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http://www.abbs.info e-mail: [email protected] ISSN
0582-9879
ACTA BIOCHIMICA et
BIOPHYSICA SINICA 2003, 35(10): 881－885
CN 31-1300/Q |
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Mini Review |
Apoptosis-inducing Factor and Apoptosis
LÜ Cui-Xian, FAN Ting-Jun*, HU Guo-Bin, CONG Ri-Shan
( Department
Marine of Biology, College of Life Sciences and Technology, Ocean University of
Abstract Apoptosis-inducing factor (AIF) is a phylogenetically conserved mitochondrial intermembrane flavoprotein which has the ability to induce apoptosis via a caspase-independent pathway. AIF plays an important role in inducing nuclear chromatin condensation as well as large-scale DNA fragmentation (approximately 50 kb), and is essential for programmed cell death during cavitation of embryoid bodies. Two homologous proteins, AIF-homologous mitochondrion-associated inducer of death (AMID) and p53-responsive gene 3 (PRG3), also have apoptosis-inducing effects. Recent studies on mechanisms of AIF-mediated apoptotic DNA degradation in Caenorhabditis elegans reveal that WAH-1(an AIF homolog in C. elegans) induced apoptosis is CED-3-dependent. AIF also interacts with cytochrome c and caspases during mammalian apoptosis processes, indicating that different apoptotic pathways may be mutually cross-regulated to activatie an apoptotic program.
Key words apoptosis-inducing factor; AMID; PRG3; WAH-1; caspase
Specific apoptotic inducing signals received by a cell trigger the open of mitochondrial permeability transition pores (MPTP) and allow apoptosis-inducing factor (AIF) to be released from mitochondria into cytosol and subsequently transported into the nucleus[1]. Within the nucleus, AIF acts directly on nuclear DNA in cooperation with a second mitochondrial protein, endonuclease G (Endo G), which results in large-scale DNA fragmentations averaging 50 kb[2]. In addition to this caspase-independent pathway, cross-reactions and cooperations among AIF, caspase and cytochrome c in the cytosol have also been characterized[3－6]. Recent studies on the molecular structure and functions of AIF and its homologous proteins are reviewed here, as well as the interactions of AIF with cytochrome c and caspase.
1 Molecular Structure and
Homologous Proteins of AIF
1.1 Molecular structure of AIF
AIF is an ancient phylogenetically conserved flavo-protein that has both NADH oxidizing and apoptosis inducing activity. Human AIF consists of 613 amino acids, and its gene, aif, is located in Xq25-26, encoding a 2.4 kb mRNA. Mouse aif is located in XA6 and codes for a protein of 612 amino acids with 92% identity to human AIF. In general, the amino acid sequence of AIF is highly conserved in mammals with homologies of over 90%. Mouse AIF has strong homology to oxidoreductases in vertebrates, non-vertebrate animals, plants, fungi and bacteria. It contains three domains: an N-terminal mitochondrial localization sequence (MLS) of 100 amino acids, a spacer of 27 amino acids and a C-terminal 485 amino acids oxidoreductase domain (including a nuclear localization sequence, NLS)[3] (Fig.1).
Fig.1 Primary
structure of mouse AIF
The overall crystal structure of mature mouse AIF has been recently elucidated at 0.20 nm (2.0 ) resolution and resembles that of NADH oxidoreductases. AIF displays a glutathione-reductase-like folding, with an FAD-binding domain (amino acids 122－262 and 400－477), an NADH-binding domain (263－399) and a C-terminal domain (478－610) that bears a small AIF-specific insertion (509－599) not found in glutathione reductase[7]. Human mature AIF has a very similar crystal structure.
1.2 AIF homologous proteins
AIF homologous proteins include AIF-homolo-gous mitochondrion-associated inducer of death (AMID) and p53-responsive gene 3 (prg3) encoded protein. BLAST searches of the GenBank database suggested that AMID and PGR3 have identical amino acid sequences of ～373 amino acids but are encoded at two different chromosomal loci. AMID/PRG3 lack mitochondrial localization sequence (MLS) but share significant homologies with AIF and NADH oxidoreductase from bacteria to mammalian species.
The amid gene is located in human chromosome 10q22.1 and encodes a protein of ～39 kD. Double immunofluorescent staining with an anti-HA (hemagglutinin) antibody and a fluorescent dye for mitochondria reveals that the majority of AMID adheres to outer mitochondrial membrane and forms a ring-like structure. Other AMID is localized in the cytosol[8]. Northern blot analysis was unable to detect amid mRNA in all human tissues tested, however amid mRNA was detected at high levels in the colon cancer cell lines DLD and HCT116 and at low levels in B lymphoma cell line RPM18226[8].
prg3 gene is located in human chromosome 11q12 and encodes a novel protein of ～40.5 kD. PRG3 is not adhered to mitochondria, but located in the cytosol[9]. The N-terminus of PRG3 contains a signature motif for the bacterial aromatic ring hydroxylases (flavoprotein monooxygenases) which is a conserved dinucleotide-binding motif found to be associated with a ‘βαβ’ fold[9].
2 Apoptogenic Function of AIF
and Its Homologous Proteins
2.1 Apoptogenic function of AIF
AIF is endowed with the unique capacity to induce caspase-independent peripheral chromatin condensation and large-scale DNA fragmentation to approximately 50 kb when specific extracellular signals trigger the opening of mitochondrial MPTP, allowing the release of AIF and other apoptogenic effectors, such as apoptosis protease activating factor-1 (Apaf-1) and cytochrome c, both of which can activate the caspase cascade. AIF in the cytosol triggers the release of more AIF from mitochondria, forming a self-amplifying loop that accelerates apoptosis. Anti-apoptotic Bcl-2 family proteins function as gatekeepers of mitochondria to prevent the release of both cytochrome c and AIF. Bcl-2 proteins on mitochondrial membranes also are involved in the regulation of mitochondrial-nuclear redistribution of AIF[10,11].
AIF is also a NADH oxidase, by means of an FAD-containing oxidase activity which can oxidize NAD(P)H while generating superoxide anion. Apoptosis is accompanied by a general shift of the cellular redox balance characterized by a depletion of NADH, NADPH and glutathione, as well as an increase of free radicals, including superoxide anion, lipid peroxidation products (such as 4-hydroxynonenal), and oxidative damage of membranes and DNA[12]. New evidence suggests that the redox-active region of AIF may actually have an anti-apoptotic activity, while its DNA binding region is apoptotic[13]. However, independent of the presence or absence of NAD(P)H and/or FAD, the essential prosthetic group of the oxidoreductase, AIF can induce apoptosis when transported into the nucleus[2]. A recent report provides new insights into mechanisms of neurodegeneration which may involve down-regulation of AIF. In a mouse mutant with progressive cerebellar and retinal degeneration, the expression of AIF was found to be down-regulated. This was correlated, paradoxically, with apoptosis of neurons associated with an imbalance in free radical metabolism and cell cycle re-entry[14].
Poly(ADP-ribose) polymerase-1 (PARP-1) protects the genome from damage by playing roles in the DNA damage surveillance network. Massive PARP-1 activation will result in energy failure and finally cell death by depleting cellular NAD+ and ATP.
At lower levels, activated PARP-1 triggers AIF release from mitochondria, and the released AIF, which is necessary for PARP-1-dependent apoptosis, also triggers the release of cytochrome c from mitochondria and subsequent caspase activation[15,16]. That AIF is an important mediator of apoptosis has been confirmed in genetic studies: AIF-deficient mouse embryonic stem cells are resistant to serum deprivation-induced apoptosis and the first wave of apoptosis during embryogenesis is defective in AIF-deficient mouse embryos[17]. Structure-based mutagenesis showed that DNA-binding-defective mutants of AIF, obtained by replacing its positively charged residues by alanines, failed to induce cell death[2]. The potential DNA-binding site identified from mutagenesis coincides with the computationally predicted site for docking of a DNA duplex to the AIF protein. Therefore, the DNA binding ability of AIF is required for its apoptogenic function, at least at the nuclear level.
2.2 Apoptogenic function of AMID and
PRG3
AMID induces chromatin condensation and accumulation at the periphery of nucleus, which is similar to that induced by AIF but distinguishable from most caspase-dependent apoptosis. AMID-induced apoptosis, independent of caspase and p53 activity, will not be inhibited by Bcl-2. The exact mechanism by which AMID induces apoptosis is still unknown[8].
The ectopic expression of PRG3 can also induce apoptosis. The expression of prg3 gene in cells undergoing p53-dependent apoptosis involves direct activation of its promoter by p53. The process of p53-dependent apoptosis requires mitochondria-dependent apoptotic machinery, including Apaf-1, caspase-9, and the released cytochrome c from mitochondria as well, and is therefore caspase-dependent[9].
3 WAH-1 Mediated Apoptosis in C.
elegans
As we know, the DNA binding ability of AIF is necessary for its apoptogenic function, but how AIF induces chromatin condensation and DNA fragmentation still remains a conundrum. Recently, some clues have been found in C. elegans. By searching the C. elegans genome database with the human AIF sequence, researchers identified a C51G7.5 sequence that was designated wah-1. The predicted 700-amino acid long protein, obtained by its full-length cDNA clone and reverse transcription-polymerase chain reaction, shows 37% sequence identity and 54% sequence similarity to the human AIF[18].
In mammals, BH3-only proteins, such as Bid and Bim, can induce the release of mitochondrial apoptogenic factors in response to apoptotic signals. The C. elegans BH3 protein EGL-1 is thought to be the most upstream cell death activator which can receive and integrate apoptotic stimuli. As a result of EGL-1 induction, WAH-1 is released from mitochondria and translocated into nucleus, resulting in a mitochondria cell death pathway. Thus C. elegans probably employs an apoptotic pathway similar to that found in mammals[18]. The fragmentation of chromosomal DNA involves a mitochondrial endonuclease G (Endo G) in mammals and its ortholog, CPS-6, a mitochondrial protein in C. elegans. WAH-1 interacts with CPS-6 to promote DNA degradation. In mammals, AIF appears to mediate a caspase-independent cell death pathway, but in worms, CED-3 (a caspase homologue) activity is important for the proper release of WAH-1 during apoptosis.
WAH-1 localizes inside mitochondria in C. elegans and is released into cytosol and in turn into the nucleus in the presence of EGL-1 in a CED-3-dependent manner. In addition, WAH-1 promoted DNA degradation and apoptosis are achieved via the association and cooperation of WAH-1 with mitochondrial endonuclease CPS-6/Endo G. Thus, AIF and Endo G define a single, mitochondria-initiated apoptotic DNA degradation pathway that is conserved between C. elegans and mammals[18].
4 Interactions
of AIF, Cytochrome c and Caspases
As discussed, the apoptosis inducing activity of WAH-1 depends on CED-3, a caspase homologue in C. elegans. In mammals AIF is a death-inducing factor independent of caspases, but there is crosstalk between AIF and the caspase cascade at several levels. Different mitochondrial proteins involved play key roles in controlling cell death pathways. Among these, Bcl-2 family proteins, together with different levels of the heat shock protein, Hsp70, participate in regulating different modes of mitochondrial membrane permeabilization (MMP), resulting in crosstalk among AIF, cytochrome c and caspases. AIF can trigger the release of cytochrome c from isolated mitochondria in vitro. In several paradigms of cell death induction, AIF is released from mitochondria before cytochrome c, and neutralization of AIF (by microinjection of an antibody or by knock out) can prevent cell death, as well as the release of cytochrome c from mitochondria[2]. So in some cases at least, AIF is required for cytochrome c-dependent caspase activation cascade.
Cytochrome c was the first characterized mitochondrial factor released from mitochondrial intermembrane space and implicated actively in apoptotic cell death. Other mitochondrial proteins, such as AIF, Smac/DIABLO, endonuclease G and Omi/HtrA2, were also found to be released from mitochondria during apoptosis and involved in various aspects of cell death process[19]. When caspase activation occurs early in apoptosis, for instance in CD95-triggered cell death, AIF is released following caspase 8 activation. Similarly, in ectoposide-induced apoptosis, caspase 2 activation occurs upstream of MMP and therefore likely upstream of AIF release. Activated caspase and the caspase-activated protein t-Bid can trigger AIF release from purified mitochondria[2]. Bax, which induces outer mitochondrial membrane permeability, induces the release of cytochrome c, but not AIF in isolated mitochondria. The association of AIF with the mitochondrial inner membrane provides a simple explanation for its lack of release upon Bax-mediated outer membrane permeabilization[20].
Heat-shock protein 70 (Hsp70) has been reported to block apoptosis by binding apoptosis protease activating factor-1 (Apaf-1), thereby preventing constitution of the apoptosome, the Apaf-1/ cytochrome c / caspase-9 activation complex[21]. According to Ravagnan et al.[22], Hsp70 prevented the AIF-induced chromatin condensation of purified nuclei in a cell-free system by specific interaction with AIF, as shown by ligand blots and co-immunoprecipitation. Cells overexpressing Hsp70 were protected against the apoptogenic effects of AIF targeted to the extramitochondrial compartment.
It appears that simultaneous neutralization of caspases and AIF is required to prevent apoptosis induced by menadione in embryonic stem cells (ES cells) as well as to prevent p53-dependent death of cortical neurons[2].
All these findings indicate AIF and caspases may thus cooperate in cell death cascade, and their contributions may depend on specific apoptosis-inducing stimuli and perhaps cell types[2]. Members of the Bcl-2 protein family control the integrity of mitochondrial outer membrane and mitochondrial response to death signals, but the molecular mechanism by which mitochondrial intermembrane space proteins are released and the regulation of mitochondrial homeostasis by Bcl-2 proteins are still elusive[20]. In the process of apoptosis, the interactions and cooperations among many different signal pathways compose a complicated network regulated by various types of signals (Fig.2).
Fig.2 Two pathways involved in the occurrence of apoptotic nuclear morphology
Left, caspase-independent apoptotic pathway mediated by mitochondrion released AIF. AIF plays three important roles in apoptosis: one is as an autonomous effector, the second as a cytosolic effector, the third as a nuclear effector. Right, caspase-dependent apoptotic pathway mediated by caspases and/or mitochondrion released cytochrome c (Cyt. c).
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Received: April 30, 2003 Accepted: July 21, 2003
This work was supported by a grant from the
Imbursement Project for Studied Abroad Returnees from Ministry of Education of
*Corresponding author: Tel, 86-532-2032459; Fax, 86-532-2032276; e-mail, [email protected]
Updated at: 2003-10-05