the role of post-translational modifications in serca2a-related...

Changwon Kho, PhDCardiovascular Research Center

Icahn School of Medicine at Mount SinaiBasic Cardiovascular Sciences 2014 Conference

Pathways to Cardiovascular TherapeuticsOutstanding Early Career Award

July 16 2014, Las Vegas, NV

The Role of Post-Translational Modifications in SERCA2a-Related Cardiac Dysfunction

Presenter Disclosure InformationChangwon Kho, PhDThe Role of Post-translational Modifications in

SERCA2a-related Cardiac Dysfunction

FINANCIAL DISCLOSURE:Grants/Research Support: K99HL116645, National Heart, Lung, And Blood Institute of the National Institutes of Health

Cardiac SR Ca2+-ATPase (SERCA2a) is a Promising Therapeutic Target in Heart Failure

• Impaired Ca2+ re-uptake resulting from (i) decreased expression and (ii) reduced SERCA2a activity is a hallmark of heart failure

• Gene therapy : Phase 2b/3 human clinical trials of SERCA2a replacement therapy (CUPID)

IB: SERCA2a

SHAM

HF

HF+S

3.8 9.2pH

Normal state

Disease state

Disease state with the gene transfer

Theoretical pIAcidic Basic

Isoelectric Point (pI) Distribution of SERCA2a is Altered in Porcine Failing Hearts

HF, Heart FailureHFS, Failing heart with SERCA2a gene transfer

Diverse Lysine Modifications:Key Role of Lysine Residues

Acetylation

SUMOylation

Ubiquitination

Methylation

Glycation Phosphoglycerylation

“Lysine”CompetitionCooperation∆ Activity

Key Findings from Small Ubiquitin-like Modifier 1(SUMO-1) Gene Transfer Experiments in Animal Models

of Heart Failure

Kho et al (2011) Nature, 477(7366):601-5Tilemann et al (2013) Science Translational Medicine, 5(211):211ra159Lee et al (2014) Antioxidant Redox & Signaling, in-press

Levels of SUMO-1 protein and SUMOylation of SERCA2a are

reduced leading to an enzymatic dysfunction and de-stabilization of

SERCA2a during HF

Increased SERCA2a SUMOylation via SUMO-1 overexpression

rescues HF phenotype in murine and porcine models of HF. This beneficial effect is SERCA2a

dependent

Combined delivery of SUMO-1 and SERCA2a suggests additional

beneficial effects on the molecular level in a porcine model of HF

Overexpression of SUMO-1 by gene transfer protects SERCA2a

from oxidative stresses

Protein stabilityEnzymatic activityPump functionContractility

S

K480

S

K585

SERCA2a

↓↓

↓↓

Hypothesis

Post-translational mechanisms causing impairment of SERCA2a function may play an important role in the

setting of heart failure

Aims

To understand the biological consequences of acetylation of SERCA2a on cardiac function and the possible relationships of acetylation to SUMOylation

SERCA2a Acetylation is Significantly Increased in Failing Hearts

A. Mouse B. Human

Sham 2M TAC

AcetylSERCA2a

SERCA2a

IP: SERCA2aIB: Acetyl-Lys

kDa

100

Ac-S

ERC

A2a/

SER

CA2

aB

and

inte

nsity

(AU

)

0

0.5

1

1.5 p < 0.05

Sham 2M TAC

100 SERCA2a

Normal HF


AcetylSERCA2a

Ac-S

ERC

A2a/

SER

CA2

aB

and

inte

nsity

(AU

)

0

1

2

3

4

Normal HF

p < 0.05

kDa

100

100

AAV9.shSUMO1

AAV9.Scramble

IP: SERCA2aIB: SUMO-1


AcetylSERCA2a

SUMOylatedSERCA2a

kDa

100

250

150

SUMO-1

GAPDH

15

37



AcetylSERCA2a

SUMOylatedSERCA2a

kDa

100

250

150

SUMO-1

GAPDH

Sham TAC

15

37

A B

SUMOylation of SERCA2a Antagonizes its Acetylation

IP : IgG SERCA2a

SERCA2a

Adult myocyte lysates

Sirt1100

100

kDa

IP : IgG SERCA2a

A C

B

IP : Flag (SERCA2a)IB : Acetyl-Lys

AcetylSERCA2a

SERCA2a

100kDa

100

in vitro assay

Sirt1 (Sirtuin 1, NAD+-dependent deacetylase) is Identified as a SERCA2a Deacetylase

2-DE gel

Substrate

AcK KSubstrate

Sirt

NAD+ NAM + O-AADPR

Sirt1 Expression and its Cofactor, NAD+, levels are Decreased in Failing Hearts

100

200

300

400

Sham TACN

AD+

(nm

ol/g

tiss

ue) p < 0.05

A B

Sirt1

GAPDH

kDa100

37

Sham 2M TAC

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Ban

d in

tens

ity (A

U)

Sham TAC

p < 0.02

Sirt1 Knockdown Increases SERCA2a Acetylation and Reduces its Function

SRC

a2+

upta

ke(%

Vmax

)

pCa7.5 7.0 6.5 6.0 5.5 5.0

100

80

60

40

20

0

Scramblesi-Sirt1

[Ca2+](μΜ)

ATPa

se a

ctiv

ity(n

mol

/min

/mg)

100

80

60

40

20

00 1 10 100

Scramblesi-Sirt1

A B

100

Scramble si-Sirt1kDa Acetyl

SERCA2aIP: SERCA2aIB: Acetyl-Lys

Sirt1

GAPDH37

100

kDa

P-PLN (Ser16)

Total PLN

25

25

SERCA2a

100

*

*

Sirt1 Silencing Increases SERCA2a Acetylation and Induces Cardiac Dysfunction in Mice

45

50

55

60

65

70

AAV9.Scr AAV9.shSirt1

FS (%

)

0

0.05

0.1

0.15

0.2

AAV9.Scr AAV9.shSirt1

LVID

s (c

m)

p < 0.001

p < 0.001AAV9.ScrAAV9.shSirt1

SRC

a2+

upta

ke(%

Vmax

)

100

80

60

40

20

0

pCa7.5 7.0 6.5 6.0 5.5 5.0

AcetylSERCA2a100

kDa

AAV9.Scr AAV9.shSirt1IP: SERCA2aIB: Acetyl-Lys

37

100 Sirt1

GAPDH

SERCA2a100

A C

B

*

Sirt1-Targeted Acetylated Residues in SERCA2a may be Important for ATP Binding and SUMOylation

Lys-451

Lys-492

TAC mouse heart treated with Sirt1 activator

SUMOylation Sites

K480K585

K451K492

ATP

Membranedomain

Nucleotide binding domain

Actuatordomain

Phosphorylationdomain

Acetylation Sites

Mutants Mimicking Acetylation in Sirt1-Targeted Lysines Decrease SERCA2a’s Function

IB: SERCA2a

SERCA2a 100


SUMOylatedSERCA2a

250

150

kDa WT K492Q K451Q

K-Q mutant: mimicking constitutive acetylation

A B

**

Summary & Conclusion

Our data suggests a pathological role of acetylated SERCA2a in the setting of heart failure

SUMOylation of SERCA2a may prevent its ability to be acetylated

We provide evidence that SERCA2a is a direct substrate of Sirt1 deacetylase

Our findings highlight the pathological importance of post-translational modification and suggest that SERCA2a activity might be manipulated by post-translational modification

Proposed Model

SERCA2a

S S

SERCA2a

Ac Ac

NORMAL

Enzymatic activity ↑

Protein stability ↑

Contractility ↑

Blocks oxidative stress↑

Enzymatic activity ↓

Contractility ↓

SERCA2a

S SSERCA2a

Ac Ac

HEART FAILURE

SERCA2a

S S

SERCA2a

Ac Ac

Therapeutic Approaches

• Small Molecule Activation of SERCA2a SUMOylation

• Gene therapy (SUMO-1 or SUMO-SERCA2a)

• Blocking SERCA2a Acetylation

Acknowledgements

Icahn School of Medicine at Mount Sinai Cardiovascular Research Center

Prof. Roger J. HajjarAhyoung LeeDongtak JeongShinichi MitsuyamaJaegyun OhPrzemyslaw Gorski

Cardiovascular Translational LaboratoryLisa TilemannKiyotake IshikawaJaume AgueroKenneth Fish

Structural and Chemical BiologyProf. Roberto Sanchez

Gwangju Institute of Science and TechnologyCollege of Life Sciences

Prof. Woo Jin ParkDong Kwon Yang Seung Phil Jang

Oslo University HospitalExperimental Medical Research Institute

Prof. Geir Christensen

RFUMSPharmaceutical Sciences

Prof. Russell Dahl

Thank you for your attention!

Sirt1-mediated Serca2a regulation could be a target for Pharmacologic Invention:

Beta-Lapachone, a Modulator of NAD Metabolism

Natural quinone compound from the bark of the lapacho tree

NAD(P)H:quinoneoxidoreductase (NQO-1)

NADH NAD+

Beta-lap futile cycle

Sirt1 expression/activity

Actyl-p53miR-34a

NAD+-Dependent Activation of Sirt1 Provides Cardioprotection in Mice

45

50

55

60

65

Veh bL Veh bL

FS (%

)

p < 0.05

0

0.05

0.1

0.15

0.2

Veh bL Veh bL

LVID

s (c

m)

Sham TAC

p < 0.05

p < 0.05

p < 0.05

Sham TAC

n = 4 n = 3 n = 3 n = 4

n = 4 n = 3 n = 3 n = 4

100

kDa


Acetyl SERCA2a

Sham TAC

Veh bL Veh bL

Ex527, small molecule Sirt1 inhibitorREV (Resveratrol), Sirt1 activator

IP : SERCA2aIB : Acetyl-Lys

SERCA2a

HL-1 cell lysates

AcetylSERCA2a

SERCA2a Acetylation is Regulated by Sirt1 Activity

100

100kDa

Antibody Test: SERCAK492Ac

Ex527, small molecule Sirt1 inhibitor

IP: Flag (SERCA2a)IB: SERCAK492Ac

IB: Flag (SERCA2a)

IP: Flag (SERCA2a)IB: Acetyl-lysine

WT WT K451A K492A

+Ex527

SERA2a

Acetyl SERA2a

Acetyl SERA2a at K492

the role of post-translational modifications in serca2a-related...

Documents