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

In spite of the large number of studies about AIF, key questions remain to be addressed…..

• 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

Abs

orba

nce

0.00

0.08

0.16

0.24 hAIF102 reoxidasedhAIF102 intermediatehAIF102 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 5.5K3Fe(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 30

0

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 30

0

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 NADHkred (s-1) Kd

NADH (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

Abso

rban

cia (U

.A.)

0,00

0,02

0,04

0,06

0,08

0,10

0,12hAIFoxhAIFred-NAD+ 0.019 shAIFred-NAD+ 0.26 shAIFred-NAD+ 0.67 shAIFred-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

NADHkred (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 formationhAIF

VariantsNADH

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

Apoptosis inducing Factor (AIF)

Kroemer et al 2009

Nazanine Modjtahedi, TRENDS in Cell Biology Vol.16 No.5 May 2006

Cellular localization of AIF

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 msec1.31 sec 67 sec 119 sec 250 sec

Reducción anaeróbica de la hAIF con NADPH

AIF como una proteína redox de señalización

Inna Y. Churbanova and Irina F. Sevrioukova, JBC 2008