RESUMO
Granzyme A (GzmA) activates a caspase-independent cell death pathway with morphological features of apoptosis. Single-stranded DNA damage is initiated when the endonuclease NM23-H1 becomes activated to nick DNA after granzyme A cleaves its inhibitor, SET. SET and NM23-H1 reside in an endoplasmic reticulum-associated complex (the SET complex) that translocates to the nucleus in response to superoxide generation by granzyme A. We now find the 3'-to-5' exonuclease TREX1, but not its close homolog TREX2, in the SET complex. TREX1 binds to SET and colocalizes and translocates with the SET complex. NM23-H1 and TREX1 work in concert to degrade DNA. Silencing NM23-H1 or TREX1 inhibits DNA damage and death of cells treated with perforin (PFN) and granzyme A, but not of cells treated with perforin and granzyme B (GzmB). After granzyme A activates NM23-H1 to make single-stranded nicks, TREX1 removes nucleotides from the nicked 3' end to reduce the possibility of repair by rejoining the nicked ends.
Assuntos
Morte Celular/efeitos dos fármacos , Dano ao DNA , DNA/metabolismo , Exodesoxirribonucleases/fisiologia , Núcleosídeo-Difosfato Quinase/fisiologia , Fosfoproteínas/fisiologia , Serina Endopeptidases/fisiologia , Linhagem Celular Tumoral , Exodesoxirribonucleases/farmacologia , Inativação Gênica , Granzimas , Células HeLa , Humanos , Células K562 , Complexos Multienzimáticos/farmacologia , Complexos Multienzimáticos/fisiologia , Nucleosídeo NM23 Difosfato Quinases , Núcleosídeo-Difosfato Quinase/farmacologia , Fosfoproteínas/farmacologia , Serina Endopeptidases/metabolismo , Especificidade por SubstratoRESUMO
Granzyme A (GzmA) induces a caspase-independent cell death pathway characterized by single-stranded DNA nicks and other features of apoptosis. A GzmA-activated DNase (GAAD) is in an ER associated complex containing pp32 and the GzmA substrates SET, HMG-2, and Ape1. We show that GAAD is NM23-H1, a nucleoside diphosphate kinase implicated in suppression of tumor metastasis, and its specific inhibitor (IGAAD) is SET. NM23-H1 binds to SET and is released from inhibition by GzmA cleavage of SET. After GzmA loading or CTL attack, SET and NM23-H1 translocate to the nucleus and SET is degraded, allowing NM23-H1 to nick chromosomal DNA. GzmA-treated cells with silenced NM23-H1 expression are resistant to GzmA-mediated DNA damage and cytolysis, while cells overexpressing NM23-H1 are more sensitive.
Assuntos
Transporte Ativo do Núcleo Celular/genética , Apoptose/genética , Proteínas Cromossômicas não Histona/metabolismo , Genes Supressores de Tumor/fisiologia , Imunidade Celular/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Núcleosídeo-Difosfato Quinase , Serina Endopeptidases/metabolismo , Fatores de Transcrição/metabolismo , Carbono-Oxigênio Liases/genética , Carbono-Oxigênio Liases/metabolismo , Proteínas Cromossômicas não Histona/genética , Fragmentação do DNA/genética , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Proteínas de Ligação a DNA , Desoxirribonucleases/genética , Desoxirribonucleases/metabolismo , Regulação da Expressão Gênica/genética , Inativação Gênica/fisiologia , Granzimas , Proteína HMGB2/genética , Proteína HMGB2/metabolismo , Células HeLa , Chaperonas de Histonas , Humanos , Células Jurkat , Células K562 , Modelos Biológicos , Proteínas Monoméricas de Ligação ao GTP/genética , Nucleosídeo NM23 Difosfato Quinases , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Serina Endopeptidases/genética , Linfócitos T Citotóxicos/metabolismo , Fatores de Transcrição/genéticaRESUMO
The cytotoxic T-lymphocyte protease granzyme A induces caspase-independent cell death in which DNA single-stranded nicking is observed instead of oligonucleosomal fragmentation. A 270- to 420-kDa endoplasmic reticulum-associated complex (SET complex) containing the nucleosome assembly protein SET, the tumor suppressor pp32, and the base excision repair enzyme APE can induce single-stranded DNA damage in isolated nuclei in a granzyme A-dependent manner. The normal functions of the SET complex are unknown, but the functions of its components suggest that it is involved in activating transcription and DNA repair. We now find that the SET complex contains DNA binding and bending activities mediated by the chromatin-associated protein HMG2. HMG2 facilitates assembly of nucleoprotein higher-order structures by bending and looping DNA or by stabilizing underwound DNA. HMG2 is in the SET complex and coprecipitates with SET. By confocal microscopy, it is observed that cytoplasmic HMG2 colocalizes with SET in association with the endoplasmic reticulum, but most nuclear HMG2 is unassociated with SET. This physical association suggests that HMG2 may facilitate the nucleosome assembly, transcriptional activation, and DNA repair functions of SET and/or APE. HMG2, like SET and APE, is a physiologically relevant granzyme A substrate in targeted cells. HMG1, however, is not a substrate. Granzyme A cleavage after Lys65 in the midst of HMG box A destroys HMG2-mediated DNA binding and bending functions. Granzyme A cleavage and functional disruption of key nuclear substrates, including HMG2, SET, APE, lamins, and histones, are likely to cripple the cellular repair response to promote cell death in this novel caspase-independent death pathway.