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Nutrient Sensing, Acetyla2on, Mitochondrial Quality Control and Pathology
Michael N. Sack Cardiovascular and Pulmonary Branch Na4onal Heart, Lung and Blood Ins4tute
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Talk Outline
• Caloric Load, Sirt3 and the Regula2on of Protein Acetyla2on • Fas2ng and Tylenol Liver Toxicity
• Sirtuin Biology and Mitochondrial Quality Control • The Role of Fas2ng and Sirt3 on NLRP3 Inflammasome Biology
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Nutrient excess, obesity and disease burden
Malik et al, Nat. Rev. Endocrinology 2013
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Nutrient overload orchestrate growth programing, in part, via protein acetyla2on and glycosyla2on
Wellen and Thompson, Nature Reviews Mol. Cell. Biol. 2012
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Lysine Acetyla2on: an emerging post-‐transla2onal modifica2on
Timeline
1968-‐acetyla4on of histones discovered
1997: 1st non-‐histone acetylated protein-‐p53
Before 2006: <90 proteins are known to be acetylated
2006: 195 acetylated proteins (Kim et al. Mol. Cell)
2009: 1750 acetylated proteins (Choudhary et al. Science)
2013: > 2000 acetylated proteins 133 mitochondrial proteins (195 total) 20% of mitochondrial proteins
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Nutrient availability dependent mitochondrial protein acetyla2on
I
Kendrick et al, Biochem. J. 2011
High Fat Feeding
Hepa2c Mitochondrial Proteins
Fed/Fas2ng Comparison
Kim et al, Molecular Cell, 2006
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Acetyla2on and deacetyla2on are enzyma2cally regulated
ScoX (2012) Essays Biochem.
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Sirtuins: NAD+-‐dependent deacetylases
Mammals express seven Sirtuins (Sirt 1-‐7)
Various cellular localiza2ons:
Sirt1 & 2: nuclear and cytosolic Sirt3-‐5: mitochondrial Sirt6 & 7: nuclear
NAD+-‐dependent deacetyla2on ADP Ribosyla2on ac2vity (Sirt4
& 6)
Yang XJ et al, Nat Review Mol. Cell Biol. 2008;9:206-18
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Sirt3 func2ons as the nutrient-‐sensi2ve mitochondrial lysine deacetylase
Hirschey et al, Nature (2010)
Bao J et al. Free Radical Biol. and Med. (2010)
Lombard et al. (2007) Mol. Biol. Cell
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Recent fasting was more common than recent alcohol use among those who suffered hepatotoxicity after a dose of 4 to 10 g of acetaminophen per day (P=.02). Recent alcohol use was more common in the group who took more than 10 g/d than in those who took 4 to 10 g/d (P=.004). Conclusion: Acetaminophen hepatotoxicity after a dose of 4 to 10 g/d was associated with fasting and less commonly with alcohol use. Patients who developed hepatoxicity after taking acetaminophen doses of greater than 10 g/d for therapeutic purposes were alcohol users. Acetaminophen hepatotoxicity after an overdose appears to be enhanced by fasting in addition to alcohol ingestion.
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Acetaminophen toxic metabolites can bind to lysine residues
Whether this plays a role in hepatotoxicity is unknown
NAPQI -‐ N-‐acetyl-‐p-‐benzoquine-‐imine
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Hypothesis
The level of mitochondrial protein acetyla2on modulates suscep2bility to acetaminophen
liver injury
This may be mediated in part by modula2ng NAPQI binding to mitochondrial proteins?
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10 x WT 10 x KO
SIRT3 KO mice are resistant to acetaminophen-‐induced liver injury
APAP 350mg/kg IP administration N-acetyl-p-aminophenol
SIRT3+/+ SIRT3-/-
*
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Iden2fica2on and characteriza2on of novel substrates of SIRT3 in the murine liver
2D gel ko
2D gel wt
2D gel wt
2D gel
membrane
x-filmmembrane
x-filmmembraneblot
blot
stain
stain
cut spots
MS
2D gel ko
2D gel wt
2D gel wt
2D gel
membrane
x-filmmembrane
x-filmmembraneblot
blot
stain
stain
cut spots
MS
2D gel and MS to ID hyper-acetylated mitochondrial proteins
ID poten2al targets
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Representa2ve 2-‐D gels employing an an2body directed against acetylated lysine-‐residues
1 2 3 4 5
6
7
8 9
10
11
12 13
14
150 75
25
kDa
pH 3 - 10
SIRT3+/+
SIRT3-/-
150 75
25
ALDH2
SIRT3+/+
SIRT3-/-
Ac- K
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ALDH2
Major ALDH2 metabolic pathways
Catalyze the oxidation of aldehydes
Acetaldehyde
Acetate
NAD+
NADH
trans-4-hydroxy-2-nonenal (4-HNE)
Lipid peroxidation produces α,β-unsaturated hydroxyalkenal
Ethanol oxidation
Carboxylates
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Mitochondrial ALDH2 is a direct target of the toxic acetaminophen metabolite -‐ NAPQI
Landin JS, et al. Toxicology and Applied Pharmacology 1996;141:299-307
Is this interaction integral to APAP hepatotoxicity?
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ALDH2 is a substrate for Sirt3 Deacetyla2on
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ALDH2 func2on is preserved in fas2ng Sirt3 KO mice
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Hepa2c shRNA knockdown of ALDH2 negates acetaminophen ‘resilience’ in SIRT3 -‐/-‐ mice
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Sirt3-‐dependent acetyla2on status modulates NAPQI binding to ALDH2
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Iden2fica2on of ALDH2-‐ K377 as the func2onal residue for NAPQI binding
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Iden2fica2on of an allosteric role of lysine deacetyla2on in fas2ng suscep2bility to acetaminophen injury
Lu et al. EMBO Reports 2011
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Talk Outline
• Caloric Load, Sirt3 and the Regula2on of Protein Acetyla2on • Fas2ng and Tylenol Liver Toxicity
• Sirtuin Biology and Mitochondrial Quality Control • The Role of Fas2ng and Sirt3 on NLRP3 Inflammasome Biology
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SuXerwala et al. (2014) Ann N Y Acad Sci
Inflammasome: Mul4protein intracellular complex that sense pathogen / damage associated molecular paXerns (PAMPS/DAMPs) and ac4vate caspase-‐1, which in turn cleaves/ac4vates pro-‐inflammatory cytokines IL-‐1β and IL-‐18.
Defining the Inflammasome Program
NLRP -‐ Nod-‐like receptor family protein ASC -‐ Adaptor apoptosis-‐associated speck-‐like protein containing a CARD and pyrin domain (PYD)
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SuXerwala et al. (2014) Ann N Y Acad Sci
NLRP3 Inflammasome: -‐ Mul4ple triggers (‘sterile inflamma2on’) –
asthma, atherosclerosis, DM and aging -‐ Regulated at the transcript and post-‐transla4onal
levels:
Priming: Transcrip4onal induc4on of genes encoding components of the NLRP3 complex. Ac2va2on: Complex ac4va4on by stress-‐signals -‐ ATP, nigericin, faXy acids & cholesterol crystals.
1) Priming: LPS
2) Ac2va2on: ATP
The NLRP3 Inflammasome Program
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Mar2non, Immunity 2012
Mitochondrial Disrup2on as a Disease Associated Molecular Pafern (DAMP) in NLRP3 Ac2va2on
Evolu2onary endosymbionts unmethylated CpG mo2fs
(mito rRNA and tRNA) N-‐formyl pep2des
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Sirt1 and Sirt3 Deacetylase Enzymes
Sack MN and Finkel T. Cold Spring Harb Perspect Biol 2012
Modulate Mitochondrial Func2on/Quality (Caloric Restric2on/Fas2ng)
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Can Mitochondrial Sirtuins Regulate the NLRP3 Inflammasome?
Is this Dependent on the
Modula2on of Mitochondrial Homeostasis?
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Fas2ng (caloric restric2on mime2c) suppresses the NLRP3 inflammasome?
F e d
F a s ted
0 .0
0 .5
1 .0
1 .5M o u s e p e rito n e a l m F
IL -1b
IL-1
b r
ele
as
e(r
ela
tiv
e t
o t
he
fe
d s
tate
)
* There is a 40 % decrease in the release of IL-‐1β afer fas4ng
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NLRP3 ac2va2on is modulated by Sirt3
* Only the mitochondrial sirtuin Sirt3 appears to regulate the NLRP3 inflammasome in human THP-‐1 macrophages.
And in mouse in vivo:
WT
Sirt3 K
O
WT faste
d
Sirt3 K
O faste
d
0.5
1.0
1.5Mouse peritoneal mΦ
IL-1β
Cyt
okin
e re
leas
e(r
elat
ive
to th
e W
T)
* + + + +-‐ + -‐ +
LPS
WT Sirt3 KO
ATP
CleavedCasp-‐1
A B C
+ + + +-‐ + -‐ +
LPS
WT Sirt3 KO
ATP
CleavedCasp-‐1
A B C
+ + + +-‐ + -‐ +
LPS
WT Sirt3 KO
ATP
CleavedCasp-‐1
A B C
+ + + +-‐ + -‐ +
LPS
WT Sirt3 KO
ATP
CleavedCasp-‐1
A B C
+ + + +-‐ + -‐ +
LPS
WT Sirt3 KO
ATP
CleavedCasp-‐1
A B C
Sirt3
IL-‐1β
NLRP3
ASC
Actin
-‐ + + -‐ + +-‐ -‐ + -‐ -‐ +
LPS
WT Sirt3 KO
ATP
A B C
Sirt3
IL-‐1β
NLRP3
ASC
Actin
-‐ + + -‐ + +-‐ -‐ + -‐ -‐ +
LPS
WT Sirt3 KO
ATP
A B CFasted Fed
Sirt3
IL-‐1β
NLRP3
ASC
Actin
-‐ + + -‐ + +-‐ -‐ + -‐ -‐ +
LPS
WT Sirt3 KO
ATP
A B CFasted Fed
C o n trol
+ Sir t
3
+ H/Y
mu ta
n t0 .0
0 .5
1 .0
1 .5 T H P -1
Cy
tok
ine
re
lea
se
(re
lati
ve
to
th
e c
on
tro
l)
*
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The role of Sirt3 in NLRP3 inflammasome regula2on is confirmed in Bone Marrow Derived Macrophages
*
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Mitochondrial Homeosta2c Role of Sirt3
Gira
lt A et a
l. Bioche
m J, 201
2
Improve Energe2cs Decrease ROS MPT Resistance
Diminished Apoptosis Enhanced Mitophagy
WT L
P S
WT L
P S + A
T P
S ir t3 K
O L
P S
S ir t3 K
O L
P S + A
T P
0
2
4
6
8
1 0
Cy
tos
olic
mtD
NA
(F
old
ch
an
ge
)
*
U n t L P S + A T P R o te n o n e0
1
2
3
4
5
Mit
os
ox
in
ten
sit
y
S c rshRNAS ir t3shRNA
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The counter-‐regulatory control of mitochondrial protein acetyla2on
Sirt3 levels are increased with fas2ng
Hirschey et al, Nature 2010
Gcn5L1 levels are decreased with fas2ng
Webster B et al. J. Cell Science (2013)
Sirt3
Gcn5L1
ScoX I, et al. Biochem J. 2012
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Counter-‐regulatory roles of Gcn5L1 and Sirt3 on mitochondrial acetyla2on and func2on
ScoX I, et al. Biochem J. 2012
Mitochondrial Protein Acetyla2on
Webster B, ScoX I. J Cell Sci 2013
Modula2on of Mitophagy
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Gcn5L1 and Sirt3 deple2on have counter-‐regulatory effects on the NLRP3 inflammasome
Traba J, et al. Unpublished Data
J774A.1 cells
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Mitochondrial phenotype in J774A.1 macrophages
Traba J, et al. Unpublished Data
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CLINICAL RESEARCH PROJECT Protocol # 14-‐H-‐0103
NHLBI Protocol: Pilot Study to Evaluate the Effect of Fas5ng on the NLRP3 Inflammasome
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00 .1 1
0
1
2
3
4
5IL -1b
L P S (n g /m L )
Cy
tok
ine
re
lea
se
(re
lati
ve
to
th
e f
as
ted
sta
te)
F a s te d
F e d 1 h
F e d 3 h
0 .1 1
0 .8
1 .0
1 .2
1 .4 IL -1b
L P S (n g /m L )
Cy
tok
ine
re
lea
se
(re
lati
ve
to
th
e f
as
ted
sta
te)
F a s te d
F e d 1 h
F e d 3 h
Fas2ng Blunts the NLRP3 inflammasome in Human Subjects
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Conclusions
• The NLRP3 inflammasome is blunted by fas2ng
• NLRP3 inflammasome ac2va2on is nutrient-‐level dependent and appears to be modulated, in part, by the Sirt3 -‐Gcn5L1 regulatory program
• Preliminary data suggest that that fas2ng and the mitochondrial acetyla2on regulatory program modifies the role of mitochondria as a DAMP in NLRP3 ac2va2on
• This nutrient-‐sensing program is opera2onal in healthy human subjects
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Mito protein
SIRT3
AC
Ac ↓
GCN5L1
Mitochondrial Homeostasis
↑ SIRT3
Ac Fas2ng or
CR
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Acknowledgements
NHLBI Laboratory of Mitochondrial Biology Kim Han
Shahin Hassanzadeh Javier Traba Komudi Singh Lingdi Wang Jing Wu Jessica Li
Miriam Kwarteng-‐Siaw
Prior Laboratory Members Zhongping Lu Iain ScoX
Brad Webster
NIAMS Richard Siegel
NHLBI
Marjan Gucek Lance Pohl
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Expert Opin. Drug Metab. Toxicol. (2008) 4(11)
NAPQI adducts covalently bind to cysteine residues on mitochondrial proteins contributing to hepatic
toxicity
NAPQI adducts increase The mass of a peptide by 149Da