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Patricia Ferreira Neila

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1 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

2 “Protein interaction and electron transfer”
Group of Structural Biology Dra. Milagros Medina Dr. Carlos Gómez-Moreno Dr. Marta Martínez-Júlvez Dra. Patricia Ferreira Thanks Dr. Susin, Dra. M. Luisa Peleato and Dra. M. Dolores Miramar for giving us the cDNA of hAIF cloned in E.coli Raquel Villanueva Ana Serrano Isaías Lans Beatriz Herguedas Sonia Arilla Ana Sánchez,

3 Apoptosis inducing Factor (AIF) AIF is a redox protein
FAD-binding domain NADH-binding C-terminal AIF oxidoreductase apoptotic hAIF crystal structure (PDB 1M6I) Lipton et al. (2002) Apoptotic insult Chromatin condensation Caspase-independent cell death AIF was discovered as the first protein that regulates caspase-independent apoptosis twenty years ago. Apoptosis-inducing factor is one of the mitochondrial proteins that are released into the cytosol during apoptosis. After that, AIF is translocated into the nucleus where cause DNA fragmentation and chromatin condensation. In addition, AIF is a flavin’dependent oxidoreductase that plays a vital and unknown role in oxidative phosphorylation and redox control. Thus, AIF seems to display a dual role in cellular death and life. AIF mature form shows three structural domains: a FAD-binding domain, a NADH-binding domain and a C-terminal-binding domain. Thus, C-terminal region is the pro-apoptotic domain, and FAD- and NADH-binding domains confer an electron transfer activity to AIF. However, The independence or linked between both functions must be clarify. AIF seems to display a dual role in cellular death and life.

4 AIF cellular localization
Kroemer et al. 2007 hAIF102 The dual role of AIF made that this protein have different cellular localization and configuration. Thus, AIF is expressed as a precursor of 67 kDa that contains a mithocondrial localization signal (MLS) in the N-terminal region. Once in mithochondrial, this precursor is processed to a mature form of 62 KDA by a proteolytic cleavage. In this configuration, AIF is located in the intermembrane space with its N-terminal portion exposed to the matrix and the C-terminal portion to the mitochondrial intermembrane space. After an apoptotic insult, AIF is cleaved at amino acid 101 by protease to yield a soluble apoptogenic (hAIF102) that is liberated into the cytosol. hAIF102 is translocated to the nucleus where provokes chromatinolysis and programmed cell dead independent of caspases. Thus, AIF seems to display a dual role in cellular death and life. MLS FAD binding NADH binding FAD binding C-terminal Anchored peptide 67 KDa 62 KDa 57 KDa

5 Vital AIF function Antioxidant defense
AIF redox activity is associated with correct behavior of the mitochondrial respiratory chain in vivo Two hypothetical models Nazanine Modjtahedi, TRENDS in Cell Biology Vol.16 No.5 May 2006 AIF as an assembly factor AIF as a maintenance factor Regarding the vital function of AIF. Cells with a deficiency of AIF are more susceptible to apoptosis induction by oxidative stress. Thus AIF participated in antioxidant defense. Moreover, AIF deficiency causes respiratory defect in complex I and III. However, It is not clear how AIF deficiency compromises oxidative phosphorilation in the cells. The imagine show hypothetical models describing the local action of AIF. AIF could be a structural component of the inner mitochondrial membrane and its redox activity could be involved in the import or assembly of the repiratory chain components. Or maybe as a maintenance factor where its redox activity might be necessary for the stability or maintenance of the repiratory chain components.

6 AIF electron transfer activity ¿Acceptor ? NADH NAD+ AIF apoptotic - H
FAD-binding domain NADH-binding C-terminal AIF oxidoreductase AIF apoptotic AIF electron transfer activity N5 C4 H - FAD NADH E-FAD E-FADH2 Oxidative half reaction Reductive half reaction NADH NAD+ ¿Acceptor ? AIF was firstly described in human and mouse. Base on amino acid sequence, both proteins share the highest homology (92%). The AIF NADH redox reaction was described at the first time in mouse protein. The protein oxidize NAD(P)H with concomitant reduction of the flavin by a hydride transfer mechanism. This activity was determined using artificial electron acceptor because the redox partner of this protein is unknown in the cellular environment.

7 In spite of the large number of studies about AIF, key questions remain to be addressed…..
With this introduction I have tried to show you that in spite of the large number of works published about AIF, key question remain to be addressed

8 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? In order to answer all these questions we start a new research project where we are studying the redox properties of hAIF. Let me show you our results. Well, we expressed and purified hAIF from e. coli. Is AIF redox activity independent or linked to the apoptotic function?

9 The hAIF102 flavin properties
Either photoreduction or sodium dithionite reduction of hAIFΔ102 produced the full reduced FAD without detection of any semiquinone intermediate. We have expressed and purified hAIF from E.coli. This is the hAIF absorption spectrum that shows the typical FAD maximum. When the protein is reduced by dithionite or phoreduced we can see a two-electron reduction process without semiquinone intermediates formation. The reduced protein was completely reoxidazed in the presence of oxygen (dashed line). That suggests AIF oxygen reactivity under experimental conditions. The photoreduced hAIF102 results completely reoxidised in the presence of oxygen.

10 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 Similar catalytic efficiency 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 Low turn-over We check different kind of redox centers as AIF electron acceptor. The experiments were performed using NADH as substrate. We do not detected NADH oxidase activity using oxygen as electron acceptor. This result suggests that, in spite of AIF show oxygen reactivity (as I previously mention), oxygen isn’t the redox partner of AIF under physiological conditions. We alos did not detected activity with oxidized iron and several quinines suggesting the proteins with a sulfo-ferric center or quinines aren’t a redox AIF partner in mitochocondria. AIF showed NADH oxidoreductase activity using Citocrome C, which has the same localization in mitochondria. However, the catalytic efficiency with Cytochrome c was similar to the detected with DCPIC ferrocianure. This together the low affinity suggests that Cytochrome c isn’t the redox partner of AIF. UP to date AIF redox partner is unknown. 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

11 hAIF hydride transfer mechanism
- FAD NADH hAIF hydride transfer mechanism Formation of very stable flavin:nicotinamide charge transfer complex (CTC). CTC Pre-steady state kinetic parameters kred (s-1) Kd (µM) NADH 1.23 ± 0.1 1260 ± 167 NADPH 0.08 ± 0.01 4848 ± 1131 Turning on to the hAIF hydride transfer mechanism hAIF102 reduction by NAD(P)H was investigated using stopped-flow. Reduction to the flavin was concomitant with the formation of very stable flavin:nicotinamide charge transfer complexes (CTC). The reduction rates were independent of the presence of molecular oxygen, confirming that oxygen is not a natural electron acceptor of hAIF. The low hAIF turnover and the formation of stable CTCs during NAD(P)H oxidation suggest a slow product dissociation that prevents protein reoxidation. The low affinities for the coenzyme suggest that the hAIF redox reaction might be activated by its electron acceptor under physiological conditions The reduction rates were independent of the presence of molecular oxygen NADH is the natural electron donor of hAIF Eox+S k1 k-1 EoxS k2 Ered-P Kd (k-1/k1) kred (k2)

12 Dimerization can modulate hAIF oxidoreductase activity.
Gel filtration profile 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. Flow (mL/min) 5 10 15 20 25 30 40 80 120 Wildtype + NADH Absorbance AIF is a monomeric protein in solution. However, we observed AIF dimerization during NADH oxidation. The molecular size of free protein and AIF:nicotinamide complex were calculated by gel filtration. This dimerization process has also observed in mouse AIF. All this observations suggest AIF redox reaction regulation, and must have some physiological relevance. This process was also observed for the mouse AIF (mAIF).

13 Dimerization can modulate hAIF oxidoreductase activity.
Crystal structure of the dimeric mAIF:NAD+ complex (pdb 3GD4) The interactions at the dimer interface R448 R429 R421 E412 The crystal structure of dimeric mouse AIF:nicotinamide complex has been recently resolved. The dimeric state of mAIF could be stabilized by salt-brigdes interaction between arginine and glutamate residues. All these residues are conserved in hAIF. We decided to construct this triple mutant. All these residues are conserved in hAIF E413A/R422A/R430A

14 Dimerization can modulate hAIF oxidoreductase activity.
E413A/R422A/R430A variant reduction with NADH Gel filtration profile CTC 40 80 120 Wildtype + NADH Flow (mL/min) 5 10 15 20 25 30 E413A/R422A/R430A Lower CTC to the wild-type Absorbance This triple mutant show similar reduction rates and lower CTC stabilization than the wild-type. Regarding its molecular size was similar to wild-type in solution. However, the mutant did not dimeriza during NADH oxidation. This result confirm the role of arginine and glutamate residues in hAIF dimerization and also suggest that CTC formation is involved in dimer stabilization. hAIF NADH kred (s-1) KdNADH (µM) Wild-type 1.2 ± 0.1 1260 ± 167 Variant 0.5 ± 0.01 2260 ± 295 Reduction rates and affinity lower to the wild-type

15 Studying hAIF redox active site
Manual docking of NADH into the hAIF redox active site F310G K177W W483G H454S NAD+ FAD E314S hAIF redox active site (pdb 1m6i) On a looking at the amino acid residues located around the FAD site suggested that several residues are potentially involved in AIF redox reaction. Therefore, five potentially involved in AIF catalysis (….) were modified by site-directed mutagenesis.

16 AIF variants reduction with NADH
CTC Wild-type W483G F310G P173G Glutamic and lysine variants are not in the graph, because these variants were not enough stable to perform stopped-flow experiment and calculate its reduction rates. The reduction of W483 was similar to wild-type with the concomitant formation of a high CTC. However, the F310G and P173 variants didn’t stabilize CTC complex during NADH reduction. In fact, the AIFreduced:nicatinamide complex is re-oxidazed at low NADH concentrations. This confirms that the formation of stable CTCs during NADH oxidation prevents the protein reoxidation by slow product dissociation.

17 AIF variants reduction with NADH
Pre-steady state kinetic parameters All residues are involved in AIF redox reaction 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 All variants show higher kred to the wild-type values W483G at least 40-times (*) Experiments performed at 12 ºC As you can see in this table, all variants had shown higher reduction constant values to the wild-type. Kd values were also accepted. These results confirm the role of mutated residues in AIF redox reaction. Regarding to the NADH affinity we can observed two different behaviors in these variant. In the case of the W483 variant shown higher Kd to the wild-type. By the constrast, P173G and F310G variants showed lower affinity to the wild-type. In fact, P173G did not form enzyme-substrate complex during redox reaction. F310G and P173G lower affinity than wild-type

18 AIF variants reduction with NADH
H454S hAIFox and rAIFred:NAD redox active site (pdb 1m6i and 3GD4) F310 K177 W483 H454S NAD+ FAD E314 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

19 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.

20 Apoptosis inducing Factor (AIF)
Kroemer et al 2009

21 Cellular localization of AIF
Nazanine Modjtahedi, TRENDS in Cell Biology Vol.16 No.5 May 2006

22 Reacción de reducción con NAD(P)H
Reducción anaeróbica de la hAIF con NADPH Reducción anaeróbica de la hAIF con NADH CTC 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

23 Inna Y. Churbanova and Irina F. Sevrioukova, JBC 2008
AIF como una proteína redox de señalización Inna Y. Churbanova and Irina F. Sevrioukova, JBC 2008


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