RESUMEN
Mitofusin 2 (Mfn2) is a key protein in mitochondrial fusion and it participates in the bridging of mitochondria to the endoplasmic reticulum (ER). Recent data indicate that Mfn2 ablation leads to ER stress. Here we report on the mechanisms by which Mfn2 modulates cellular responses to ER stress. Induction of ER stress in Mfn2-deficient cells caused massive ER expansion and excessive activation of all three Unfolded Protein Response (UPR) branches (PERK, XBP-1, and ATF6). In spite of an enhanced UPR, these cells showed reduced activation of apoptosis and autophagy during ER stress. Silencing of PERK increased the apoptosis of Mfn2-ablated cells in response to ER stress. XBP-1 loss-of-function ameliorated autophagic activity of these cells upon ER stress. Mfn2 physically interacts with PERK, and Mfn2-ablated cells showed sustained activation of this protein kinase under basal conditions. Unexpectedly, PERK silencing in these cells reduced ROS production, normalized mitochondrial calcium, and improved mitochondrial morphology. In summary, our data indicate that Mfn2 is an upstream modulator of PERK. Furthermore, Mfn2 loss-of-function reveals that PERK is a key regulator of mitochondrial morphology and function.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Mitocondrias/metabolismo , Respuesta de Proteína Desplegada/fisiología , eIF-2 Quinasa/metabolismo , Factor de Transcripción Activador 6/genética , Factor de Transcripción Activador 6/metabolismo , Animales , Apoptosis/genética , Autofagia/genética , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Estrés del Retículo Endoplásmico , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , GTP Fosfohidrolasas/genética , Técnicas de Inactivación de Genes , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Mitocondrias/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción del Factor Regulador X , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína 1 de Unión a la X-Box , eIF-2 Quinasa/genéticaRESUMEN
Brown adipose tissue (BAT) thermogenesis affects energy balance, and thereby it has the potential to induce weight loss and to prevent obesity. Here, we document a macroautophagic/autophagic-dependent mechanism of peroxisome proliferator-activated receptor gamma (PPARG) activity regulation that induces brown adipose differentiation and thermogenesis and that is mediated by TP53INP2. Disruption of TP53INP2-dependent autophagy reduced brown adipogenesis in cultured cells. In vivo specific-tp53inp2 ablation in brown precursor cells or in adult mice decreased the expression of thermogenic and mature adipocyte genes in BAT. As a result, TP53INP2-deficient mice had reduced UCP1 content in BAT and impaired maximal thermogenic capacity, leading to lipid accumulation and to positive energy balance. Mechanistically, TP53INP2 stimulates PPARG activity and adipogenesis in brown adipose cells by promoting the autophagic degradation of NCOR1, a PPARG co-repressor. Moreover, the modulation of TP53INP2 expression in BAT and in human brown adipocytes suggests that this protein increases PPARG activity during metabolic activation of brown fat. In all, we have identified a novel molecular explanation for the contribution of autophagy to BAT energy metabolism that could facilitate the design of therapeutic strategies against obesity and its metabolic complications.
Asunto(s)
Tejido Adiposo Pardo , PPAR gamma , Ratones , Humanos , Animales , Tejido Adiposo Pardo/metabolismo , PPAR gamma/metabolismo , Autofagia , Obesidad/metabolismo , Termogénesis/genética , Proteínas Nucleares/metabolismo , Co-Represor 1 de Receptor Nuclear/metabolismoRESUMEN
Transcription factors are among the most attractive therapeutic targets but are considered largely 'undruggable' in part due to the intrinsically disordered nature of their activation domains. Here we show that the aromatic character of the activation domain of the androgen receptor, a therapeutic target for castration-resistant prostate cancer, is key for its activity as transcription factor, allowing it to translocate to the nucleus and partition into transcriptional condensates upon activation by androgens. On the basis of our understanding of the interactions stabilizing such condensates and of the structure that the domain adopts upon condensation, we optimized the structure of a small-molecule inhibitor previously identified by phenotypic screening. The optimized compounds had more affinity for their target, inhibited androgen-receptor-dependent transcriptional programs, and had an antitumorigenic effect in models of castration-resistant prostate cancer in cells and in vivo. These results suggest that it is possible to rationally optimize, and potentially even to design, small molecules that target the activation domains of oncogenic transcription factors.
Asunto(s)
Neoplasias de la Próstata Resistentes a la Castración , Neoplasias de la Próstata , Masculino , Humanos , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/genética , Receptores Androgénicos/genética , Receptores Androgénicos/química , Andrógenos/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Dominios Proteicos , Factores de Transcripción , Línea Celular TumoralRESUMEN
Cancer cachexia is a multifactorial syndrome characterized by anorexia, weight loss and muscle wasting that impairs patients' quality of life and survival. Aim of this work was to evaluate the impact of either autophagy inhibition (knocking down beclin-1) or promotion (overexpressing TP53INP2/DOR) on cancer-induced muscle wasting. In C26 tumor-bearing mice, stress-induced autophagy inhibition was unable to rescue the loss of muscle mass and worsened muscle morphology. Treating C26-bearing mice with formoterol, a selective ß2-agonist, muscle sparing was paralleled by reduced static autophagy markers, although the flux was maintained. Conversely, the stimulation of muscle autophagy exacerbated muscle atrophy in tumor-bearing mice. TP53INP2 further promoted atrogene expression and suppressed mitochondrial dynamics-related genes. Excessive autophagy might impair mitochondrial function through mitophagy. Consistently, tumor-induced mitochondrial dysfunction was detected by reduced ex vivo muscle fiber respiration. Overall, the results evoke a central role for muscle autophagy in cancer-induced muscle wasting.
Asunto(s)
Caquexia/complicaciones , Mitocondrias/patología , Atrofia Muscular/complicaciones , Neoplasias/complicaciones , Síndrome Debilitante/complicaciones , Animales , Autofagia , Caquexia/patología , Línea Celular Tumoral , Femenino , Humanos , Masculino , Ratones Endogámicos C57BL , Músculo Esquelético/patología , Atrofia Muscular/patología , Neoplasias/patología , Síndrome Debilitante/patologíaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Molecular chaperones such as Hsp40 and Hsp70 hold the androgen receptor (AR) in an inactive conformation. They are released in the presence of androgens, enabling transactivation and causing the receptor to become aggregation-prone. Here we show that these molecular chaperones recognize a region of the AR N-terminal domain (NTD), including a FQNLF motif, that interacts with the AR ligand-binding domain (LBD) upon activation. This suggests that competition between molecular chaperones and the LBD for the FQNLF motif regulates AR activation. We also show that, while the free NTD oligomerizes, binding to Hsp70 increases its solubility. Stabilizing the NTD-Hsp70 interaction with small molecules reduces AR aggregation and promotes its degradation in cellular and mouse models of the neuromuscular disorder spinal bulbar muscular atrophy. These results help resolve the mechanisms by which molecular chaperones regulate the balance between AR aggregation, activation and quality control.
Asunto(s)
Andrógenos/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Receptores Androgénicos/metabolismo , Animales , Técnicas de Sustitución del Gen , Células HEK293 , Humanos , Ligandos , Masculino , Ratones , Ratones Transgénicos , Resonancia Magnética Nuclear Biomolecular , Agregado de Proteínas , Dominios Proteicos , Multimerización de Proteína , Receptores Androgénicos/química , Receptores Androgénicos/genética , SolubilidadRESUMEN
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.