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Marica BakovicDepartment of Human Health and Nutritional Sciences,

University of Guelph, Guelph, ON, Canada

August 2015

The phospholipid enzyme Pcyt2 is a new target for oxidative stress and

heart disease

AGPAT

EPT

EK

DAG/TAG PathwayKennedy Pathway

Ethanolamine

P-Eth

CDP-Eth

PE

CTP

ATP

G3P

LPA

GPAT

TAG

PAPAP

DGAT

FA

FA

FA

DAGPcyt2

Pcyt2 regulates DAG partitioning between PE and TAG

8a

Pcyt2α

7a

GT

7-13

TT

Pcyt2β

Pcyt2γ

1297TGA

1232TGA 74ATG

85ATG

57ATG 960TGA

1-6

1-6

1-6

7 8 9-14

A. Pcyt2 Splicing

1486

1881

1228

B. Pcyt2 Proteins

21 156 301

Pcyt2 a

Pcyt2 b

Pcyt2γ

N-Domain Linker C-Domain

21 156 213 350 386

21 156 231 368 404

180 – 197 of Exon 7

35HYGH

35HYGH

35HYGH

244HIGH

226HIGH

Exon-skipping

Intron-retention

N

S191

S197 (215a)

S205 (223a)S282

C

αD

αLαA

αF

αB

αEαE’

αB’

αD’

αL’

αA’

αC’

S145,T146T147

J Biol Chem 2014

Meclizine(RS)-1-[(4-chlorophenyl)(phenyl)methyl]-4-(3-methylbenzyl)piperazine

J Biol Chem 2013

Meclizine Story

6h at 37C80% Methanol extraction

Separation and Identification of Metabolites by LC-MS

Control 50 uM Meclizine

Meclizine is a non-competitive inhibitor of Pcyt2 with respect to CTP

14C-

PE (p

mol

)

****Ctrl Mec Ctrl Mec 0.0

2.5

5.0

7.5

10.0

A

B

Ctrl_6h Mec_6hCtrl 24hMec 24h

Etn PEtn CDP-Etn

14C-

Etn

(pm

ol)

*

*

01234567

**

**

C

D

PS195PS192

A. Pcyt2 specific [PS192EVPS195SQCPG200]

(-Serum)

PS223

B. Pcyt2 shared [PS223GKEP227]

(-Serum)

Pcyt2 a

Pcyt2 b

linker

N-CAT C-CAT

145, 146, 147

157, 162, 167, 168

191, 205* 282

N-CAT C-CAT

180 – 197spliced out in b

300, 308

209, 215,* 223*

192, 195, 196

Proteomics

PKC

J Biol Chem 2014

0 30min 1h 2h0.0

2.5

5.0

7.5

10.0

12.5

Py

ct2

en

zym

e a

cti

vit

y

(pm

ol/

mg

/min

) ****

Pcyt2total

pSer

PMA 0 30 min 1h 2hA.

B. P

cy

t2 e

nzy

me

ac

tiv

ity

(p

mo

l/m

g/m

in)

PMA PMA+GO6976PMA+EGF-R

5

10

15

20

****

D.

PMA PMA+GO6976 PMA+EGF-RC.

Pcyt2total

pSer

F. Pcyt2 phosphorylation with PKC 207GVSQFLQTpS215QKI 18

(b1—11) – (b1-8) =pSPS215

G. Pcyt2 phosphorylation with PKC TpS215Q

PS215

(b1-4)-(b1-3)=pS

J Biol Chem 2014

B.

S215.

223A

Pcyt2

α

S215A

S223A

Anti-PSer

Anti-V5

A.

D.Endogenous Overexpressed

Pc

yt2

ac

tiv

ity

(p

mo

l/m

g/m

in)

PMA (nM)

C.PMA

EGF-R

Pcyt2α S215.223A

Anti-PSer

Anti-V5

- + + - + +

- - + - - +

J Biol Chem 2014

N-Domain Linker C-Domain

21 156 231 368 404

180 – 197 of Exon 7

343PKRRGIF349Pcyt2 a

21 156 301

Pcyt2 γ 291RGD293

A

B

Pcyt2 γPcyt2 a

N

N

C

C

Gene 2014

Pcyt2γ37kDa

β-tubulin

Pcyt2 is a dominant negative isoform 1 2 3 4 5 6 7 8

Pcyt2γ Pcyt2αPcyt2βCtrl

Pcyt

2α+P

cyt2

γ

Pcyt

2α

Pcyt

2γ

Pcyt

2α+P

cyt2

γ

Pcyt2α 50kDa

Pcyt2γ37 kDa

Pcyt2α50kDa

Pcyt2γ37kDa

IP: Pcyt2α IP: Pcyt2γ

Pcyt

2α+P

cyt2

γPc

yt2α

+Pcy

t2γ

Pcyt2αDimer

Pcyt

2α

Pcyt

2α

Dimerization

(100kDa)

Gene 2014

Pcy

t2α

+H

24

4Y

Pcy

t2α

+H

35

Y

β-tubilin

Pcy

t2α

50kDa

α-Myc

224 bp

Pcyt2α mRNA

Pcy

t2α

H2

44

Y

H3

5Y Pcy

t2α

Pcy

t2α

+

H2

44

Y

Pcy

t2α

+

H

35

Y

Pc

yt2

α+

H2

44

Y

Pc

yt2

α+

H2

44

Y

Pc

yt2

α+

H3

5Y

Pc

yt2

α+

H3

5Y

Dimerization

Pc

yt2

α

Pc

yt2

α

100kDa

Mutants of active isoforms are also inhibitors

Gene 2014

Activity

Degradation

c

γα

α

ααγ

Heterodimers and aggregates are

inactive

γ

ααPcyt2α homodimers are active

γ

γ

γ γ

γ

?

Gene 2014

Epi. Subcut. Renal Liver0

1

2

3

4

5

6

7

**

*

*P=0.01

P=0.029

P=0.04

P=0.02

Weigh

t (% of

body)

0 5 10 15 20 25 30 35 40 45 500

10

20

30

40

50

Female +/-

Male +/- Male +/+

Female +/+P<0.05

Time (wks)

Weigh

t (g)

A B

Pcyt2+/+ Pcyt2+/-

Pcyt2+/-WT

Pcyt2 deficient mouse ETKO

J Biol. Chem 2009

LD

Pcyt2 +/-Pcyt2 +/+

m

m

rER

LD

E

lec

tro

n m

icro

sc

op

y o

f l

ive

r

Liv

er

lip

id d

rop

lets

Pcyt2 +/+ Pcyt2 +/-

Liv

er

fib

ros

is

Pan

cre

atic

is

lets

Pcyt2 +/+ Pcyt2 +/-

A. B.

C. D.

Pcyt2 +/+

Pcyt2 +/-Pcyt2 +/+

Pcyt2 +/-E.

1µm 1µm 200µm 200µm

200µm 200µm 100µm 100µm

Ma

le h

eart

s

m

m

m

Mol Cell Biol 2015

p<0.05

Acox

lFa

ds1

Acls

1mR

NA

(fo

ld c

han

ge)

Aldh3

a2

Acad1

1Ac

adm

Cyt

p450

Fads

2

Cpt1a

p<0.05m

RN

A (

fold

ch

ang

e)

Hmgc

s2

Cyp2c

70H

sd3b

4Sq

leEb

pPgr

mc1

Tm7s

f2

Cyp7a

1Cyp

2a5

Com

t 1

Cyp3a

25

Hsd17

b1

Microarray analysis Mol Cell Biol 2015

Gender effect on ETKO heart genes

Scd1

Pcyt2+/+

Pgc1α

Pcyt2+/- Pcyt2+/+ Pcyt2+/-

Male Female

Gapdh

Mct1

Ace1

A.

p<0.001

Male Female

B.p<0.05

**

**

**

Pcyt2 +/+ Pcyt2 +/- Pcyt2 +/+ Pcyt2 +/-

Arb

itra

ry u

nit

Male Female

***

***

Pcy

t2 +

/+

Pcy

t2 +

/+

Pcy

t2 +

/-

Pcy

t2 +

/-

*

FemaleMale

p<0.05

**

Pcy

t2 +

/+

Pcy

t2 +

/+

Pcy

t2 +

/-

Pcy

t2 +

/-

Arb

itra

ry u

nit

Arb

itra

ry u

nit

C. D. E.

FemaleMale

p<0.01

******

**

Pcy

t2/+

/+

Pcy

t2 +

/+

Pcy

t2 +

/-

Pcy

t2 +

/-

Arb

itra

ry u

nit

Mol Cell Biol 2015

Pcyt2 +/+

Pcyt2 +/+

Pcyt2 +/-

Pcyt2 +/-

Female Male

Fa

tty

ac

ids

(%

Ph

osp

ho

lipid

s)

16:018:018:118:222:6

B.

Pcyt2 +/- Female

Pcyt2 +/+ Male

Pcyt2 +/- Male

Pcyt2 +/+ Female

16:016:118:018:118:2

Fa

tty

ac

ids

(%

TA

G)

C.

Pcyt2: +/+ +/- +/+ +/- Male Female

% T

ota

l P

E

** ** ** *

Arb

itra

ry u

nit

s

Pcyt2: +/+ +/- +/+ +/- Male Female

PC/PE Ratio

*

% T

ota

l P

I

Pcyt2: +/+ +/- +/+ +/- Male Female

A

Heart Lipid DimorphismMol Cell Biol 2015

*** **

** **

Linoleic acid

Arachidonic acid

Dihomo γ linolenic acid

****

Docosatetranoic acid

% 1

8:2

n-6

% 2

0:3

n-6

% 2

0:4

n-6

% 2

2:4

n-6

***

*

N-6 PUFA synthesis and oxidative stress

** **

Docosatrienoic acid

Docosapentaneoic acid

Docosahesaneoic acid

% 2

2:3

n

-6%

22

:5 n

-6

% 2

2:6

n

-3

** ***

*

** ***

*

Male Female

*

**

TAG

mg

/g

Male Female

Male Female Male Female

B.

Pcyt2+/+ Pcyt2+/- ***

*****

Male Female

TB

AR

S (

µM

) H

ear

t

TB

AR

S (

µM

) A

ort

a

***

**

Male Female

C.

A.

Mol Cell Biol 2015

Impaired heart functionHypertension, Hypertrophy

Normal heart function

Male

Cd36 Glut4

Female

TAG

PI3KpAktpAMPK

Pcyt2+/- Heart

PI3KpAktpAMPK

Glucose Glucose

Scd1Mct1

Ace1Mct1

Fatty acids Fatty acids

Cd36 Glut4

20:320:422:322:423:4

TAG

Enriched in n-6 fatty acids Unmodified n-6 fatty acids

PC/PEPC/PE

Male-specific heart diseaseMol Cell Biol 2015

AlbertRatnesh

Maida Leanne Laila

ZvezdanSugash

Pulami

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