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From CellBiology

Individual Project

Apoptosis Inducing Factor (AIF)

What is AIF?

  • AIF was originally discovered to be a positive intrinsic regulator of apoptosis(Cited in Feraud et al, 2006).
  • It is a mitochondrial oxidoreductase, an electron donar/acceptor, and has recently been shown to have anti-apoptotic function (Krantic et al, 2007).
  • It is flavoprotein because it is able to be stably bound to FAD (flavin adenine dinucleotide) (Cande et al, 2002).
  • The AIF gene has been conserved throughout the eukaryotes (Cande et al 2002).
  • There are thought to be three isoforms; AIF, AIF-exB (different exon), and AIFsh (short) (Cande et al, 2002).

Topology and Gene location

Crystal structure:

Fig. 1.Crystal structure of AIF, clearly showing the three domains.

AIF consists of a three domains,

  • Mitochondrial localization sequence (MLS).
  • Spacer sequence.
  • Oxidoreductase domain (Cande et al, 2002).

Gene location:

  • Xq25-q26. The gene which encodes for AIF is located on the X chromosome and is on the long arm at position 25-26.

How it works

  • Cell Death -The release of AIF can trigger a caspase independent pathway via which apoptosis is induced. AIF in this pathway can induce nuclear apoptosis by contributing to the fragmentation of DNA and chromatin condensation (Millan et al, 2007).
  • Cell survival- As its name suggests apoptosis inducing factor has primarily been associated with its apoptosis inducing capabilities. It does however have a cell survival function as well. AIF has been shown to be a free radical scavengers and oxidative stress can occur from too many free radicals (Klein et al, 2002).

Cell Death

Arriving in the mitochondria

In the nucleus AIF is transcribed from the X chromosome at Xq25-q26. It is translated to the cytosol of the cell where if forms a precursor protein, still containing the mitochondrial localization sequence (MLS) domain (Fig.2.) (Modjtahedi et al, 2006). The AIF resides within the mitochondrion, and is translocated to the inner membranous region of the organelle via its N-terminus. Here the AIF is responsible for metabolite redox and vital bioenergetics within the mitochondria (Modjtahedi et al, 2006). It remains within the mitochondria until sufficient stimulus has altered the mitochondrial outer membrane. Once the membrane becomes permeable, the protein is transformed into its mature form. AIF becomes truncated, as it loses its MLS N-terminus domain. AIF is now able to be translted of back into the cytosol via its C-terminus.

Fig. 2. Schematic of the structural chnages of AIF.

Getting out of the Mitochondria

The exact mechanism by which the outer membrane of the mitochondria becomes more permeable to AIF is not fully understood. It has been suggested that death stimuli, such as oxidative stress and DNA damage begin the cascade. Two mechanisms have been hypothesised for which AIF translocation from the mitochondria is induced by death stimuli (Vosler et al, 2009). Recent studies by Volser et al (2009) show that a calcium dependent cystien protease calprins and a calcium indepenten cathespins are the triggers of AIF release from the mitochondria be cleaving AIF off the inner membrane making it soluble (Wang et al., 2004; Culmsee et al., 2005; Moubarak et al., 2007). This study showed that calprin activity is dependent on that of PARP-1. PARP-1 is said to become activated within the nuclease and down regulates calprin within the cytosol. Calprin in turn triggers Bax on the outer membrane of the mitochondria (Moubarak et al., 2007). Bax activation increases mitochondrial membrane permeability to AIF. This downstream regulation has been shown to be modulated by calcium levels within the mitochondria however this exact mechanism is not known (Vosler et al, 2009).

In the cytosol

Once in the cytosol AIF translocates to the nucleus. It should be emphasised that many of these AIF pathways are speculations. One hypothesis made by Daugas et al (2000) suggested that this translocation may be due to ATP depletion or NAD+ depletion. Heat shock protein 70 family (Hsp70) can inhibit this action.

To the nucleus

Fig 3. Immunostaining of the colocalization of DNA and AIF

Once the AIF is within the nucleus it has been shown to bind to a endonuclease cyclophilians A (Cyp A). The AIF and the Cyp A bind to DNA, where the formation of a trimolecular complex occurs (Fig. 4). This attraction could be as a result of their electrostatic attraction, AIF being positively charged and DNA negatively (Millan et al, 2009). This interaction allows for the degeneration of the DNA (Modjtahedi et al, 2006). Images from Ye et al (2002) immunostaining showed colocalization of AIF with DNA during the first stage of chromatin condensation (see Fig.3). The process converts chromatin to high molecular weight segments (Bajti et al, 2006). This idea that AIF binding to DNA is significant for cell death has been shown in AIF mutant mice. Studies have indicated that AIF mutant mice are defective in DNA binding and are consequently defective at inducing cell death (Klein et al, 2002). The overall mechanism of DNA degeneration via AIF has not been fully uncovered. Like many of the other processes, ideas of their workings have been hypothesised. One idea is that the AIF binding to DNA recruits the enzymes proteases and nucleases – inducing the chromatin condensation (Hong et al, 2004). Another idea is that the binding of AIF to DNA may displace specific chromatin-associated proteins, disrupting the normal structure and leading to a collapse of the DNA (Ye et al, 2002).

Fig. 4. Schematic drawing of the pathways involved in AIF induced apoptosis.

Cell survival

It has been hypothesised that AIF also has anti-apoptotic properties (Lipton et al, 2002). The oxidoreductase domain on AIF (see Fig. 2.) shares unique similarities to the hydrogen peroxide scavengers found in bacteria (Porter et al, 2006). The function of AIF has been research in vivo, being well documented in AIF knockout mice (Klein et al, 2002). This study by Klein et al (2002) demonstrated that without AIF expression, oxidative stress occurred as a result of over expression of free radicals. Subsequent effects of an increase in oxidative stress were shown to lead to neuronal degeneration. These findings suggest that his region has the ability to influence the cells sensitivity to free radicals and consequently the cell's life (Klein et al, 2002).

Current studies

AIF and Diabetes in Animal models

A recent study by Schulthess et al (2009) demonstrated the importance of AIF in maintaining beta cell mass in mice. It is this decline of beta cell mass which is a major contributor to diabetes within humans. Harlequin (Hq) mutant mice have been used in previous studies to demonstrate the role of AIF in scavenging for free radicals to prevent apoptosis (Modjtahedi et al, 2006). Hq mutant mice had a provirus inserted into the gene which resulted in 80% less AIF production. As a consequence these mice showed evidence of a significant reduction in oxidative phosphorylation (OXPHOS) and subsequently a reduction of ATP. The effect of reduced OXPHOS and mitochondrial function has been shown to influence insulin resistance (Schulthess et al, 2009). In this current study by Schulthess et al (2009) the depletion of AIF in mice showed a significant decrease of beta cell mass and a continual decline with age. It was shown that the proliferation of the beta cells did not reduce, but it was the cell cycle that was disrupted. It was hypothesised that the G2 phase of the cell’s development was interrupted by the presence of oxidative stress. It could then be hypothesised that it is the AIF which scavenges for oxidants. A G2 check point is triggered by the oxidative stress and this severe disruption induces apoptosis. The effect of a depletion of AIF could be a significant factor in beta cell mass loss.

AIF in Colon Cancer

It is known that some cellular changes that inhibit apoptosis can induce the development of abnormal growths such as cancer. It has been observed that the blockage of the AIF signalling pathway could be implicated in chemo-resistance in some cancer types such as non small cell lung carcinoma. Unlike these cancers, colon cancer has shown to have a different interaction with AIF. AIF has been seen to reduce chemical induced apoptosis and it sustains the abnormal formation of the malignant cell. Although this concept has some unsolved issues, it has been shown by Urbano et al (2009) that AIF deficient cells were excessively susceptible to apoptosis. As such AIF could, in the future, be a potential target for cancer drug therapy. This enables a potential drug alternative to those patients whom have developed drug resistance to regular chemoradiotherapeutic intervention.

Terms

  • oxidoreductase: An enzymes which is known to dehydrogenases or oxidase, catalyzing the removal of hydrogen atoms and electrons from the substance.
  • free radical: a highly unstable and reactive atom (or group of atoms), due to at least one unpaired electron.
  • redox: stands for reducation-oxidation reaction,that results in a change of oxidation state (and number). Oxidation is the gain of and electron and reduction is the lose of an electron
  • bioenergetics: the stuy of the flow of energy through a living thing.
  • PARP-1: Poly (ADP-ribose) polymerase family, member 1.
  • calprins: Ca2+-dependent protease.
  • cathepsin: proteolytic enzymes that reside in endolysosomal vesicles.
  • ATP: adenosine triphosphate, transports chemical energy within cells for metabolism.
  • NAD+: Nicotinamide adenine dinucleotide,is involved in redox reactions, transporting electrons.
  • immunostaining:method to detect a specific protein in a sample using antibody-based techniques.
  • chromatin:the loosley coiled form of DNA and associated protiens within the cell.
  • Oxidative phosphorylation: the synthesis of ATP by phosphorylation of ADP, where energy is attained from the transport of electrons.this process takes place in mitochondria during aerobic respiration.

Reference

  • Candé C, Cohen I, Daugas E, Ravagnan L, Larochette N, Zamzami N, Kroemer G (2002) Apoptosis-inducing factor (AIF): a novel caspase-independent death effector released from mitochondria. Biochimie. 84(2-3):215-22.
  • Candé C, Vahsen N, Kouranti I, Schmitt E, Daugas E, Spahr C, Luban J, Kroemer RT, Giordanetto F, Garrido C, Penninger JM, Kroemer G (2004) AIF and cyclophilin A cooperate in apoptosis-associated chromatinolysis. Oncogene. 26;23(8):1514-21.
  • Feraud O, Debili N, Penninger PN and Kroemer G (2006) Cavitation of embryoid bodies requires optimal oxidative phosphorylation and AIF Cell Death and Differentiation.14; 385–387. doi:10.1038/sj.cdd.4402041
  • Hong SJ, Dawson TM, Dawson VL (2004) Nuclear and mitochondrial conversations in cell death: PARP-1 and AIF signaling. Trends Pharmacol Sci. 25(5):259-64.
  • Joza N, Susin SA, Daugas E, Stanford WL, Cho SK, Li CY, Sasaki T, Elia AJ, Cheng HY, Ravagnan L, Ferri KF, Zamzami N, Wakeham A, Hakem R, Yoshida H, Kong YY, Mak TW, Zúñiga-Pflücker JC, Kroemer G, Penninger JM.(2000)Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death.Nature.410(6828):549-54
  • Krantic S, Mechawar N, Reix S, Quirion R (2007) Apoptosis-inducing factor: a matter of neuron life and death. Prog Neurobiol.81(3):179-96.
  • Lorenzo HK, Susin SA, Penninger J, Kroemer G.(1999). Apoptosis inducing factor (AIF): a phylogenetically old, caspase-independent effector of cell death.Cell Death Differ.6(6):516-24.
  • Millan A, Huerta S (2009) Apoptosis-inducing factor and colon cancer. J Surg Res. 151(1):163-70. Epub 2007 Dec 3
  • Modjtahedi N, Giordanetto F, Madeo F, Kroemer G (2006) Apoptosis-inducing factor: vital and lethal. Trends Cell Biol. 16(5):264-72. Epub 2006 Apr 18.
  • Moubarak RS, Yuste VJ, Artus C, Bouharrour A, Greer PA, Menissier-de Murcia J, Susin SA (2007) Sequential Activation of Poly(ADP-Ribose) Polymerase 1, Calpains and Bax Is Essential in Apoptosis-Inducing Factor-Mediated Programmed Necrosis. Mol Cell Biol. 27(13):4844-62.
  • Porter AG, Urbano AGL. (2006) Does apoptosis-inducing factor (AIF) have both life and death functions in cells?. BioEssays 28:834-843.
  • Vosler PS, Sun D, Wang S, Gao Y, Kintner DB, Signore AP, Cao G, Chen J (2009) Calcium Dysregulation Induces Apoptosis-inducing Factor Release: Cross-talk Between PARP-1-and Calpain-Signaling Pathways. Exp Neurol. doi:10.1016/j.expneurol.2009.04.032
  • Ye H, Cande C, Stephanou NC, Jiang S, Gurbuxani S, Larochette N, Daugas E, Garrido C, Kroemer G, Wu H (2002) DNA binding is required for the apoptogenic action of apoptosis inducing factor. Nat Struct Biol.9(9):680-4.