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Research Units - Molecular Oncology

Structural Biology Unit

Laboratory 8

We study the structure-function of proteins relevant for the onset and progression of cancer. Our main work consists on the structural characterization of proteins involved in chromatin dynamics and cell adhesion by means of NMR and other biophysical techniques.

The proteins of the family INhibitors of Growth (ING1-5) form part of chromatin remodelling complexes modulating the transcription levels of their target genes. Their primary biological function is the inhibition of cell growth and proliferation, and enhance apoptosis in response to genotoxic stress. We have characterised the structure of ING4, which has a conserved N-terminal coiled-coil dimerization domain, a central flexible region, and a C-terminal PlantHomeoDomain (PHD). ING4 recruits the histone acetylation complex HBO1 to chromatin sites with the histone H3 trimethylated at lysine 4 (H3K4me3), which results in the acetylation of histone H3 at lysine 9. The PHD binds H3K4me3 peptides with a Kd=1μM, but because ING4 is a dimer (and therefore a bivalent "reader" of this "histone code" mark) the global affinity of the molecular recognition event is larger, and could be even larger in the context of the nucleosome. Mutations in the PHD abolish the tumour suppression activity of ING4, and dimerization through its N-terminal domain is essential for apoptosis induction upon DNA damage. Because of the strong sequence conservation at the N-terminal dimerization domain among the ING proteins, ING4 could form heterodimers with other members of the family, especially with ING5, the closest in sequence similarity.

Proliferating cellular nuclear antigen (PCNA) is an essential factor for DNA replication and repair and the effector through which several cell cycle and apoptosis signals are realized. PCNA forms a homotrimeric ring that embraces the DNA duplex and acts as a docking platform for multiple proteins in a coordinated way. We have assigned the backbone NMR spectrum of the 98 kDa human PCNA molecule and are currently investigating the interaction of PCNA with other proteins.

We have characterized the binding of peptides and small molecules designed to compete for the binding to the I-domain of the integrin lymphocyte function-associated antigen-1 (LFA-1) to the intercell adhesion molecule-1 (ICAM-1). These interactions plays a key role in autoimmune diseases and cancer. We have found that these ligands bind to the I-domain of LFA-1, but not to MIDAS (metal ion dependent adhesion site), the site of binding of ICAM-1. They bind instead to the IDAS (I-domain allosteric site), suggesting that they act as allosteric inhibitors, in a similar way as lovastatin.

Meganucleases recognize long DNA sequences and produce double strand breaks (DSB) at single sites in whole genomes. These rare cutting endonucleases can be engineered to repair defective genes ex vivo by promoting efficient gene targeting through DSB-induced homologous recombination. We study the structure of novel meganucleases designed to repair genes causing human monogenic diseases.

Ikerbasque Research Professor

Francisco Blanco
  • Email: fblanco@cicbiogune.es
  • Teléfono: 946.572.521 (EXT. 310)
  • Address: Unidad de Biologa Estructural - Ed. 800
    Derio (Vizcaya)
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Laboratory members

Nekane Merino

Technician

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Georgina Ormaza

PhD Student

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Projects

Histone H3 methyl recognition by the tumor suppressor ING5

Funded by Spanish MINECO grant CTQ2014-56966-R.

We propose to experimentally address the following hypothesis on ING5 structure and function: 1) The asymmetric structure of the ING5 N-t domain is a crystallization artefact and in solution it forms a symmetric dimer similar to ING4. To check this we propose two main strategies: to assign the NMR spectrum of the domain and to crystallize two mutants designed to eliminate possible crystallization artefacts. 2) Two cancer mutations in the N-t domain that strongly destabilize the protein will reduce its tumour suppressor activity, while a third mutation with little effect on the protein thermal stability will not. To validate this hypothesis we will measure the dissociation constant of the wild type and mutant domains at the physiological temperature and evaluate the impact of the mutations on cell proliferation and/or apoptosis induction in response to DNA damage. 3) The N-t domain of ING5 is involved in the binding to JADE1L protein, one of the components of the histone acetylation complex HBO1. To test this hypothesis we will map by NMR the binding site of the domain IIb of JADE1L to the N-t domain of ING5. 4) The dimeric ING5 protein can bind simultaneously two H3K4me3 tails on the same nucleosome and may bind other regions on its surface. To explore these possibilities we will investigate the binding of ING5 to DNA and to reconstituted nucleosomes with the H3K4me3 mark.

Publications

De Biasio A, Ibáñez de Opakua A, Mortuza GB, Molina R, Cordeiro TN, Castillo F, Villate M, Merino N, Delgado S, Gil-Cartón D, Luque I, Diercks T, Bernadó P, Montoya G, Blanco FJ.; Structure of the p15PAF-PCNA complex and implications for clamp sliding on the DNA during replication and repair. Nature Commun 6, 6439 (2015)


Scheiba RM, Ibáñez de Opakua A, Díaz-Quintana A, Martínez-Cruz LA, Martínez-Chantar ML, Blanco FJ, Díaz-Moreno I; The C-terminal RNA Binding Motif of HuR is a multi-functional domain leading to HuR oligomerization and binding to U-rich RNA targets . RNA Biology 11, 10 1250-1261 (2015)


A De Biasio, A Ibáñez de Opakua, T N Cordeiro, M Villate, N Merino, N Sibille, M Lelli, T Diercks, P Bernadó, FJ Blanco; p15PAF is an Intrinsically Disordered Protein with Non-random Structural Preferences at Sites of Interaction with Other Proteins. Biophys J, 106 865-874. (2014)


P Redondo, Nekane Merino, Maider Villate, FJ Blanco, G Montoya, R Molina; Crystallization and preliminary X-ray diffraction analysis on the homing endonuclease I-CvuI in complex with its target DNA. Acta Cryst F 70, 256-259 (2014)


Blanco-Toribio A, Lacadena J, Nuñez-Prado N, Alvarez-Cienfuegos A, Villate M, Compte M, Sanz L, Blanco FJ, Alvarez-Vallina L; Efficient production of single-chain fragment variable-based N-terminal trimerbodies in Pichia pastoris. Microb Cell Fact 13, 116 (2014)


Mortuza GB, Cavazza T, Garcia-Mayoral MF, Hermida D, Peset I, Pedrero JG, Merino N, Blanco FJ, Lyngs J, Bruix M, Pedersen JS, Vernos I, Montoya G; XTACC3-XMAP215 association reveals an asymmetric interaction promoting microtubule Elongation. Nature Commun 5, 50-72 (2014)


Blanco-Toribio A, Sainz-Pastor N, Alvarez-Cienfuegos A, Merino N, Cuesta AM, Sánchez-Martín D, Bonet J, Santos-Valle P, Sanz L, Oliva B, Blanco FJ, Alvarez-Vallina L ; Generation and characterization of monospecific and bispecific hexavalent trimerbodies . mABs 5, 1 70-79 (2013)


San Sebastián E, Zimmerman T, Zubia A, Vara Y, Martin E, Sirockin F, Dejaegere A, Stote RH, Lopez X, Pantoja-Uceda D, Valcárcel M, Mendoza L, Vidal-Vanaclocha, F, Cossío FP, Blanco FJ; Design, Synthesis, and Functional Evaluation of Leukocyte Function Associated Antigen-1 Antagonists in Early and Late Stages of Cancer Development FA-1. J Med Chem 56, 3 735-747 (2013)


E Fernández, ML Artiguez, I Martínez, M Villate, FJ Blanco, JC Arboleya; Effect of pulsed-light processing on the surface and foaming properties of b-lactoglobulin. Food Hydrocolloids 27, 154-160 (2012)


Ángel M. Cuesta, David Sánchez-Martín, Ana Blanco-Toribio, Maider Villate, Kelly Enciso-Álvarez, Ana Alvarez-Cienfuegos, Noelia Sainz-Pastor, Laura Sanz, Francisco J. Blanco and Luis Álvarez-Vallina; Improved stability of multivalent antibodies containing the human collagen XV trimerization domain. mAbs 4, 2 226 - 232 (2012)


Santos-Valle P, Guijarro-Muñoz I, Cuesta AM, Alonso-Camino V, Villate M, Alvarez-Cienfuegos A, Blanco FJ, Sanz L, Alvarez-Vallina L.; The Heterotrimeric Laminin Coiled-Coil Domain Exerts Anti-Adhesive Effects and Induces a Pro-Invasive Phenotype. PloS One 7, 6 e39097 (2012)


Maider Villate, Nekane Merino, Francisco J. Blanco; Production of Meganucleases by Cell-Free Protein Synthesis for Functional and Structural Studies. Protein Expression and Purification 85, 2 246-9 (2012)


Culurgioni S, Muñoz IG, Moreno A, Palacios A, Villate M, Palmero I, Montoya G, Blanco FJ; The crystal structure of the ING4 dimerization domain reveals the functional organization of the ING family of chromatin binding proteins. J Biol Chem 287, 14 10876-10884 (2012)


A De Biasio, R Campos-Olivas, R Sanchez, JP Lopez-Alonso, D Pantoja-Uceda, N Merino, M Villate, JM Martin-Garcia, F Castillo, I Luque, FJ Blanco; Proliferating Cell Nuclear Antigen (PCNA) Interactions in Solution Studied by NMR. PLoS One 7, e48390 (2012)


I GMuñoz, J Prieto, S Subramanian, J Coloma, P Redondo, M Villate, N Merino, M Marenchino, M D'Abramo, F L Gervasio, S Grizot, F Daboussi, J Smith, I Chion-Sotinel, F Pâques, P Duchateau, A Alibés, F Stricher, L Serrano, FJ Blanco, G Montoya; Molecular basis of engineered meganuclease targeting of the endogenous human RAG1 locus. Nucleic Acids Res 39, 2 729-743 (2011)


M Abad, A Moreno, A Palacios, M Narita, FJ Blanco, G Moreno-Bueno, M Narita, I Palmero ; The tumor suppressor ING1 contributes to epigenetic control of cellular senescence. Aging Cell 10, 158-171 (2011)


Alfredo De Biasio, Ricardo Sánchez, Jesús Prieto, Maider Villate, Ramón Campos-Olivas, Francisco J. Blanco; Reduced Stability and Increased Dynamics in the Human Proliferating Cell Nuclear Antigen (PCNA) Relative to the Yeast Homolog. PLoS ONE 6, 2 e16600 (2011)


FJ Blanco, G Montoya
; Transient DNA/RNA-protein interactions.. FEBS Journal 278, 10 1643-50 (2011)


P Manikwar, BA Tejo, Heather Shinogle, DS Moore, T Zimmerman, F Blanco, TJ Siahaan; Utilization of I-domain of LFA-1 to Target Drug and Marker Molecules to Leukocytes. Theranostics 1, 277-289 (2011)


Manikwar P, Zimmerman T, Blanco FJ, Williams TD, Siahaan TJ.; Rapid Identification of Fluorochrome Modification Sites in Proteins by LC ESI-Q-TOF Mass Spectrometry.. Bioconjugate Chemistry (2011)


Marcaida MJ, Muñoz IG, Blanco FJ, Prieto J, Montoya G; Homing endonucleases: from basics to therapeutic applications. Cellular and Molecular Life Sciences 67, 5 727-748 (2010)


Ibañez de Opakua A, Diercks T, Viguera AR, Blanco FJ; NMR assignment and backbone dynamics of the pore-forming domain of colicin A. Biomol NMR Assign 4, 33-36 (2010)


Palacios A, Moreno A, Oliveira BL, Rivera T, Prieto J, García P, Fernández-Fernández MR, Bernadó P, Palmero I, Blanco FJ; The Dimeric Structure and the Bivalent Recognition of H3K4me3 by the Tumor Suppressor ING4 Suggests a Mechanism for Enhanced Targeting of HBO1 Complex to Chromatin. J Mol Biol 396, 4 1117-1127 (2010)


Blanco FJ, Villegas S, Benítez S, Bancells C, Diercks T, Ordóñez-Llanos J & Sánchez-Quesada JL; 2D-NMR reveals different populations of exposed lysine residues in the apoB-100 protein of electronegative and electropositive fractions of LDL particles. J Lipid Res 51, 1560-1565 (2010)


Culurgioni S, Muñoz IG, Palacios A, Redondo P, Blanco FJ, Montoya G; Crystallization and preliminary X-ray diffraction analysis on the dimerization domain of the tumour suppressor ING4. Acta Cryst F66, 567-570 (2010)


Sánchez R, Pantoja-Uceda D, Prieto J, Diercks T, Marcaida MJ, Montoya G, Campos-Olivas R, Blanco FJ; Solution structure of human Growth Arrest and DNA Damage 45 alpha(Gadd45 alpha) and its interactions with Proliferating Cell Nuclear Antigen (PCNA) and Aurora. J Biol Chem 285, 29 22196-22210 (2010)


C Bancells, Sandra Villegas, FJ Blanco, S Benítez, I Gállego, L Beloki, M Pérez-Cuellar, J Ordóñez-Llanos, JL Sánchez-Quesada; Aggregated electronegative low-density lipoprotein in human plasma shows high tendency to phospholipolysis and particle fusion. J Biol Chem 285, 42 32425-35 (2010)


Moreno A, Palacios, Orgaz JL, Jiménez B, Blanco FJ, Palmero I.; Functional impact of cancer-associated mutations in the tumor suppressor protein ING4. Carcinogenesis 20, 39 5503-10 (2010)


Cuesta AM, Sánchez-Martín D, Sanz L, Bonet J, Compte M, Kremer L, Blanco FJ, Oliva B, Alvarez-Vallina L.; In vivo tumor targeting and imaging with engineered trivalent antibody fragments containing collagen-derived sequences. PLoS ONE 4, 4 e5381 (2009)


Grizot S, Smith J, Daboussi F, Prieto J, Redondo P, Merino N, Villate M, Thomas S, Lemaire L, Montoya G, Blanco FJ, Pâques F, Duchateau P; Efficient targeting of a SCID gene by an engineered single-chain homing endonuclease. Nucleic Acids Research 37, 16 5405-5419 (2009)


Palacios A, Muñoz IG, Pantoja-Uceda D, Marcaida MJ, Torres D, Martín-García JM, Luque I, Montoya G, Blanco FJ. ; Molecular basis of histone H3K4me3 recognition by ING4. J. Biol. Chem. 283, 15956-15964 (2008)


Muñoz IG, Blanco FJ. & Montoya G; On the relevance of defining protein structures in cancer research. Clin. Transl. Oncol. 10, 204-212 (2008)


Pantoja-Uceda D, Arolas JL, Garcia P, Lopez-Hernandez E, Padro D, Aviles FX, & Blanco FJ; The NMR Structure and Dynamics of the Two-Domain Tick Carboxypeptidase Inhibitor Reveal Flexibility in Its Free Form and Stiffness upon Binding to Human Carboxypeptidase B. Biochemistry 47, 7066-7078 (2008)


Arolas JL, Pantoja-Uceda D, Ventura, S, Blanco FJ, Aviles FX; The NMR structures of the major intermediates of the two-domain tick carboxypeptidase inhibitor reveal symmetry in its folding and unfolding pathways. J. Biol. Chem. 283, 27110-27120 (2008)


Contreras LM, Gómez J, Prieto J, Clemente-Jiménez JM, Las Heras-Vázquez FJ, Rodríguez-Vico F, Blanco FJ, Neira JL; The family 52 -xylosidase from Geobacillus stearothermophilus is a dimer: structural and biophysical characterization of a glycoside hydrolase. Biochim Biophys Acta 1784, 12 1924-1934 (2008)


Zimmerman T, Blanco FJ; Inhibitors targeting the LFA-1/ICAM-1 cell-adhesion interaction: design and mechanism of action. Curr Pharm Des 14, 22 2128-2139 (2008)


Sánchez R, Pantoja-Uceda D, Torres D, Prieto J, Campos-Olivas R, Blanco FJ; NMR assignment and secondary structure of human growth arrest and DNA damage α protein (Gadd45α). Biomolecular NMR assignments 2, 245-247 (2008)


MJ Marcaida, J Prieto, P Redondo, AD Nadra, A Alibés, Luis Serrano, Sylvestre Grizot, P Duchateau, F Pâques, FJ Blanco & GMontoya; The crystal structure of I-DmoI in complex with its target DNA provides new insights into meganuclease engineering. PNAS 105, 16888-16893 (2008)


Redondo P, Prieto J, Muñoz IG, Alibés A, Stricher F, Serrano L, Cabaniols JP, Daboussi F, Arnould S, Perez C, Duchateau P, Pâques F, Blanco FJ, Montoya G.; Molecular basis of xeroderma pigmentosum group C DNA recognition by engineered meganucleases. Nature 456, 107-111 (2008)


J Prieto, J-C Epinat, P Redondo, E Ramos, D Padró, G Montoya, F. Pâques, FJ Blanco; Generation and analysis of mesophilic variants of the thermostable I-DmoI homing endonuclease. J Biol Chem 283, 4364-4374 (2008)