Presentation on theme: "Patricia Ferreira Neila Departamento de Bioquímica y Biología Molecular y Celular Instituto de Biocomputación y Física de Sistemas Complejos Universidad."— Presentation transcript:
Patricia Ferreira Neila Departamento de Bioquímica y Biología Molecular y Celular Instituto 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 Medina Dr. Carlos Gómez-Moreno Dr. Marta Martínez-Júlvez Dra. Patricia Ferreira Raquel Villanueva Ana Serrano Isaías Lans Beatriz Herguedas Sonia Arilla Ana 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 condensation Caspase-independent cell death AIF seems to display a dual role in cellular death and life. FAD-binding domain NADH-binding domain C-terminal AIF oxidoreductase AIF apoptotic hAIF crystal structure (PDB 1M6I) AIF is a redox protein
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 factorAIF as a maintenance factor Vital AIF function Two hypothetical models Antioxidant defense
FAD-binding domain NADH-binding domain C-terminal AIF oxidoreductase AIF apoptotic E-FADE-FADH 2 Oxidative half reaction Reductive half reaction NADH NAD + ¿Acceptor ? AIF electron transfer activity N5 C4 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 hAIF 102 flavin properties Either photoreduction or sodium dithionite reduction of hAIF Δ102 produced the full reduced FAD without detection of any semiquinone intermediate. 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 No activity was detected using 1,4-benzoquinone, 1,2-naptoquinone or Fe 3+ - EDTA as electron acceptors. k cat (s -1 ) K m (µM) k cat /K m (s -1 ·mM -1 ) DCPIP 1.5 ± ± K 3 Fe(CN) ± ± Cytochrome c 1.3 ± ± Steady-state kinetic parameters of hAIF 102 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 k red (s-1)K d (µM) NADH1.23 ± ± 167 NADPH0.08 ± ± 1131 Pre-steady state kinetic parameters K d (k -1 /k 1 )k red (k 2 ) E ox +S k1k1 k -1 E ox S k2k2 E red -P CTC The reduction rates were independent of the presence of molecular oxygen NADH is the natural electron donor of hAIF N5 C4 H - FAD NADH
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. Dimerization can modulate hAIF oxidoreductase activity. This process was also observed for the mouse AIF (mAIF). Gel filtration profile Absorbance
Dimerization can modulate hAIF oxidoreductase activity. R448 R429 R421 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
CTC Dimerization can modulate hAIF oxidoreductase activity. Gel filtration profile Absorbance Reduction rates and affinity lower to the wild-type Lower CTC to the wild-type E413A/R422A/R430A variant reduction with NADH hAIF NADH k red (s -1 )K d NADH (µM) Wild-type 1.2 ± ± 167 Variant 0.5 ± ± 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
hAIF Variants NADH k red (s -1 ) K d NADH (µM) Wild-type 1.2 ± ± 167 W483G (*) 39.4 ± 1245 ± 26 F310G 17.3 ± ± 89 P173G 4.7 ± ± 1548 AIF variants reduction with NADH All variants show higher k red 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
AIF variants reduction with NADH hAIF ox and rAIF red :NAD redox active site (pdb 1m6i and 3GD4) F310 K177 W483 H454S NAD + FAD E314 H454S No CTC formation hAIF Variants NADH k red (s -1 )K d NADH (µM) Wild-type 1.2 ± ± 167 H454S 3.7 ± 12743± 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 AIF ox -NADH se evidencia en los ensayos con un incremento del espectro de la proteína Reducción anaeróbica de la hAIF con NADH CTC Reducción anaeróbica de la hAIF con NADPH
AIF como una proteína redox de señalización Inna Y. Churbanova and Irina F. Sevrioukova, JBC 2008