Allosteric Conversation in the Androgen Receptor Ligand-Binding Domain Surfaces

Androgen receptor (AR) is a major therapeutic target that plays pivotal roles in prostate cancer (PCa) and androgen insensitivity syndromes. Wepreviously proposed that compounds recruited to ligand-binding domain (LBD) surfaces could regulate AR activity in hormone-refractory PCa and discovered several surface modulators of AR function. Surprisingly, the most effective compounds bound preferentially to a surface of unknown function [binding function 3 (BF-3)] instead of the coactivator-binding site [activation function 2 (AF-2)]. Different BF-3 mutations have been identified in PCa or androgen insensitivity syndrome patients, and they can strongly affect AR activity. Further, comparison of AR x-ray structures with and without bound ligands at BF-3 and AF-2 showed structural coupling between both pockets. Here, we combine experimental evidence and molecular dynamic simulations to investigate whether BF-3 mutations affect AR LBD function and dynamics possibly via allosteric conversation between surface sites. Our data indicate that AF-2 conformation is indeed closely coupled to BF-3 and provide mechanistic proof of their structural interconnection. BF-3 mutations may function as allosteric elicitors, probably shifting the AR LBD conformational ensemble toward conformations that alter AF-2 propensity to reorganize into subpockets that accommodate N-terminal domain and coactivator peptides. The induced conformation may result in either increased or decreased AR activity. Activating BF-3 mutations also favor the formation of another pocket (BF-4) in the vicinity of AF-2 and BF-3, which we also previously identified as a hot spot for a small compound. We discuss the possibility that BF-3 may be a protein-docking site that binds to the N-terminal domain and corepressors. AR surface sites are attractive pharmacological targets to develop allosteric modulators that might be alternative lead compounds for drug design. © 2012 by The Endocrie Society

From neural stem cells to glioblastoma: A natural history of GBM recapitulated in vitro

Due to its aggressive and invasive nature glioblastoma (GBM), the most common and aggressive primary brain tumour in adults, remains almost invariably lethal. Significant advances in the last several years have elucidated much of the molecular and genetic complexities of GBM. However, GBM exhibits a vast genetic variation and a wide diversity of phenotypes that have complicated the development of effective therapeutic strategies. This complex pathogenesis makes necessary the development of experimental models that could be used to further understand the disease, and also to provide a more realistic testing ground for potential therapies. In this report, we describe the process of transformation of primary mouse embryo astrocytes into immortalized cultures with neural stem cell characteristics, that are able to generate GBM when injected into the brain of C57BL/6 mice, or heterotopic tumours when injected IV. Overall, our results show that oncogenic transformation is the fate of NSC if cultured for long periods in vitro. In addition, as no additional hit is necessary to induce the oncogenic transformation, our model may be used to investigate the pathogenesis of gliomagenesis and to test the effectiveness of different drugs throughout the natural history of GBMThis study was supported in part by Xunta de Galicia (2014-PG029), University of Santiago de Compostela (2018-PU001), Ministerio de Economía y Competitividad (PI15/01129, José A. Costoya), the Centro Singular de Investigación de Galicia accreditation 2016–2019, ED431G/05), and the European Regional Development Fund (ERDF)S

EPI-001, a compound active against castration-resistant prostate cancer, targets transactivation unit 5 of the androgen receptor

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

Antibody cooperative adsorption onto AuNPs and its exploitation to force natural killer cells to kill HIV-infected T cells

HIV represents a persistent infection which negatively alters the immune system. New tools to reinvigorate different immune cell populations to impact HIV are needed. Herein, a novel nanotool for the specific enhancement of the natural killer (NK) immune response towards HIV-infected T-cells has been developed. Bispecific Au nanoparticles (BiAb-AuNPs), dually conjugated with IgG anti-HIVgp120 and IgG anti-human CD16 antibodies, were generated by a new controlled, linker-free and cooperative conjugation method promoting the ordered distribution and segregation of antibodies in domains. The cooperatively-adsorbed antibodies fully retained the capabilities to recognize their cognate antigen and were able to significantly enhance cell-to-cell contact between HIV-expressing cells and NK cells. As a consequence, the BiAb-AuNPs triggered a potent cytotoxic response against HIV-infected cells in blood and human tonsil explants. Remarkably, the BiAb-AuNPs were able to significantly reduce latent HIV infection after viral reactivation in a primary cell model of HIV latency. This novel molecularly-targeted strategy using a bispecific nanotool to enhance the immune system represents a new approximation with potential applications beyond HIV.This study was supported by the Spanish Secretariat of Science and Innovation and FEDER funds (grants SAF2015-67334-R and RTI2018-101082-B-I00 [MINECO/FEDER]), American National Institutes of Health (grant R21AI118411 to M.B), an unrestricted research grant from Bristol-Myers Squibb S.A.U (PfC-2015-AI424-564) to M.B, the Spanish “Ministerio de Economía y Competitividad, Instituto de Salud Carlos III” (ISCIII, PI17/01470) to M.G and the Spanish “Ministerio de Economía y Competitividad, Instituto de Salud Carlos III” (ISCIII, PI14/01058) to J.G.P, a research grant from Gilead Sciences (GLD17-00204 and GLD19-00084) to M.B, GeSIDA and the Spanish AIDS network “Red Temática Cooperativa de Investigación en SIDA” (RD16/0025/0007). The Miguel Servet program funded by the Spanish Health Institute Carlos III (CP17/00179) to M.B and J.G.P (CPII15/00014). The “Pla estratègic de recerca i innovació en salut” (PERIS), from the Catalan Government to M.G. The Spanish Secretariat of Science and Innovation Ph.D. fellowship to A.A-G (BES-2016-076382), AGAUR-FI-B-00582 Ph.D. fellowship from the Catalan Government to O.BL, and PIF-UAB Ph.D. fellowship from Universitat Autònoma de Barcelona to R.SL.Peer reviewe

The Oncoprotein BCL11A Binds to Orphan Nuclear Receptor TLX and Potentiates its Transrepressive Function

Nuclear orphan receptor TLX (NR2E1) functions primarily as a transcriptional repressor and its pivotal role in brain development, glioblastoma, mental retardation and retinopathologies make it an attractive drug target. TLX is expressed in the neural stem cells (NSCs) of the subventricular zone and the hippocampus subgranular zone, regions with persistent neurogenesis in the adult brain, and functions as an essential regulator of NSCs maintenance and self-renewal. Little is known about the TLX social network of interactors and only few TLX coregulators are described. To identify and characterize novel TLX-binders and possible coregulators, we performed yeast-two-hybrid (Y2H) screens of a human adult brain cDNA library using different TLX constructs as baits. Our screens identified multiple clones of Atrophin-1 (ATN1), a previously described TLX interactor. In addition, we identified an interaction with the oncoprotein and zinc finger transcription factor BCL11A (CTIP1/Evi9), a key player in the hematopoietic system and in major blood-related malignancies. This interaction was validated by expression and coimmunoprecipitation in human cells. BCL11A potentiated the transrepressive function of TLX in an in vitro reporter gene assay. Our work suggests that BCL11A is a novel TLX coregulator that might be involved in TLX-dependent gene regulation in the brain

Estudi de la interacció del pèptid de fusió de la proteïna gp41 del VIH amb membranes model

El Virus de la Immunodeficiència Humana, VIH, és un virus amb envolta de la família Retroviridae. El primer pas en el procés d'infecció del virus és la seva entrada dins de la cèl·lula hoste. En el cas d'infecció de limfòcits T, primer es produeix el reconeixement del receptor CD4 del limfòcit per part de la proteïna gp120 del virus. Aquest proteïna està unida mitjançant interaccions de tipus febles a una altra proteïna de l'envolta lipídica del virus, la proteïna gp41. Aquesta proteïna és la responsable final de la fusió entre la membrana plasmàtica de la cèl·lula hoste i l'envolta lipídica del virus, permetent l'entrada d'aquest últim al citosol de la cèl·lula i iniciant el procés d'infecció.La proteïna gp41 presenta en el seu extrem amino terminal una seqüència molt hidrofòbica i rica en residus de glicina i alanina, la qual cosa fa que sigui conformacionalment polimòrfca, i que es coneix com pèptid de fusió. Un cop produït el reconeixement, la gp120 sofreix un canvi conformacional que es transmet a la gp41, de manera que el pèptid de fusió interacciona amb la membrana del limfòcit, desencadenant el procés de fusió.En el present treball s'ha estudiat la interacció de pèptids de fusió de la proteïna gp41 del VIH amb membranes model. Els resultats obtinguts han permès proposar un model cinètic dels diferents processos que tenen lloc durant la interacció d'aquests pèptids amb membranes model, contribuint de manera remarcable a resoldre una qüestió fins ara controvertida, referent al tipus d'estructura que adopta el pèptid de fusió quan interacciona amb les membranes i que desencadena la fusió. El treball també ha permès determinar la importància del potencial dipolar de membrana, un paràmetre físico-químic de les membranes, en aquests processos així com la importància de la presència de colesterol en les membranes model. Segons els resultats obtinguts, el pèptid de fusió primerament interaccionaria amb la membrana. Un cop unit a aquesta, el pèptid de fusió experimentaria un canvi conformacional, passant d'una barreja d'estructura helicoïdal, desordenada i de fulla ?, a més estrcutura ?, la qual finalment seria l'estructura responsable de la fusió. A més, el pèptid durant la interacció provocaria una desestabilització de la membrana, una agregació de membranes model i una deshidratació de la seva interfície aigua-lípid. El potencial dipolar de membrana, afectaria tan sols al procés d'unió del pèptid de fusió a les membranes, mentre que no afectaria al canvi conformacional ni a la fusió. Aquests últims estarien afectats per la hidratació de les membranes i/o per la seva fluïdesa, tal i com mostren els resultats obtinguts amb el colesterol. Finalment el treball ens ha permès formular la qüestió de si els agregats peptídics amorfs detectats en el cas dels pèptids de fusió del VIH podrien correspondre a un tipus conformacional més general, implicat en d'altres processos amb rellevància biològica, com l'efecte citotòxic dels pèptids amiloïdes relacionat amb certes malalties neurodegeneratives.El conexeixement dels processos moleculars de la infecció vírica podria permetre el disseny de noves estratègies farmacològiques que podrien inhibir la entrada del virus a la cèl·lula.The Human Immunodeficiency Virus, HIV, is an enveloped virus of the Retroviridae family. The first step in the sequence of events leading to the virus entry into the target cell is the binding of gp120 to the CD4 receptor on T-lymphocytes membranes. The gp120 protein is noncovalently associated to gp41 protein, which is the protein responsible of fusion between cellular and viral membranes, allowing virus entry in the target cell.The gp41 contains in its amino terminus a sequence rich in glycine and alanine residues, the so-called fusion peptide. Following the interaction of gp120 with the CD4 receptor, a series of conformational rearrangements takes place, which result in the interaction of the N-terminal part of gp41 with the cell membrane. This process has been described to trigger the fusion of the viral and the cellular membranes.In the present work we have studied the interaction of HIV gp41 fusion peptide with model membranes. The results obtained allow us to propose a kinetic model for the different steps that take place during interaction of fusion peptide with model membranes, contributing to resolve a controversial question about the secondary structure that adopts the fusion peptide upon interaction with membranes and that trigger membrane fusion. Results also allowed us to determine the significance of membrane dipole potential on the interaction of fusion peptides with the membranes, an effect which is due to the presence of cholesterol in the model membranes. The results showed that membrane fusion depends on the transformation of unordered and probably helical structures into ? aggregates structures and that such a conformational change occurs upon binding to the membrane. Moreover, this interaction would induce membrane destabilization, vesicle aggregation and the dehydration of the membrane surface. The membrane dipole potential would only affect the binding process but would not affect either the conformational change or the fusion. These two processes would be affected by surface membrane hydration and/or membrane fluidity. Finally this work allowed us hypothesize whether amorphous peptidic aggregates detected in the case of HIV fusion peptides would correspond to a more general structure, that could be involved in others relevant biological processes, such as the cytotoxic effect of amyloid peptides which is related with certain neurodegenerative diseases.The knowledge of molecular mechanisms underlying viral infection could facilitate the design of novel pharmacological strategies against the viral entry into the target cell

GPE Promotes the Proliferation and Migration of Mouse Embryonic Neural Stem Cells and Their Progeny In Vitro

This study was designed to investigate a possible role of the N-terminal tripeptide of insulin-like growth factor-1 (IGF-I), Gly-Pro-Glu (GPE), physiologically generated in neurons following IGF-I-specific cleavage, in promoting neural regeneration after an injury. Primary cultures of mouse neural stem cells (NSCs), obtained from 13.5 Days post-conception (dpc) mouse embryos, were challenged with either GPE, growth hormone (GH), or GPE + GH and the effects on cell proliferation, migration, and survival were evaluated both under basal conditions and in response to a wound healing assay. The cellular pathways activated by GPE were also investigated by using specific chemical inhibitors. The results of the study indicate that GPE treatment promotes the proliferation and the migration of neural stem cells in vitro through a mechanism that involves the activation of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase PI3K-Akt pathways. Intriguingly, both GPE effects and the signaling pathways activated were similar to those observed after GH treatment. Based upon the results obtained from this study, GPE, as well as GH, may be useful in promoting neural protection and/or regeneration after an injuryS

The oncoprotein BCL11A binds to human TLX.

<p>(A) Schematic diagram of the different isoforms of BCL11A (XL, L, S) and corresponding exons (1, E2, E3, E4, E5L, E5S). All BCL11A identified clones share a 159 amino acid overlapping region (residues 586–744), whose amino acid sequence is shown. Previously BCL11A regions identified to bind to COUP-FT and named ID-1 and ID-2 are highlighted in light grey. Clone 1 features a novel combination of exons, while clones 3, and 4 contain an intronic sequence. All the identified clones contain ID-2, but only some of them also have ID-2 as well. (B) Validation of the interaction between TLX and the identified BCL11A clones by forward one-to-one Y2H assay. TLX constructs FL-TLX (1–385), TLX-LBD (172–385), TLX-H-LBD (94–385) and TLX-DBD (1–95) (baits) were tested for interaction with the BCL11A clones identified (preys). Yeast transformants were plated on a control plate (lacking Trp and Leu) and plated on a selective plate (lacking Trp, Leu, His supplemented with 50 mM 3AT).</p

Human TLX recruits ATN1.

<p>Y2H screens using TLX-FL and TLX-LBD domain as baits against an adult brain cDNA library identified ATN1 as a TLX-interactor. (A) Schematic diagram of the identified ATN1 clones. All clones share a 378 amino acid overlapping region (residues 813–1190) featuring the ATRO-BOX region. (B) Validation of the interaction between TLX and the identified ATN1 clones by forward one-to-one Y2H assay. TLX constructs FL-TLX (1–385), TLX-LBD (172–385), TLX-H-LBD (94–385) and TLX-DBD (1–95) (baits) were tested for interaction with the ATN1 clones identified (preys). Yeast transformants were plated on a control plate (lacking Trp and Leu) and plated on a selective plate (lacking Trp, Leu, His supplemented with 50 mM 3AT).</p