RESUMEN
The breast and ovarian cancer-specific tumor suppressor BRCA1, along with its heterodimer partner BRCA1-associated RING domain protein (BARD1), plays important roles in DNA repair, centrosome regulation, and transcription. To explore further functions of BRCA1/BARD1, we performed mass spectrometry analysis and identified Obg-like ATPase 1 (OLA1) as a protein that interacts with the carboxy-terminal region of BARD1. OLA1 directly bound to the amino-terminal region of BRCA1 and γ-tubulin. OLA1 localized to centrosomes in interphase and to the spindle pole in mitotic phase, and its knockdown resulted in centrosome amplification and the activation of microtubule aster formation. OLA1 with a mutation observed in breast cancer cell line, E168Q, failed to bind BRCA1 and rescue the OLA1 knockdown-induced centrosome amplification. BRCA1 variant I42V also abrogated the binding of BRCA1 to OLA1. These findings suggest that OLA1 plays an important role in centrosome regulation together with BRCA1.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteína BRCA1/metabolismo , Neoplasias de la Mama/metabolismo , Centrosoma/metabolismo , Proteínas de Unión al GTP/metabolismo , Adenosina Trifosfatasas/genética , Sustitución de Aminoácidos , Proteína BRCA1/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Proteínas de Unión al GTP/genética , Técnicas de Silenciamiento del Gen , Humanos , Mutación Missense , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
IgG4-related disease (IgG4-RD) is characterized by multi-organ irreversible damage resulting from tissue-specific infiltration of IgG4+ plasma cells and cytotoxic T lymphocytes (CTLs). However, whether IgG4 antibody contributes to the inflammation remains unclear. In this study, we established a mouse model that enabled us to evaluate the pathogenic function of IgG4 antibodies in response to a tissue-specific autoantigen using recombinant ovalbumin (OVA)-specific human IgG4 monoclonal antibody (rOVA-hIgG4 mAb) and the mice expressing OVA of the pancreatic islets (RIP-mOVA mice). We found no inflammatory effect of rOVA-hIgG4 mAb transfer alone; however, co-transfer with OVA-specific CD8 CTLs (OT-I T cells) induced tissue damage with dense lymphocytic inflammation in the pancreas of RIP-mOVA mice. rOVA-hIgG4 mAb caused accumulation of conventional DC1 cells (cDC1s) in the lymphoid tissues, and the dendritic cells (DCs) activated the OT-I T cells via cross-presentation. We also revealed that the synergistic effects of CTLs and antibodies were observed in the other subclasses including endogenous antibodies if they recognized the same antigen. The transfer of OVA-specific CD4 helper T cells (OT-II T cells) into RIP-mOVA mice induced the production of anti-OVA antibody, which had a synergistic effect, through acquisition of a T follicular helper (TFH) phenotype. Moreover, using OT-II T cells deficient in Bcl6 caused lower anti-OVA antibody production and inflammation with OT-I T cells. Our results indicated that autoreactive IgG4 antibodies play an important role of the tissue-specific CTL response in IgG4-RD.
Asunto(s)
Enfermedad Relacionada con Inmunoglobulina G4/inmunología , Inmunoglobulina G/inmunología , Inflamación/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas Recombinantes/inmunologíaRESUMEN
Breast cancer gene 1 (BRCA1) is a tumor suppressor that is associated with hereditary breast and ovarian cancer. BRCA1 functions in DNA repair and centrosome regulation together with BRCA1-associated RING domain protein (BARD1), a heterodimer partner of BRCA1. Obg-like ATPase 1 (OLA1) was identified as a protein that interacts with BARD1. OLA1 regulates the centrosome by binding to and collaborating with BRCA1 and BARD1. We identified receptor for activated C kinase (RACK1) as a protein that interacts with OLA1. RACK1 directly bound to OLA1, the N-terminal region of BRCA1, and γ-tubulin, associated with BARD1, and localized the centrosomes throughout the cell cycle. Knockdown of RACK1 caused abnormal centrosomal localization of BRCA1 and abrogated centriole duplication. Overexpression of RACK1 increased the centrosomal localization of BRCA1 and caused centrosome amplification due to centriole overduplication. The number of centrioles in cells with two γ-tubulin spots was higher in cell lines derived from mammary tissue compared to those derived from other tissues. The effects of aberrant RACK1 expression level on centriole duplication were observed in cell lines derived from mammary tissue, but not in those derived from other tissues. Two BRCA1 variants, R133H and E143K, and a RACK1 variant, K280E, associated with cancer, which weakened the BRCA1-RACK1 interaction, interfered with the centrosomal localization of BRCA1 and reduced centrosome amplification induced by overexpression of RACK1. These results suggest that RACK1 regulates centriole duplication by controlling the centrosomal localization of BRCA1 in mammary tissue-derived cells and that this is dependent on the BRCA1-RACK1 interaction.