HGNC Approved Gene Symbol: APAF1
Cytogenetic location: 12q23.1 Genomic coordinates (GRCh38): 12:98,645,290-98,735,433 (from NCBI)
APAF1 plays an essential role in apoptosis. In the presence of cytochrome c (123970) and dATP, APAF1 assembles into an oligomeric apoptosome, which is responsible for activation of procaspase-9 (CASP9; 602234) and maintenance of the enzymatic activity of processed caspase-9 (Bao et al., 2007).
From HeLa cell cytosol, Zou et al. (1997) purified and cloned a cDNA of APAF1, a 130-kD protein that participates in the cytochrome c-dependent activation of caspase-3 (CASP3; 600636). The NH2-terminal 85 amino acids of APAF1 show 21% identity to the NH2-terminal prodomain of the C. elegans caspase CED3. The next 320 amino acids show 22% identity to CED4, a protein that is believed to initiate apoptosis in C. elegans. The COOH-terminal region of APAF1 comprises multiple WD repeats, which are proposed to mediate protein-protein interactions. Cytochrome c binds to APAF1, an event that may trigger the activation of CASP3, leading to apoptosis.
In an RT-PCR screen of various normal and tumor cell lines and tissues, Hahn et al. (1999) identified 3 variants of APAF1. These forms varied in length, but sequence analysis indicated that each contained all of the functional domains previously characterized by Zou et al. (1997).
Activation of procaspase-9 by APAF1 in the cytochrome c/dATP-dependent pathway requires proteolytic cleavage to generate the mature caspase molecule. Srinivasula et al. (1998) showed that deletion of the APAF1 WD40 repeats makes APAF1 constitutively active and capable of processing procaspase-9 independent of cytochrome c and dATP. APAF1-mediated processing of procaspase-9 occurs at asp315 by an intrinsic autocatalytic activity of procaspase-9 itself. Srinivasula et al. (1998) provided evidence that APAF1 can form oligomers and may facilitate procaspase-9 autoactivation by oligomerizing its precursor molecules. Once activated, caspase-9 can initiate a biochemical cascade involving the downstream executioners caspase-3, -6, and -7.
Metastatic melanoma is a deadly cancer that fails to respond to conventional chemotherapy. Mutations in p53 (191170) often occur in aggressive and chemoresistant cancers but are rarely observed in melanoma. Soengas et al. (2001) showed that metastatic melanomas often lose APAF1. Loss of APAF1 expression was accompanied by allelic loss in metastatic melanomas, but could be recovered in melanoma cell lines by treatment with the methylation inhibitor 5-aza-2-prime-deoxycytidine (5aza2dC). APAF1-negative melanomas were invariably chemoresistant and were unable to execute a typical apoptotic program in response to p53 activation. Restoring physiologic levels of APAF1 through gene transfer or 5aza2dC treatment markedly enhanced chemosensitivity and rescued the apoptotic defects associated with APAF1 loss. Soengas et al. (2001) concluded that APAF1 is inactivated in metastatic melanomas, leading to defects in the execution of apoptotic cell death.
Robles et al. (2001) identified a classic p53-responsive element upstream of the APAF1 transcription start site that bound p53 and induced APAF1 gene expression. Apoptosis in a lymphoblastoid cell line, caused by DNA damage due to exposure to ionizing radiation or to doxorubicin, induced APAF1 mRNA and protein expression and was strictly dependent on wildtype p53 function. Robles et al. (2001) concluded that APAF1 is an essential downstream effector of p53-mediated apoptosis.
Fortin et al. (2001) identified 2 p53 consensus binding sites in the mouse Apaf1 promoter. By electrophoretic mobility shift assays and transient transfections of Apaf1 promoter reporter constructs into mouse neuronal cells, they demonstrated that both sites were utilized by p53. Primary cultures of Apaf1-deficient neurons were significantly protected from p53-induced apoptosis.
Furukawa et al. (2002) identified an E2F1 (189971)-binding element within the promoter region of human APAF1 and confirmed binding in a chromatin immunoprecipitation assay. They found that E2F1-induced apoptosis was accompanied by caspase-9 activation and enhanced expression of APAF1 without the cytosolic accumulation of cytochrome c. Overexpression of APAF1 resulted in direct activation of caspase-9 without mitochondrial damage and initiated a caspase cascade.
Marsden et al. (2002) established that the cell death pathway controlled by BCL2 (151430) does not require caspase-9 or its activator APAF1. In keeping with their evidence that neither is required for hematopoietic homeostasis, in which the BCL2 family has major roles, deletion of thymocytes with self-reactivity depends on BIM (603827) but not on APAF1. Because apoptosis was at most slightly delayed by the absence of APAF1 or caspase-9, Marsden et al. (2002) concluded that the apoptosome is not an essential trigger for apoptosis but is rather a machine for amplifying the caspase cascade. They found that BCL2 overexpression increased lymphocyte numbers in mice and inhibited many apoptotic stimuli, but the absence of APAF1 and caspase-9 did not. Caspase activity was still discernible in cells lacking APAF1 or caspase-9 and a potent caspase antagonist both inhibited apoptosis and retarded cytochrome c release. Marsden et al. (2002) concluded that BCL2 regulates a caspase activation program independently of the cytochrome c/APAF1/caspase-9 apoptosome, which seems to amplify rather than initiate the caspase cascade.
Bao et al. (2007) found that physiologic concentrations of calcium ion negatively affect the assembly of apoptosome by inhibiting nucleotide exchange in the monomeric autoinhibited APAF1 protein. Consequently, calcium blocked the ability of APAF1 to activate caspase-9. Bao et al. (2007) concluded that calcium homeostasis has an important role in the APAF1-dependent apoptotic pathway.
Crystal Structure
Riedl et al. (2005) reported the 2.2-angstrom crystal structure of an ADP-bound, WD40-deleted APAF1, which revealed the molecular mechanism By which APAF1 exists in an inactive state before ATP binding. The N-terminal caspase recruitment domain packs against a 3-layered alpha/beta fold, a short helical motif, and a winged-helix domain, resulting in the burial of the CASP9 (602234)-binding interface. The deeply buried ADP molecule serves as an organizing center to strengthen interactions between these 4 adjoining domains, thus locking APAF1 in an inactive conformation. APAF1 binds to and hydrolyzes ATP/dATP and their analogs. Riedl et al. (2005) concluded that the binding and hydrolysis of nucleotides seem to drive conformational changes that are essential for the formation of the apoptosome and the activation of CASP9.
By fluorescence in situ hybridization, Kim et al. (1999) mapped the APAF1 gene to chromosome 12q23.
Harlan et al. (2006) studied APAF1 variants to determine molecular phenotypes using an in vitro reconstruction of the apoptosome complex in which Apaf-1 activates caspase-9 and thus initiates a cascade of proteolytic events leading to apoptotic destruction of the cell. Cellular phenotypes were measured using a yeast heterologous expression assay. Human variants encoded by APAF1 alleles that segregate with major depression in families linked to chromosome 12q (MDD1; 608520) demonstrated a common gain-of-function phenotype in both assay systems. In contrast, other APAF1 variants showed neutral or loss-of-function phenotypes. Harlan et al. (2006) concluded that depression-associated alleles have a common phenotype that is distinct from that of nonassociated variants, suggesting an etiologic role for enhanced apoptosis in major depression.
The cytosolic protein APAF1, human homolog of C. elegans CED-4, participates in the CASP9-dependent activation of CASP3 in the general apoptotic pathway. Cecconi et al. (1998) generated by gene trap a null allele of the murine Apaf1. Homozygous mutants died at embryonic day 16.5. Their phenotype included severe craniofacial malformations, brain overgrowth, persistence of the interdigital webs, and dramatic alterations of the lens and retina. Homozygous embryonic fibroblasts exhibited reduced response to various apoptotic stimuli. In situ immunodetection showed that the absence of Apaf1 protein prevented the activation of Casp3 in vivo. In agreement with the reported function of CED-4 in C. elegans, this phenotype can be correlated with a defect of apoptosis. Cecconi et al. (1998) suggested that Apaf1 is essential for Casp3 activation in embryonic brain and is a key regulator of developmental programmed cell death in mammals.
Yoshida et al. (1998) also produced Apaf1-deficient mice which exhibited reduced apoptosis in the brain and striking craniofacial abnormalities with hyperproliferation of neuronal cells. Apaf1-deficient cells were resistant to a variety of apoptotic stimuli, and the processing of caspases-2, -3, and -8 was impaired. However, both Apaf1 -/- thymocytes and activated T lymphocytes were sensitive to Fas-induced killing, showing that Fas-mediated apoptosis in these cells is independent of Apaf1. These data indicated that Apaf1 plays a central role in the common events of mitochondria-dependent apoptosis in most death pathways and that this role is critical for normal development.
Honarpour et al. (2000) extended previous findings of perinatal mortality and exencephaly in Apaf1 -/- mice by showing that neurogenesis was aberrant at both the neural progenitor and the mature neuron stage in these mice. They noted that 5% of Apaf1 mutant mice survived to adulthood without brain pathology, but their male germ-cell development exhibited spermatogonia degeneration similar to that observed in Bax (600040)-deficient mice or mice overexpressing Bcl2 (151430) or Bclxl (BCL2L1; 600039). Honarpour et al. (2000) concluded that compensatory alternative apoptotic pathways exist in the absence of cytochrome c- and caspase 3-mediated apoptosis in brain development, but these pathways are essential for spermatogenesis.
The 'forebrain overgrowth' mutation (fog) in mice was originally described as a spontaneous autosomal recessive mutation mapping to chromosome 10 that produces forebrain defects, facial defects, and spina bifida. Because of its phenotypic resemblance to apoptotic protease activating factor-1 (Apaf1) knockout mice, Honarpour et al. (2001) investigated the possibility that the fog mutation is in the Apaf1 gene. They showed that fog mutant mice lack Apaf1 activity, and that Apaf1 mRNA is aberrantly processed, resulting in greatly reduced expression levels of normal mRNA. These findings strongly suggested that the fog mutation is a hypomorphic Apaf1 defect and implicated neural progenitor cell death in the pathogenesis of spina bifida. Because a complete deficiency in Apaf1 usually results in perinatal lethality and fog/fog homozygous mice more readily survive into adulthood, these mutants serve as a valuable model with which apoptotic cell death can be studied in vivo.
Huntington disease (HD; 143100) is caused by expansion of a polyglutamine tract near the N terminus of the huntingtin (HTT; 613004) protein. Mutant huntingtin forms aggregates in striatum and cortex, where extensive cell death occurs. Sang et al. (2005) reported that polyglutamine-induced cell death was dramatically suppressed in flies lacking Dark, the fly homolog of human APAF1. Dark appeared to play a role in the accumulation of polyglutamine-containing aggregates. Suppression of cell death, caspase activation, and aggregate formation were also observed when mutant huntingtin exon 1 was expressed in homozygous Dark-mutant flies. Expanded polyglutamine induced a marked increase in expression of Dark, and Dark was observed to colocalize with ubiquitinated protein aggregates. APAF1 was found to colocalize with huntingtin-containing aggregates in a murine model and HD brain, suggesting a common role for Dark/APAF1 in polyglutamine pathogenesis in invertebrates, mice, and man. These findings suggest that limiting APAF1 activity may alleviate both pathologic protein aggregation and neuronal cell death in HD.
Bao, Q., Lu, W., Rabinowitz, J. D., Shi, Y. Calcium blocks formation of apoptosome by preventing nucleotide exchange in Apaf-1. Molec. Cell 25: 181-192, 2007. [PubMed: 17244527] [Full Text: https://doi.org/10.1016/j.molcel.2006.12.013]
Cecconi, F., Alvarez-Bolado, G., Meyer, B. I., Roth, K. A., Gruss, P. Apaf1 (CED-4 homolog) regulates programmed cell death in mammalian development. Cell 94: 727-737, 1998. [PubMed: 9753320] [Full Text: https://doi.org/10.1016/s0092-8674(00)81732-8]
Fortin, A., Cregan, S. P., MacLaurin, J. G., Kushwaha, N., Hickman, E. S., Thompson, C. S., Hakim, A., Albert, P. R., Cecconi, F., Helin, K., Park, D. S., Slack, R. S. APAF1 is a key transcriptional target for p53 in the regulation of neuronal cell death. J. Cell. Biol. 155: 207-216, 2001. [PubMed: 11591730] [Full Text: https://doi.org/10.1083/jcb.200105137]
Furukawa, Y., Nishimura, N., Furukawa, Y., Satoh, M., Endo, H., Iwase, S., Yamada, H., Matsuda, M., Kano, Y., Nakamura, M. Apaf-1 is a mediator of E2F-1-induced apoptosis. J. Biol. Chem. 277: 39760-39768, 2002. [PubMed: 12149244] [Full Text: https://doi.org/10.1074/jbc.M200805200]
Hahn, C., Hirsch, B., Jahnke, D., Durkop, H., Stein, H. Three new types of Apaf-1 in mammalian cells. Biochem. Biophys. Res. Commun. 261: 746-749, 1999. [PubMed: 10441496] [Full Text: https://doi.org/10.1006/bbrc.1999.1124]
Harlan, J., Chen, Y., Gubbins, E., Mueller, R., Roch, J.-M., Walter, K., Lake, M., Olsen, T., Metzger, P., Dorwin, S., Ladror, U., Egan, D. A., and 17 others. Variants in Apaf-1 segregating with major depression promote apoptosome function. Molec. Psychiat. 11: 76-85, 2006. [PubMed: 16231040] [Full Text: https://doi.org/10.1038/sj.mp.4001755]
Honarpour, N., Du, C., Richardson, J. A., Hammer, R. E., Wang, X., Herz, J. Adult Apaf-1-deficient mice exhibit male infertility. Dev. Biol. 218: 248-258, 2000. [PubMed: 10656767] [Full Text: https://doi.org/10.1006/dbio.1999.9585]
Honarpour, N., Gilbert, S. L., Lahn, B. T., Wang, X., Herz, J. Apaf-1 deficiency and neural tube closure defects are found in fog mice. Proc. Nat. Acad. Sci. 98: 9683-9687, 2001. [PubMed: 11504943] [Full Text: https://doi.org/10.1073/pnas.171283198]
Kim, H., Jung, Y. K., Kwon, Y. K., Park, S. H. Assignment of apoptotic protease activating factor-1 gene (APAF1) to human chromosome band 12q23 by fluorescence in situ hybridization. Cytogenet. Cell Genet. 87: 252-253, 1999. [PubMed: 10702682] [Full Text: https://doi.org/10.1159/000015436]
Marsden, V. S., O'Connor, L., O'Reilly, L. A., Silke, J., Metcalf, D., Ekert, P. G., Huang, D. C. S., Cecconi, F., Kuida, K., Tomaselli, K. J., Roy, S., Nicholson, D. W., Vaux, D. L., Bouillet, P., Adams, J. M., Strasser, A. Apoptosis initiated by Bcl-2-regulated caspase activation independently of the cytochrome c/Apaf-1/caspase-9 apoptosome. Nature 419: 634-637, 2002. [PubMed: 12374983] [Full Text: https://doi.org/10.1038/nature01101]
Riedl, S. J., Li, W., Chao, Y., Schwarzenbacher, R., Shi, Y. Structure of the apoptotic protease-activating factor 1 bound to ADP. Nature 434: 926-933, 2005. [PubMed: 15829969] [Full Text: https://doi.org/10.1038/nature03465]
Robles, A. I., Bemmels, N. A., Foraker, A. B., Harris, C. C. APAF-1 is a transcriptional target of p53 in DNA damage-induced apoptosis. Cancer Res. 61: 6660-6664, 2001. [PubMed: 11559530]
Sang, T.-K., Li, C., Liu, W., Rodriguez, A., Abrams, J. M., Zipursky, S. L., Jackson, G. R. Inactivation of Drosophila Apaf-1 related killer suppresses formation of polyglutamine aggregates and blocks polyglutamine pathogenesis. Hum. Molec. Genet. 14: 357-372, 2005. [PubMed: 15590702] [Full Text: https://doi.org/10.1093/hmg/ddi032]
Soengas, M. S., Capodieci, P., Polsky, D., Mora, J., Esteller, M., Opitz-Araya, X., McCombie, R., Herman, J. G., Gerald, W. L., Lazebnik, Y. A., Cordon-Cardo, C., Lowe, S. W. Inactivation of the apoptosis effector Apaf-1 in malignant melanoma. Nature 409: 207-211, 2001. [PubMed: 11196646] [Full Text: https://doi.org/10.1038/35051606]
Srinivasula, S. M., Ahmad, M., Fernandes-Alnemri, T., Alnemri, E. S. Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization. Molec. Cell 1: 949-957, 1998. [PubMed: 9651578] [Full Text: https://doi.org/10.1016/s1097-2765(00)80095-7]
Yoshida, H., Kong, Y.-Y., Yoshida, R., Elia, A. J., Hakem, A., Hakem, R., Penninger, J. M., Mak, T. W. Apaf1 is required for mitochondrial pathways of apoptosis and brain development. Cell 94: 739-750, 1998. [PubMed: 9753321] [Full Text: https://doi.org/10.1016/s0092-8674(00)81733-x]
Zou, H., Henzel, W. J., Liu, X., Lutschg, A., Wang, X. APAF-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 90: 405-413, 1997. [PubMed: 9267021] [Full Text: https://doi.org/10.1016/s0092-8674(00)80501-2]