32 research outputs found
EPI-001, A Compound Active against Castration-Resistant Prostate Cancer, Targets Transactivation Unit 5 of the Androgen Receptor
ACKNOWLEDGEMENTS We thank J. M. Valverde (IRB) as well as the NMR facilities of the University of Barcelona (CCiT UB) and the Instituto de Química Física Rocasolano (IQFR, CSIC) for their assistance in, respectively, protein production and NMR. This work was supported by IRB, ICREA (X.S.), Obra Social “la Caixa” (Fellowship to E.D.M. and CancerTec grants to X.S.) MICINN (CTQ2009-08850 to X.S.), MINECO (BIO2012-31043 to X.S.; CTQ2014-56361-P to A.R), Marató de TV3 (102030 to X.S. and 102031 to E.E.P) the COFUND programme of the European Commission (C.T.W.P., A. R. and X.S.), the European Research Council (CONCERT, contract number 648201, to X.S.), the Ramón y Cajal program of MICINN (RYC-2011-07873 to C.W.B.) the Serra Hunter Programme (E.E.P.) and AGAUR (SGR-2014-56RR14 to E.E.P). IRB Barcelona is the recipient of a Severo Ochoa Award of Excellence from MINECO (Government of Spain)Peer reviewedPostprin
RARRES3 suppresses breast cancer lung metastasis by regulating adhesion and differentiation
In estrogen receptor-negative breast cancer patients, metastatic relapse usually occurs in the lung and is responsible for the fatal outcome of the disease. Thus, a better understanding of the biology of metastasis is needed. In particular, biomarkers to identify patients that are at risk of lung metastasis could open the avenue for new therapeutic opportunities. Here we characterize the biological activity of RARRES3, a new metastasis suppressor gene whose reduced expression in the primary breast tumors identifies a subgroup of patients more likely to develop lung metastasis. We show that RARRES3 downregulation engages metastasis-initiating capabilities by facilitating adhesion of the tumor cells to the lung parenchyma. In addition, impaired tumor cell differentiation due to the loss of RARRES3 phospholipase A1/A2 activity also contributes to lung metastasis. Our results establish RARRES3 downregulation as a potential biomarker to identify patients at high risk of lung metastasis who might benefit from a differentiation treatment in the adjuvant programme.We would like to thank the Functional Genomics, Microscopy, and Cytometry core facilities of IRB Barcelona, and the UB. We thank C. Caelles for the 3AOX-luc construct. We thank Angel Nebreda for his scientific suggestions. EJA is supported by "La Caixa" PhD fellowship programme, and JU is a Juan de la Cierva Researcher (MICINN). JM is a Howard Hughes investigator. The work of A. C. and S.F-R is supported by the Ramon y Cajal award to AC (Spanish Ministry of Education) and the ERC (336343). JM was supported by HHMI. RRG and XS are ICREA Research Professors (Institucio Catalana de Recerca i Estudis Avancats). Support and structural funds were provided by the Associacion Espanola Contra el Cancer (AECC), Fundacion BBVA, Generalitat de Catalunya (2009 SGR 1429), and Spanish Ministerio de Ciencia e Innovacion (MICINN) (SAF2010-21171) to RRG
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
Understanding biomolecular motion, recognition, and allostery by use of conformational ensembles
We review the role conformational ensembles can play in the analysis of biomolecular dynamics, molecular recognition, and allostery. We introduce currently available methods for generating ensembles of biomolecules and illustrate their application with relevant examples from the literature. We show how, for binding, conformational ensembles provide a way of distinguishing the competing models of induced fit and conformational selection. For allostery we review the classic models and show how conformational ensembles can play a role in unravelling the intricate pathways of communication that enable allostery to occur. Finally, we discuss the limitations of conformational ensembles and highlight some potential applications for the future
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Integrated description of protein dynamics from room-temperature X-ray crystallography and NMR
Detailed descriptions of atomic coordinates and motions are required for an understanding of protein dynamics and their relation to molecular recognition, catalytic function, and allostery. Historically, NMR relaxation measurements have played a dominant role in the determination of the amplitudes and timescales (picosecond-nanosecond) of bond vector fluctuations, whereas high-resolution X-ray diffraction experiments can reveal the presence of and provide atomic coordinates for multiple, weakly populated substates in the protein conformational ensemble. Here we report a hybrid NMR and X-ray crystallography analysis that provides a more complete dynamic picture and a more quantitative description of the timescale and amplitude of fluctuations in atomic coordinates than is obtainable from the individual methods alone. Order parameters (S(2)) were calculated from single-conformer and multiconformer models fitted to room temperature and cryogenic X-ray diffraction data for dihydrofolate reductase. Backbone and side-chain order parameters derived from NMR relaxation experiments are in excellent agreement with those calculated from the room-temperature single-conformer and multiconformer models, showing that the picosecond timescale motions observed in solution occur also in the crystalline state. These motions are quenched in the crystal at cryogenic temperatures. The combination of NMR and X-ray crystallography in iterative refinement promises to provide an atomic resolution description of the alternate conformational substates that are sampled through picosecond to nanosecond timescale fluctuations of the protein structure. The method also provides insights into the structural heterogeneity of nonmethyl side chains, aromatic residues, and ligands, which are less commonly analyzed by NMR relaxation measurements
Modeling of Hidden Structures Using Sparse Chemical Shift Data from NMR Relaxation Dispersion
NMR relaxation dispersion measurements report on conformational changes occurring on the μs-ms timescale. Chemical shift information derived from relaxation dispersion can be used to generate structural models of weakly populated alternative conformational states. Current methods to obtain such models rely on determining the signs of chemical shift changes between the conformational states, which are difficult to obtain in many situations. Here, we use a "sample and select" method to generate relevant structural models of alternative conformations of the C-terminal-associated region of Escherichia coli dihydrofolate reductase (DHFR), using only unsigned chemical shift changes for backbone amides and carbonyls (1H, 15N, and 13C'). We find that CS-Rosetta sampling with unsigned chemical shift changes generates a diversity of structures that are sufficient to characterize a minor conformational state of the C-terminal region of DHFR. The excited state differs from the ground state by a change in secondary structure, consistent with previous predictions from chemical shift hypersurfaces and validated by the x-ray structure of a partially humanized mutant of E. coli DHFR (N23PP/G51PEKN). The results demonstrate that the combination of fragment modeling with sparse chemical shift data can determine the structure of an alternative conformation of DHFR sampled on the μs-ms timescale. Such methods will be useful for characterizing alternative states, which can potentially be used for in silico drug screening, as well as contributing to understanding the role of minor states in biology and molecular evolution
Slow Dynamics of Tryptophan–Water Networks in Proteins
Water has a profound effect on the
dynamics of biomolecules and
governs many biological processes, leading to the concept that function
is slaved to solvent dynamics within and surrounding the biomolecule.
Protein conformational changes on μs–ms time scales are
frequently associated with protein function, but little is known about
the behavior of protein-bound water on these time scales. Here we
have used NMR relaxation dispersion measurements to probe the tryptophan
indoles in the enzyme dihydrofolate reductase (DHFR). We find that
during structural changes on the μs–ms time scale, large
chemical shift changes are often observed for the NH proton on the
indole ring, while relatively smaller chemical shift changes are observed
for the ring nitrogen atom. Comparison with experimental chemical
shifts and density functional theory-based chemical shift predictions
show that during the structural change the tryptophan indole NHs remain
bound to water, but the geometry of the protein-bound water networks
changes. These results establish that relaxation dispersion measurements
can indirectly probe water dynamics and indicate that water can influence,
or be influenced by, protein conformational changes on the μs–ms
time scale. Our data show that structurally conserved bound water
molecules can play a critical role in transmitting information between
functionally important regions of the protein and provide evidence
that internal protein motions can be coupled through the mediation
of hydrogen-bonded water bound in the protein structure