Sirtuin 3: A Mitochondrial Watchdog
Protein
David Gius, M.D., Ph.D.
Professor, Departments of Cancer Biology,
Pediatrics, and Radiation Oncology
Vanderbilt University School of Medicine
Vanderbilt Medical School
The Human Sirtuins
Nuclear
Mitochondrial
Cytoplasmic
• Sirt1
• Sirt6
• Sirt7
• Sirt3
• Sirt4
• Sirt5
• Sirt2
Protein Deacetylation as a Post-Translation Protein Modification
Non-Histone
Protein
In active protein
Active
protein
Rational for Sirtuins as Tumor Suppressors
Decreases
aging
Increasing
age
? Carcinogenesis
Activation of Sirtuin
genes increase longevity
Caloric
Restriction
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.
Two Gene Transformation Model for MEFs
TSG Gene
Myc Loss
CMV Promoter
Ras
CMV Promoter
Oncogene 1
Proliferative gene
Pro-proliferative
Pro-survival genes
I
P
Normal cell
P
P
P
Transformed cell
Oncogene 2
Sirt3-/- MEFs are immortalized by a Single Oncogene
-/-
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+/+ Myc/Ras cells
Sirt3-/- Myc/Ras cells
Kim et al, 2010 Cancer Cell
Colonies/Hpf
In Vitro Transformation Sirt3-/- MEFs by a Single Oncogene
4
SIRT3 +/+
3
SIRT3 -/-
2
*
*
*
1
Myc/Ras
Myc
Ras Myc/Ras
Mammary Carcinogenesis in the Sirt3 knockout mice
Tumor #1
Tumor #2
Tumor #3
Tumor #4
Tumor #5
Tumor #6
Tumor #7
*
6
0
SIRT3 +/+ SIRT3 -/-
% Tumor Free
# of tumors
PR
++
+
+
+
++
+
++
++
+
+
+
++
+
+
100
10
2
ER
WT
KO
50
6
9
12 15 18 21 24 27 30
SIRT3 is Decreased in Human Breast Cancers
Tissue Array (IHC)
RNA Array (RT-PCR)
Tumors
Normal
SIRT3 mRNA
Fold expression
% Total Samples
100
8
31
17
50
10
3
Low Med High
4
** P < 0.0025
6
* P < 0.02
4
*
2
**
*
IIA
IIB
1
Low Med High
Stage
NB
I
III
Kim et al, 2010 Cancer Cell
P = 1.0E-9
1.0
1.0
0.5
P = 2.4E-13
0.5
0.0
0.5
1.0
1.0
Normalized expression units
1.5
Normalized expression units
Normalized expression units
SIRT3 is Decreased in Human Breast Cancers
P = 3.8E-10
0.5
0.0
0.0
Normal
Breast
Breast
Cancer
0.5
1.5
G-1
G-2
G-3
Well
Mod
Poor
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?
4.0
SIRT3 -/3.0
2.0
SIRT3 +/+
SIRT3 +/+
SIRT3 -/-
*
*
*
*
1.0
Cont
5 Gy
Antimycin
Mito-SOX Fluorescence
Fold change from WT
Mito-SOX Fluorescence
Fold change from WT
Transformed Sirt3 KO MEFs exhibit mt Superoxide
**
3.0
**
2.0
*
1.0
Myc/Ras
Myc
Ras
Myc/Ras
Kim et al, 2010 Cancer Cell
Primary Mitochondrial O2- Detoxification Pathway
O2-
H2O2
MnSOD
H2O + O2
Catalase
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.
1.0
*
0.5
Sirt3+/+
Sirt3-/-
MEFs Relative MnSOD
Activity (% Cont)
Liver Relative MnSOD
Activity (% Cont)
MnSOD contains a reversible lysine
1.0
*
0.5
Sirt3+/+
Sirt3-/-
Tao et al., 2010, Molecular Cell
MnSOD’s reversible is deacetylated by Sirt3
Targeting domain
Catalytic Domain
IgG IP : MnSOD
Deacetylase Domain
SIRT3-DN
Deacetylase Domain
-MnSOD
248 - H-Y
lenti-Sirt3
1.0
Sirt3-/-
0.5
Lenti- Con
Sirt3dn
Sirt3
-acetyl
Mito-SOX Fluorescence
Fold change from WT
MEFs Relative MnSOD
Activity (% Cont)
SIRT3-WT
3
wt
Con
Sirt3 +/+
Sirt3 -/-
*
2
1
dn
wt
*
**
Cont
Cont
lentilentiSirt3-dn Sirt3-wt
MnSOD K122 is an evolutionarily conserved reversible lysine
GELLEAIK*RDF
MnSOD
50
Human
Mouse
Rat
Bovine
Guinea pig
Horse
PIG
B. Orangutan
S. Orangutan
C. elegans
100
115
150
122
127
GELLEAIKRDFGS
GELLEAIKRDFGS
GELLEAIKRDFGS
GELLEAIKRDFGS
GELLEAIKRDFGS
GKLLDAIKRDFGS
GELLDAIKRDFGS
GELLDAIKRDFGS
GELLDAIKRDFGS
114
121
126
AELLTAIKSDFGS
200
Rhesus macaque
Callithrix jacchus
Common gibbon
Chimpanzee
Xenopus tropicalis
Zebrafish
Rhesus Monkey
Chimpanzee
91
98
103
GELLEAIKRDFGS
GELLEAIKRDFGS
GELLEAIKRDFGS
GELLEAIKRDFGS
117
124
129
102
109
114
GELLDAIKRDFGS
GELLEAIKRDFGS
GELLEAIKRDFGS
GELLEAIKRDFGS
Tao et al., 2010, Molecular Cell
MnSOD K122 is Deacetylated by Sirt3 in vitro and in vivo
In vitro
TSA
MnSOD
SIRT3
NAD+
20kD
+
+
+
-
In vivo
+
+
+
+
-K122
MnSOD
-acetyl lysine
122 MnSOD
-MnSOD
Sirt3
+/+
+/+
-/ -
-/ -
Wild-type
K122
O
H 2N
K122
NH2
COOH
Acetylated
Lysine (Lys, K)
lenti-MnSODK122
NH 3 +
H 2N
De-acetylated
N2H
COOH
Glutamine ( Gln
Gln, Q)
lenti -MnSOD K122 -Q
NH2+
NH
H2N
COOH
Arginine (Arg, R)
Lenti-MnSODK122-R
K122
K122
MnSODK122 acetylation status directs dismutase activity
*
30
20
10
MnSOD
*
cont
K122wt K122-R K122-Q
MitoSOX Fluorescence
Ratio / Mock infection
MnSOD Activity
(U/mg) in MnSDO-/-
MnSOD-/- MEFs
50
30
**
*
10
MnSOD pCMV K122wt K122-R K122-Q
De-Acetylated
Acetylated MnSOD
Tao et al., 2010, Molecular Cell
MnSODK122-R prevents in vitro Immortalization
TABLE 1. MnSOD prevents Immortalization of SIRT3
-/-
MEFs by a single oncogene
__________________________________________________________________________________________________________________________________________________________
Myc/Ras
Ras
Myc
Control
MEFs
_____________________________________________________________________________
SIRT3+/+
None
None
None
Immort
SIRT3-/-
None
Immort
Immort
Immort
SIRT3-/- + lenti-MnSODK122-Q
None
Immort
Immort
Immort
Immortt
None
None
None
SIRT3-/- + lenti-MnSODK122-R
_____________________________________________________________________________
None , no MEF immortalization.
Immort, immortalization.
lenti-MnSOD 10 MOI.
Immortalization experiments were done in triplicate.
Tao et al., 2010, Molecular Cell
MnSOD De-Acetylation Responds to Exercise and CR
Sirt3+/+
Cont
EX
Sirt3-/Cont
EX
AL
CR
MnSOD
Ac-MnSODK122
MnSODK122
MnSODK68
MnSOD
Ac-MnSODK68
OSCP
Ac-OSCPK139
SIRT3
COXIV
K122
O2- O2-
O2O
O2
2
+
K122
H 2O 2
K122
K122
How does MnSOD fit into this model??
Feasting Metabolic State
Fasting Metabolic State
ATP and Oxidative damage
Repair of Oxidative damage
MnSOD
AC
AC AC AC
AC AC AC
AC AC AC
AC
Aging
Cancer
Pro-metabolism
AC
FOXO
Longevity
Hibernation
Energy Conservation
AC
The Gius Lab