David Gius, M.D., Ph.D.

David Gius, M.D., Ph.D.Professor, Departments of Cancer Biology,

Pediatrics, and Radiation OncologyVanderbilt University School of Medicine

Sirtuin 3: A Mitochondrial Watchdog Protein

Vanderbilt Medical School

The Human Sirtuins

Nuclear

• Sirt1• Sirt6• Sirt7

Mitochondrial

• Sirt3• Sirt4• Sirt5

Cytoplasmic

• Sirt2

In active protein

Protein Deacetylation as a Post-Translation Protein Modification

Active protein

Non-HistoneProtein

CarcinogenesisIncreasing age

Caloric Restriction

Decreases aging

Activation of Sirtuin genes increase longevity

Rational for Sirtuins as Tumor Suppressors

?

Mice lacking Sirt2 and Sirt3 were constructed to determine if they prevent cancer / TSGs?

Tumor Suppressor Gene

Loss of function results in an in vitro tumor permissive cellular phenotype (two hit tissue culture immortalization.

Genetic knockout in mice results in the formation of murine tumors.

There is a loss of function or decrease in protein levels in human malignancies and this matches human samples.

Oncogene 1CMV Promoter Oncogene 2CMV Promoter

Two Gene Transformation Model for MEFs

I PP P

Normal cell Transformed cell

P

Pro-proliferativePro-survival genes

I PP P

Normal cell Transformed cell

P

Proliferative genePro-proliferative

Pro-survival genes

Myc RasTSG Gene Loss

Kim et al, 2010 Cancer Cell

Sirt3+/+ Myc/Ras cells

Sirt3-/- Myc/Ras cells

TABLE 1. Immortalization of Sirt3-/-

MEFs only requires a single oncogene __________________________________________________________________________________________________________________________ _______________________

Control Myc Ras Myc/Ras

_________________________________________________________________________

MEF Sirt3+/+

None None None Immort

MEF Sirt3-/-

None Immort Immort Immort __________________________________________________________________________ None , no MEF immortalization. Immort, immortalization.

Sirt3-/- MEFs are immortalized by a Single Oncogene

In Vitro Transformation Sirt3-/- MEFs by a Single Oncogene

*

Col

onie

s/H

pf

1

2

3

4

Myc/Ras Myc Ras Myc/Ras

*

*

SIRT3 +/+

SIRT3 -/-

*

Col

onie

s/H

pf

1

2

3

4

Myc/Ras Myc Ras Myc/Ras

*

*

SIRT3 +/+

SIRT3 -/-

SIRT3 +/+

SIRT3 -/-

Mammary Carcinogenesis in the Sirt3 knockout mice

# of

tu

mor

s

2

10

SIRT3+/+ SIRT3 -/-

*6

0

# of

tu

mor

s

2

10

SIRT3+/+ SIRT3 -/-

*6

0

Tumor #1 ++ ++Tumor #2 + +Tumor #3 + +Tumor #4 + +Tumor #5 ++ ++Tumor #6 + +Tumor #7 ++ +

ER PR

Tumor #1 ++ ++Tumor #2 + +Tumor #3 + +Tumor #4 + +Tumor #5 ++ ++Tumor #6 + +Tumor #7 ++ +

ER PR

6 15129 242118

% T

um

or F

ree

WTKO

50

100

30276 15129 242118

% T

um

or F

ree

WTKO

50

100

3027

100

Low

50

Med High Low Med High

3

1710

14

31Normal

Tumors

% T

otal

Sam

ple

s

100

Low

50

Med High Low Med High

3

1710

14

31Normal

Tumors

% T

otal

Sam

ple

s

SIRT3 is Decreased in Human Breast Cancers

SIR

T3

mR

NA

Fol

d e

xpre

ssio

n

8

6

4

2

** P < 0.0025

* P < 0.02

** *

*

Stage NB I IIA IIB IIIS

IRT

3 m

RN

AF

old

exp

ress

ion

8

6

4

2

** P < 0.0025

* P < 0.02

** *

*

Stage NB I IIA IIB III

Tissue Array (IHC) RNA Array (RT-PCR)


SIRT3 is Decreased in Human Breast CancersN

orm

aliz

ed e

xpre

ssio

n u

nit

s 1.5

1.0

0.5

0.0NormalBreast

BreastCancer

P = 1.0E-9

Nor

mal

ized

exp

ress

ion

un

its 1.5

1.0

0.5

0.0NormalBreast

BreastCancer

Nor

mal

ized

exp

ress

ion

un

its 1.5

1.0

0.5

0.0NormalBreast

BreastCancer

P = 1.0E-9 1.0

0.5

0.0

1.5

0.5

1.0

Nor

mal

ized

exp

ress

ion

uni

ts

G-1 G-2 G-3

P = 2.4E-131.0

0.5

0.0

1.5

0.5

1.0

Nor

mal

ized

exp

ress

ion

uni

ts

G-1 G-2 G-3

1.0

0.5

0.0

1.5

0.5

1.0

Nor

mal

ized

exp

ress

ion

uni

ts

G-1 G-2 G-3

P = 2.4E-131.0

0.5

0.5

0.0

Nor

mal

ized

exp

ress

ion

uni

ts

Well Mod Poor

P = 3.8E-101.0

0.5

0.5

0.0

Nor

mal

ized

exp

ress

ion

uni

ts

Well Mod Poor

1.0

0.5

0.5

0.0

Nor

mal

ized

exp

ress

ion

uni

ts

Well Mod Poor

P = 3.8E-10

Oncomine, UMich

Sirt3 is a mitochondrial tumor suppressor but…

• Mechanism? • Is it a sensing protein?• What are the targets of Sirt3 ?• Or what dysregulated proteins play a

role in the Sirt3-/- tumor permissive?

1.0

2.0

3.0

Myc/Ras Ras Myc/RasMyc

SIRT3+/+

SIRT3-/-

Mit

o-SO

X F

luor

esce

nce

Fol

d c

han

ge f

rom

WT

*

****

1.0

2.0

3.0

Myc/Ras Ras Myc/RasMyc

SIRT3+/+

SIRT3-/-

SIRT3+/+

SIRT3-/-

Mit

o-SO

X F

luor

esce

nce

Fol

d c

han

ge f

rom

WT

*

****

Transformed Sirt3 KO MEFs exhibit mt SuperoxideM

ito-

SOX

Flu

ores

cen

ceF

old

ch

ange

fro

m W

T

1.0

3.0

SIRT3+/+

SIRT3-/-

Cont

2.0

Antimycin

4.0

*

5 Gy

*

**

Mit

o-SO

X F

luor

esce

nce

Fol

d c

han

ge f

rom

WT

1.0

3.0

SIRT3+/+

SIRT3-/-SIRT3+/+

SIRT3-/-

Cont

2.0

Antimycin

4.0

*

5 Gy

*

**


MnSOD

O2- H2O2 H2O + O2

Catalase

Primary Mitochondrial O2- Detoxification Pathway

Criteria for Potential Sirt3 physiological Target

A protein that contain at least one reversible acetyl lysine that is altered by either caloric restriction, feasting, or other type of stress.

A Protein is hyperacetylated in the Sirt3 knockout livers or MEFs.

A protein contains at least one lysine that is deacetylated by Sirt3 both in vitro and in vivo.

The reversible acetyl lysine is evolutionary through out multiple species including less complex species.

Acetylation of the target lysine regulates enzymatic activity.

MnSOD contains a reversible lysine

0.5

1.0

Sirt3+/+ Sirt3-/-

*

Liv

er R

elat

ive

MnS

OD

Act

ivit

y (%

Con

t)

0.5

1.0

Sirt3+/+ Sirt3-/-

*

Liv

er R

elat

ive

MnS

OD

Act

ivit

y (%

Con

t)

0.5

1.0

Sirt3+/+ Sirt3-/-

*

ME

Fs

Rel

ativ

e M

nSO

DA

ctiv

ity

(% C

ont)

0.5

1.0

Sirt3+/+ Sirt3-/-

*

ME

Fs

Rel

ativ

e M

nSO

DA

ctiv

ity

(% C

ont)

Tao et al., 2010, Molecular Cell

MnSOD’s reversible is deacetylated by Sirt3

0.5

1.0 Sirt3-/-

ME

Fs

Rel

ativ

e M

nSO

DA

ctiv

ity

(% C

ont)

Lenti- Con Sirt3dn Sirt3

0.5

1.0 Sirt3-/-

ME

Fs

Rel

ativ

e M

nSO

DA

ctiv

ity

(% C

ont)

Lenti- Con Sirt3dn Sirt3

-acetyl

IP : MnSOD

lenti-Sirt3 Con dn wt

-MnSOD

IgG

wt

-acetyl

IP : MnSOD

lenti-Sirt3 Con dn wt

-MnSOD

IgG

wt

Mit

o-SO

X F

luor

esce

nce

Fol

d c

han

ge f

rom

WT

1

3 Sirt3+/+

Sirt3-/-

Cont

2 *

Cont

*

**

lenti-Sirt3-dn

lenti-Sirt3-wt

Mit

o-SO

X F

luor

esce

nce

Fol

d c

han

ge f

rom

WT

1

3 Sirt3+/+

Sirt3-/-Sirt3+/+

Sirt3-/-Sirt3+/+

Sirt3-/-

Cont

2 *

Cont

*

**

lenti-Sirt3-dn

lenti-Sirt3-wt

Targeting domain Catalytic Domain

Deacetylase DomainSIRT3-WT

Deacetylase DomainSIRT3-DN

248 - H-Y






248 - H-Y


248 - H-Y

MnSOD

GELLEAIK*RDF

50 100 150 200

MnSOD

GELLEAIK*RDF

50 100 150 200

115 122 127 91 98 103Human GELLEAIKRDFGS Rhesus macaque GELLEAIKRDFGSMouse GELLEAIKRDFGS Callithrix jacchus GELLEAIKRDFGSRat GELLEAIKRDFGS Common gibbon GELLEAIKRDFGSBovine GELLEAIKRDFGS Chimpanzee GELLEAIKRDFGS Guinea pig GELLEAIKRDFGSHorse GKLLDAIKRDFGS 117 124 129PIG GELLDAIKRDFGS Xenopus tropicalis GELLDAIKRDFGSB. Orangutan GELLDAIKRDFGS Zebrafish GELLEAIKRDFGSS. Orangutan GELLDAIKRDFGS

102 109 114114 121 126 Rhesus Monkey GELLEAIKRDFGS

C. elegans AELLTAIKSDFGS Chimpanzee GELLEAIKRDFGS

MnSOD K122 is an evolutionarily conserved reversible lysine


MnSOD K122 is Deacetylated by Sirt3 in vitro and in vivo

TSA + +MnSOD + +

SIRT3 + +NAD+ - +

20kD-acetyl lysine

122 MnSOD

TSA + +MnSOD + +

SIRT3 + +NAD+ - +

20kD-acetyl lysine

122 MnSOD

-K122MnSOD

-MnSOD

Sirt3 -/-+/+ +/+ -/-

-K122MnSOD

-MnSOD

Sirt3 -/-+/+ +/+ -/-

In vitro In vivo

K122

K122

K122K122

lenti-MnSODK122

Wild-type

H2N COOH

NH2O

Lysine (Lys, K)

lenti-MnSODK122

Wild-type

H2N COOH

NH2O

Lysine (Lys, K)

Lenti-MnSODK122-R

De-acetylated

H2N COOH

NH

N2H NH2+

Arginine (Arg, R)

Lenti-MnSODK122-R

De-acetylated

H2N COOH

NH

N2H NH2+

Arginine (Arg, R)

lenti -MnSOD K122 -Q

Acetylated

H2N COOH

NH3+

Glutamine ( Gln, Q)


Acetylated

H2N COOH

NH3+

Glutamine ( Gln


Acetylated

H2N COOH

NH3+

Glutamine ( Gln, Q)


Acetylated

H2N COOH

NH3+

Glutamine ( Gln

Mn

SOD

Act

ivit

y(U

/mg)

in M

nSD

O-/

-

10

20

30

MnSOD K122wt K122-Rcont K122-Q

*

*

Mn

SOD

Act

ivit

y(U

/mg)

in M

nSD

O-/

-

10

20

30

MnSOD K122wt K122-Rcont K122-Q

*

* 10

50

MnSOD

30

Mit

oSO

X F

luor

esce

nce

R

atio

/ M

ock

infe

ctio

n

K122wt K122-RpCMV K122-Q

*

**

10

50

MnSOD

30

Mit

oSO

X F

luor

esce

nce

R

atio

/ M

ock

infe

ctio

n

K122wt K122-RpCMV K122-Q

*

**

MnSODK122 acetylation status directs dismutase activity

MnSOD-/- MEFs


De-Acetylated MnSODAcetylated MnSOD

TABLE 1. MnSOD prevents Immortalization of SIRT3-/-

MEFs by a single oncogene __________________________________________________________________________________________________________________________________________________________

MEFs Control Myc Ras Myc/Ras

_____________________________________________________________________________

SIRT3+/+ None None None Immort SIRT3-/- None Immort Immort Immort SIRT3-/- + lenti-MnSODK122-Q None Immort Immort Immort SIRT3-/- + lenti-MnSODK122-R None None None Immortt _____________________________________________________________________________ None , no MEF immortalization. Immort, immortalization. lenti-MnSOD 10 MOI. Immortalization experiments were done in triplicate.

MnSODK122-R prevents in vitro Immortalization


AL CRMnSOD

Ac-MnSODK122

Ac-MnSODK68

OSCP

Ac-OSCPK139

SIRT3

COXIV

AL CRMnSOD

Ac-MnSODK122

Ac-MnSODK68

OSCP

Ac-OSCPK139

SIRT3

COXIV

MnSOD De-Acetylation Responds to Exercise and CR

Sirt3+/+

Cont EX Cont EX

Sirt3-/-

MnSODK122

MnSODK68

MnSOD

Sirt3+/+

Cont EX Cont EX

Sirt3-/-

MnSODK122

MnSODK68

MnSOD

K122

K122

K122K122

O2- O2

-

O2- O2

-

O2-

+-

H2O2

How does MnSOD fit into this model??

LongevityHibernation

Energy Conservation

FOXOACAC

ACAC ACAC

AC AC AC

Fasting Metabolic StateFeasting Metabolic State

AC

ACAC ACAC

AgingCancer

Pro-metabolism

ATP and Oxidative damage Repair of Oxidative damage

MnSOD

The Gius Lab

David Gius, M.D., Ph.D.

Documents

targets of sirt3

sirt3 knockout micesirt3

sirt3 knockout livers

molecular cell mnsod

cr o2o2

reversible acetyl lysine

human malignancies

molecular cell mnsodk122r