIKLWK
RED
NOS
0
10.6
CYS 155 A
8.01
155
NIKLWKTFTDCFNCLPIAAIV
RED
SOD
0.26
9.78
CYS 158 A
10.65
158
LWKTFTDCFNCLPIAAIVDEK
RED
NOS
0
8.52
CYS 171 A
8.97
171
IAAIVDEKIFCCHGGLSPDLQ
RED
SOD
0
11.98
CYS 172 A
12.59
172
AAIVDEKIFCCHGGLSPDLQS
RED
NOS
0
12.72
CYS 202 A
11.56
202
PTDVPDQGLLCDLLWSDPDKD
RED
NOS
0.21
10.73
CYS 245 A
18.55
245
FLHKHDLDLICRAHQVVEDGY
RED
SOD
0
14.28
CYS 273 A
9.76
273
LVTLFSAPNYCGEFDNAGAMM
RED
SEP
17
8.1
CYS 291 A
11.94
291
AMMSVDETLMCSFQILKPA--
RED
NOS
4.45
9.67
C
D
Diamide (µM)
PKA RI
C
1
10
102
103
104
kDa
Fig. 1. Redox regulation of ß–adregenic signaling pathway in cardiac myocytes (adapted from Bers DM, 2002). ROS alters the balance between kinase and phosphatase activity in dose dependent manner (Humphries KM et al, 2007).
C
1
10 102 103 104
5. Redox- induced Disulfide Formation and Gluthathionylation in rPP-1
A
H2O2 SOH
SH
PP-1
PP-1
active
Inactive
PDB id: 3v4y Reducing Agent
Fig. 2: (A) Crystal structure of the nuclear PP-1 holoenzyme; B) schematic depicting the proposed model which shows H2O2 can oxidize free thiol groups in PP-1, which leads to inactive form.
Diamide µM
PP-1
B
A
.
kDa
Non-Reduced Non-Reduced
G
+ DTT kDa
B
37 Non-Reduced
SERCA2a
Reduced
PP-1
No H2O2 Non-Reduced
37
Reduced
37 Monomer
E
*Oxidation Status Codes: RED – Reduced, DSB - Disulfide Bond, OXI – Oxidized.
48
F
Fig. 6: (A) Prediction of all cysteine’s Pka via Propka 3.1; (B) COPP* for PP-1 (PDB id : 4MOV); (C) immunoblots of NRCMs exposed to increasing concentration of diamide (15 min) under nonreducing and reducing condition probed with antiPKA RI and (D) anti-PP-1; (E) densitometric analysis showing the percentage of PKA-RI that exists as monomer or dimer and (F) PP-1 expression after diamide treatment; (G) SERCA2a and PP-1 expression under non-reducing and reducing conditions. n=2. The values compared by one-way ANOVA, followed by Bonferroni’s multiple comparison test, *P < 0.05 vs Control.
Prediction Codes: SOD - Susceptible to Oxidation by Sulfur Distance, SEP - Susceptible to Oxidation by Exposure and pKa, NOS - Not Oxidation Susceptible ,N/P - No Prediction
M183_189
Mn C155_158, C171_172
Glutathion Peptides
Various other possible reasons for oxidation of PP-1
Fig. 8: (A) Pictorial representation of various disulfide bond formation possible in rPP-1 with a chart showing distance between various cysteine in angstrom; (B) results from the first round of massspectrometry on rPP-1 via LTQ Orbitrap XL, 60 min standard gradients; (C) other possible outcome for oxidation of PP-1.
Conclusions The physiologically relevant concentrations of H2O2 reduce PP-1 activity is reversible in presence of a reducing agent. Difficult to allocate changes in PP-1 activity to the phosphorylation status of downstream proteins due to a mutual effect of H2O2 on kinase activity. Established PKA’s dimersation and Serca’s post translational modification (PTM) in immunoblots, but cannot replicate same in the PP-1. Also, decrease in expression of PP-1 with higher concentration of H2O2.
C
15min H2O2 (500 µM)
Buffer –Mn
48
Monomer
110
Buffer +Mn
100
Dimer
-DTT
Future prospect
Mn2+ in the buffer protects cysteines from oxidative stress and also prevents PP-1 from forming disulfide bridges. H2O2 induces oxidative stress at almost all cysteines, the inter-peptide disulfide bridges are protected best from it. Oxidative stress might induce disulfide bridges due to structural changes in the protein. Gluthathionylation can be induced at outerbound cysteines in consequence of oxidative stress.
