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The phospholipid enzyme Pcyt2 is a
new target for oxidative stress and
heart disease
Marica Bakovic
Department of Human Health and Nutritional Sciences,
University of Guelph, Guelph, ON, Canada
August 2015
Kennedy Pathway
DAG/TAG Pathway
Ethanolamine
G3P
ATP
EK
LPA
P-Eth
Pcyt2
CTP
CDP-Eth
AGPAT
FA
DAG
EPT
PE
GPAT
FA
PAP
PA
DGAT
FA
TAG
Pcyt2 regulates DAG partitioning between PE and TAG
A. Pcyt2 Splicing
Exon-skipping
74ATG
Pcyt2β
1232TGA
1-6
7-13
1486
1297TGA
85ATG
Pcyt2α
7
1-6
8
9-14
1881
57ATG
TT
GT
Pcyt2γ
960TGA
1-6
1228
7a
8a
Intron-retention
B. Pcyt2 Proteins
N-Domain
Pcyt2 a
21
Linker
C-Domain
156
231
35HYGH
368
404
350
386
244HIGH
180 – 197 of Exon 7
Pcyt2 b
21
156
226HIGH
35HYGH
21
Pcyt2γ
35HYGH
213
156
301
N
S205 (223a)
S282
S197 (215a)
αD’
αF
αL’
αA’
αD
αA
αL
αC’
S191
αB’
αB
αE’
S145,T146
T147
αE
C
J Biol Chem 2014
Control
50 uM Meclizine
6h at 37C
80% Methanol extraction
Separation and Identification
of Metabolites by LC-MS
Meclizine Story
Meclizine
(RS)-1-[(4-chlorophenyl)(phenyl)
methyl]-4-(3-methylbenzyl)piperazine
J Biol Chem 2013
14C-Etn
A
(pmol)
Meclizine is a non-competitive inhibitor of Pcyt2 with respect to CTP
14C-PE
(pmol)
B
7
6
5
4
3
2
1
0
**
*
*
Etn PEtn
**
CDP-Etn
10.0
7.5
5.0
2.5
0.0
B
Ctrl_6h
Mec_6h
Ctrl 24h
Mec 24h
**
Mec Ctrl
Ctrl
**
Mec
250
200
D
1/[V]
No inhibitor
150
30 μM Mec
100
120 μM Mec
50
150 μM Mec
0
600
850
1000
-0.005
0
0.005
1/[S]
Figure 3
0.01
C
Proteomics
linker
Pcyt2 a
J Biol Chem 2014
180 – 197
spliced out in b
N-CAT
C-CAT
192, 209,
195, 215,* PKC
196 223*
300,
308
Pcyt2 b
N-CAT
B. Pcyt2a shared [PS223GKEP227]
PS223
(-Serum)
C-CAT
145, 157,
146, 162,
147 167,
168
191,
205*
282
A. Pcyt2a specific [PS192EVPS195SQCPG200]
PS192
(-Serum)
PS195
F. Pcyt2a phosphorylation with PKCa
207GVSQFLQTpS215QKI 18
PS215
G. Pcyt2a phosphorylation with PKCa
TpS215Q
(b1—11) – (b1-8) =pS
PS215
(b1-4)-(b1-3)=pS
0
30 min
1h
2h
C.
PMA+GO6976 PMA+EGF-R
pSer
pSer
Pcyt2total
Pcyt2total
Pyct2 enzyme activity
(pmol/mg/min)
B.
PMA
**
12.5
**
10.0
7.5
5.0
2.5
0.0
0
30min 1h
2h
D.
Pcyt2 enzyme activity
(pmol/mg/min)
A. PMA
20
15
*
***
10
5
PMA PMA+GO6976PMA+EGF-R
J Biol Chem 2014
A.
C.
PMA
+
EGF-R -
Anti-PSer
Pcyt2α
+
-
S215.223A
+
+
-
+
-
+
Anti-PSer
Anti-V5
Anti-V5
B.
Endogenous
Overexpressed
Pcyt2 activity
(pmol/mg/min)
D.
PMA (nM)
J Biol Chem 2014
A
N-Domain
Pcyt2 a
21
Linker
156
C-Domain
231
368
404
343PKRRGIF349
180 – 197 of Exon 7
21
156
Pcyt2 γ
B
301
291RGD293
Pcyt2 a
N
Pcyt2 γ
N
C
C
Gene 2014
Pcyt2 is a dominant negative isoform
1 2 3 4 5 6 7 8
Pcyt2γ
Pcyt2α
Pcyt2β
Ctrl
Pcyt2γ
37kDa
β-tubulin
IP: Pcyt2α
IP: Pcyt2γ
Pcyt2α
50kDa
Pcyt2γ
37kDa
Pcyt2γ
37 kDa
Pcyt2α
50kDa
Dimerization
Pcyt2α
Dimer
(100kDa)
Gene 2014
224 bp
Gene 2014
Pcyt2α+H35Y
Pcyt2α+H35Y
Pcyt2α+H244Y
Pcyt2α+H244Y
Pcyt2α
Pcyt2α
Pcyt2α +
H35Y
Pcyt2α
+H244Y
Pcyt2α
H35
Y
H244Y
Pcyt2α
Pcyt2α+H35Y
Pcyt2α+H244Y
Pcyt2α
Mutants of active isoforms are also inhibitors
Activity
50kDa
α-Myc
β-tubilin
Dimerization
Pcyt2α mRNA
100kDa
α α
Pcyt2α homodimers
are active
γ
Heterodimers and
aggregates are
inactive
α
c
γ
γ
γ
α
?
γ
α α
γ
γ
γ
Gene 2014
Pcyt2 deficient
mouse
ETKO
A
Male +/-
50
Male +/+
Weight (g)
40
30
P<0.05
Female +/Female +/+
20
10
WT
Pcyt2+/-
0
0
5
10 15 20 25 30 35 40 45 50
Time (wks)
J Biol. Chem 2009
B
Pcyt2 +/-
m
rER
m
m
B.
m
LD
m
LD
Pcyt2 +/+
1µm
200µm
200µm
D.
Pcyt2 +/+
Pcyt2 +/-
Pcyt2 +/+
200µm
100µm
100µm
Pcyt2 +/-
Male hearts
E.
Pcyt2 +/-
200µm
Pcyt2 +/-
Liver fibrosis
C.
1µm
Pcyt2 +/+
Liver lipid droplets
Pcyt2 +/+
Pancreatic islets
Electron microscopy of liver
A.
Mol Cell Biol 2015
mRNA (fold change)
mRNA (fold change)
p<0.05
Microarray analysis
p<0.05
Mol Cell Biol 2015
Gender effect on ETKO heart genes
A.
B.
Male
Pcyt2+/- Pcyt2+/+ Pcyt2+/-
**
**
Mct1
Ace1
Pgc1α
Scd1
Pcyt2 +/+ Pcyt2 +/- Pcyt2 +/+ Pcyt2 +/-
Gapdh
Male
C.
D.
Female
p<0.001
E.
***
p<0.05
***
***
**
Male
Male
***
Arbitrary unit
Arbitrary unit
**
Arbitrary unit
*
p<0.01
p<0.05
Arbitrary unit
Pcyt2+/+
**
Female
Female
Male
Female
Female
Mol Cell Biol 2015
PC/PE Ratio
**
% Total PE
Arbitrary units
**
**
B.
+/+
+/Male
+/+
+/Female
Pcyt2: +/+
+/Male
Pcyt2: +/+
+/Male
+/+
+/Female
+/+
+/Female
16:0
18:0
18:1
18:2
22:6
Female
Pcyt2 +/-
Pcyt2 +/+
Male
Pcyt2 +/-
Fatty acids (% TAG)
Pcyt2 +/+
C.
*
Fatty acids
(% Phospholipids)
Pcyt2:
*
% Total PI
A
16:0
16:1
18:0
18:1
18:2
Pcyt2 +/- Female
Pcyt2 +/+ Male
Pcyt2 +/- Male
Pcyt2 +/+ Female
Heart Lipid Dimorphism
Mol Cell Biol 2015
N-6 PUFA synthesis and oxidative stress
A.
B.
**
*
TBARS (µM) Heart
***
Male
**
***
% 22:5 n-6
**
Female
Docosahesaneoic acid
***
**
*
% 22:6 n-3
Female
Male
Female
Docosapentaneoic acid
Docosatetranoic acid
***
Male
*
TBARS (µM) Aorta
% 20:4 n-6
Arachidonic acid
C.
% 22:3 n-6
***
% 22:4 n-6
**
Docosatrienoic acid
*
**
*
*
% 20:3 n-6
**
% 18:2 n-6
***
Male
Female
**
TAG mg/g
Dihomo γ linolenic acid
**
*
Linoleic acid
*
Male
Female
***
**
Male
Female
Pcyt2+/+
Pcyt2+/-
Mol Cell Biol 2015
Pcyt2+/- Heart
Female
Male
20:3
20:4
22:3
22:4
23:4
Fatty acids
Cd36
PC/PE
TAG
Glucose
Glut4 PI3K
pAkt
pAMPK
Ace1
Mct1
Enriched in n-6 fatty acids
Impaired heart function
Hypertension, Hypertrophy
Fatty acids
Cd36
Glucose
Glut4
PC/PE
TAG
PI3K
pAkt
pAMPK
Scd1
Mct1
Unmodified n-6 fatty acids
Normal heart function
Male-specific heart disease
Mol Cell Biol 2015
Pulami
Albert
Maida Leanne Zvezdan
Sugash
Ratnesh
Laila
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