RESUMEN
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.
Asunto(s)
Muerte Celular/efectos de los fármacos , Daño del ADN , ADN/metabolismo , Exodesoxirribonucleasas/fisiología , Nucleósido-Difosfato Quinasa/fisiología , Fosfoproteínas/fisiología , Serina Endopeptidasas/fisiología , Línea Celular Tumoral , Exodesoxirribonucleasas/farmacología , Silenciador del Gen , Granzimas , Células HeLa , Humanos , Células K562 , Complejos Multienzimáticos/farmacología , Complejos Multienzimáticos/fisiología , Nucleósido Difosfato Quinasas NM23 , Nucleósido-Difosfato Quinasa/farmacología , Fosfoproteínas/farmacología , Serina Endopeptidasas/metabolismo , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Transporte Activo de Núcleo Celular/genética , Apoptosis/genética , Proteínas Cromosómicas no Histona/metabolismo , Genes Supresores de Tumor/fisiología , Inmunidad Celular/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Nucleósido-Difosfato Quinasa , Serina Endopeptidasas/metabolismo , Factores de Transcripción/metabolismo , Liasas de Carbono-Oxígeno/genética , Liasas de Carbono-Oxígeno/metabolismo , Proteínas Cromosómicas no Histona/genética , Fragmentación del ADN/genética , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Proteínas de Unión al ADN , Desoxirribonucleasas/genética , Desoxirribonucleasas/metabolismo , Regulación de la Expresión Génica/genética , Silenciador del Gen/fisiología , Granzimas , Proteína HMGB2/genética , Proteína HMGB2/metabolismo , Células HeLa , Chaperonas de Histonas , Humanos , Células Jurkat , Células K562 , Modelos Biológicos , Proteínas de Unión al GTP Monoméricas/genética , Nucleósido Difosfato Quinasas NM23 , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleosomas/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Serina Endopeptidasas/genética , Linfocitos T Citotóxicos/metabolismo , Factores de Transcripción/genéticaRESUMEN
The cytolytic T lymphocyte protease granzyme A (GzmA) initiates a caspase-independent cell death pathway. Here we report that the rate-limiting enzyme of DNA base excision repair, apurinic endonuclease-1 (Ape1), which is also known as redox factor-1 (Ref-1), binds to GzmA and is contained in the SET complex, a macromolecular complex of 270-420 kDa that is associated with the endoplasmic reticulum and is targeted by GzmA during cell-mediated death. GzmA cleaves Ape1 after Lys31 and destroys its known oxidative repair functions. In so doing, GzmA may block cellular repair and force apoptosis. In support of this, cells with silenced Ape1 expression are more sensitive, whereas cells overexpressing noncleavable Ape1 are more resistant, to GzmA-mediated death.
Asunto(s)
Apoptosis/fisiología , Liasas de Carbono-Oxígeno/metabolismo , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Serina Endopeptidasas/metabolismo , Animales , Secuencia de Bases , Liasas de Carbono-Oxígeno/química , Liasas de Carbono-Oxígeno/genética , ADN/genética , ADN/metabolismo , Silenciador del Gen , Granzimas , Células HeLa , Humanos , Células K562 , Sustancias Macromoleculares , Glicoproteínas de Membrana/metabolismo , Oxidación-Reducción , Perforina , Proteínas Citotóxicas Formadoras de Poros , Interferencia de ARN , Proteínas Recombinantes/metabolismo , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismoRESUMEN
RNA interference silences gene expression through short interfering 21 23-mer double-strand RNA segments that guide mRNA degradation in a sequence-specific fashion. Here we report that siRNAs inhibit virus production by targeting the mRNAs for either the HIV-1 cellular receptor CD4, the viral structural Gag protein or green fluorescence protein substituted for the Nef regulatory protein. siRNAs effectively inhibit pre- and/or post-integration infection events in the HIV-1 life cycle. Thus, siRNAs may have potential for therapeutic intervention in HIV-1 and other viral infections.
Asunto(s)
Silenciador del Gen , VIH-1/fisiología , ARN no Traducido/fisiología , Secuencia de Bases , Antígenos CD4/fisiología , Línea Celular , VIH-1/efectos de los fármacos , VIH-1/genética , Células HeLa , Humanos , ARN sin Sentido/farmacología , ARN Mensajero/genética , ARN Interferente Pequeño , ARN no Traducido/farmacología , Receptores CCR5/fisiología , Transfección , Integración Viral/efectos de los fármacosRESUMEN
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.
