Presentation on theme: "Patricia Ferreira Neila"— Presentation transcript:
1 Unraveling the mitochondrial role of the human apoptosis inducing factor (hAIF) Patricia Ferreira NeilaDepartamento de Bioquímica y Biología Molecular y CelularInstituto de Biocomputación y Física de Sistemas ComplejosUniversidad de ZaragozaBIFI2011: V National Conference
2 “Protein interaction and electron transfer” Group of Structural BiologyDra. Milagros MedinaDr. Carlos Gómez-MorenoDr. Marta Martínez-JúlvezDra. Patricia FerreiraThanks Dr. Susin, Dra. M. Luisa Peleato and Dra. M. Dolores Miramar for giving us the cDNA of hAIF cloned in E.coliRaquel VillanuevaAna SerranoIsaías LansBeatriz HerguedasSonia ArillaAna Sánchez,
3 Apoptosis inducing Factor (AIF) AIF is a redox protein FAD-bindingdomainNADH-bindingC-terminalAIFoxidoreductaseapoptotichAIF crystal structure(PDB 1M6I)Lipton et al. (2002)Apoptotic insultChromatin condensationCaspase-independent cell deathAIF 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. 2007hAIF102The 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 (hAIF102) that is liberated into the cytosol. hAIF102 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.MLSFAD bindingNADH bindingFADbindingC-terminalAnchoredpeptide67 KDa62 KDa57 KDa
5 Vital AIF function Antioxidant defense AIF redox activity is associated with correct behavior of themitochondrial respiratory chain in vivoTwo hypothetical modelsNazanine Modjtahedi, TRENDS in Cell Biology Vol.16 No.5 May 2006AIF as an assembly factorAIF as a maintenance factorRegarding 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-bindingdomainNADH-bindingC-terminalAIF oxidoreductaseAIFapoptoticAIF electrontransfer activityN5C4H-FADNADHE-FADE-FADH2Oxidative half reactionReductive half reactionNADHNAD+¿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 cellularenvironment?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 hAIF102 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 hAIF102 results completely reoxidised in the presence of oxygen.
10 Screening hAIF102 redox acceptor NADH oxidase activity was not detected using oxygen as electron acceptorSteady-state kinetic parameters of hAIF102 with different electron acceptors using NADH substrateSimilar catalytic efficiencykcat(s-1)Km(µM)kcat/Km(s-1·mM-1)DCPIP1.5 ± 0.1272.9 ± 31.35.5K3Fe(CN)66.4 ± 0.41219 ± 191.65.2Cytochrome c1.3 ± 0.1202.6 ± 37.66.4Low turn-overWe 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 -FADNADHhAIF hydride transfer mechanismFormation of very stable flavin:nicotinamide charge transfer complex (CTC).CTCPre-steady state kinetic parameterskred (s-1)Kd (µM)NADH1.23 ± 0.11260 ± 167NADPH0.08 ± 0.014848 ± 1131Turning on to the hAIF hydride transfer mechanismhAIF102 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 conditionsThe reduction rates were independent of the presence of molecular oxygenNADH is the natural electron donor of hAIFEox+Sk1k-1EoxSk2Ered-PKd (k-1/k1)kred (k2)
12 Dimerization can modulate hAIF oxidoreductase activity. Gel filtration profilehAIF102 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)510152025304080120Wildtype+ NADHAbsorbanceAIF 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 interfaceR448R429R421E412The 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 hAIFE413A/R422A/R430A
14 Dimerization can modulate hAIF oxidoreductase activity. E413A/R422A/R430A variant reduction with NADHGel filtration profileCTC4080120Wildtype+ NADHFlow (mL/min)51015202530E413A/R422A/R430ALower CTC tothe wild-typeAbsorbanceThis 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.hAIFNADHkred (s-1)KdNADH (µM)Wild-type1.2 ± 0.11260 ± 167Variant0.5 ± 0.012260 ± 295Reduction rates and affinity lower to the wild-type
15 Studying hAIF redox active site Manual docking of NADH into the hAIF redox active siteF310GK177WW483GH454SNAD+FADE314ShAIF 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 CTCWild-typeW483GF310GP173GGlutamic 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 parametersAll residues are involved in AIF redox reactionhAIFVariantsNADHkred(s-1)KdNADH(µM)Wild-type1.2 ± 0.11260 ± 167W483G(*)39.4 ± 1245 ± 26F310G17.3 ± 15585 ± 89P173G4.7 ± 0.311932 ± 1548All variants show higher kred to the wild-type valuesW483G at least 40-times(*) Experiments performed at 12 ºCAs 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 thanwild-type
18 AIF variants reduction with NADH H454ShAIFox and rAIFred:NAD redox active site(pdb 1m6i and 3GD4)F310K177W483H454SNAD+FADE314No CTC formationhAIFVariantsNADHkred (s-1)KdNADH (µM)Wild-type1.2 ± 0.11260 ± 167H454S3.7 ± 12743± 295
19 Future work Explore new acceptor as AIF redox partner Analyse the AIF oligomerization state into the cellStudy the effect of AIF variants in the efficiency of oxidative phosphorylation in mitochondriaStudy 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 NADPHReducción anaeróbica de la hAIF con NADHCTCReducción completa de la flavina mediada por dos electronesSimilares espectros de reducción con NAD(P)H en presencia y ausencia de oxigenoFormación de complejos de transferencia de carga altamente establesLa 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 señalizaciónInna Y. Churbanova and Irina F. Sevrioukova, JBC 2008