RESUMO
The phase equilibria of intrinsically disordered proteins are exquisitely sensitive to changes in solution conditions and this can be used to investigate the driving forces of phase separation in vitro as well as the biological roles of phase transitions in live cells. Here we investigate how using D2 O as co-solvent in an aqueous buffer changes the phase equilibrium of a fragment of the activation domain of the androgen receptor, a transcription factor that plays a role in the development of the male phenotype and is a therapeutic target for castration resistant prostate cancer. We show how replacing even small fractions of H2 O with D2 O increases the propensity of this fragment to undergo liquid-liquid phase separation, likely reflecting a stabilization of the hydrophobic interactions that drive condensation. Our results indicate that it is necessary to take this effect into consideration when studying phase separation phenomena with biophysical methods that require using D2 O as a co-solvent. In addition, they suggest that additions of D2 O may be used to enhance phase separation phenomena in cells, facilitating their observation.
Assuntos
Óxido de Deutério/química , Proteínas Intrinsicamente Desordenadas/química , Receptores Androgênicos/química , Humanos , Domínios ProteicosRESUMO
The androgen receptor is a transcription factor that plays a key role in the development of prostate cancer, and its interactions with general transcription regulators are therefore of potential therapeutic interest. The mechanistic basis of these interactions is poorly understood due to the intrinsically disordered nature of the transactivation domain of the androgen receptor and the generally transient nature of the protein-protein interactions that trigger transcription. Here, we identify a motif of the transactivation domain that contributes to transcriptional activity by recruiting the C-terminal domain of subunit 1 of the general transcription regulator TFIIF. These findings provide molecular insights into the regulation of androgen receptor function and suggest strategies for treating castration-resistant prostate cancer.
Assuntos
DNA/química , Proteínas Intrinsicamente Desordenadas/química , Receptores Androgênicos/química , Fatores de Transcrição TFII/química , Motivos de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Masculino , Modelos Moleculares , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Transcrição TFII/genética , Fatores de Transcrição TFII/metabolismo , Ativação TranscricionalRESUMO
Castration-resistant prostate cancer is the lethal condition suffered by prostate cancer patients that become refractory to androgen deprivation therapy. EPI-001 is a recently identified compound active against this condition that modulates the activity of the androgen receptor, a nuclear receptor that is essential for disease progression. The mechanism by which this compound exerts its inhibitory activity is however not yet fully understood. Here we show, by using high resolution solution nuclear magnetic resonance spectroscopy, that EPI-001 selectively interacts with a partially folded region of the transactivation domain of the androgen receptor, known as transactivation unit 5, that is key for the ability of prostate cells to proliferate in the absence of androgens, a distinctive feature of castration-resistant prostate cancer. Our results can contribute to the development of more potent and less toxic novel androgen receptor antagonists for treating this disease.
