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
China, Qingdao
266003, China )

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 China
(No. 980418)

*Corresponding author: Tel, 86-532-2032459;
Fax, 86-532-2032276; e-mail, [email protected]

Updated at: 2003-10-05