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Identification of Regulatory and Catalytic Domains in the Apoptosis Nuclease DFF40/CAD

1999; Elsevier BV; Volume: 274; Issue: 1 Linguagem: Inglês

10.1074/jbc.274.1.270

ISSN

1083-351X

Autores

Naohiro Inohara, Takeyoshi Koseki, Shu Chen, Mary Benedict, Gabriel Núñez,

Tópico(s)

Trace Elements in Health

Resumo

The DNA fragmentation factor (DFF) is composed of two subunits, the 40-kDa caspase-3-activated nuclease (DFF40/CAD) and its 45-kDa inhibitor (DFF45/ICAD). During apoptosis, DFF-40/CAD is activated by caspase-3-mediated cleavage of DFF45/ICAD. Mutational analysis of DFF40/CAD revealed that DFF40/CAD is composed of a C-terminal catalytic domain and an N-terminal regulatory domain. Deletion of the catalytic domain (residues 290–345) abrogated the caspase-3-induced nuclease activity of DFF40/CAD but not its ability to interact with DFF45/ICAD. Conversely, removal of the regulatory domain (residues 1–83) yielded a constitutively active DFF40/CAD nuclease that neither bound to its inhibitor nor required caspase-3 for activation. Amino acid alignment revealed that the regulatory domain of DFF40/CAD has homology to the N-terminal region of mammalian andDrosophila DFF45/ICAD and CIDE-N, a regulatory domain previously identified in pro-apoptotic CIDE proteins. Mutational analysis of the N-terminal region revealed mutants with diminished nuclease activity but with intact ability to bind DFF45/ICAD. Thus, CIDE-N represents a new type of domain that is associated with the regulation of the apoptosis/DNA fragmentation pathway. The DNA fragmentation factor (DFF) is composed of two subunits, the 40-kDa caspase-3-activated nuclease (DFF40/CAD) and its 45-kDa inhibitor (DFF45/ICAD). During apoptosis, DFF-40/CAD is activated by caspase-3-mediated cleavage of DFF45/ICAD. Mutational analysis of DFF40/CAD revealed that DFF40/CAD is composed of a C-terminal catalytic domain and an N-terminal regulatory domain. Deletion of the catalytic domain (residues 290–345) abrogated the caspase-3-induced nuclease activity of DFF40/CAD but not its ability to interact with DFF45/ICAD. Conversely, removal of the regulatory domain (residues 1–83) yielded a constitutively active DFF40/CAD nuclease that neither bound to its inhibitor nor required caspase-3 for activation. Amino acid alignment revealed that the regulatory domain of DFF40/CAD has homology to the N-terminal region of mammalian andDrosophila DFF45/ICAD and CIDE-N, a regulatory domain previously identified in pro-apoptotic CIDE proteins. Mutational analysis of the N-terminal region revealed mutants with diminished nuclease activity but with intact ability to bind DFF45/ICAD. Thus, CIDE-N represents a new type of domain that is associated with the regulation of the apoptosis/DNA fragmentation pathway. DNA fragmentation factor hemaglutinin caspase-3-activated DNase cell death-inducing DFF45-like effector inhibitor of caspase-3-activated DNase monoclonal antibody wild type tumor necrosis factor receptor-1. Apoptosis, a morphologically defined form of programmed cell death plays an essential role in animal development and tissue homeostasis (1Jacobson M.D. Weil M. Raff M.C. Cell. 1997; 88: 347-354Abstract Full Text Full Text PDF PubMed Scopus (2393) Google Scholar). Apoptotic cells undergo multiple changes including membrane blebbing, nuclear condensation, and fragmentation of genomic DNA into nucleosomal fragments. These morphological and biochemical changes are promoted by caspases, a family of cysteine proteases that are activated by apoptotic stimuli. Activated caspases can cleave multiple cytoplasmic and nuclear substrates, a process that appears to play a pivotal role in the execution phase of apoptosis (2Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4104) Google Scholar).DNA fragmentation associated with apoptosis is induced by the DNA fragmentation factor (DFF)1, which is activated by caspases, mainly caspase-3 (3Liu X. Zou H. Slaughter C. Wang X. Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1638) Google Scholar, 4Enari M. Sakahira H. Yokoyama H. Okawa K. Iwamatsu A. Nagata S. Nature. 1998; 391: 43-50Crossref PubMed Scopus (2795) Google Scholar, 5Sakahira H. Enari M. Nagata S. Nature. 1998; 391: 96-99Crossref PubMed Scopus (1416) Google Scholar, 6Halenbeck R. MacDonald H. Roulston A. Chen T.T. Conroy L. Williams L.T. Curr. Biol. 1998; 8: 537-540Abstract Full Text Full Text PDF PubMed Google Scholar, 7Liu X. Li P. Widlak P. Zou H. Luo X. Garrard W.T. Wang X. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8461-8466Crossref PubMed Scopus (500) Google Scholar, 8Mukae N. Enari M. Sakahira H. Fukuda Y. Inazawa J. Toh H. Nagata S. Proc. Natl. Acad. Sci. U. S. A . 1998; 95: 9123-9128Crossref PubMed Scopus (171) Google Scholar). DFF is composed of two protein subunits, a 40-kDa caspase-activated nuclease (DFF40/CAD), also called CPAN, and its 45-kDa inhibitor (DFF45/ICAD) (3Liu X. Zou H. Slaughter C. Wang X. Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1638) Google Scholar, 4Enari M. Sakahira H. Yokoyama H. Okawa K. Iwamatsu A. Nagata S. Nature. 1998; 391: 43-50Crossref PubMed Scopus (2795) Google Scholar, 5Sakahira H. Enari M. Nagata S. Nature. 1998; 391: 96-99Crossref PubMed Scopus (1416) Google Scholar, 6Halenbeck R. MacDonald H. Roulston A. Chen T.T. Conroy L. Williams L.T. Curr. Biol. 1998; 8: 537-540Abstract Full Text Full Text PDF PubMed Google Scholar, 7Liu X. Li P. Widlak P. Zou H. Luo X. Garrard W.T. Wang X. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8461-8466Crossref PubMed Scopus (500) Google Scholar, 8Mukae N. Enari M. Sakahira H. Fukuda Y. Inazawa J. Toh H. Nagata S. Proc. Natl. Acad. Sci. U. S. A . 1998; 95: 9123-9128Crossref PubMed Scopus (171) Google Scholar). Cleavage of DFF45/ICAD by caspase-3 releases DFF40/CAD from its inhibitor leading to the induction of nuclease activity, nuclear condensation, and DNA fragmentation in vitro (3Liu X. Zou H. Slaughter C. Wang X. Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1638) Google Scholar, 4Enari M. Sakahira H. Yokoyama H. Okawa K. Iwamatsu A. Nagata S. Nature. 1998; 391: 43-50Crossref PubMed Scopus (2795) Google Scholar, 5Sakahira H. Enari M. Nagata S. Nature. 1998; 391: 96-99Crossref PubMed Scopus (1416) Google Scholar, 6Halenbeck R. MacDonald H. Roulston A. Chen T.T. Conroy L. Williams L.T. Curr. Biol. 1998; 8: 537-540Abstract Full Text Full Text PDF PubMed Google Scholar, 7Liu X. Li P. Widlak P. Zou H. Luo X. Garrard W.T. Wang X. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8461-8466Crossref PubMed Scopus (500) Google Scholar, 8Mukae N. Enari M. Sakahira H. Fukuda Y. Inazawa J. Toh H. Nagata S. Proc. Natl. Acad. Sci. U. S. A . 1998; 95: 9123-9128Crossref PubMed Scopus (171) Google Scholar). However, the molecular basis for DFF40/CAD function and its regulation by DFF45/ICAD remains poorly understood.In a previous study, we identified a conserved family of proteins named CIDEs, that include CIDE-A, CIDE-B, and Fsp27, based on amino acid homology to the N-terminal region of DFF45/ICAD (9Inohara N. Koseki T. Chen S. Wu X. Nunez G. EMBO J. 1998; 17: 2526-2533Crossref PubMed Scopus (279) Google Scholar). Unlike DFF45/ICAD, CIDEs are pro-apoptotic proteins that induce DNA fragmentation as well as other apoptotic features such as membrane blebbing and nuclear condensation (9Inohara N. Koseki T. Chen S. Wu X. Nunez G. EMBO J. 1998; 17: 2526-2533Crossref PubMed Scopus (279) Google Scholar). The activity of CIDEs is inhibited by DFF45/ICAD (9Inohara N. Koseki T. Chen S. Wu X. Nunez G. EMBO J. 1998; 17: 2526-2533Crossref PubMed Scopus (279) Google Scholar). Mutational analysis revealed that CIDEs contains two domains, CIDE-N and CIDE-C (9Inohara N. Koseki T. Chen S. Wu X. Nunez G. EMBO J. 1998; 17: 2526-2533Crossref PubMed Scopus (279) Google Scholar). The CIDE-C domain is necessary and sufficient for apoptosis, whereas CIDE-N acts as a regulatory domain required for the inhibitory activity of DFF45/ICAD (9Inohara N. Koseki T. Chen S. Wu X. Nunez G. EMBO J. 1998; 17: 2526-2533Crossref PubMed Scopus (279) Google Scholar).Here we report that DFF40/CAD has two domains with distinct biological functions. A catalytic domain located in the C-terminal region mediates nuclease activity, whereas a regulatory domain is located in the N-terminal half of DFF40/CAD. The regulatory domain of DFF40/CAD has homology to a conserved CIDE-N domain that was previously identified in the pro-apoptotic CIDE proteins. These results identify the N-terminal region of DFF40/CAD as a conserved domain that regulates the activation and activity of this apoptosis-associated nuclease. Apoptosis, a morphologically defined form of programmed cell death plays an essential role in animal development and tissue homeostasis (1Jacobson M.D. Weil M. Raff M.C. Cell. 1997; 88: 347-354Abstract Full Text Full Text PDF PubMed Scopus (2393) Google Scholar). Apoptotic cells undergo multiple changes including membrane blebbing, nuclear condensation, and fragmentation of genomic DNA into nucleosomal fragments. These morphological and biochemical changes are promoted by caspases, a family of cysteine proteases that are activated by apoptotic stimuli. Activated caspases can cleave multiple cytoplasmic and nuclear substrates, a process that appears to play a pivotal role in the execution phase of apoptosis (2Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4104) Google Scholar). DNA fragmentation associated with apoptosis is induced by the DNA fragmentation factor (DFF)1, which is activated by caspases, mainly caspase-3 (3Liu X. Zou H. Slaughter C. Wang X. Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1638) Google Scholar, 4Enari M. Sakahira H. Yokoyama H. Okawa K. Iwamatsu A. Nagata S. Nature. 1998; 391: 43-50Crossref PubMed Scopus (2795) Google Scholar, 5Sakahira H. Enari M. Nagata S. Nature. 1998; 391: 96-99Crossref PubMed Scopus (1416) Google Scholar, 6Halenbeck R. MacDonald H. Roulston A. Chen T.T. Conroy L. Williams L.T. Curr. Biol. 1998; 8: 537-540Abstract Full Text Full Text PDF PubMed Google Scholar, 7Liu X. Li P. Widlak P. Zou H. Luo X. Garrard W.T. Wang X. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8461-8466Crossref PubMed Scopus (500) Google Scholar, 8Mukae N. Enari M. Sakahira H. Fukuda Y. Inazawa J. Toh H. Nagata S. Proc. Natl. Acad. Sci. U. S. A . 1998; 95: 9123-9128Crossref PubMed Scopus (171) Google Scholar). DFF is composed of two protein subunits, a 40-kDa caspase-activated nuclease (DFF40/CAD), also called CPAN, and its 45-kDa inhibitor (DFF45/ICAD) (3Liu X. Zou H. Slaughter C. Wang X. Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1638) Google Scholar, 4Enari M. Sakahira H. Yokoyama H. Okawa K. Iwamatsu A. Nagata S. Nature. 1998; 391: 43-50Crossref PubMed Scopus (2795) Google Scholar, 5Sakahira H. Enari M. Nagata S. Nature. 1998; 391: 96-99Crossref PubMed Scopus (1416) Google Scholar, 6Halenbeck R. MacDonald H. Roulston A. Chen T.T. Conroy L. Williams L.T. Curr. Biol. 1998; 8: 537-540Abstract Full Text Full Text PDF PubMed Google Scholar, 7Liu X. Li P. Widlak P. Zou H. Luo X. Garrard W.T. Wang X. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8461-8466Crossref PubMed Scopus (500) Google Scholar, 8Mukae N. Enari M. Sakahira H. Fukuda Y. Inazawa J. Toh H. Nagata S. Proc. Natl. Acad. Sci. U. S. A . 1998; 95: 9123-9128Crossref PubMed Scopus (171) Google Scholar). Cleavage of DFF45/ICAD by caspase-3 releases DFF40/CAD from its inhibitor leading to the induction of nuclease activity, nuclear condensation, and DNA fragmentation in vitro (3Liu X. Zou H. Slaughter C. Wang X. Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1638) Google Scholar, 4Enari M. Sakahira H. Yokoyama H. Okawa K. Iwamatsu A. Nagata S. Nature. 1998; 391: 43-50Crossref PubMed Scopus (2795) Google Scholar, 5Sakahira H. Enari M. Nagata S. Nature. 1998; 391: 96-99Crossref PubMed Scopus (1416) Google Scholar, 6Halenbeck R. MacDonald H. Roulston A. Chen T.T. Conroy L. Williams L.T. Curr. Biol. 1998; 8: 537-540Abstract Full Text Full Text PDF PubMed Google Scholar, 7Liu X. Li P. Widlak P. Zou H. Luo X. Garrard W.T. Wang X. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8461-8466Crossref PubMed Scopus (500) Google Scholar, 8Mukae N. Enari M. Sakahira H. Fukuda Y. Inazawa J. Toh H. Nagata S. Proc. Natl. Acad. Sci. U. S. A . 1998; 95: 9123-9128Crossref PubMed Scopus (171) Google Scholar). However, the molecular basis for DFF40/CAD function and its regulation by DFF45/ICAD remains poorly understood. In a previous study, we identified a conserved family of proteins named CIDEs, that include CIDE-A, CIDE-B, and Fsp27, based on amino acid homology to the N-terminal region of DFF45/ICAD (9Inohara N. Koseki T. Chen S. Wu X. Nunez G. EMBO J. 1998; 17: 2526-2533Crossref PubMed Scopus (279) Google Scholar). Unlike DFF45/ICAD, CIDEs are pro-apoptotic proteins that induce DNA fragmentation as well as other apoptotic features such as membrane blebbing and nuclear condensation (9Inohara N. Koseki T. Chen S. Wu X. Nunez G. EMBO J. 1998; 17: 2526-2533Crossref PubMed Scopus (279) Google Scholar). The activity of CIDEs is inhibited by DFF45/ICAD (9Inohara N. Koseki T. Chen S. Wu X. Nunez G. EMBO J. 1998; 17: 2526-2533Crossref PubMed Scopus (279) Google Scholar). Mutational analysis revealed that CIDEs contains two domains, CIDE-N and CIDE-C (9Inohara N. Koseki T. Chen S. Wu X. Nunez G. EMBO J. 1998; 17: 2526-2533Crossref PubMed Scopus (279) Google Scholar). The CIDE-C domain is necessary and sufficient for apoptosis, whereas CIDE-N acts as a regulatory domain required for the inhibitory activity of DFF45/ICAD (9Inohara N. Koseki T. Chen S. Wu X. Nunez G. EMBO J. 1998; 17: 2526-2533Crossref PubMed Scopus (279) Google Scholar). Here we report that DFF40/CAD has two domains with distinct biological functions. A catalytic domain located in the C-terminal region mediates nuclease activity, whereas a regulatory domain is located in the N-terminal half of DFF40/CAD. The regulatory domain of DFF40/CAD has homology to a conserved CIDE-N domain that was previously identified in the pro-apoptotic CIDE proteins. These results identify the N-terminal region of DFF40/CAD as a conserved domain that regulates the activation and activity of this apoptosis-associated nuclease. We are grateful to M. Garcia-Calvo and N. Thorberry of Merck for recombinant caspase-3; V. M. Dixit for pcDNA3-TNFR1-Flag; and L. del Peso, V. Gonzalez, D. Ekhterae, and Y. Hu for critical review of the manuscript.

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