RESUMEN
The MKK1/2 kinase tumour progression locus 2 (TPL-2) is critical for the production of tumour necrosis factor alpha (TNFα) in innate immune responses and a potential anti-inflammatory drug target. Several earlier pharmaceutical company screens with the isolated TPL-2 kinase domain have identified small-molecule inhibitors that specifically block TPL-2 signalling in cells, but none of these have progressed to clinical development. We have previously shown that TPL-2 catalytic activity regulates TNF production by macrophages while associated with NF-κB1 p105 and ABIN-2, independently of MKK1/2 phosphorylation via an unknown downstream substrate. In the present study, we used a positional scanning peptide library to determine the optimal substrate specificity of a complex of TPL-2, NF-κB1 p105 and ABIN-2. Using an optimal peptide substrate based on this screen and a high-throughput mass spectrometry assay to monitor kinase activity, we found that the TPL-2 complex has significantly altered sensitivities versus existing ATP-competitive TPL-2 inhibitors than the isolated TPL-2 kinase domain. These results imply that screens with the more physiologically relevant TPL-2/NF-κB1 p105/ABIN-2 complex have the potential to deliver novel TPL-2 chemical series; both ATP-competitive and allosteric inhibitors could emerge with significantly improved prospects for development as anti-inflammatory drugs.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Antiinflamatorios/farmacología , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Subunidad p50 de NF-kappa B/antagonistas & inhibidores , Péptidos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Antiinflamatorios/síntesis química , Expresión Génica , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Subunidad p50 de NF-kappa B/genética , Subunidad p50 de NF-kappa B/metabolismo , Biblioteca de Péptidos , Péptidos/síntesis química , Unión Proteica , Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
High-risk human papillomavirus (HPV) types cause cervical lesions of varying severity, ranging from transient productive infections to high-grade neoplasia. Disease stratification requires the examination of lesional pathology, and possibly also the detection of biomarkers. P16(INK4a) and MCM are established surrogates of high-risk HPV E6/E7 activity, and can be extensively expressed in high-grade lesions. Here we have combined these two cellular biomarkers with detection of the abundant HPV-encoded E4 protein in order to identify both productive and transforming lesions. This approach has allowed us to distinguish true papillomavirus infections from similar pathologies, and has allowed us to divide the heterogeneous CIN2 category into those that are CIN1-like and express E4, and those that more closely resemble nonproductive CIN3. To achieve this, 530 lesional areas were evaluated according to standard pathology criteria and by using a multiple staining approach that allows us to superimpose biomarker patterns either singly or in combination onto an annotated hematoxylin and eosin (H&E) image. Conventional grading of neoplasia was established by review panel, and compared directly with the composite molecular pathology visualized on the same tissue section. The detection of E4 coincided with the onset of vacuolation, becoming abundant in koilocytes as the MCM marker declined and cells lost their defined nuclear margins as visualized by standard H&E staining. Of the dual marker approaches, p16(INK4a) and E4 appeared most promising, with E4 generally identifying areas of low-grade disease even when p16(INK4a) was present. Extensive p16(INK4a) expression usually coincided with an absence of E4 expression or its focal retention in sporadic cells within the lesion. Our results suggest that a straightforward molecular evaluation of HPV life-cycle deregulation in cervical neoplasia may help improve disease stratification, and that this can be achieved using complementary molecular biomarker pairs such as MCM/E4 or, more promisingly, p16(INK4a)/E4 as an adjunct to conventional pathology.
Asunto(s)
Cuello del Útero/patología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Infecciones por Papillomavirus/patología , Displasia del Cuello del Útero/patología , Neoplasias del Cuello Uterino/patología , Biomarcadores de Tumor/metabolismo , Cuello del Útero/virología , Femenino , Humanos , Clasificación del Tumor , Papillomaviridae , Infecciones por Papillomavirus/virología , Neoplasias del Cuello Uterino/virología , Displasia del Cuello del Útero/virologíaRESUMEN
NF-kappaB1 p105 functions both as a precursor of NF-kappaB1 p50 and as a cytoplasmic inhibitor of NF-kappaB. Following the stimulation of cells with tumor necrosis factor alpha (TNF-alpha), the IkappaB kinase (IKK) complex rapidly phosphorylates NF-kappaB1 p105 on serine 927 in the PEST region. This phosphorylation is essential for TNF-alpha to trigger p105 degradation, which releases the associated Rel/NF-kappaB subunits to translocate into the nucleus and regulate target gene transcription. Serine 927 resides in a conserved motif (Asp-Ser(927)-Gly-Val-Glu-Thr-Ser(932)) homologous to the IKK target sequence in IkappaBalpha. In this study, TNF-alpha-induced p105 proteolysis was revealed to additionally require the phosphorylation of serine 932. Experiments with IKK1(-/-) and IKK2(-/-) double knockout embryonic fibroblasts demonstrate that the IKK complex is essential for TNF-alpha to stimulate phosphorylation on p105 serines 927 and 932. Furthermore, purified IKK1 and IKK2 can each phosphorylate a glutathione S-transferase-p105(758-967) fusion protein on both regulatory serines in vitro. IKK-mediated p105 phosphorylation generates a binding site for betaTrCP, the receptor subunit of an SCF-type ubiquitin E3 ligase, and depletion of betaTrCP by RNA interference blocks TNF-alpha-induced p105 ubiquitination and proteolysis. Phosphopeptide competition experiments indicate that betaTrCP binds p105 more effectively when both serines 927 and 932 are phosphorylated. Interestingly, however, betaTrCP affinity for the IKK-phosphorylated sequence on p105 is substantially lower than that on IkappaBalpha. Thus, it appears that reduced p105 recruitment of betaTrCP and subsequent ubiquitination may contribute to delayed p105 proteolysis after TNF-alpha stimulation relative to that for IkappaBalpha.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , FN-kappa B/metabolismo , Precursores de Proteínas/metabolismo , Serina , Secuencia de Aminoácidos , Animales , Unión Competitiva , Células Cultivadas , Fibroblastos , Proteínas de Unión al GTP/genética , Humanos , Quinasa I-kappa B , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , FN-kappa B/efectos de los fármacos , FN-kappa B/genética , Subunidad p50 de NF-kappa B , Fosforilación , Precursores de Proteínas/efectos de los fármacos , Precursores de Proteínas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Ubiquitina/metabolismo , Proteínas con Repetición de beta-TransducinaRESUMEN
Grading cervical intraepithelial neoplasia (CIN) determines clinical management of women after abnormal cytology with potential for overdiagnosis and overtreatment. We studied a novel biomarker of human papillomavirus (HPV) life-cycle completion (panHPVE4), in combination with the minichromosome maintenance (MCM) protein cell-cycle marker and the p16INK4a transformation marker, to improve CIN diagnosis and categorization. Scoring these biomarkers alongside CIN grading by 3 pathologists was performed on 114 cervical specimens with high-risk (HR) HPV. Interobserver agreement for histopathology was moderate (κ=0.43 for CIN1/negative, 0.54 for CIN2/≤CIN1, and 0.36 for CIN3). Agreement was good or excellent for biomarker scoring (E4: κ=0.896; 95% confidence interval [CI]: 0.763-0.969; p16INK4a : κ=0.798; 95% CI: 0.712-0.884; MCM: κ=0.894; 95% CI: NC (this quantity cannot be calculated). Biomarker expression was studied by immunofluorescence and immunohistochemistry and was correlated with 104 final CIN diagnoses after histologic review. All 25 histologically negative specimens were p16INK4a and panHPVE4 negative, although 9 were MCM-positive. There were variable extents of p16INK4a positivity in 11/11 CIN1 and extensive panHPVE4 staining in 9/11. Ten CIN2 lesions expressed panHPVE4 and p16INK4a, and 13 CIN2 expressed only p16INK4a. CIN3 showed extensive p16INK4a positivity with no/minimal panHPVE4 staining. PanHPVE4, unlike MCM, distinguished CIN1 from negative. PanHPVE4 with p16INK4a separated CIN2/3 showing only expression of p16INK4a, indicating transforming HR-HPV E7 expression, from CIN1/2 showing completion of HR-HPV life cycle by E4 expression and variable p16INK4a expression. PanHPVE4 and p16INK4a staining are complementary markers that could provide simple, reliable support for diagnosing CIN. Their value in distinguishing CIN1/2 that supports HR-HPV life-cycle completion (and which might ultimately regress) from purely transforming CIN2/3 needing treatment warrants further research.
Asunto(s)
Biomarcadores de Tumor/análisis , Inhibidor p16 de la Quinasa Dependiente de Ciclina/análisis , Proteínas Oncogénicas Virales/análisis , Papillomaviridae/química , Infecciones por Papillomavirus/diagnóstico , Displasia del Cuello del Útero/diagnóstico , Neoplasias del Cuello Uterino/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Europa (Continente) , Femenino , Técnica del Anticuerpo Fluorescente , Pruebas de ADN del Papillomavirus Humano , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Clasificación del Tumor , Variaciones Dependientes del Observador , Papillomaviridae/genética , Infecciones por Papillomavirus/patología , Infecciones por Papillomavirus/virología , Valor Predictivo de las Pruebas , Reproducibilidad de los Resultados , Neoplasias del Cuello Uterino/química , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/virología , Adulto Joven , Displasia del Cuello del Útero/química , Displasia del Cuello del Útero/patología , Displasia del Cuello del Útero/virologíaRESUMEN
The E4 (also called E1--E4) and E2 proteins of human papillomavirus type 16 are thought to be expressed within the same cells of a lesion, and their open reading frames overlap, suggesting that they may have a functional relationship. We have examined the effect of co-expression of these two proteins and found that each enhances the level of the other. We also identified the N-terminus of E2 as the first example of a viral protein that directly binds the HPV16 E1--E4 protein. This appears to result in the E2 becoming less soluble and promotes its relocation from the nucleus to the cytoplasm. In addition, the turnover of the E2 protein is decreased in the presence of E1--E4. All this raises the possibility that E1--E4 acts to influence E2 activity by varying the amount of available E2 in the cell.