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SummaryTranscription factors are among the most attractive therapeutic targets but are considered largely undruggable 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 a transcription factor by allowing it to partition into transcriptional condensates. Based on this knowledge we optimized the structure of a small molecule inhibitor, previously identified by phenotypic screening, that targets a specific transactivation unit within the domain that is partially folded and rich in aromatic residues. The optimized compounds had more affinity for their target, inhibited androgen receptor-dependent transcriptional programs, and had antitumorigenic effect in models of castration-resistant prostate cancer in cells andin vivo. These results establish a generalizable framework to target small molecules to the activation domains of oncogenic transcription factors and other disease-associated proteins with therapeutic intent.

32 pags., 17 figs., 1 tab., The regular functioning of the nucleolus and nucleus-mitochondria crosstalk are considered unrelated processes, yet cytochrome c (Cc) migrates to the nucleus and even the nucleolus under stress conditions. Nucleolar liquid-liquid phase separation usually serves the cell as a fast, smart mechanism to control the spatial localization and trafficking of nuclear proteins. Actually, the alternative reading frame (ARF), a tumor suppressor protein sequestered by nucleophosmin (NPM) in the nucleoli, is shifted out from NPM upon DNA damage. DNA damage also triggers early translocation of respiratory Cc to nucleus before cytoplasmic caspase activation. Here, we show that Cc can bind to nucleolar NPM by triggering an extended-to-compact conformational change, driving ARF release. Such a NPM-Cc nucleolar interaction can be extended to a general mechanism for DNA damage in which the lysine-rich regions of Cc-rather than the canonical, arginine-rich stretches of membrane-less organelle components-controls the trafficking and availability of nucleolar proteins., We thank the staf at the NMR facility at CITIUS (University of Seville), the microscopy facility at CABIMER (Seville) and the ALBA Synchrotron (Barcelona). We are grateful to the Spanish Government (grant nos. PID2021-126663NB-I00, PGC2018-096049-B-I00/FEDER, BIO2015-70092-R, BFU2015-71017/BMC, BFU2016-75984/BMC, PID2019-105872GB and BFU2017-90030-P, FEDER/Ministerio de Ciencia e Innovación–Agencia Estatal de Investigación), European Regional Development Fund (FEDER), the Regional Government of Andalusia (grant nos. BIO-198; US-1254317, US-1257019, P18-FR-3487 and P18-HO-4091, US/JUNTA/FEDER, UE), the European Commission: European Regional Development Fund and European Research Council (CONCERT, contract number 648201) and the Ramón Areces Foundation. This work has been supported by Infrastructure for NMR, EM and X-rays for Translational Research (iNEXT, grant no. PID 3407) funded by the Horizon 2020 program of the European Commission, Cámara Foundation (C.A.E.R.´s fellowship), the Spanish Ministry of Education, Culture and Sports (grant nos. FPU18/06577, FPU16/01513 and FPU013/04373; M.A.C.-C., A.V.-C. and F.R.-R.’s fellowships, respectively), La Caixa Foundation (E.S.’s fellowship), Severo Ochoa Award of Excellence from MINECO (Government of Spain, IRB Barcelona and CNB Madrid) and the Spanish Association Against Cancer Scientific Foundation (FC AECC, M.P.G.’s postdoctoral grant)

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 D(2)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 H(2)O with D(2)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 D(2)O as a co‐solvent. In addition, they suggest that additions of D(2)O may be used to enhance phase separation phenomena in cells, facilitating their observation.

The alternative reading frame (ARF) protein is crucial in the cellular response to oncogenic stress, being likewise the second most frequently inactivated gene in a wide spectrum of human cancers. ARF is usually sequestered in the nucleolus by the well-known oncogenic nucleophosmin (NPM) protein and is liberated in response to cell damage to exhibit its tumor-suppressor ability. However, the mechanism underlying ARF activation is unknown. Here we show that mitochondria-to-nucleus translocation of cytochrome c upon DNA damage leads to the break-off of the NPM-ARF ensemble and subsequent release of ARF from the nucleoli. Our structural and subcellular data support a molecular model in which the hemeprotein triggers the extended-to-compact conformation of NPM and competes with ARF for binding to NPM.

Summary Prion-like proteins form multivalent assemblies and phase separate into membraneless organelles. Heterogeneous ribonucleoprotein D-like (hnRNPDL) is a RNA-processing prion-like protein with three alternative splicing (AS) isoforms, which lack none, one, or both of its two disordered domains. It has been suggested that AS might regulate the assembly properties of RNA-processing proteins by controlling the incorporation of multivalent disordered regions in the isoforms. This, in turn, would modulate their activity in the downstream splicing program. Here, we demonstrate that AS controls the phase separation of hnRNPDL, as well as the size and dynamics of its nuclear complexes, its nucleus-cytoplasm shuttling, and amyloidogenicity. Mutation of the highly conserved D378 in the disordered C-terminal prion-like domain of hnRNPDL causes limb-girdle muscular dystrophy 1G. We show that D378H/N disease mutations impact hnRNPDL assembly properties, accelerating aggregation and dramatically reducing the protein solubility in the muscle of Drosophila, suggesting a genetic loss-of-function mechanism for this muscular disorder., Graphical Abstract, Highlights • hnRNPDL requires both N- and C-terminal IDRs to phase separate • The absence of N-terminal IDR facilitates aggregation • The unique combination of IDRs in each isoform determines its cellular behavior • D378N/H mutations accelerate hnRNPDL aggregation and compromise its solubility, Batlle et al. show that alternative splicing controls heterogeneous ribonucleoprotein D-like (hnRNPDL) phase separation, aggregation, and solubility. Mutations that cause LGMD1G accelerate hnRNPDL aggregation and promote insolubility in Drosophila.

The aim of the paper is to discuss the course of development of methodology of economic convergence analyses, which points up the necessity of taking into consideration spatial connections among regions in regional growth models. It presents empirical models of ß-convergence concerning the economic growth of European regions using various methodological conceptions. In the paper the models offered by spatial econometrics are recommended. The empirical data refer to per capita GDP across the European Union regions at a NUTS-2 level over the period 1995–2009 (annual data).

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. Fil: de Mol, Eva. Barcelona Institute of Science and Technology. Institute for Research in Biomedicine; España Fil: Fenwick, R. Bryn. Barcelona Institute of Science and Technology. Institute for Research in Biomedicine; España Fil: Phang, Christopher T. W.. Barcelona Institute of Science and Technology. Institute for Research in Biomedicine; España Fil: Buzón, Victor. Universidad de Barcelona; España Fil: Szulc, Elzbieta. Barcelona Institute of Science and Technology. Institute for Research in Biomedicine; España Fil: de la Fuente, Alex. Barcelona Institute of Science and Technology. Institute for Research in Biomedicine; España Fil: Escobedo, Albert. Barcelona Institute of Science and Technology. Institute for Research in Biomedicine; España Fil: García, Jesús. Barcelona Institute of Science and Technology. Institute for Research in Biomedicine; España Fil: Bertoncini, Carlos Walter. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Barcelona Institute of Science and Technology. Institute for Research in Biomedicine; España Fil: Estébanez Perpiñá, Eva. Universidad de Barcelona; España Fil: McEwan, Iain J.. University of Aberdeen; Reino Unido Fil: Riera, Antoni. Barcelona Institute of Science and Technology. Institute for Research in Biomedicine; España. Universidad de Barcelona; España Fil: Salvatella, Xavier. Barcelona Institute of Science and Technology. Institute for Research in Biomedicine; España. Institució Catalana de Recerca i Estudis Avancats; España

The paper concerns the measurement of the dependence between economic spatial processes at various levels of data aggregation. The considerations refer to the investigations confirming efficiency of the so-called quasi-congruent spatial model as a tool of the measurement of the dependence between economic processes, on condition that the model description of the structures of the processes is correct and sufficient. The main problem of the paper is to discuss such a description of the spatial connections, which would be adequate to express the autodependence of the investigated processes. The application of the economic distance, which characterizes the similarity of the regions on the ground of the values of the analyzed processes, is proposed.

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. Identifying ways to inhibit the androgen receptor (AR) is key for developing treatments for castration-resistant prostate cancer. Here, De Mol, Szulc et al. show that AR activity relies on transient interactions of a disordered motif with the transcription machinery and suggest therapeutic strategies for this disease. Fil: De Mol, Eva. Institute for Research in Biomedicine; España Fil: Szulc, Elzbieta. Institute for Research in Biomedicine; España Fil: Di Sanza, Claudio. Institute for Research in Biomedicine; España Fil: Martínez Cristóbal, Paula. Institute for Research in Biomedicine; España Fil: Bertoncini, Carlos Walter. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Institute for Research in Biomedicine; España Fil: Fenwick, R. Bryn. Institute for Research in Biomedicine; España. The Scripps Research Institute; Estados Unidos Fil: Frigolé-Vivas, Marta. Institute for Research in Biomedicine; España Fil: Masin, Marianela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Institute for Research in Biomedicine; España Fil: Hunter, Irene. University of Aberdeen; Reino Unido Fil: Buzón, Víctor. Institute for Research in Biomedicine; España Fil: Brun Heath, Isabelle. Institute for Research in Biomedicine; España Fil: García, Jesús. Institute for Research in Biomedicine; España Fil: De Fabritiis, Gianni. Universitat Pompeu Fabra; España. Institució Catalana de Recerca i Estudis Avancats; España Fil: Estébanez Perpiñá, Eva. Universidad de Barcelona; España Fil: McEwan, Iain J.. University of Aberdeen; Reino Unido Fil: Nebreda, Ángel R.. Institute for Research in Biomedicine; España. Institució Catalana de Recerca i Estudis Avancats; España Fil: Salvatella, Xavier. Institució Catalana de Recerca i Estudis Avancats; España. Institute for Research in Biomedicine; España

Summary Mutations in the tumor suppressor SPOP (speckle-type POZ protein) cause prostate, breast, and other solid tumors. SPOP is a substrate adaptor of the cullin3-RING ubiquitin ligase and localizes to nuclear speckles. Although cancer-associated mutations in SPOP interfere with substrate recruitment to the ligase, mechanisms underlying assembly of SPOP with its substrates in liquid nuclear bodies and effects of SPOP mutations on assembly are poorly understood. Here, we show that substrates trigger phase separation of SPOP in vitro and co-localization in membraneless organelles in cells. Enzymatic activity correlates with cellular co-localization and in vitro mesoscale assembly formation. Disease-associated SPOP mutations that lead to the accumulation of proto-oncogenic proteins interfere with phase separation and co-localization in membraneless organelles, suggesting that substrate-directed phase separation of this E3 ligase underlies the regulation of ubiquitin-dependent proteostasis.