RESUMEN
Fbw7, a substrate receptor for Cul1-RING-ligase (CRL1), facilitates the ubiquitination and degradation of several proteins, including Cyclin E and c-Myc. In spite of much effort, the mechanisms underlying Fbw7 regulation are mostly unknown. Here, we show that Glomulin (Glmn), a protein found mutated in the vascular disorder glomuvenous malformation (GVM), binds directly to the RING domain of Rbx1 and inhibits its E3 ubiquitin ligase activity. Loss of Glmn in a variety of cells, tissues, and GVM lesions results in decreased levels of Fbw7 and increased levels of Cyclin E and c-Myc. The increased turnover of Fbw7 is dependent on CRL and proteasome activity, indicating that Glmn modulates the E3 activity of CRL1(Fbw7). These data reveal an unexpected functional connection between Glmn and Rbx1 and demonstrate that defective regulation of Fbw7 levels contributes to GVM.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cullin/metabolismo , Proteínas F-Box/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/genética , Proteínas Cullin/genética , Ciclina E/genética , Ciclina E/metabolismo , Proteínas F-Box/genética , Proteína 7 que Contiene Repeticiones F-Box-WD , Tumor Glómico/genética , Tumor Glómico/metabolismo , Células HEK293 , Células HeLa , Humanos , Paraganglioma Extraadrenal/genética , Paraganglioma Extraadrenal/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
CONTEXT: - Excision repair cross-complementation 1 (ERCC1) is a key enzyme in nuclear excision repair pathway and has a critical role in helping remove DNA adducts caused by cross-linking agents, such as platinum-containing cancer chemotherapies and other DNA-damaging therapeutic modalities. ERCC1 expression, evaluated by techniques such as immunohistochemistry, has been associated with clinical response; ERCC1+ tumors are more resistant to cisplatin treatment than are ERCC1- tumors. Although several immunohistochemistry, anti-ERCC1 antibodies are available, the 8F1 clone, in particular, has been used in many studies. Recent evidence has suggested that the 8F1 antibody cross-reacts with at least one other protein, raising concerns about the specificity of this clone. OBJECTIVE: - To design an immunohistochemistry assay to detect ERCC1 levels that show dynamic range and consistent analytic performance. DESIGN: - Two different primary antibodies to ERCC1, clones 4F9 and D6G6, were evaluated on formalin-fixed, paraffin-embedded tissue. We then performed a fit-for-purpose assay validation with the 4F9 clone, which included sensitivity assessment across several solid tumor types and evaluation of analytic parameters, such as precision and reproducibility. RESULTS: - The 4F9 clone was consistently superior to the D6G6 clone in the optimization phase. A range of expression was seen in ovarian, head and neck, non-small cell lung, and esophageal cancer samples when tested with the 4F9 clone. The antibody showed acceptable reproducibility (31.02%) and precision (16.06%). CONCLUSIONS: - This assay can be used to assess ERCC1 levels during clinical studies of patient tumors from a variety of tumor types.