RESUMO
The general transcription factor II H (TFIIH) plays an essential role in transcription and nucleotide excision DNA repair (NER). TFIIH is a complex 10 subunit containing molecular machine that harbors three enzymatic activities while the remaining subunits assume regulatory and/or structural functions. Intriguingly, the three enzymatic activities of the CDK7 kinase, the XPB translocase, and the XPD helicase exert different impacts on the overall activities of TFIIH. While the enzymatic function of the XPD helicase is exclusively required in NER, the CDK7 kinase is deeply involved in transcription, whereas XPB is essential to both processes. Recent structural and biochemical endeavors enabled unprecedented details towards the molecular basis of these different TFIIH functions and how the enzymatic activities are regulated within the entire complex. Due to its involvement in two fundamental processes, TFIIH has become increasingly important as a target in cancer therapy and two of the three enzymes have already been addressed successfully. Here we explore the possibilities of recent high resolution structures in the context of TFIIH druggability and shed light on the functional consequences of the different approaches towards TFIIH inhibition.
Assuntos
Antineoplásicos/farmacologia , Reparo do DNA , Neoplasias/metabolismo , Fator de Transcrição TFIIH/antagonistas & inibidores , Fator de Transcrição TFIIH/metabolismo , Antineoplásicos/uso terapêutico , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , DNA/metabolismo , DNA Helicases/antagonistas & inibidores , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Humanos , Neoplasias/tratamento farmacológico , Proteína Grupo D do Xeroderma Pigmentoso/metabolismo , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
The identification of molecules, which could modulate protein-protein interactions (PPIs), is of primary interest to medicinal chemists. Using biophysical methods during the current study, we have screened 76 compounds (grouped into 16 mixtures) against the p8 subunit of the general transcription factor (TFIIH), which has recently been validated as an anti-cancer drug target. 10% of the tested compounds showed interactions with p8 protein in STD-NMR experiments. These results were further validated by molecular docking studies where interactions between compounds and important amino acid residues were identified, including Lys20 in the hydrophobic core of p8, and Asp42 and 43 in the ß3 strand. Moreover, these compounds were able to destabilize the p8 protein by negatively shifting the Tm (≥2 °C) in thermal shift assay. Thus, this study has identified 8 compounds which are likely negative modulators of p8 protein stability, and could be further considered as potential anticancer agents.
Assuntos
Antineoplásicos/química , Bibliotecas de Moléculas Pequenas/química , Fator de Transcrição TFIIH/antagonistas & inibidores , Antineoplásicos/metabolismo , Antineoplásicos/toxicidade , Linhagem Celular , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Ligação Proteica , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/toxicidade , Eletricidade Estática , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/metabolismoRESUMO
Disruption of the enzymatic activities of the transcription factor TFIIH by the small molecules Triptolide (TPL) or THZ1 could be used against cancer. Here, we used the MCF10A-ErSrc oncogenesis model to compare the effect of TFIIH inhibitors between transformed cells and their progenitors. We report that tumour cells exhibited highly increased sensitivity to TPL or THZ1 and that the combination of both had a synergic effect. TPL affects the interaction between XPB and p52, causing a reduction in the levels of XPB, p52 and p8, but not other TFIIH subunits. RNA-Seq and RNAPII-ChIP-Seq experiments showed that although the levels of many transcripts were reduced, the levels of a significant number were increased after TPL treatment, with maintained or increased RNAPII promoter occupancy. A significant number of these genes encode for factors that have been related to tumour growth and metastasis, suggesting that transformed cells might rapidly develop resistance to TPL/THZ inhibitors. Some of these genes were also overexpressed in response to THZ1, of which depletion enhances the toxicity of TPL, and are possible new targets against cancer.
Assuntos
Neoplasias da Mama/metabolismo , Transformação Celular Neoplásica/efeitos dos fármacos , Diterpenos/farmacologia , Fenantrenos/farmacologia , Fenilenodiaminas/farmacologia , Pirimidinas/farmacologia , Fator de Transcrição TFIIH/antagonistas & inibidores , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Compostos de Epóxi/farmacologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Modelos Biológicos , Simulação de Dinâmica Molecular , Análise de Sequência de RNARESUMO
OBJECTIVE: Atherosclerosis is a disease characterized by abnormal lipid metabolism, and the formation of foam cells is considered an early event of atherosclerosis. Intracellular cholesterol efflux mediated by ABCA1 and ABCG1 helps to reduce lipid accumulation in foam cells. Related studies have shown that autophagy and mTOR are involved in cholesterol efflux, but the role of p62, an autophagy substrate protein, has not been evaluated. METHODS: THP-1 derived macrophages were incubated with ox-LDL to establish a foam cell model and treated with different autophagy inducers. The effects of p62 on cholesterol efflux were investigated using overexpression vectors, gene silencing and western blotting. RESULTS: This study showed a blockage of autophagy and decreased expression of ABCA1 and ABCG1 under the stress of excess ox-LDL in a concentration-dependent manner in THP-1â¯cells. Furthermore, the activation of autophagy led to increased expression of ABCA1 and ABCG1, as well as their upstream transcription factor LXRα, thereby promoting cholesterol efflux from foam cells. We also demonstrated that accumulated p62 played an important role during autophagy blockage, which was achieved by activating mTOR and then inhibited the expression of LXRα and its downstream target proteins ABCA1 and ABCG1. CONCLUSION: In conclusion, our experiments demonstrated that a p62/mTOR/LXRα signaling pathway was involved in cholesterol efflux mediated by ABCA1 and ABCG1 when autophagy blockage occurred. Our study offers a rationale for the development of autophagy and p62 as a new target for the treatment of atherosclerosis.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Colesterol/metabolismo , Receptores X do Fígado/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Autofagia/efeitos dos fármacos , Humanos , Receptores X do Fígado/antagonistas & inibidores , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/efeitos dos fármacos , Células THP-1 , Serina-Treonina Quinases TOR/antagonistas & inibidores , Fator de Transcrição TFIIH/antagonistas & inibidores , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/metabolismoRESUMO
Transcription factor IIH (TFIIH) is a multiprotein complex involved in both eukaryotic transcription and DNA repair, revealing a tight connection between these two processes. Composed of 10 subunits, it can be resolved into a 7-subunits core complex with the XPB translocase and the XPD helicase, and the 3-subunits kinase complex CAK, which also exists as a free complex with a distinct function. Initially identified as basal transcription factor, TFIIH also participates in transcription regulation and plays a key role in nucleotide excision repair (NER) for opening DNA at damaged sites, lesion verification and recruitment of additional repair factors. Our understanding of TFIIH function in eukaryotic cells has greatly benefited from studies of the genetic rare diseases xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD), that are not only characterized by cancer and aging predispositions but also by neurological and developmental defects. Although much remains unknown about TFIIH function, significant progresses have been done regarding the structure of the complex, the functions of its catalytic subunits and the multiple roles of the regulatory core-TFIIH subunits. This review provides a non-exhaustive survey of key discoveries on the structure and function of this pivotal factor, which can be considered as a promising target for therapeutic strategies.
Assuntos
Reparo do DNA , Fator de Transcrição TFIIH/metabolismo , Transcrição Gênica , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Fator de Transcrição TFIIH/antagonistas & inibidores , Transcrição Gênica/efeitos dos fármacosRESUMO
TFIIH is a 10-subunit complex involved in transcription and DNA repair. It contains several enzymatic activities including a ATP-dependent DNA translocase in XPB and a cyclin-dependent kinase in CDK7. Recently the discovery of several XPB and CDK7 inhibitors with specific impact on the transcriptional addiction of many tumors pinpointed these activities as potential target in cancer chemotherapy. Unexpectedly a basal transcription factor involved in global mRNA expression now emerges a one of the most clinically promising Achilles heels of cancerous cells. These inhibitors also proved to be useful tools to unveil new functions of TFIIH in gene expression.
Assuntos
Reparo do DNA/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Fator de Transcrição TFIIH/antagonistas & inibidores , Fator de Transcrição TFIIH/metabolismo , Transcrição Gênica/efeitos dos fármacos , Quinases Ciclina-Dependentes/antagonistas & inibidores , DNA Helicases/antagonistas & inibidores , Proteínas de Ligação a DNA/antagonistas & inibidores , Humanos , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/patologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Transcription factor IIH (TFIIH) is a multiprotein complex involved in both transcription and DNA repair, revealing a striking functional link between these two processes. Some of its subunits also belong to complexes involved in other cellular processes, such as chromosome segregation and cell cycle regulation, emphasizing the multitasking capabilities of this factor. This review aims to depict the structure of TFIIH and to dissect the roles of its subunits in different cellular mechanisms. Our understanding of the biochemistry of TFIIH has greatly benefited from studies focused on diseases related to TFIIH mutations. We address the etiology of these disorders and underline the fact that TFIIH can be considered a promising target for therapeutic strategies.