chiarugi mitocon 2011
TRANSCRIPT
Strategie teraputiche
Modulazione farmacologica dell'omeostasi del NADmitocondriale: implicazioni terapeutiche
Alberto ChiarugiI° Convegno Nazionale sulle Malattie Mitocondriali
Roma, 21-22 Maggio 2011
4 Nobel nella “storia del NAD”
Destino metabolico del NAD
NAD
De novopathway
Tryptophan
Kynurenine pathway
cADPR
NAADP
NADK
NADP
NADases
PARPARPs PARG
AADPR
Sirtuins Nam
ADPR
MARTs
Rescuepathway
ATP
NMN
NPRT
NMNAT
ATP
ADPRPP
Reazioni metaboliche che distruggono e riformano il NAD
Alcuni ruoli metabolici aggiuntivi del NAD mitocondriale
Organizzazione dei complessi Fe/S
Rigenerazione del glutatione ridotto
Attività della piruvato deidrogenasi
Origine del NAD mitocondriale: un enigma ancora irrisolto
NAD
NAD
NAD support therapy
NAD depletion therapy
….nascono quindi 2 nuovi settori della farmacologia
Occorre però porre attenzione a:
Equilibri omeostaticiFarmacocinetica
direct
indirect
Effetti della deplezione di NAD cellularesulla funzione mitocondriale
C 1 10 100
60
70
80
90
100
110
M, FK 866
C6MT2FibroblastsGliaNeurons
HeLa
NA
D(%
of c
ontr
ol)
Effetti del blocco della resintesi del NAD sulla concentrazioni di NAD
Pittelli M. et al, JBC 2010
ATP
ATP
FK866Nam
NMN
NMNAT
ARTSIRT1-7
CD38PARPs
NAD
Nampt
C FK C FK0
30
60
90
120
150
**
NA
D%
of c
ontr
ol
Ma la deplezione di NAD non avviene nei mitocondri
MitocondriCytosol
C FK100µM/1h FK100nM/24h
Cell autofluorescence (NADH)
0
100
200
300
400 FK866
C PYR PYRADP
AT
P
(arb
itra
ry u
nits)
C PYR PYRADP
******
* *
TMRE Merge
NamptGFP
cytGFP
MitGFP
Nampt
MitGFP
TMRE
TMRE
Nampt is localized in citosol and not in mitochondria of HeLa cells
Pittelli M. et al, JBC 2010
Effetti del NAD esogeno sulla funzione mitocondriale
0 1 2 6 120
50
100
150
200
250
300
350
400
1µM
10µM
100µM
1mM
hrs
1mM 4°C
% o
f int
race
llula
r N
AD
CRL NAD 1mM (6h)
C 0 5 C 0 50
50
100
150
200
250 Mit-Lucif
h
NAD 1mMNAD 1mM
Cyt-Lucif
*
** **
**
Ph
oto
n e
mis
sio
n%
of
con
tro
l
C NAD C NAD0
2
4
6
8
10 mitochondria
nuclei
**
*nmol
NA
D/ m
g pr
ot
Effetti del NAD esogeno sulla funzione mitocondriale
CTRL NAD0
1
2
3
4 **
Oxy
ge
n c
on
sum
ptio
n r
ate
(nm
ol/m
l/min
)
0 2 4 6 8 10
205.0
210.0
215.0
220.0
225.0
230.0
235.0
min
nm
ol o
xyge
n/m
l
Control
NAD 1mM
BA
Effetti del NAD esogeno sul consumo di ossigeno
STP + NAD 1 mMSTPCTRL
Effetti del NAD esogeno sulla sopravvivenza cellulare
Total NAD+ cellular content in HEK293 shNMNAT3 stable cell line(3 exp in duplicate)
shSCRAM
BLED
shNM
NAT30
50
100
150
200
**
NA
D+
(A
BS
/mg
prot
.)Total ATP cellular content in HEK 293 shNMNAT3 stable cell line
(2 exp in triplicate)
shSCRAM
BLED
shNM
NAT30
50
100
150
200
*
ATP
/mg
prot
.
NAD tot NMNAT3-flag (var.2) overexpression 48 hHEK293 (1 exp. in duplicate)
Control
NMNAT3-
flag
0
1
2
3
**
NA
D+
(A
BS
/mg
prot
.)
Total ATP cellular content overexpression NMNAT3-flag 48h HEK 293 (1 exp in triplicate)
Control
NMNAT3
+0
1.0107
2.0107
3.0107
4.0107
ATP
/mg
prot
.
Ruolo dell’enzima mitocondriale NMNAT3
NMN + ATP NAD + PPi
Strategie teraputiche
Potenziali precursori del NAD
Acido Nicotinico
Nicotinamide
Nicotinamide riboside
Nicotinamide adenin mononucleotide
Intermedi della via delle chinurenine
Potenziale terapeutico dei precursori del NAD
Conclusioni
Laura FormentiniFrancesco BlasiMirko Muzzi Giuseppe FaracoAndrea LapucciDaniela BuonvicinoLeonardo Cavone
Elisa LanducciTania ScartabelliElisabetta GeraceAlessio MasiMaria SiliAndrea CozziVincenzo Carlà
Ringraziamenti
Fondazione Giuseppe Tomasello
Roberta Felici
PARP-1 and necrosis(the suicide hypothesis)
glucose
GAP
ATPADP
1,3-DPG
pyruvate
NADNADH
ATP
DNAdamage
NAD
NADH
NA+PRPP
NMN
NAD
NMNAT
PARP1
ADP
H+
H+
III
IIIIV
ATP
PRT
ATP
CELL NECROSIS
NAD e NAMPT e degenerazione assonale
CRL NAD 1mM (6h)
NAD e mitocondri concetto di saturazione
NADH(autofluorescenza cellulare)
A key role of PARP-1 in epigenetic chromatin remodeling
Homeostatic role
NAD
III
IIIIV
nPARP1
mPARP1
ROS
I II III IV
ANT
nPARP1
Detrimental role
Poly(ADP-ribose) polymerase-1 (PARP-1) in nucleus-mitochondrial cross-talk under homeostatic or pathogenetic conditions
1
2
3
4
5
6
BER
I IIIIV
1 - nPARP-1 regulates nuclear respiratory chain gene expression2 - nPARP-1 regulates mitochondrial DNA repair gene expression3 - mPARP-1 regulates mitochondrial respiratory chain gene expression4 - mPARP-1 assists mitochondrial genome repair
5 - mitochondrial ROS hyperactivate nuclear PARP-1 and prompt energy failure6 – mitochondrial ROS hyperactivate nuclear PARP-1 and alter epigenetic regulation
ATP
Ca2+
Ca2+
Ca2+
IP3
PLC
GPR
0 15 30 45 600
102030405060708090
100110
NAD
ATP
MNNG 100µM
min hrs2 4 86
% o
f co
ntro
l
MNNG
0 1h
Wash
Working model
10 -7 10 -6 10 -5 10 -4 10 -3 10 -2
0
25
50
75
100 PHEBZD
NA
D d
eple
tion
% o
f in
hibi
tion
10 -7 10 -6 10 -5 10 -4 10 -3 10 -2
0
25
50
75
100 PHEBZD
AT
P d
eple
tion
% o
f in
hibi
tion
NH 2
O N H
O
Phenanthridinone (PHE)IC50 = 3µM
Benzamide (BZD)IC50 = 30µM
The DNA alkylating agent Methyl-Nitro-Nitrosoguanidine (MNNG) induces PARP-1 The DNA alkylating agent Methyl-Nitro-Nitrosoguanidine (MNNG) induces PARP-1
hyperactivation and a long-lasting depletion of NAD and ATP in HeLa cellshyperactivation and a long-lasting depletion of NAD and ATP in HeLa cells
5’ 10’ 15’C
Cel
l cou
nts
TMRE fluorescence intensity
MNNG 2h
MNNG 3h
MNNG 1h
MNNG 4h
MNNGControl
0 1 2 3 4 5 60
20
40
60
80
100
120
140
160
MNNG
*
******
hrs
TM
RE
fluo
resc
ence
(% o
f con
trol)
Mitochondrial membrane potential () in HeLa cells undergoing PARP-1 hyperactivation
Poly(ADP-ribose) metabolism
NAD
NA
PAR
ATP
ATP
NADNMNAT
NMN
PARP
ADPRPARG
NAD rescue pathway
PPi
5-P ribose
ATP
ADP-ribosepyrophosphorylase
ATPmitochondria
ATPglycolysis
and catabolism
PRT
5-P ribose
AMP
ADP-ribosepyrophosphatases
(NUDIX hydrolases)
5-P ribose
AMP
Phosphodiesterases ?
From McLennan A.G., Cell. Mol. Life Sci. 63 (2006) 123–143
Summary of the human Nudix genes and hydrolases
ADP
AMP
ADPAMP
= 6.3±0.9
Control
ADP
AMP
ADPAMP
= 0.35±0.05
PARP-1 hyperactivation
Inverted ADP/AMP ratio underlies PARP-1-dependent energy failure
AMP reduces ADP uptake and ATP output from isolated mitochondria
ADP
ADP
ATP
ATP
INTERMEMBRANESPACE
MATRIX
The Adenine NucleotideTranslocator
Amino acid residues involved in the orientation and molecular constraintof ADP during its journey down to the ANT cavity
- Non fix anti conformation- NH in C6- No sobstistution in C2 - 2 or 3 PO4 groups
Transported Nucleotides
Ser227
Gly224
Arg279
Glu29
Lys22
Arg137
Arg235
Lys32
Ser227
Gly224
Arg279
Glu29
Lys22
Arg137
Arg235
Lys32
Ser227
Gly224
Arg279
Glu29
Lys22
Arg137
Arg235
Lys32
Ser227
Gly224
Arg279
Glu29
Lys22
Arg137
Arg235
Lys32ADP AMP
0
25
50
75
100
125
CRL ATR
CP
MA
(% o
f con
trol
)
0 1 10 100 1000
[AMP] M
ADP AMP
Bioactive Conformation (ΔE) = +7.60 kcal/mole Bioactive Conformation (ΔE) = +3.25 kcal/mole
Conformational Energy Evaluation of ADP or AMP bound to ANT
PARP NAD PAR AMP
ADP
ATPADP
glycolysis
metabolism
AK
ATP
NADrescue
Conclusion: The Nudix Hypothesis
ANT
AKAK
AK
AK
EVOLUTIONARY IMPLICATIONSPARP-1-dependent energy failure helps disclosing some of the ancestral strategies adopted
by eukaryotic cells to preserve bioenergetic exchanges with the protomitochondrion