patricia ferreira neila departamento de bioquímica y biología molecular y celular instituto de...
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Patricia Ferreira Neila
Departamento de Bioquímica y Biología Molecular y CelularInstituto de Biocomputación y Física de Sistemas Complejos
Universidad de Zaragoza
Unraveling the mitochondrial role of the human apoptosis
inducing factor (hAIF)
BIFI2011: V National Conference
Dra. Milagros MedinaDr. Carlos Gómez-Moreno Dr. Marta Martínez-JúlvezDra. Patricia Ferreira
Raquel Villanueva Ana SerranoIsaías LansBeatriz Herguedas Sonia ArillaAna Sánchez,
“Protein interaction and electron transfer”
Group of Structural Biology
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)
Lipton et al. (2002)
Apoptotic insult
Chromatin condensationCaspase-independent cell death
AIF seems to display a dual role in cellular death and life.
FAD-binding domain
NADH-bindingdomain
C-terminal
AIFoxidoreductase
AIF apoptotic
hAIF crystal structure(PDB 1M6I)
AIF is a redox protein
AIF cellular localization
Kroemer et al. 2007
hAIF102
MLS FAD binding NADH bindingFAD
binding C-terminalAnchoredpeptide
67 KDa
62 KDa
57 KDa
• AIF redox activity is associated with correct behavior of the mitochondrial respiratory chain in vivo
Nazanine Modjtahedi, TRENDS in Cell Biology Vol.16 No.5 May 2006
AIF as an assembly factor AIF as a maintenance factor
Vital AIF function
Two hypothetical models
• Antioxidant defense
FAD-binding domain
NADH-bindingdomain
C-terminal
AIF oxidoreductase
AIF apoptotic
E-FAD E-FADH2
Oxidative half reaction
Reductive half reactionNADH NAD+
¿Acceptor ?
AIF electron transfer activity
N5C4
H -
FAD
NADH
• Is AIF an oxidoreductase?
• Who is AIF redox partner in the cellular environment?
• Which is the biological role of AIF in a healthy cell?
• Is AIF redox activity independent or linked to the apoptotic function?
In spite of the large number of studies about AIF, key questions remain to be addressed…..
The hAIF102 flavin properties
Wavelength (nm)
300 400 500 600 700
Ab
sorb
an
ce
0.00
0.08
0.16
0.24 hAIF102 reoxidasedhAIF102 intermediate
hAIF102 reduced
hAIF102 oxidasedEither photoreduction or sodium dithionite reduction of hAIFΔ102 produced the full reduced FAD without detection of any semiquinone intermediate.
The photoreduced hAIF102 results completely reoxidised in the presence of oxygen.
Screening hAIF102 redox acceptor
NADH oxidase activity was not detected using oxygen as electron acceptor
No activity was detected using 1,4-benzoquinone, 1,2-naptoquinone or Fe3+-EDTA as electron acceptors.
kcat (s-1)
Km (µM)
kcat/Km
(s-1·mM-1)
DCPIP 1.5 ± 0.1 272.9 ± 31.3 5.5
K3Fe(CN)6 6.4 ± 0.4 1219 ± 191.6 5.2
Cytochrome c 1.3 ± 0.1 202.6 ± 37.6 6.4
Steady-state kinetic parameters of hAIF102 with different electron acceptors using NADH substrate
Similar catalytic efficiency
Low turn-over
The low affinities for the coenzyme suggest that the hAIF redox reaction might be activated by its electron acceptor under physiological conditions
Formation of very stable flavin:nicotinamide charge transfer complex (CTC).
hAIF hydride transfer mechanism
kred (s-1) Kd (µM)
NADH 1.23 ± 0.1 1260 ± 167
NADPH 0.08 ± 0.01 4848 ± 1131
Pre-steady state kinetic parameters
Kd (k-1/k1) kred (k2)
Eox+Sk1
k-1EoxS
k2Ered-P
Wavelength (nm)
400 500 600 700 800
Abs
orba
nce
0.00
0.04
0.08
0.120 sec0.3 sec6.88 sec 3.6 sec 16.71 sec 26.54 sec 36.37 sec 46.2 sec
CTC
The reduction rates were independent of the presence of molecular oxygen
NADH is the natural electron donor of hAIF
N5C4
H -
FAD
NADH
hAIF102 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.
Dimerization can modulate hAIF oxidoreductase activity.
This process was also observed for the mouse AIF (mAIF).
Gel filtration profile
Flow (mL/min)
0 5 10 15 20 25 300
40
80
120
0
40
80
120 Wildtype Wildtype + NADH
Abs
orba
nce
Dimerization can modulate hAIF oxidoreductase activity.
R448
R448
R429
R429
R421
R421
E412
E412
Crystal structure of the dimeric mAIF:NAD+ complex (pdb 3GD4)
The interactions at the dimer interface
All these residues are conserved in hAIF
E413A/R422A/R430A
Wavelength (nm)
400 500 600 700 800
Abs
orba
nce
0.00
0.02
0.04
0.06
0.08
0.10
CTC
0
40
80
120 Wildtype Wildtype + NADH
Flow (mL/min)
0 5 10 15 20 25 300
40
80
120 E413A/R422A/R430AE413A/R422A/R430A + NADH
Dimerization can modulate hAIF oxidoreductase activity.
Gel filtration profile
Abs
orba
nce
Reduction rates and affinity lower to the wild-type
Lower CTC to the wild-type
E413A/R422A/R430A variant reduction with NADH
hAIF NADH
kred (s-1) KdNADH (µM)
Wild-type 1.2 ± 0.1 1260 ± 167
Variant 0.5 ± 0.01 2260 ± 295
Studying hAIF redox active site
hAIF redox active site (pdb 1m6i)
Manual docking of NADH into the hAIF redox active site
F310G
K177W
W483G
H454S
NAD+FAD
E314S
Longitud de onda (nm)
400 500 600 700
Abs
orba
nci
a (U
.A.)
0,00
0,02
0,04
0,06
0,08
0,10
0,12
hAIFox
hAIFred-NAD+ 0.019 s
hAIFred-NAD+ 0.26 s
hAIFred-NAD+ 0.67 s
hAIFred-NAD+ 1.6 shAIFred-NAD+ 4.09 s
Wavelength (nm)
400 500 600 700 800
Abs
orba
nce
0.00
0.03
0.06
0.09 0 sec9 msec60 msec0.16 sec3 sec
W483G
F310G
Wavelength (nm)
400 500 600 700 800
Abs
orba
nce
0.00
0.04
0.08
0.120 sec8 sec11 sec17 sec21 sec25 sec
P173G
AIF variants reduction with NADH
Wavelength (nm)
400 500 600 700 800
Abs
orba
nce
0.00
0.04
0.08
0.120 sec0.3 sec6.88 sec 3.6 sec 16.71 sec 26.54 sec 36.37 sec 46.2 sec
CTC
Wild-type
hAIF Variants
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
AIF variants reduction with NADH
All variants show higher kred to the wild-type values
W483G at least 40-times
All residues are involved in AIF redox reaction
Pre-steady state kinetic parameters
(*) Experiments performed at 12 ºC
F310G and P173G lower affinity than wild-type
[NADH] mM0 2 4 6
k obs
(s-1
)
0
5
10
15
20
WTF310G
W483G
AIF variants reduction with NADH
hAIFox and rAIFred:NAD redox active site (pdb 1m6i and 3GD4)
F310
K177
W483
H454S
NAD+
FAD
E314
Wavelength (nm)
400 500 600 700 800
Abs
orba
nce
0.00
0.05
0.10
0.159 msec 0.1 sec 0.2 sec 0.4 sec 0.7 sec 1.5 sec 2 sec
H454S
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
• 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.
• Study the effect of AIF variants in the efficiency of oxidative phosphorylation in mitochondria
• Analyse the AIF oligomerization state into the cell
• Explore new acceptor as AIF redox partner
Reacción de reducción con NAD(P)H
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
hAIF1-102 + 612.5M NADH en condiciones aeróbicas
Wavelength (nm)
400 500 600 700 800
Abs
orba
nce
0.00
0.04
0.08
AIFox
AIFox-NADH 1 msec
AIFred-NAD+ 40 msec
AIFred-NAD+ 1.27 sec
AIFred-NAD+ 3.3 sec
AIFred-NAD+ 5.36 sec
Reducción anaeróbica de la hAIF con NADH
CTC
Reduccion de hAIF1-102 con 625 M NADPH
Wavelength (nm)
400 500 600 700 800
Abs
orba
nce
0.00
0.04
0.08
0.12
AIFoxAIFox-NADPH 2 msec
1.31 sec 67 sec 119 sec 250 sec
Reducción anaeróbica de la hAIF con NADPH