Unraveling the mitochondrial
role of the human apoptosis
inducing factor (hAIF)
Patricia Ferreira Neila
Departamento de Bioquímica y Biología Molecular y Celular
Instituto de Biocomputación y Física de Sistemas Complejos
Universidad de Zaragoza
BIFI2011: V National Conference
“Protein interaction and electron transfer”
Group of Structural Biology
Dra. Milagros Medina
Dr. Carlos Gómez-Moreno
Dr. Marta Martínez-Júlvez
Dra. Patricia Ferreira
Raquel Villanueva
Ana Serrano
Isaías Lans
Beatriz Herguedas
Sonia Arilla
Ana Sánchez,
Thanks Dr. Susin, Dra. M. Luisa Peleato and Dra.
M. Dolores Miramar for giving us the cDNA of
hAIF cloned in E.coli
Apoptosis inducing
Factor (AIF)
Apoptotic insult
AIF is a redox protein
NADH-binding
domain
C-terminal
AIF
apoptotic
FAD-binding
domain
AIF
oxidoreductase
hAIF crystal structure
(PDB 1M6I)
Lipton et al. (2002)
Chromatin condensation
Caspase-independent cell death
AIF seems to display a dual role in
cellular death and life.
AIF cellular localization
hAIF102
Kroemer et al. 2007
MLS
Anchored
peptide
FAD binding
NADH binding
FAD
binding
C-terminal
67 KDa
62 KDa
57 KDa
Vital AIF function
• Antioxidant defense
• AIF redox activity is associated with correct behavior of the
mitochondrial respiratory chain in vivo
Two hypothetical models
AIF as an assembly factor
Nazanine Modjtahedi, TRENDS in Cell Biology Vol.16 No.5 May 2006
AIF as a maintenance factor
NADH-binding
domain
AIF electron
transfer activity
C-terminal
AIF
apoptotic
HN5
C4
FAD
FAD-binding
domain
AIF
oxidoreductase
NADH
NADH
Reductive half reaction
E-FAD
E-FADH2
Oxidative half reaction
¿Acceptor ?
NAD+
In spite of the large number of studies about
AIF, key questions remain to be addressed…..
In spite of the large number of studies about
AIF, key questions remain to be addressed…..
• Which is the biological role of AIF in a healthy cell?
• Is AIF an oxidoreductase?
• Who is AIF redox partner in the cellular
environment?
• Is AIF redox activity independent or linked to the
apoptotic function?
The hAIF102 flavin properties
Absorbance
0.24
hAIF 102 reoxidased
hAIF 102 intermediate
hAIF 102 reduced
hAIF 102 oxidased
0.16
0.08
0.00
300
400
500
600
Either
photoreduction
or
sodium dithionite reduction of
hAIFΔ102 produced the full
reduced FAD without detection
of
any
semiquinone
intermediate.
700
Wavelength (nm)
The photoreduced hAIF102 results completely
reoxidised in the presence of oxygen.
Screening hAIF102 redox acceptor
NADH oxidase activity was not detected using oxygen as electron acceptor
Steady-state kinetic parameters of hAIF102 with different
electron acceptors using NADH substrate
kcat
Km
DCPIP
K3Fe(CN)6
(s-1)
1.5 ± 0.1
6.4 ± 0.4
(µM)
272.9 ± 31.3
1219 ± 191.6
Cytochrome c
1.3 ± 0.1
202.6 ± 37.6
kcat/Km
(s-1·mM-1)
5.5
5.2
Similar catalytic
efficiency
Low turn-over
6.4
No activity was detected using 1,4-benzoquinone, 1,2-naptoquinone or Fe3+EDTA as electron acceptors.
The low affinities for the coenzyme suggest that the hAIF redox reaction might be
activated by its electron acceptor under physiological conditions
H-
hAIF hydride transfer mechanism
0 sec
0.3 sec
6.88 sec
3.6 sec
16.71 sec
26.54 sec
36.37 sec
46.2 sec
Absorbance
0.12
0.08
CTC
0.04
N5
C4
FAD
NADH
Formation
of
flavin:nicotinamide
complex (CTC).
very
charge
stable
transfer
0.00
400
500
600
700
800
Wavelength (nm)
The reduction rates were independent of the
presence of molecular oxygen
Pre-steady state kinetic
parameters
kred (s-1)
Kd (µM)
NADH
1.23 ± 0.1
1260 ± 167
NADPH
0.08 ± 0.01
4848 ± 1131
k1
NADH is the natural electron donor of hAIF
Eox+S
k-1
EoxS
Kd (k-1/k1)
k2
Ered-P
kred (k2)
Dimerization can modulate hAIF
oxidoreductase activity.
Gel filtration profile
Wildtype
Wildtype
+ NADH
Absorbance
120
hAIF102 is a monomeric protein
that evolves to a dimeric state
during NADH oxidation.
This observation suggests that the
AIF redox reaction is regulated,
and must have some physiological
relevance.
80
40
0
0
5
10
15
20
25
30
Flow (mL/min)
This process was also observed for the mouse AIF (mAIF).
Dimerization can modulate hAIF
oxidoreductase activity.
Crystal
structure of the dimeric
mAIF:NAD+ complex (pdb 3GD4)
The interactions at the
dimer interface
R448
E412
R421
R429
R448
R421
All these residues are conserved in hAIF
E412
R429
E413A/R422A/R430A
Dimerization can modulate hAIF
oxidoreductase activity.
E413A/R422A/R430A variant
reduction with NADH
Gel filtration profile
0.10
Wildtype
Wildtype
+ NADH
80
40
Absorbance
Lower CTC to
the wild-type
0.08
Absorbance
120
0.06
0.04
CTC
0.02
0.00
0
400
120
E413A/R422A/R430A
E413A/R422A/R430A
+ NADH
500
600
700
800
Wavelength (nm)
NADH
hAIF
80
40
kred (s-1)
KdNADH (µM)
Wild-type
1.2 ± 0.1
1260 ± 167
Variant
0.5 ± 0.01
2260 ± 295
0
0
5
10
15
20
Flow (mL/min)
25
30
Reduction rates and affinity lower to the wild-type
Studying hAIF redox active site
Manual docking of NADH into
the hAIF redox active site
W483G
K177W
hAIF redox active site
(pdb 1m6i)
F310G
H454S
E314S
NAD+
FAD
AIF variants reduction with NADH
W483G
Wild-type
Absorbance
0.08
CTC
0.04
0.00
0.06
0.03
0.00
400
500
600
700
400
800
500
600
700
P173G
F310G
0.12
0 sec
8 sec
11 sec
17 sec
21 sec
25 sec
hAIFox
hAIFred-NAD+ 0.67 s
hAIFred-NAD+ 1.6 s
0,08
hAIFred-NAD+ 4.09 s
0,06
0,04
Absorbance
hAIFred-NAD+ 0.019 s
hAIFred-NAD+ 0.26 s
0,10
Absorbancia (U.A.)
800
Wavelength (nm)
Wavelength (nm)
0,12
0 sec
9 msec
60 msec
0.16 sec
3 sec
0.09
Absorbance
0 sec
0.3 sec
6.88 sec
3.6 sec
16.71 sec
26.54 sec
36.37 sec
46.2 sec
0.12
0.08
0.04
0,02
0.00
0,00
400
500
600
Longitud de onda (nm)
700
400
500
600
Wavelength (nm)
700
800
AIF variants reduction with NADH
Pre-steady state kinetic parameters
hAIF
Variants
All residues are involved in AIF redox
reaction
NADH
kred
(s-1)
KdNADH
(µM)
Wild-type
1.2 ± 0.1
1260 ± 167
W483G(*)
39.4 ± 1
245 ± 26
F310G
17.3 ± 1
5585 ± 89
P173G
4.7 ± 0.3
11932 ± 1548
All variants show higher kred to the wildtype values
W483G at least 40-times
20
(*) Experiments performed at 12 ºC
F310G and P173G lower affinity than
wild-type
kobs (s-1)
15
WT
F310G
W483G
10
5
0
0
2
4
[NADH] mM
6
AIF variants reduction with NADH
H454S
E314
F310
9 msec
0.1 sec
0.2 sec
0.4 sec
0.7 sec
1.5 sec
2 sec
0.15
Absorbance
NAD+
FAD
0.10
0.05
K177
0.00
W483
400
500
600
700
800
Wavelength (nm)
H454S
hAIFox and rAIFred:NAD redox active site
(pdb 1m6i and 3GD4)
No CTC formation
hAIF
Variants
NADH
kred (s-1)
KdNADH (µM)
Wild-type
1.2 ± 0.1
1260 ± 167
H454S
3.7 ± 1
2743± 295
Future work
• Explore new acceptor as AIF redox partner
• Analyse the AIF oligomerization state into the cell
• Study the effect of AIF variants in the efficiency of oxidative
phosphorylation in mitochondria
• Study the effect of AIF variants in isolated nuclei to evaluate the role of the
hAIF redox function, and the derived conformational changes of the NADH
interaction, in the apoptotic hAIF function.
Apoptosis inducing Factor (AIF)
Kroemer et al 2009
Cellular localization of AIF
Nazanine Modjtahedi, TRENDS in Cell Biology Vol.16 No.5 May 2006
Reacción de reducción con NAD(P)H
Reducción anaeróbica de la hAIF con
NADPH de hAIF 1-102 con 625 M NADPH
Reduccion
Reducción anaeróbica de la hAIF con NADH
hAIF
1-102
+ 612.5 M NADH en condiciones aeróbicas
0.12
AIFox
AIFox-NADPH 2 msec
AIFox
AIFox-NADH 1 msec
0.08
1.31 sec
67 sec
119 sec
250 sec
AIFred-NAD+ 40 msec
+
AIFred-NAD 1.27 sec
0.08
Absorbance
Absorbance
AIFred-NAD+ 3.3 sec
AIFred-NAD+ 5.36 sec
CTC
0.04
0.00
0.04
0.00
400
500
600
700
800
400
Wavelength (nm)
500
600
Wavelength (nm)
Reducción completa de la flavina mediada por dos electrones
Similares espectros de reducción con NAD(P)H en presencia y ausencia de oxigeno
Formación de complejos de transferencia de carga altamente estables
La formación del complejo AIFox-NADH se evidencia en los ensayos con un incremento del
espectro de la proteína
700
800
AIF como una proteína redox de señalización
Inna Y. Churbanova and Irina F. Sevrioukova, JBC 2008