Biophysics.

Biomolecular Interactions

Head of the Research Line:

Adrian Velazquez-Campoy

Researchers:

José Luis Neira (Universidad Miguel Hernández, Elche)
David Ortega Alarcón (doctorando FPI)
Ana Jiménez Alesanco (investigadora contratada)

SUMMARY OF THE RESEARCH LINE

All biological processes can be described as a sequence of interaction events between biomolecules and conformational changes coupled to those interactions. We work on three main aspects, from both basic and applied perspectives, related to biological molecules:

– Biophysical characterization of proteins with biotechnological and/or biomedical relevance: conformational landscape, interactions with other biomolecules, conformational changes, phase diagrams, cooperative phenomena…

– Development of experimental methodologies for the study of biomolecular interactions, cooperative phenomena and functional regulation in biological systems.

– Development and implementation of high-throughput screening procedures for the identification of bioactive compounds able to modulate the function of pharmacological protein targets.

The biophysical characterization of a protein provides

– Information on the protein conformational landscape. The conformational landscape is the set of conformational states, structurally and energetically distinguishable, that are accessible and significantly populated. The distribution of the protein molecules among these conformational states depends on Gibbs free energy of each state, which in turn is a function of the environmental variables (temperature, pH, ionic strength,…), as well as the interaction with other biomolecules or mutations in the sequence. Because each conformational state may exhibit particular functional properties, the conformation and the function of a given protein are regulated through changes in the environmental variables and biochemical signaling (presence of interacting biomolecules) or mutations. This connection between conformation and function constitutes the basis for the allosteric behavior in proteins: modulation and control of protein conformation (and, therefore, protein function) through interactions with other biomolecules (e.g. ions, cofactors, inhibitors, etc.).

We apply biophysical tools and methods for studying the conformational landscape of proteins of biotechnological and and/or biomedical interest.

– Information on the protein function, regulation and evolution. The physiological function of proteins, and its regulation, always relies on the interaction with other molecules. Therefore, the detailed structural and energetic characterization of its interactions is fundamental for understanding its function and regulation. The interaction with structural metal ions and cofactors is of special interest for protein function regulation. In addition, this information might shed light into evolutive aspects: mutation-induced and/or ligand-induced stabilization of alternative conformational and functional states may give rise to new functions.

We apply biophysical tools and methods to study binding interactions and its coupling with protein stability and the influence on the conformational landscape.

– Information on the molecular and energetic basis of a disease. Very often defective proteins are causative agents for diseases. The mutation-induced and/or ligand-induced stabilization of non-native partially (or completely) unfolded states may lead to improper folding, inactivation and/or early degradation of a given protein, or to the formation of toxic aggregates. Likewise, mutations may distort or impair required interactions for proper function. The biophysical study of its conformational landscape provides information on conformational states that may be relevant regarding its function and regulation, as well as information on the impact of mutations on the interactions required for proper protein function.

We apply biophysical tools and methods to assess the impact of mutations and ligand binding on the stability and function of pharmacological protein targets.-

– Information for establishing strategies for the identification of bioactive compounds. In both infectious diseases (in which the inactivation of a key protein of the life cycle of the pathogen with an inhibitor is the usual therapeutic strategy) as well as in genetic or metabolic disorders (in which both the inactivation of an increased aberrant protein activity with an inhibitor or the rescue of a defective protein activity with a pharmacological chaperone represents alternative therapeutic strategies) it is necessary to modulate and control the function of a given protein. In order to achieve this goal we must identify low-molecular weight compounds: 1) able to interact with the protein; 2) able to modulate appropriately the protein function; 3) avoiding interactions with unwanted targets minimizing side-effects; and 4) exhibiting an optimal pharmacokinetic (ADMET) profile. The biophysical characterization of a protein target provides relevant information about points 1-3, representing a crucial element for designing, validating and optimizing molecular screening procedures.

We develop and implement high-throughput screening procedures for identifying bioactive compounds against pharmacological protein targets.

– Information for optimizing bioactive compounds (affinity, selectivity, induced-conformational changes in the target…). The biophysical study of protein interactions provides information on the relevant underlying intermolecular interactions (i.e. hydrogen bonds, van der Waals, electrostatic and hydrophobic interactions). The thermodynamic dissection of a given interaction allows estimating the enthalpic and entropic contributions to the Gibbs free energy of interaction. The partition of the Gibbs energy into its enthalpic and entropic terms (thermodynamic profile or signature of the binding) is fundamental for describing that interaction, because those two energetic terms reflect intermolecular interactions of different nature (regarding strength, specificity, susceptibility to mutations, and ability to redesign and engineering). It is known that ligands of similar affinity interacting with a protein will bind differently and will display different binding properties if they exhibit different thermodynamic signature; in particular, their interaction will be driven by different intermolecular forces, they will show different susceptibility to mutations in the protein target, and they will induce different conformational changes. Therefore, not only the binding affinity, but also the complete thermodynamic signature is relevant for understanding the mode of interaction and as a key decision criterion for the optimization of binding affinity and selectivity.
We develop and implement procedures for the thermodynamic characterization of binding interactions. A biophysical thermodynamic characterization of lead compound binding provides invaluable information for compound optimization.

Cooperative phenomena are inherent to the behavior of biomolecules and constitute the molecular and energetic basis for protein regulation and allostery. We may distinguish different cooperativity types or levels in biomolecules, which are intimately interrelated:

– Intrinsic protein structural stability. The conformational landscape and the intrinsic structural and energetic features of the protein conformation are key determinants of its behavior and function. Many proteins fold spontaneously and adopt a structured well-defined native state. Other proteins may populate non-native partially folded states with biological relevance. Moreover, many proteins do not fold into a well-defined structured conformational state unless they interact with another molecule (intrinsically disordered proteins), which may have implications regarding their function and regulation. Interestingly, conformational landscape and intrinsic structural stability are not conserved among structurally homologous proteins.
We study the structural stability of proteins, with particular interest on intrinsically (partially) unfolded proteins.

– Coupling between binding and protein conformational changes. Very often protein interactions are accompanied by conformational changes. This is a reflection of the modulation of the conformational landscape of the protein through the interaction with a given ligand. The extent of the conformational change will depend on many factors, in particular, the intrinsic structural stability of the protein and the specific structural and functional features of the ligand. For example, it has been demonstrated the structural determinants in a ligand for binding affinity do not necessarily coincide with those determinants for allosteric regulation (which is mediated through conformational changes coupled to binding). Also, ligand binding induces protein stabilization giving rise to the distinction between strong or weak coupling between conformational changes and binding, as well as between induced-fit and conformational selection. In fact, induced-fit can be considered as a limit case of conformational selection when a very large Gibbs free energy gap exists between the two conformational states interconnected by ligand binding.
We study conformational changes coupled to ligand binding in proteins, as well as the interplay between folding and binding in protein function regulation and protein binding specificity.

– Binding cooperativity. Homotropic cooperativity (binding cooperativity between molecules of the same ligand binding to several sites in a protein) and heterotropic cooperativity (binding cooperativity between molecules of different ligands binding to one or several sites in a protein) constitute the basis for protein function regulation and allostery. In a broad sense, allostery can be defined as the control and modulation of protein conformation (conformational landscape) through ligand binding. Therefore, any protein can be considered an allosterically regulated protein. The most common example is the pH dependency of the structural stability or the binding affinity of a ligand in a given protein; in this particular case, the binding or dissociation of protons taking place at ionizable functional groups in the protein (or in the ligand) alters the energetics of unfolding or ligand binding. Binding affinity and binding cooperativity are not conserved properties among structurally similar ligands.
We develop and implement methodologies for characterizing and assessing cooperative (homotropic and heterotropic) binding in proteins and its relation to the biological function.

Relevant publications

1.- J.L. Neira, J. Bintz, M. Arruebo, B. Rizzuti, T. Bonacci, S. Vega, A. Lanas, A. Velazquez-Campoy, J.L. Iovanna, O. Abian. Identification of a drug targeting an intrinsically disordered protein involved in pancreatic adenocarcinoma. Scientific Reports 2017 7:39732

2.- R. Claveria-Gimeno, P.M. Lanuza, I. Morales-Chueca, O.C. Jorge, S. Vega, O. Abian, M. Esteller, A. Velazquez-Campoy. The intervening domain from MeCP2 enhances the DNA affinity of the methyl binding domain and provides an independent DNA interaction site. Scientific Reports 2017 7:41635

3.- Biophysical screening for identifying pharmacological chaperones and inhibitors against conformational and infectious diseases. Velazquez-Campoy, J. Sancho, O. Abian, S. Vega. Current Drug Targets 2016 17:1492-1505.

4.- R. Sant’Anna, P. Gallego, L. Robinson, A. Pereira, Ne. Ferreira, F. Pinheiro, S. Esperante, I. Pallares, O. Huerta, R. Almeida, N. Reixach, R. Insa, A. Velazquez-Campoy, D. Reverter, N. Reig, S. Ventura. Repositioning Tolcapone as a potent inhibitor of transthyretin amyloidogenesis and associated cellular toxicity. Nature Communications 2016 7:10787.

5.- S. Vega, O. Abian, A. Velazquez-Campoy. A unified framework based on the binding polynomial for characterizing biological systems by isothermal titration calorimetry. Methods 2015, 76:99-115

6.- R.M. Buey, R. Ledesma-Amaro, A. Velazquez-Campoy, M. Balsera, M. Chagoyen, J.M. de Pereda, J.L. Revuelta. Guanine nucleotide binding to the Bateman domain mediates the allosteric inhibition of eukaryotic IMP dehydrogenases. R.M. Buey, R. Ledesma-Amaro, A. Velazquez-Campoy, M. Balsera, M. Chagoyen, J.M. de Pereda, J.L. Revuelta. Nature Communications 2015 6:8923.

7.- K. Gonzalez-Arzola, I. Diaz-Moreno, A. Cano-Gonzalez, A. Diaz-Quintana, A. Velazquez-Campoy, B. Moreno-Beltrán, A. Lopez-Rivas, M.A. De la Rosa. Structural basis for inhibition of the histone chaperone activity of SET/TAF-Iβ by cytochrome c.  Proceedings of the National Academy of Sciences USA 2015 112:9908-9913.

8.- J.J. Galano, M. Alias, R. Perez, A. Velazquez-Campoy, P.S. Hoffman, J. Sancho. Improved flavodoxin inhibitors with potential therapeutic effects against Helicobacter pylori infection. Journal of Medicinal Chemistry 2013 56:6248-6258.

9.- O. Abian, S. Vega, J. Sancho, A. Velazquez-Campoy. Allosteric inhibitors of the NS3 protease from the hepatitis C virus. PLoS ONE 2013 8:e69773.

10.- S. Chopra, A. Palencia, C. Virus, A. Tripathy, B.R. Temple, A. Velazquez-Campoy, S. Cusack, J.S. Reader. Plant tumour biocontrol agent employs a tRNA-dependent mechanism to inhibit leucyl-tRNA synthetase. Nature Communications 2013 4:1417.

11.- P.T. Martins, A. Velazquez-Campoy, W.L. Vaz, R.M. Cardoso, J. Valerio, M.J. Moreno. Kinetics and thermodynamics of chlorpromazine interaction with lipid bilayers: Effect of charge and cholesterol. Journal of the American Chemical Society 2012 134:4184-4195.

12.- O. Abian, S. Vega, J.L. Neira, A. Velazquez-Campoy. Conformational stability of hepatitis C virus NS3 protease. Biophysical Journal 2010 99:3811-3820.

13. N. Cremades, A. Velazquez-Campoy, M. Martinez-Julvez, J.L. Neira, I. Perez-Dorado, J. Hermoso-Dominguez, P. Jimenez, A. Lanas, P.S. Hoffman, J. Sancho. Discovery of specific flavodoxin inhibitors as potential therapeutic agents against Helicobacter pylori infection. ACS Chemical Biology 2009 4:928-938.

14.- O. Abian, J.L. Neira, A. Velazquez-Campoy. Thermodynamics of zinc binding to hepatitis C virus NS3 protease: A folding by binding event. Proteins: Structure, Function and Bioinformatics 2009 77:624-636.

15.- A.L. Pey, M. Ying, N. Cremades, A. Velazquez-Campoy, T. Scherer, B. Thöny, J. Sancho, A. Martinez. Identification of pharmacological chaperones as new therapeutic agents to treat phenylketonuria. Journal of Clinical Investigation 2008 118:2858-2867.

Main research projects

1.- Structure, energetics, and simulation of (partially) unfolded conformations in proteins. Towards quantitative atomic models for the protein stability. Ministerio de Economía y Competitividad (BFU2016-78232-P). 2016-2019. PI: Adrián Velázquez Campoy / Javier Sancho (co-IPs) (Universidad de Zaragoza – BIFI).

2.- New infrastructure for the Laboratorio Avanzado de Cribado e Interacciones Moleculares de Aragon (LACRIMA). Diputación General de Aragón y Ministerio de Economía y Competitividad (Proyecto de Infraestructuras Científico-Tecnológicas UNZA15-EE-3250). Universidad de Zaragoza. 2016-2018. PI: Javier Sancho (Universidad de Zaragoza – BIFI).

3.- Between atom and cell: Integrating molecular biophysics approaches for biology and healthcare (MOBIEU). European Cooperation in Science and Technology (eCOST, COST Action CA15126). ARBRE (Association of Resources for Biophysical Research in Europe). 2016-2020. PI: Patrick England (Institute Pasteur). Adrián Velázquez-Campoy (Management Committee).

4.- Validation of a new, quick, non-invasive diagnostic method in serum for early detection of pancreatic cancer (PANCal). Asociación Española de Gastroenterología – Club Español del Páncreas. Instituto Investigación Sanitaria de Aragón, Hospital Clínico Universitario Lozano Blesa, Hospital Universitario Miguel Servet, Hospital de Donosti, Hospital de Barbastro, Universidad de Zaragoza – Instituto BIFI. 2015. PI: Olga Abián (Instituto de Investigación Sanitaria de Aragón (IIS) – Universidad de Zaragoza – BIFI).

5.- Protein stability: Basic principles of (partially) unfolded states and molecular studies on conformational disorders. Ministerio de Economía y Competitividad (BFU2013-47064-P). Universidad de Zaragoza. 2014-2017. PI: Adrián Velázquez Campoy / Javier Sancho (co-IPs) (Universidad de Zaragoza – BIFI).

6.- NEUROMED – Diagnosis and treatment of three neurodegenerative diseases (Parkinson, Phenylketonuria and TTR amyloidosis). ERDF – SUDOE Interreg IVB (SOE4/P1/E831 – Neuromed). Universidad de Zaragoza, Universidad Autónoma de Barcelona, Instituto de Biologia Molecular e Celular (Porto), Universidade de Coimbra, CNRS, INSERM (Languedoc-Roussillon). 2014-2015. PI: Javier Sancho (Universidad de Zaragoza – BIFI).

7.- Bioavailability of amphiphilic ligands – Drugs and metabolites. Ministerio de Ciencia e Innovación (Proyectos de Movilidad – Acciones Integradas, PRI-AIBPT-2011-1025). Universidad de Zaragoza, Universidad de Coimbra. 2012-2013. PI: Adrián Velázquez Campoy (Universidad de Zaragoza – BIFI).

8.- NS3 protease from hepatitis C virus: Identification of competitive and allosteric inhibitors. Ministerio de Ciencia e Innovación (BFU2010-19451). Universidad de Zaragoza. 2010-2013. PI: Adrián Velázquez Campoy (Universidad de Zaragoza – BIFI).

9.- NS3 protease from hepatitis C virus: Competitive and allosteric inhibitors, and molecular basis of drug resistance. Universidad de Zaragoza (UZ2009-BIO-05). Universidad de Zaragoza. 2010. PI: Adrián Velázquez Campoy (Universidad de Zaragoza – BIFI).

10.- Conformational equilibrium of the NS3 protease from hepatitis C virus: Non-native states and identification of a new type of allosteric inhibitors. Diputación General de Aragón (PI044/09). Universidad de Zaragoza. 2009-2011. PI: Adrián Velázquez Campoy (Universidad de Zaragoza – BIFI).

Collaborators

• Olga Abian (Instituto Aragones de Ciencias de la Salud and BIFI, Spain)
• Juan Ausio (University of Victoria, Canada)
• Rui Brito (Universidade de Coimbra, Portugal)
• Pierpaolo Bruscolini (Universidad de Zaragoza and BIFI, Spain)
• Jose A. Carrodeguas (Universidad de Zaragoza and BIFI, Spain)
• Irene Diaz-Moreno (Instituto de Bioquimica Vegetal y Fotosintesis – CSIC, Spain)
• Maria Fillat (Universidad de Zaragoza and BIFI, Spain)
• Marcos R. Fontes (São Paulo State University, Brazil)
• Ernesto Freire (The Johns Hopkins University, USA)
• Enrique Garcia-Hernandez (Universidad Nacional Autonoma de Mexico, Mexico)
• Ruben Martinez-Buey (Universidad de Salamanca, Spain)
• Milagros Medina (Universidad de Zaragoza and BIFI, Spain)
• Maria João Moreno (Universidade de Coimbra, Portugal)
• Arturo Muga (Universidad del Pais Vasco, Spain)
• Jose A. Navarro (Instituto de Bioquimica Vegetal y Fotosintesis – CSIC, Spain)
• Julian Pardo (Universidad de Zaragoza and IIS-Aragon, Spain)
• Santiago Ramon-Maiques (CNIO, Spain)
• David Reverter (Universidad Autonoma de Barcelona, Spain)
• Javier Sancho (Universidad de Zaragoza and BIFI, Spain)
• Jayaraman Sivaraman (National University of Singapore, Singapore)
• Maria A. Urbaneja (Universidad del Pais Vasco, Spain)
• Salvador Ventura (Universidad Autonoma de Barcelona, Spain)

Protein glycosylation and its role in disease

Head of the Research Line:

Ramón Hurtado Guerrero

Researchers:

Ana García García
Javier Macías León
Andrés Manuel González Ramírez
Víctor Taleb
Erandi Lira Navarrete

SUMMARY OF THE RESEARCH LINE

Our group is interested in the study of glycosyltransferases, glycosylhydrolases and carbohydrate-binding proteins/modules involved in human diseases. We use protein X-ray crystallography as the main tool complemented with enzymology, inhibition studies, etc, in order to study the molecular mechanisms of enzymes that are involved in the synthesis, modification and degradation of glycoconjugates, oligo and polysaccharides (see more relevant publications below).

We majorly work in Protein O-glycosylation and glycosyltransferases responsible of this post-translational modification. In particular we are currently working in several glycosyltransferases such as Protein O-fucosyltransferases 1 and 2 (POFUT1 and 2), FUT8 and the large family of GalNAc-Ts. While POFUT1 and 2 fucosylate folded domains such as EFG and TSR domains, respectively, GalNAc-Ts mainly glycosylate unstructured regions present in a large number of proteins such as mucins . FUT8 is responsible of the so-called core fucosylation (Figure 1).

Figure 1. Scheme summarizing the structure of FUT8 complexed to GDP and a biantennary N-glycan.

Furthermore we are interested in the elucidation of the reaction coordinates and the molecular mechanism by using transition state analogues or Michaelis complexes. Finally these studies will be important for the design of new future drugs with potential therapeutic applications.

Although not related to the Glycobiology field, we have a close collaboration with Prof. Guadix and Dr Conejo-García from University of Granada in the development of new inhibitors against human choline kinase α1.

Relevant publications

1.- Structural insights into mechanism and specificity of O-GlcNAc transferase. Clarke AJ*, Hurtado-Guerrero R*, Pathak S*, Schüttelkopf AW*, et al. EMBO J. 2008, 27(20): 2780-8. *equal contributation as first author.

2.- Molecular mechanisms of O-GlcNAcylation. Hurtado-Guerrero R, Dorfmueller HC, van Aalten DM. Curr Opin Struct Biol. 2008, 18(5): 551-7.

3.- Recent structural and mechanistic insights into post-translational enzymatic glycosylation. Ramon Hurtado-Guerrero* and Gideon J Davies. Current Opinion in Chemical Biology. 16(5-6):479-87, 2012. *corresponding author

4.- The Vibrio cholerae colonization factor GbpA possesses a modular structure that governs binding to different host surfaces. Wong E, Vaaje-Kolstad G, Ghosh A, Ramon Hurtado-Guerrero, et al. PLoS Pathogen, 2012.

5.- The mechanism of allosteric coupling in choline kinase α1 revealed by a rationally designed inhibitor. María Sahún-Roncero, Belén Rubio-Ruiz, Giorgio Saladino, Ana Conejo-García, Antonio Espinosa, Adrián Velázquez-Campoy, Francesco Luigi Gervasio, Antonio Entrena and Ramon Hurtado-Guerrero*. Angewandte Chemie (selected as VIP), 52(17):4582-6, 2013. *corresponding author

6.- Combined structural snapshots and metadynamics reveal a substrate-guided front-face reaction for polypeptide GalNAc-transferase T2. Erandi Lira-Navarrete, Javier Iglesias-Fernández, Wesley F. Zandberg, Ismael Compañón, Yun Kong, Francisco Corzana, B. Mario Pinto, Henrik Clausen, Jesús M. Peregrina, David Vocadlo, Carme Rovira* and Ramon Hurtado-Guerrero*. Angewandte Chemie International Edition, 53(31):8206-10, 2014. *corresponding author

7.- Dynamic interplay between catalytic and lectin domains of GalNAc transferases modulates protein O-glycosylation. Erandi Lira-Navarrete, Matilde de las Rivas, Ismael Compañón, María Carmen Pallarés, Yun Kong, Javier Iglesias-Fernández, Gonçalo J. L. Bernardes, Jesús M. Peregrina, Carme Rovira, Pau Bernadó, Pierpaolo Bruscolini, Henrik Clausen, Anabel Lostao, Francisco Corzana, and Ramon Hurtado-Guerrero*. Nature Communications, 5;6:6937, 2015. *corresponding author

8.- X-ray Structures decipher the Non-equivalence of Serine and Threonine O-glycosylation points: Implications for the Molecular Recognition of the Tn Antigen by an anti-MUC1 Antibody. Nuria Martínez-Sáez,‡ Jorge Castro-López,‡ Jessika Valero-González,‡ David Madariaga, Ismael Compañón, Víctor J. Somovilla, Míriam Salvadó, Juan L. Asensio, Jesús Jiménez-Barbero, Alberto Avenoza, Jesús H. Busto, Gonçalo J. L. Bernardes, Jesús M. Peregrina,* Ramón Hurtado-Guerrero,* and Francisco Corzana*. Angewandte Chemie International, 54(34):9830-9834, 2015. *corresponding author

9.- Mucin Architecture behind the Immune Response: Design, Evaluation and Conformational Analysis of an Antitumor Vaccine Derived from an Unnatural MUC1 Fragment. Martínez-Sáez N, Supekar NT, Wolfert MA, Bermejo IA, Hurtado-Guerrero R, Asensio JL, Jiménez-Barbero J, Busto JH, Avenoza A, Boon G-J, Peregrina JM, and Corzana F. Chemical Science, 2016. DOI: 10.1039/C5SC04039F

10.- A proactive role of water molecules in acceptor recognition by Protein-O-fucosyltransferase 2. Jessika Valero-González, Christina Leonhard-Melief , Erandi Lira-Navarrete, Gonzalo Jiménez-Osés, Cristina Hernández-Ruiz, María Carmen Pallarés, Inmaculada Yruela, Deepika Vasudevan, Anabel Lostao, Francisco Corzana, Hideyuki Takeuchi, Robert S. Haltiwanger, and Ramon Hurtado-Guerrero*. Nature Chemical Biology, accepted manuscript, 2016. DOI:10.1038/nchembio.2019. * corresponding author

11.- A trapped covalent intermediate of a glycoside hydrolase on the pathway to transglycosylation. Insights from experiments and QM/MM simulations. Lluís Raich, Vladimir Borodkin, Wenxia Fang, Jorge Castro-López, Daan van Aalten*, Ramon Hurtado-Guerrero* and Carme Rovira*. Journal of the American Chemical Society (JACS), 2016. DOI: 10.1021/jacs.5b10092. * corresponding authors

12.- The interdomain flexible linker of the polypeptide GalNAc transferases (GalNAc-Ts) dictates their long-range glycosylation preferences. Matilde de las Rivas, Erandi Lira-Navarrete, et al., and Ramon Hurtado-Guerrero*.Nature Communications, 2017. DOI: 10.1038/s41467-017-02006-0. *corresponding author.

13.- The use of fluoroproline in MUC1 antigen enables efficient detection of antibodies in patients with prostate cancer. Somovilla Víctor, et al., Hurtado-Guerrero Ramon, et al., Corzana Francisco. JACS, 2017. DOI:10.1021/jacs.7b09447.

14.– Water Sculpts the Distinctive Shapes and Dynamics of the Tn Antigens: Implications for their Molecular Recognition. Iris A. Bermejo, et al., Ramón Hurtado-Guerrero*. JACS, 2018 DOI: 10.1021/jacs.8b04801. *corresponding authors.

15.- Dynamic acetylation of cytosolic phosphoenolpyruvate carboxykinase toggles enzyme activity between gluconeogenic and anaplerotic reactions. Pedro Latorre-Muro, Josue Baeza, Eric A. Armstrong, Ramón Hurtado-Guerrero, et al., José A. Carrodeguas* and John M. Denu*. Molecular Cell, 2018, just accepted. Authors.

16.- Structural basis for arginine glycosylation of host substrates by bacterial effector proteins. Jun Bae Park, Young Hun Kim, Youngki Yoo, Juyeon Kim, Sung-Hoon Jun, Jin Won Cho, Samir El Qaidi, Samuel Walpole, Serena Monaco, Ana A. García-García, Miaomiao Wu, Michael P. Hays, Ramon Hurtado-Guerrero*, Jesus Angulo*, Philip R. Hardwidge, Jeon-Soo Shin* and Hyun-Soo Cho*. Nature Communications, 2018, just accepted. *joint corresponding authorship.

17.- Structural and mechanistic insights into the catalytic domain-mediated short-range glycosylation preferences of GalNAc-T4. de las Rivas Matilde, Paul Daniel Earnest James, Coelho Helena, Lira-Navarrete Erandi, Raich Lluís, Companon Ismael, Diniz Ana, Lagartera Laura, Jiménez-Barbero Jesús, Clausen Henrik, Rovira Carme, Marcelo Filipa, Corzana Francisco, Gerken Thomas* and Hurtado-Guerrero, Ramón*. ACS Central Science, 2018, just accepted. *joint corresponding authorship.

18. Polypeptide GalNAc-Ts: from redundancy to specificit.  de las Rivas Matilde, Lira-Navarrete, Erandi, Gerken Thomas* and Hurtado-Guerrero, Ramon*. . Current Opinion in Structural Biology, 2019. doi: 10.1016/j.sbi.2018.12.007 *joint corresponding authorship.

19. Structure-based design of potent tumor-associated antigens: modulation of peptide presentation by single atom O/S or O/Se substitutions at the glycosidic linkage. Compañón Ismael, Guerreiro Ana, Mangini Vincenzo Castro-López Jorge, Escudero-Casao Margarita, Avenoza Alberto, Busto Jesús, Castillón Sergio, Jiménez-Barbero Jesús, Asensio Juan, Jiménez-Osés Gonzalo, Boutureira Omar, Peregrina Jesús, Hurtado-Guerrero Ramón, Fiammengo Roberto, Bernardes Gonçalo and Corzana Francisco. . JACS, 2019. doi: 10.1021/jacs.8b13503

20. Mechanisms of redundancy and specificity of the Aspergillus fumigatus Crh transglycosylases. Fang Wenxia, Sanz Ana Belén, Galan Bartual Sergio, Wang Bin, Ferenbach Andrew, Farkaš Vladimír, Hurtado-Guerrero Ramon, Arroyo Javier and van Aalten DMF.  Nature Communications, 2019. doi: 10.1038/s41467-019-09674-0.

21. Molecular basis for Fibroblast growth factor 23 (FGF23) O-glycosylation and regulation by polypeptide GalNAc-T3. Matilde de las Rivas, Earnest Daniel, Yoshiki Narimatsu, Ismael Compañón, Kentaro Kato, Pablo Hermosilla, Aurélien Thureau, Laura Ceballos-Laita, Helena Coelho, Pau Bernado, Filipa Marcelo, Lars Hansen, Ryota Maeda, Anabel Lostao, Francisco Corzana, Henrik Clausen, Thomas Gerken and Ramón Hurtado-Guerrero*.  Nature Chemical Biology, 2020. doi: 10.1038/s41589-019-0444-x * corresponding autor.

22.  Structural basis for substrate specificity and catalysis of α1,6-fucosyltransferase. Ana García-García, Laura Ceballos-Laita, Sonia Serna, Raik Artschwager, Niels C. Reichardt, Francisco Corzana and Ramon Hurtado-Guerrero*. Just accepted in Nature Communications, 2020. doi: 10.1038/s41467-020-14794-z. * corresponding autor.

23. Recent advances in the design of Choline kinase α inhibitors and the molecular basis of their inhibition. Belén Rubio-Ruiz, Lucía Serán-Aguilera, Ramón Hurtado-Guerrero*, and Ana Conejo-García*.  Medicinal Research Reviews. doi: 10.1002/med.21746. *Joint corresponding authorship

Main research projects

1.- Study of glycosyltransferases involved in the Notch signalling pathway
MICINN, 2011-2014. 120,000 euros. PI: Ramón Hurtado-Guerrero

2.- Study of protein-carbohydrate interactions involved in human diseases. MEC, 2014-2016. 76,000 euros. PI: Ramón Hurtado-Guerrero

3.- BFU2016-75633-P (MICINN), 2017-2019. 220,000 euros plus extra funding for one PhD student. PI: Ramón Hurtado-Guerrero

4.- FEDER proyects for infrastructure and in particular for LACRIMA (2016-2017). UNZA15-EE-3250. PI: Javier Sancho. coPIs: Ramón Hurtado-Guerrero and others. 386,872 euros

5.- FEDER proyects for infrastructure and in particular for the adquistion of a FRET machine (2016-2017). UNZA15-EE-2922. PI: Nunilo Cremades. coPIs: Ramón Hurtado-Guerrero and others. 323,049 euros

6.- Crowdfunding for development of antifungal drugs. PI: Ramón Hurtado-Guerrero. 6600 euros, 2018-2019.

7.- Collaborative research network in Glycobiology “GLYCOBIOCHEM”. CTQ2017-90719-REDT. PI: Niels Reichardt, coPI: Ramón Hurtado-Guerrero and others. 17,000 euros, 01072018-30062020.

8.- DGA Group. Chemical Biology and Computation. E34_17R. PI: Pedro Merino, coPI: Ramón Hurtado-Guerrero. 45,971 euros, 2018-2019.

9.- Bases moleculares de la O-glicosilación tipo mucina y su aplicación en el tratamiento de tumores. LMP58_18 (DGA). 85,500 euros, 2019-2020. PI: Ramón Hurtado-Guerrero

10.- FEDER proyect for infrastructure (EQC2019-005847-P). Implementation of complex cellular models for drug discovery: Advanced equipment for the production, purification and analysis of proteins. PI: Milagros Medina. coPIs: Ramón Hurtado-Guerrero and others. 463,086.87 euros (2017-2020).

11.- Desarrollo de nanobodies y “adhirons” frente al dominio de unión de la glicoproteína “spike” del virus SARS-CoV-2 como tratamiento para la enfermedad CoVid19. DGA, 275,000 euros, 2020-2021. PI: Ramón Hurtado-Guerrero, coPI: Julián Pardo

12.- Desarrollo y validación de un test de inmunocromatografía para su aplicación en el diagnóstico serológico frente a SARS‐CoV‐2 en animales de compañía. Proyecto Santander-Unizar. 8500 euros, 2020-2021. PI: Pérez Cabrejas, M. Dolores, coPI among others: Ramón Hurtado-Guerrero.

13.- PID2019-105451GB-I00 (MICINN), 2020-2022. 266,200 euros plus extra funding for one PhD student. PI: Ramón Hurtado-Guerrero

14. Collaborative research network in Glycobiology “GLYCOBIOCHEM”. CTQ2017-90719-REDT. PI: Niels Reichardt, coPI: Ramón Hurtado-Guerrero and others. 17,000 euros, 01072018-30062020.

15.- COST “Innogly”. PI: Luigi Lay, coPIs: Ramón Hurtado-Guerrero and others. 2019-2022

Collaborators

Robert Haltiwanger, The University of Georgia
Henrik Clausen, University of Copenhagen
Daan van Aalten, University of Dundee
Philip Hardwidge, Kansas State University
Tom Gerken, Case Western Reserver University
Pedro Merino, Universidad de Zaragoza
Francisco Corzana, Universidad de La Rioja
Carme Rovira, Universidad de Barcelona
Filipa Marcelo, New University of Lisbon
Julián Pardo, Universidad de Zaragoza
Anabel Lostao, Universidad de Zaragoza

Flavoenzymes: action mechanisms and biotecnology

Head of the Research Line:

Dr. Milagros Medina

Researchers:

Dr. Marta Martínez Júlvez
Dr. Patricia Ferreira Neila
Silvia Romero Tamayo
Martha Minjárez Sáenz
Nerea Novo Huerta
Sergio Boneta Martinez
Andrea Moreno Maldonado
Olga Arjona

SUMMARY OF THE RESEARCH LINE

Flavoenzymes are versatile and diverse biomolecules that contribute to maintain and build cellular structures, to move molecules among subcellular compartments, to eliminate toxins, to facilitate chemical transformations in biosynthetic pathways, while are particularly in charge of cellular bioenergetics and signaling. They have as cofactors the riboflavin (RF, vitamin B2) derivatives flavin mononucleotide (FMN) and/or flavin adenine dinucleotide (FAD), which confer them unique and versatile properties. All organisms contain key flavoproteins and flavoenzymes, and many of them are becoming interesting as therapeutic targets or biotechnological tools. Understanding of their mechanisms for their biotechnological applications is the main goal of the “Flavoenzymes: mechanisms of action and biotechnology” group.

Our hypothesis is that we need to understand the molecular mechanisms of flavoenzymes with key metabolic functions in bacteria, parasites, plants or animals, in order to exploit their potential in several areas of biotechnology. In our group, we apply an interdisciplinary combination of tools of biochemistry, biophysics, cell biology and computational biology in order to investigate the mechanisms and factors that provide versatility to several flavoenzyme-dependent systems, and to use this information in new biotechnological and therapeutic applications. The group’s publications reflect our contributions in key areas in the understanding of flavoenzymes. Given the wide range of interest and methodologies in our research, we maintain close collaborations with specialists from other disciplines, both within BIFI and UNIZAR, as well as with others from outside institutions.

HUMAN FLAVOENZYMES IN HEALTH AND DISEASE: THE HUMAN APOPTOSIS INDUCING FACTOR (AIF)

The human body uses more than 80 flavoenzymes that perform very different functions. If an error, mutation, occurs in one of them, it might aberrantly affect its activity, stability and/or interplay with other biomolecules, altering cellular functions. Two thirds of flavin-dependent human proteins are associated with disorders caused by allelic variants, making of interest the development of molecules able to rescue native conformation and function. To treat the cause of these diseases, it is advisable the use of personalized molecular therapies able to rescue each variant functionality. Implementation of such actions requires the in advance understanding of relationships between the pathological and molecular mechanisms in which the protein is involved. The human AIF (hAIF) family consists of three flavoproteins that have in common: i) mitochondrial localization, ii) FAD- and NADH-dependent oxidoreductase domains involved in an -albeit yet not well understood- oxidoreductase function and iii) an apoptosis-inducing activityAIF1, also known as AIF, if the family prototype.

It is a phylogenetically preserved mitochondrial flavoenzyme that shares homology with different reductase families. In intact mitochondria, AIF exhibits NADH-oxide-reductase activity through its flavin cofactor, FAD, which has been linked to the stability and biogenesis of respiratory complexes I and III through interaction with other proteins, such as CHCHD4 (homolog in humans to MIA40). However, neither their role as reductase nor the molecular mechanisms underlying the biogenesis of mitochondrial complexes are currently understood. In addition, when mitochondria receive an apoptotic stimulus, AIF is translocated to the nucleus, where it interacts with other nuclear proteins forming the degradosome that induces chromatinolysis. Likewise, the involvement of mutations in the sequence of AIF as direct responsible for various neurodegenerative disorders that lead to a decrease in the efficiency of the mitochondrial oxidative phosphorylation system has been described.
Thus, both the interest in the design of new therapies to modulate the independent caspase apoptosis and the understanding of the molecular basis of the pathologies related to single mutations in AIF has increased in recent years. This has turned AIF into a potential target for the treatment of pathological alterations (cancer or degenerative diseases) in which this protein either causes a defect or an excess of apoptosis, or alters the mitochondrial bioenergetics. The redox state of AIF influences its conformation and its monomer-dimer equilibrium and, by extension, its pro-apoptotic function because these factors modulate the interaction with other proteins.
AIF1, also known as AIF, if the family prototype. It is a phylogenetically preserved mitochondrial flavoenzyme that shares homology with different reductase families. In intact mitochondria, AIF exhibits NADH-oxide-reductase activity through its flavin cofactor, FAD, which has been linked to the stability and biogenesis of respiratory complexes I and III through interaction with other proteins, such as CHCHD4 (homolog in humans to MIA40). However, neither their role as reductase nor the molecular mechanisms underlying the biogenesis of mitochondrial complexes are currently understood. In addition, when mitochondria receive an apoptotic stimulus, AIF is translocated to the nucleus, where it interacts with other nuclear proteins forming the degradosome that induces chromatinolysis. Likewise, the involvement of mutations in the sequence of AIF as direct responsible for various neurodegenerative disorders that lead to a decrease in the efficiency of the mitochondrial oxidative phosphorylation system has been described.

Since one of the possibilities of modulating the pro-apoptotic activity of AIF is the regulation of its redox activity, it is imperative to answer multiple questions related to the mechanism and cellular function of the mentioned activity.

Our research on AIF aims to provide some of these responses by understanding the molecular mechanisms linked to the processes of oxide-reduction of AIF and the consequences of its redox activity in the interaction with its protein partners in the different cellular compartments where it’s found

FLAVOPROTEINS FROM PATHOGENIC BACTERIA AS POTENTIAL ANTIMICROBIAL TARGETS.

Pathogenic bacteria usually survive in intracellular niches deprived of nutrients when infecting mammalian cells. Among those nutrients is RF, a vitamin precursor of FMN and FAD.

Thus, flavins and flavoproteins are relevantcomponents in regulating bacterial physiology against stresses and in the biology of host cells infection and colonization, closely relating flavin metabolism of pathogens to survive in situations of oxidative stress, microanaerobic conditions or lack of nutrients. Some flavoproteins are involved in the pathogen:host cell interaction, or as primary photoreceptors in signalling response of bacterium against stress. Cell free FMN levels also control expression of particular genes through FMN riboswitches, while many pathogens are unable to acquire FMN and FAD from their environment. Finally, most flavoprotein traits and mechanisms are species-specific, and the flavoprotein content appears widely divergent. These facts make flavin homeostasis and bacterial flavoproteomes interesting targets for the development of chemotherapies to fight infectious diseases. In this context, it is important to compare flavoproteins from different species with respect to ligand binding and reaction mechanism, because, even when substrates are invariant, significant differences may prevail. Such comparative bacterial flavoenzymology will help in the development of species-specific antimicrobials. We are making our contributions by identifying potential inhibitors of a phytopathogenic bacteria reductase, as well as of the FAD synthases (FADS) from Streptococcus pneumoniae and Mycobacterium tuberculosis.

THE BIOSYNTHESIS OF FMN AND FAD AND THEIR DISTRIBUTION TO APOPROTEIN CLIENTS.

The biosynthesis of FMN and FAD, their cell homeostasis and their distribution to client apo-proteins are critical and coordinated processes for cell energy efficiency, being also of interest for their sanitary and/or biotechnological (providing FMN and FAD to flavoproteins for heterogeneous and multi-functional biocatalysts) exploitation. Bacterial FADSs have both ATP:riboflavin kinase (RFK, FMN production) and ATP:FMN:adenylyltransferase (FMNAT, FAD production) activities, which, as we have shown, are performed by almost independent protein modules. These two activities are performed by two independent enzymes in eukaryotes, known as HsRFK and HsFADS in humans. The bacterial RFK module shows sequence and structural homology with HsRFK, while the FMNAT module neither presents sequence nor structural similarity with the proteins that produce FAD in mammals. The monofunctional mammalian FADS/FMNAT has been suggested to operate as a chaperone of the flavin, providing physical interaction with its client proteins. Such a possibility has not been explored in the bifunctional bacterial FADSs, and nothing is known regarding HsRFK. Since the understanding of the molecular bases of such canalization might be fundamental to achieve the identification of molecules inhibiting the FMN and FAD production, as well as positively or negatively modulating their canalization to the client protein, we consider further efforts must be done to clarify these facts. We are using different human and bacterial proteins to address this topic.

LONG-TERM COLLABORATIONS IN OTHER DEPENDENT SYSTEMS OF FLAVOENZYMES

FAD dependent reductases of bacteria: In collaboration with the groups of Dr. E. Ceccarelli and Dr. E. Orellano of the National University of Rosario, Argentina, we are comparatively studying bacterial and plastidic enzymes. Since in many bacteria, these enzymes are essential or are involved in the response to oxidative stress, they can be treated as interesting pharmacological targets for the treatment of infections caused by pathogens.

In collaboration with Dr. M. Balsera of the Instituto de Recursos Naturales y Agrobiología de Salamanca we are kinetically evaluating the catalytic mechanisms of a variety of bacterial thioredoxin reductases that control the redox state of thioredoxins—widely occurring proteins that regulate a spectrum of enzymes by dithiol-disulfide exchange.

Oxidoreductases in human metabolism: In collaboration with Dr. A. Pey of the University of Granada, we are studying the catalytic mechanism of the antioxidant and cancer-associated NAD(H):quinone oxidoreductase 1 enzyme. Kinetic studies are being used to evaluate conformational dynamics and functional cooperativity, as well as to better understand the effects of cancer-associated single amino acid variants and post-translational modifications in this protein of high relevance in cancer progression and treatment.

Dehydrogenases and oxidases: In collaboration with Dr. Martínez, from Centro de Investigaciones Biológicas, CSIC, Madrid, we have described the catalytic mechanism of the aryl-alcohol oxidase fungal flavoenzyme and identified its key residues. This enzyme is involved in the degradation of lignin and the use of plant biomass for biotechnological purposes. The structure-function studies carried out have allowed to describe in molecular detail its stereoselective mechanism of oxidation of aromatic alcohols and aldehydes and to explore its potential for the production of enantiomers or the synthesis of biopolymers of interest. In collaboration with Dr. Nonato of the Universidade de São Paulo, Brazil, we are characterizing other flavoenzymes of biomedical interest such as the dihydroorotate dehydrogenase of Leshmania major and the L-amino acid oxidase of snake venom.

In addition, we also maintain specific collaborations with various groups with activity in the field of flavoenzymes for the kinetic and structural characterization of their flavoproteins or systems.

Keywords: flavoenzymes as biocatalysts • flavoenzyme mechanisms • rapid kinetic methods • enzymatic structure and dynamics • chemical biology

METHODOLOGIES USED IN OUR RESEARCH:

• Production of native and mutant proteins through the use of protein engineering techniques.
• Homologous and heterologous protein expression in different microorganisms.
• Purification of proteins (electrophoresis, chromatographic methods, HPLC, FPLC, …).
• Absorption spectrometry: kinetic studies in steady-state, differential spectroscopy, binding affinity, midpoint reduction potentials determination.
• Use of transient kinetic techniques such as stopped-flow and laser flash photolysis.
• Interaction and kinetic assays under anaerobic conditions.
• Fluorescence spectroscopy and circular dichroism.
• Protein crystallization and x-ray diffraction for the determination of 3D protein and protein complexes structures.
• Advance Electron Paramagnetic Resonance related techniques (ESEEM, HYSCORE, ENDOR).
• Isothermal Titration and Differential Scanning Calorimetries.
• Computational Biology Methods: Docking, Molecular Dynamics and QM/MM simulations.

RELEVANT PUBLICATIONS

1.- The catalytic cycle of the antioxidant and cancer-associated human NQO1 enzyme: hydride transfer, conformational dynamics and functional cooperativity. Ernesto Anoz-Carbonell, David J. Timson, Angel Pey and Milagros Medina. Antioxidants 9, 972. DOI: 10.3390/antiox9090772 (2020)

2.- The apoptosis-inducing factor family: Moonlighting proteins in the crosstalk between mitochondria and nuclei. Nerea Novo, Patricia Ferreira, Milagros Medina M. IUBMB Life 2020,1–14. DOI: 10.1002/iub.2390 (2020)

3.- In silico discovery and biological validation of ligands of FAD synthase, a promising new antimicrobial target. Isaias Lans, Ernesto Anoz-Carbonell, Karen Palacio-Rodríguez, José Antonio Aínsa, Milagros Medina and Pilar Cossio. PLOS Computational Biology, 19. DOI: 10.1371/journal.pcbi.1007898 (2020)

4.- Human riboflavin kinase: Species‐specific traits in the biosynthesis of the FMN cofactor. Ernesto Anoz‐Carbonell, Maribel Rivero, Victor Polo, Adrián Velázquez‐Campoy, Milagros Medina. The FASEB Journal, 34, 8. DOI: 10.1096/fj.202000566R (2020)

5.- Redox and ligand binding dependent conformational ensembles in the human apoptosis-inducing factor regulate its pro-life and cell death functions. Raquel Villanueva, Silvia Romero-Tamayo, Ruben Laplaza, Juan Martínez-Olivan, Adrián Velázquez-Campoy, Javier Sancho, Patricia Ferreira and Milagros Medina. Antioxidants and Redox Signaling. 20;30(18):2013-2029. DOI: 10.1089/ars.2018.7658. (2019)

6.- Towards the competent conformation for catalysis in the ferredoxin-NADP⁺ reductase from the Brucella ovis pathogen. Daniel Pérez-Amigot, Victor Taleb, Sergio Boneta, Ernesto Anoz-Carbonell, Maria Sebastián, Adrián Velázquez-Campoy, Victor Polo, Marta Martínez-Júlvez, and Milagros Medina. Biochim Biophys Acta Bioenerg, 1860:148058. DOI: 10.1016/j.bbabio.2019.148058. (2019)

7.- Self-sustained enzymatic cascade for the production of 2, 5-furandicarboxylic acid from 5-methoxymethylfurfural. Juan Carro, Elena Fernández-Fueyo, Carmen Fernández-Alonso, Javier Cañada, René Ullrich, Martin Hofrichter, Miguel Alcalde, Patricia Ferreira, Ángel T. Martínez. Biothechnology for Biofuels 11 – 1, pp. 86 (2018)

8.- Discovery of antimicrobial compounds targeting bacterial FAD synthetase. María Sebastián, Ernesto Anoz-Carbonel, Begoña Gracia, Pilar Cossio, José Antonio Aínsa, Isaías Lans and Milagros Medina. J. Enz. Inhib. Med. Chem. 33, 241-254 (2018)

9.- Identification of inhibitors targeting the Ferredoxin-NADP⁺ reductase from the Xanthomonas citri subsp citri phytopathogenic bacteria. Marta Martínez-Júlvez, Guillermina Goñi, Daniel Pérez- Amigot, Rubén Laplaza, Irina Ionescu, Silvana Petrocelli, María Laura Tondo, Javier Sancho, Elena Orellano and Milagros Medina. Molecules 23. E29. (2018)

10.- Multiple implications of an active site phenylalanine in the catalysis of aryl-alcohol oxidase. Juan Carro, Pep Amengual-Rigo, Ferran Sancho, Milagros Medina, Víctor Guallar, Patricia Ferreira, Ángel T. Martínez. Scientific Reports 8(1):8121 (2018)

11.- Proline dehydrogenase from Thermus thermophilus does not discriminate between FAD and FMN as cofactor. Mieke M.E.  Huijbers, Marta Martínez-Júlvez, Adrie H. Westphal, Estela Delgado-Arciniega, Milagros Medina and Willem J.H. van Berkel. Scientific Reports 7:43880 (2017)

12.- The trimer interface in the quaternary structure of the bifunctional prokaryotic FAD synthetase from Corynebacterium ammoniagenes. Ana Serrano, María Sebastián, Sonia Arilla-Luna, Silvia Baquedano, Beatriz Herguedas, Adrián Velázquez-Campoy, Marta, Martínez-Júlvez and Milagros Medina. Scientific Reports 7:404 (2017)

13.- Protein dynamics promote hydride tunnelling in substrate oxidation by aryl- alcohol oxidase. Juan Carro, Marta Martínez-Júlvez, Milagros Medina, Angel T. Martínez and Patricia Ferreira. Phys.Chem.Chem.Phys. 19, 28666 (2017)     

14.- Redox proteins of hydroxylating bacterial dioxygenases establish a regulatory cascade that prevents gratuitous induction of tetralin biodegradation genes. Laura Ledesma-García, Ana Sánchez-Azqueta, Milagros Medina, Francisca Reyes-Ramírez and Eduardo Santero. Scientific Reports 6:23848 (2016)

15.- Key residues regulating the reductase activity of the human mitochondrial apoptosis inducing factor. Raquel Villanueva, Carlos Marcuello, Alejandro Usón, M. Dolores Miramar, Maria Luisa Peleato, Ana Lostao, Santos A. Susin, Patricia Ferreira, and Milagros Medina. Biochemistry 54, 5175-5184 (2015)

16.- Dynamics of the active site architecture in plant-type Ferredoxin-NADP⁺ reductases catalytic complexes. Ana Sánchez-Azqueta, Daniela L. Catalano-Dupuy, Arleth López-Rivero, María Laura Tondo, Elena G. Orellano, Eduardo A. Ceccarelli, and Milagros Medina. BBA-Bioenergetics 1837, 1730-1738 (2014)

17.- Structural insights into the coenzyme mediated monomer-dimer transition of the pro-apoptotic Apoptosis Inducing Factor. Patricia Ferreira, Raquel Villanueva, Marta Martínez-Júlvez, Beatriz Herguedas, Carlos Marcuello, Patricio Fernandez-Silva, Lauriane Cabon, Juan A. Hermoso, Anabel Lostao, Santos A. Susin and Milagros Medina. Biochemistry 53, 4204-4215 (2014)

18.- Theoretical study of the mechanism of the hydride transfer between Ferredoxin NADP⁺ reductase and NADP⁺. The role of Tyrosine 303. Isaías Lans, Milagros Medina, Edina Rosta, Gerhard Hummer, Mireia García-Viloca, José M. Lluch, and Àngels González-Lafont. J. Am. Chem. Soc. 134, 20544-20553 (2012)

MAIN RESEARCH PROJECTS AND CONTRACTS

1.- Flavoenzymes in Health, Disease and Drug Discovery (FIHDADD). PID2019-103901GB-I00. Spanish Ministry of Science, Innovation and Universities. June 2020-June 2023. University of Zaragoza. Research Leaders: Milagros Medina and Patricia Ferreira.

2.- Paramagnetic Species in Catalysis Research. A Unified Approach Towards Heterogeneous, Homogeneous and Enzyme Catalysis (PARACAT). 813209. Program Marie Sklodovswka Curie, EU. Innovative Training Networks (ITN). January 2019-December 2022. Universities of Zaragoza, Turín, Amberes, Cardiff y Leipzig. Research Leader: Inés García Rubio from UNIZAR.

3.- Structural Biology Reference Group. E35_20R. Diputación General de Aragón (DGA). January 2020-December 2022. University of Zaragoza. Research Leader: Milagros Medina.

4.- Flavoenzymes: molecular mechanisms and targets, pathologies and Biotechnological applications. BIO2016-75183-P. Spanish Ministry of Economy and Competitiviness (MINECO). January 2017-December 2019. University of Zaragoza. Research Leader: Milagros Medina.

5.- Flavoenzyme dependent systems: from action mechanisms to biotechnological and sanitary applications. BIO2013-42978-P. Spanish Ministry of Economy and Competitiviness (MINECO). January2014-December 2016. University of Zaragoza. Research Leader: Milagros Medina.

6.- Seguimiento de los cambios conformacionales del dominio apoptótico del Factor de Inducción de Apoptosis Humano (hAIF) con marcaje selectivo de espín y espectroscopia de EPR. University of Zaragoza. January 2015-December 2015. Research Leader: Patricia Ferreira.

7.- Retos enzimáticos, químicos y de ingeniería para la utilización de los recursos agroforestales no alimentarios (lignocelulosa) en una bio-economía más sostenible y menos contaminante. AC2015-00008-00-00. Spanish Ministry of Economy and Competitiviness (MINECO). July 2015-March 2017. CIB-CSIC/ University of Zaragoza and others. Research Leader: Susana Camarero Fernández.

8.- Structure-based drug design for diagnosis and treatment of neurological diseases: dissecting and modulating complex function in the monoaminergic systems of the brain. COST ACTION CM1103. EU. November 2011-November 2015. University of St. Andrews+18. Research Leader: Rona Ramsay.

9.- Catalytic mechanisms in flavoenzymes: keys for their biotechnological and therapeutic use. BIO2010-14983. Spanish Ministry of Science and Innovation. January 2011- September 2014. University of Zaragoza. Research Leader: Milagros Medina.

10.- Propiedades pro-apoptótica y oxido-reductasa de la proteína mitocondrial AIF (factor de inducción de apoptosis): ¿Funciones biológicas independientes o complementarias? Ref. 212363 (JIUZ-2012-BIO-01). University of Zaragoza. January 2013-December 2013. Research Leader: Patricia Ferreira.

PATENTS

Enzymatic Composition and Enzymatic process for the production of 2,5-furandicarboxylic acid from 5-methoxymethylfurfural using said enzymatic composition. J. Carro, E. Fernández-Fueyo, M. Alcalde, P. Ferreira, R. Ullrich, M. Hofrichter, AT. Martinez. CSIC, University of Zaragoza and Technical University of Dresden. 2017.

COLLABORATORS

From BIFI

Dr. Adrián Velázquez-Campoy
Dr. Pier Paolo Bruscolini
Dr. Ramón Hurtado-Guerrero
Dr. José Alberto Carrodeguas
Dr. José Antonio Aínsa
Dr. Patricio Fernández
Dr. Raquel Moreno
Dr. Víctor Polo
Dr. Javier Sancho

From other Institutions

Instituto de Nanociencia de Aragón (INA), Zaragoza
– Dr. Ana Isabel Gracia Lostao
Estación Experimental de Aula Dei, Zaragoza
– Dr. Inmaculada Yruela
Universidad Nacional de Rosario, Rosario, Argentina
– Prof. Néstor Carrillo
– Dr. Eduardo Ceccarelli
– Dr. Elena Orellano
Centro de Investigaciones Biológicas, CSIC, Madrid
– Dr. Ángel Martínez
Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC. Sevilla
– Dr. Manuel Hervás
– Dr. José A. Navarro
Centre de Recherche des Cordeliers, Paris, France 
– Dr. Santos Susín
Universita degli Studi di Bari
– Dr. Maria Barile
Universidad de Sao Paolo, Brasil
– Dr. M Cristina Nonato
Clark University, Biology Department, USA
– Dr. David Scott Hibbett
Universidad de Granada
– Dr. Angel L. Pey
Centro de Investigación y Tecnología Agroalimentaria de Aragón
– Dr. Pilar María Muñoz Álvaro
Instituto de Recursos Naturales y Agrobiología de Salamanca  
– Dr. Mónica Balsera Diéguez
University of Antioquia UdeA, Medellin, Colombia.
– Dr. Pilar Cossio
Universitat Jaume I, Castellón
– Dr. Vicent Moliner

Protein misfolding and amyloid aggregation

Head of the Research Line:

Dr. Nunilo Cremades

Researchers:

José Daniel Camino, PhD student
Pablo Gracia, PhD student
Diego De la Fuente, PhD student
David Polanco Irisarri, PhD student

MAIN LINE OF RESEARCH

The phenomenon of protein misfolding and amyloid aggregation has emerged in recent years as a subject of fundamental importance in a wide range of scientific disciplines such as physics, chemistry, biology and medicine. This explosion of interest on the process of protein misfolding and amyloid aggregation has primarily resulted from the recognition that approximately 50 human diseases and disorders are associated with this process, some of them among the most common and debilitating medical conditions in the modern world, including Alzheimer’s, Parkinson’s and type II diabetes.

Despite the social and economical impact of some of these diseases, little is known about the molecular origins and mechanisms of protein amyloid aggregation and its associated toxicity. The research conducted in the group led by Dr. Nunilo Cremades aims to address these fundamental questions by combining a wide range of biophysical techniques, including state-of-the-art single-molecule fluorescence, with cell biology experiments. In addition, we aim to identify new protein targets for the development of early diagnostic tools as well as more effective therapeutics for this type of diseases.

Figure 1. The development and application of single-molecule fluorescence techniques have allowed us to investigate in unprecedented detail amyloid aggregation and to discover new possible therapeutic targets. Cremades N. et al. Cell 2012.

Figure 2. We have recently been able to purify highly stable amyloid oligomeric species of alpha-synuclein, the protein whose aggregation and deposition is linked with the development of Parkinson’s disease, and show that these species are highly cytotoxic and have general properties common to other amyloid oligomers. By combining a wide range of biophysical methods with cryo-EM image reconstruction techniques we were able to obtain three-dimensional structural models and reveal the quaternary structural architectures of toxic alpha-synuclein amyloid oligomers. Chen SW. et al. PNAS USA 2015.

Figure 3. We have studied the role of water in the modulation of the energetic barrier of alpha-synuclein amyloid nucleation, and we have found that under conditions of low water activity homogeneous nucleation is orders of magnitude faster than homogeneous or heterogeneous nucleation under high protein hydration conditions. We have also demonstrated that alpha-synuclein homogeneous nucleation is highly favoured in the interior of biomolecular condensates, also referred to as membrane-less organelles, generated by liquid-liquid phase separation. Camino et al. Chem. Sci. 2020.

OTHERS LINES OF RESEARCH

The group is also interested in understanding the role of intrinsic structural disorder in protein function and disease.

Figure 4. Typical phase diagram for a natively ordered and intrinsically disordered protein. We recently characterised the energy landscape of a naturally occurring intrinsically disordered enzyme. This protein acquires a wide range of molten globule-like, pre-molten globule-like and random coil-like conformations depending on the solution conditions. Zambelli and Cremades et al. Mol. Biosyst. 2012.

Figure 5. The presence of a specific type of structural disorder (molten globule-like conformations) correlates with the ability of human lysozyme to form amyloid fibrils (I56T mutant responsible for a hereditary form of systemic amyloidosis). Dhulesia and Cremades et al. J. Am. Chem. Soc. 2010

Relevant publications

1.The extent of protein hydration dictates the preference for heterogeneous or homogeneous nucleation generating either parallel or antiparallel b-sheet alpha-synuclein aggregates. Camino J.D., Gracia P., Chen S.W., Sot J., de la Arada I., Sebastián V., Arrondo J.L.R., Goñi F.M., Dobson C.M. and Cremades N. Chem. Sci. 2020, DOI: 10.1039/D0SC05297C.

2. Defining alpha-synuclein species responsible for Parkinson´s disease phenotypes in mice. Froula J.M., Castellana-Cruz M., Anabtawi N.M., Camino J.D., Chen S.W., Thrasher D.R., Freire J., Yazdi AA., Fleming S., Dobson C.M., Kumita J.R., Cremades N. (co-corresponding author) and Volpicelli-Daley L.A. J. Biol. Chem. 2019, 294(27):10392-10406. Selected paper of the year in JBC in the area “Molecular basis of disease”.

3. The contribution of biophysical and structural studies of protein self-assembly to the design of therapeutic strategies for amyloid diseases. Cremades N. and Dobson C.M. Neurobiol. Dis. 2018, 109:178-190.

4. Structural basis of membrane disruption and cellular toxicity by alpha-synuclein oligomers. Fusco G., Chen S.W., Williamson P.T.F., Cascella R., Perni M., Jarvis J.A., Cecchi C., Vendruscolo M., Chiti F., Cremades N., Ying L., Dobson C.M. and De Simone A. Science 2017, 358(6369): 1440-1443.

5. Inhibition of alpha-synuclein fibril elongation by Hsp70 is governed by a kinetic binding competition between alpha-synuclein species. Aprile F.A., Arosio P., Fusco G., Chen S.W., Kumita J.R., Dhulesia A., Tortora P., Knowles T.P., Vendruscolo M., Dobson C.M. and Cremades N. Biochemistry 2017, 56(9): 1177-1180.

6. Amyloid-b and a-synuclein decreases the level of metal-catalyzed reactive oxygen species by radical scavenging and redox silencing. Pedersen J.T., Chen S.W., Borg C.B., Ness S., Bahl J.M., Heegaard N.H., Dobson C.M., Hemmingsen L., Cremades N. (co-corresponding author) and Teilum K. J. Am. Chem. Soc. 2016, 138(12):3966-9.

7. Alpha-synuclein oligomers interact with metal ions to induce oxidative stress and neuronal death in Parkinson’s disease. Deas E., Cremades N., Angelova P.R., Ludtmann M.H., Yao Z., Chen S.W., Horrocks M.H., Banushi B., Little D., Devine M.J., Gissen P., Klenerman D., Dobson C.M., Wood N.W., Gandhi S. & Abramov AY. Antioxid. Redox Signal. 2016, 24(7):376-91.

8. Structural characterization of toxic oligomers that are kinetically trapped during α-synuclein fibril formation. Chen S.W., Drakulic S., Deas E., Ouberai M., Aprile F.A., Arranz R., Ness S., Roodveldt C., Guilliams T., De-Genst E.J., Klenerman D., Wood N.W., Knowles T.P.J., Alfonso C., Rivas G., Abramov A.Y., Valpuesta J.M., Dobson C.M. and Cremades N. Proc. Natl. Acad. Sci U.S.A. 2015, 112(16):E1994-2003.

9. Direct observation of the interconversion of normal and toxic forms of alpha-synuclein. Cremades N., Cohen S.I., Deas E., Abramov A.Y., Chen A.Y., Orte A., Sandal M., Clarke R.W., Dunne P., Aprile F.A., Bertoncini C.W., Wood N.W., Knowles T.P.J., Dobson C.M. & Klenerman D. Cell 2012, 149(5): 1048-59.

Main research projects

Main current research projects

• PGC2018-096335-B-100: “Understanding Parkinson´s disease alpha-synuclein amyloid formation in the cell: From single-molecule biophysics in cell-like test tubes to organs-on-chips”. Sponsor: Ministerio Español de Ciencia, Innovación y Universidades, Agencia Estatal de Investigación / EU (Fondos FEDER). IP: Dra. Nunilo Cremades y Dr. José A. Carrodeguas.1/01/2019 – 31/12/2022
• UZCUD2020-BIO-01: “Caracterización de las primeras interacciones entre moléculas de alfa-sinucleína que llevan a la formación de agregados tóxicos involucrados en la enfermedad de Párkinson”. Sponsor: Universidad de Zaragoza. IP: Dra. Nunilo Cremades y Dra. Inés García. 1/10/2020 – 30/09/2021

Collaborators

Laura Volpicelli-Daley (University of Alabama, USA)
Fabrizio Chiti (University of Florence, Italy)
Janet Kumita (University of Cambridge, UK)
Douglas V. Laurents (IQFR-CSIC, Spain)
Felix Goñi (Basque Centre for Biophysics, Spain)
Arturo Muga (Basque Centre for Biophysics, Spain)
Paola Picotti (ETH Zurich, Switzerland)

Contact

Dra. Nunilo Cremades

Instituto de Biocomputación y Física de Sistemas Complejos
Universidad de Zaragoza
Mariano Esquillor S/N. Edificio I+D+i
Zaragoza 50018, Spain
Teléfono: +34-876555417
Email: ncc@unizar.es

Clinical diagnosis and drug delivery

Head of the Research Line:

Olga Abian Franco

Researchers:

Rafael Clavería, Predoctoral Student
María Arruebo, Predoctoral Student
Alberto Rodrigo, Predoctoral Student
Arturo Vinuesa, Predoctoral Student

SUMMARY OF THE RESEARCH LINE

Clinical Diagnosis

Differential Scanning Calorimetry (DSC) has recently emerged as a promising technique that provides useful information about serum/plasma interactomics (composition in proteins and metabolites, as well as their interactions). As a result of the illness, serum/plasma composition is altered and it is possible to discriminate between healthy individuals and patients with certain diseases.

A previous study performed in our group (25 healthy subjects and 60 gastric adenocarcinoma patients) showed that there were significant differences in the variables obtained from the calorimetric profiles of healthy and gastric adenocarcinoma patients and also between patients with different disease stages.

The validation and implementation of a methodology (DIGCAL) as a useful tool in diagnosing and monitoring relevant tumor pathologies (pancreatic ductal adenocarcinoma, preneoplasic pancreatic cystic lesions and stomach cancer), as well as in the isolation and identification of potential tumor biomarkers, are proposed in this project.

Calorimetric profiles of serum from healthy subjects and patients with the three types of cancer at different stages will be obtained before and after 6 months of clinical treatment. The multiparametric analysis of the thermal profiles will allow us to establish a clinical protocol for: 1/ screening a certain tumor process; 2/ classifying patients according to their tumour stage; 3/ monitoring the progression or control of the illness; 4/ identifying specific biomarkers for each disease type.
DIGCAL could reach the clinical level not only as a diagnosis tool, but also as a monitoring and tracking system of patients during the therapeutic treatment, adding value in prognostic or pharmacologic decisions. At the same time, identified potential biomarkers could be part of a user-friendly diagnosis kit for primary points of care.

Drug Delivery

Drug delivery is the method or process of administering a pharmaceutical compound to achieve a therapeutic effect in humans or animals. Drug delivery technologies modify drug release profile, absorption, distribution and elimination for the benefit of improving product efficacy and safety, as well as patient convenience and compliance. Drug release is from: diffusion, degradation, swelling, and affinity-based mechanisms. Most common routes of administration include the preferred non-invasive peroral (through the mouth), topical (skin), transmucosal (nasal, buccal/sublingual, vaginal, ocular and rectal) and inhalation routes. Many medications such as peptide and protein, antibody, vaccine and gene based drugs, in general may not be delivered using these routes because they might be susceptible to enzymatic degradation or cannot be absorbed into the systemic circulation efficiently due to molecular size and charge issues to be therapeutically effective. For this reason many protein and peptide drugs have to be delivered by injection or a nanoneedle array. For example, many immunizations are based on the delivery of protein drugs and are often done by injection.

Current efforts in the area of drug delivery include the development of targeted delivery in which the drug is only active in the target area of the body (for example, in cancerous tissues) and sustained release formulations in which the drug is released over a period of time in a controlled manner from a formulation. In order to achieve efficient targeted delivery, the designed system must avoid the host’s defense mechanisms and circulate to its intended site of action. Types of sustained release formulations include liposomes, drug loaded biodegradable microspheres and drug polymer conjugates. In this sense, Nanoparticles (NP) in Biomedicine represent a promising technology for drug transport and release. There are many possibilities for NP´s surface functionalization and so, many strategies for including drugs in them (allowing NP going mainly to their acting site) can be developed.

This research line represents a new strategy for including some antiviral compounds active against hepatitis C virus (HCV), that were previously developed in this group.

Several materials have been used:

1/ Cyclodextrins:

The chemical structure of CDs, cyclic oligosaccharides composed of α-1,4-glycosidic-linked glycosyl residues, provides them structural and physico-chemical properties that allow their use as molecular carriers.

In their hydrophobic cavity a wide range of compounds ranging from ions to proteins can be trapped. In addition, CDs exhibit low toxicity and low immunogenicity, and they have been used in the pharmaceutical field by promoting CD-drug complexes in order to improve the absorption, distribution, metabolism, excretion, and toxicity (ADMET)-related properties of a drug (eg, solubility, stability, delivery and release, membrane permeability and absorption, toxicity). Currently, more than 30 products can be found in the market based on CD complexes.

2/ Shell Cross-Linked Polymeric Micelles

Cross-linked polymeric micelles (CLPM), formed by amphiphilic block copolymers, have been successful for biomedical applications. irst, they can fulfill those general requirements for drug delivery systems: water solubility, low toxicity, to increase the stability of the drug inside the living organisms, to facilitate cellular uptake compared to free drug, and to produce its controlled release at a specific location. Second, the amphiphilic nature of the constituent polymer results in a hydrophobic core and a hydrophilic shell that allows encapsulation of both types of drug. Third, these nanoparticles offer further stability under high dilution conditions, below the critical micellar concentration, compared to other polymeric micelles.

Indeed, cross-linking avoids its disintegration in the bloodstream and the release of the drug before reaching the target cell. Particularly, fixation of the micelle structure by light-induced covalent cross-linking, mostly employing acrylate reactive groups, represents a clean and effective procedure to prepare stable polymer micelles that can hold either water-soluble and non-water soluble molecules and transport them through the bloodstream.

3/ Block Copolymers Micelles

Polymeric drug carriers are one of the current challenges of nanomedicine. Since the concept of physical drug encapsulation within polymeric aggregates was introduced, a significant number of polymer assemblies have been identified. In particular, the construction of amphiphilic block copolymer-based drug carriers is a subject of great interest and a stimulating topic of interdisciplinary research in chemistry, biology and materials science. In aqueous media, self-assembly of amphiphilic block copolymers (BCs) to minimize energetically unfavorable hydrophobic water interactions can lead to a variety of polymeric nanostructures including especially appealing spherical micelles and vesicles.

Relevant publications

1.- Polymeric micelles from block copolymers containing 2,6-diacylaminopyridine units for encapsulation of hydrophobic drugs.
Concellón A, Clavería-Gimeno E, Velázquez-Campoy A, Abian O, Piñol M, Oriol L.RSC Advances, 2016, 6, 24066–24075.

2.- Biophysical Screening for Identifying Pharmacological Chaperones and Inhibitors against Conformational and Infectious Diseases. Velazquez-Campoy A, Sancho J, Abian O, Vega S. Curr Drug Targets. 2016 Jan 31. [Epub ahead of print]

3.- Cysteine Mutational Studies Provide Insight into a Thiol-Based Redox Switch Mechanism of Metal and DNA Binding in FurA from Anabaena sp. PCC 7120. Botello-Morte L, Pellicer S, Sein-Echaluce VC, Contreras LM, Neira JL, Abian O, Velázquez-Campoy A, Peleato ML, Fillat MF, Bes MT. Antioxid Redox Signal. 2016, 24, 173-185.

4.- On the link between conformational changes, ligand binding and heat capacity. Vega S, Abian O, Velazquez-Campoy A. Biochim Biophys Acta. 2015 Oct 14. pii: S0304-4165(15)00274-3.

5.- Shell Cross-Linked Polymeric Micelles as Camptothecin Nanocarriers for anti-HCV Therapy. Jiménez-Pardo I, González-Pastor R, Lancelot A, Claveria-Gimeno R, Velázquez-Campoy A, Abian O, Ros MB, and Sierra T. Macromol. Biosci. 2015, 15, 1381–1391.

6.- Su1990 A New Technology for the Classification of Patients With Gastric Adenocarcinoma Based on Differential Scanning Calorimetry Serum Thermograms. Vega S, Garcia-Gonzalez MA, Lanas A, Velazquez-Campoy A, Abian O.*. Gastroenterology 148(4): S-569 · April 2015

7.- Rescuing compound bioactivity in a secondary cell-based screening by using γ-cyclodextrin as a molecular carrier. Clavería-Gimeno R, Vega S, Grazu V, De la Fuente JM, Lanas A, Velazquez-Campoy A, Abian O.*. International Journal of Nanomedicine 2015, 10: 2249-2259.

8.- Deconvolution Analysis for Classifying Gastric Adenocarcinoma Patients Based on Differential Scanning Calorimetry Serum Thermograms. Vega S, Garcia-Gonzalez MA, Lanas A, Velazquez-Campoy A, Abian O.*. Scientific reports, 5:7988 (2015).

9.- Ionic liquids in water: a green and simple approach to improve activity and selectivity of lipases.
Filice M, Romero O,  Abian O, De las Rivas B and Palomo J.M. RSC Advances 4: 49115- 49122 (2014).

10.- A unified framework based on the binding polynomial for characterizing biological systems by isothermal titration calorimetry. Vega S., Abian O.*and Velazquez-Campoy A. Methods. 2014 pp: S1046-2023(14) 00316-8.

11.- Allosteric Inhibitors of the NS3 Protease From the Hepatitis C Virus. Abian O.*, Vega S., Sancho J., Velazquez-Campoy A. PLOSone 2013, 8 (7): 69773.

12.- NS3 protease from hepatitis C virus: Biophysical studies on an intrinsically disordered protein domain. Vega S., Neira J.L., Marcuello C.,  Lostao A., Abian O. * and Velazquez-Campoy A. International Journal of Molecular Sciences 2013, 14: 13282-13306.

13.- Experimental Validation of In Silico Target Predictions on Synergistic Protein Targets. Cortes-Ciriano I, Koutsoukas A, Abian O, Velazquez-Campoy A and Bender A. MedChemComm 2013, 4, 278–288.

14.- Altering the interfacial activation mechanism of a lipase by solid-phase selective chemical modification. López-Gallego F, Abian O, Guisán JM. Biochemistry. 2012 Sep 4;51(35):7028-36.

15.- Semisynthetic peptide-lipase conjugates for improved biotransformations. Romero O, Filice M, de las Rivas B, Carrasco-Lopez C, Klett J, Morreale A, Hermoso JA, Guisan JM, Abian O, Palomo JM. Chem Commun (Camb). 2012 Sep 18;48(72):9053-5.

Main research projects

Ongoing Projects:

1.- Analysis of protein/metabolites interactions in plasma serum using calorimetry: application as a quick and noninvasive diagnostic method for early detection and monitoring of tumoral digestive diseases (DIGCAL). Funding Institution: Health Institute Carlos III. From: January 2016 To: December 2018. Principal Investigator (PI): Olga Abian Franco.

2.- Validación de un nuevo método diagnóstico en suero, rápido no invasivo para detección precoz de cáncer de páncreas (PANCal). Funding Institution: Asociación Española de Gastroenterología (AEG). From: 2015 To: 2016. Principal Investigator (PI): Olga Abian Franco.

Former Projects:

3.- Adapted Nanoparticles for transport and specific release of drugs against hepatitis C virus (VHC).
Funding Institution: Health Institute Carlos III. From: 2011 To: 2013. Principal Investigator (PI): Olga Abian Franco.

4.- Implementation of in vitro e in vivo studies of anti-infectious compounds effective against Helicobacter pylori y el HCV (hepatitis C virus). Funding Institution: Health Institute Carlos III. From: 2008 To: 2011. Principal Investigator (PI): Olga Abian Franco.

Collaborators

Collaborations from BIFI

Dr. Adrián Velázquez-Campoy
Prof. Javier Sancho
Dr. Jose Luis Neira

Collaborators from other Institutions

James Graham Brown Cancer Center, University of Louisville, Louisville, KY, EEUU
PhD. Nichola Garbett

Instituto Aragonés de Ciencias de la Salud (I+CS), Zaragoza
Prof. Angel Lanas
Dra. Trinidad Serrano
Dra. Estela Solanas

Instituto de Ciencia de Materiales de Aragón (ICMA). Química Orgánica. Facultad de Ciencias.
Dra. Teresa Sierra
Prof. Luis Oriol
Prof. Milagros Piñol

Instituto de Nanociencia (INA), Universidad de Zaragoza, Zaragoza
Dr. Jesús Martínez de Lafuente
Dra. Valeria Grazú
Dra. Berta Saez

Universidad de San Jorge (USJ)
Prof. Victor López
Prof. Elisa Langa

Instituto de Catálisis y Petroleoquímica, CSIC, Madrid.
Dr. Jose Miguel Palomo
Dr. Fernando López Gallego
Dr. Jose Manuel Guisan

Universidad de Zaragoza
Dr. Jose Antonio Ainsa

Unidad de Investigación Traslacional, Hospital Universitario Miguel Servet, Zaragoza
Dra. Pilar Alfonso
Dra. Pilar Giraldo
Dr. Miguel Pocovi

Servicio de Microbiología-INIBIC. Complejo Hospitalario Universitario A Coruña, La Coruña
Dr. Francisco José Pérez-Llarena

Signal transduction and membrane protein therapies

Head of the Research Line:

Javier García Nafría

Personal group webpage:
https://sites.google.com/view/signal-transduction-lab/homepage

Researchers:

Sandra Arroyo Urea, PhD student
Ángela Carrión Antolí, PhD stuednt
Iris del Val García, PhD student
Natalia Garré Ramo Researcher
Andrés Manuel González Ramirez, Postdoctoral Researcher
Kleopatra Papadouli, Erasmus+ Researcher

SUMMARY OF THE RESEARCH LINE

Brain membrane protein complexes

Membrane proteins represent 30% of the human genome, mediate intercellular communication and are the target for 60% of all the drugs currently used to treat disease. Membrane proteins play a especial role in the brain, where cellular communication is the basis for brain function including consciousness and memory formation. Our goal is to understand how neuronal receptors work, understanding signal detection and transduction to the intracellular millieu. We pose special emphasis on the integration of signals at the membrane by membrane protein complexes. For this purpose we use an integrated approach of structural biology techniques (Cryo-electron microscopy and X-ray crystallography), biophysics and biochemical assays. High-resolution cryo-electron microscopy currently plays a central role since we can now determine structures of membrane protein complexes that were previously intractable by X-ray crystallography.

Protein engineering tools

Membrane proteins are intrinsically unstable outside the membrane environment and to overcome this problem we develop our own protein engineering tools. We have develop a molecular cloning system that simplifies all cloning protocols (García-Nafría, Sci. Rep, 2016 and Watson, JBC, 2019) , allowing to create in a cheap, quick and simple manner an array of membrane protein constructs that can be tested for expression and stability. However, this system can be applied in any biomedical research laboratory in any type of project.

Methodologies used in our research

• Molecular cloning.
• Protein engineering.
• Protein production in bacteria, insect and mammalian cells.
• Protein purification.
• Surface plasmon resonance (Biacore).
• Microscale thermophoresis.
• Macromolecular X-ray crystallography.
• Single-particle Cryo-electron microscopy.
• Biochemical cell assays (Western Blot, fluorescence, functional assays).

Relevant publications

1. Structural insights into promiscuous GPCR-G protein coupling.Carrión-Antolí A., Mallor-Franco J., Arroyo-Urea S., García-Nafría J.Progress in Molecular Biology and Translational Science. 2022. In press.

2. Structure determination of GPCRs: cryo-EM compared with X-ray crystallography. García-Nafría J, Tate CG. Biochem Soc Trans. 2021 Sep 28:BST20210431. doi: 10.1042/BST20210431.

3. Molecular determinants of β-arrestin coupling to formoterol-bound β1-adrenoceptor. Lee Y., Warne T., Nehme R., Pandey S., Chaturvedi M., Dwivedi-Agnihotri H., Edwards P., García-Nafría J., Leslie A., Shukla AK., Tate CG. Nature. 2020 Jun 17. doi: 10.1038/s41586-020-2419-1.

4. In vivo DNA assembly using common laboratory bacteria: a re-emerging tool to simplify molecular cloning. Jake F. Watson and García-Nafría J. Journal of Biological Chemistry. 2019 Oct 18;294(42):15271-15281.

5.Cryo-Electron Microscopy: Moving Beyond X-ray Crystal Structures for Drug receptors and Drug development.García-Nafría J.* and Tate CG*.  Annual Reviews in Pharmacology and Toxicology. 2020. In press. (*corresponding author)

6. Architecture of the heteromeric GluA1/2 AMPA receptor in complex with the auxiliary subunit TARP γ8. Herguedas B, Watson JF, Ho H, Cais O, García-Nafría J, Greger IH. Science. 2019 Mar 14. pii: eaav9011

7. Cryo-EM structure of the serotonin 5-HT1B receptor coupled to heterotrimeric Go. García-Nafría J., Nehme R., Edwards P., Tate CG. Nature. 2018, 558 (7711). 620-62.

8. Cryo-EM structure of the adenosine A_2A receptor coupled to an engineered heterotrimeric G protein.García-Nafría J., Lee Y., Bai X., Carpenter B. & Tate CG. Elife. 2018, May 4;7. pii: e35946.

9. Structure and organization of heteromeric AMPA-type glutamate receptors. Herguedas B*, García-Nafria J*, Cais O, Fernandez-Leiro R, Ho H, Krieger J, Greger IH. Science. 2016, Apr 29;352(6285):aad3873.

10. IVA cloning: A single-tube universal cloning system exploiting bacterial In Vivo Assembly. García-Nafria J*, Watson JF, Greger IH. Scientific Reports.  2016, Jun 6;6:27459. (*corresponding author).

Main research projects

1.- Ministerio de Ciencia, Innovacion y Universidades. 163.350 euros.
PID2020-113359GA-I00. 2021-2024.

Collaborators

Ramón Hurtado Guerrero (BIFI, Zaragoza, Spain).
Jake Watson (MRC Laboratory of Molecular Biology, Cambridge, UK).
Maria José Sanchez Barrena (CSIC, Madrid, Spain).
Beatriz Herguedas Francés (BIFI, Zaragoza, Spain).
Nunilo Cremades Casasín (BIFI, Zaragoza, Spain)

Structural Biology of neuronal membrane receptors.<br />

Head of the Research Line:

Beatriz Herguedas Francés

Researchers

Beatriz Herguedas Francés (IP)
Carlos Vega Gutiérrez (PhD student)
Irene Sánchez Valls (PhD student)

SUMMARY

AMPA type Glutamate receptors (AMPARs) are ligand-gated ion channels that mediate fast excitatory neurotransmission between neurons and play a role in neuronal plasticity. Their disfunction is associated with different disease conditions, including ALS, stroke and epilepsy.  AMPARs are a diverse group of protein complexes with different kinetic, trafficking and pharmacological properties. Their diversity is achieved at different levels. The receptor core is a tetrameric ion channel composed of different combinations of four subunits (GluA1 to GluA4) that assemble as preferential heterotetramers (Herguedas et al 2013). Subunit composition is regionally and developmentally regulated (Schwenk et al 2014).  There are two splicing forms for each AMPAR gene –called flip and flop- that modulate receptor kinetics, while two RNA editing sites exist in some subunits (Traynellis et al 2010). Receptor heterogeneity is further increased by the interaction with more than 30 proteins, which form both transient and stable complexes (Schwenk et al 2012). In our group we aim to explore the architecture of different AMPAR complexes using cryo-EM, getting insights into their molecular mechanism and the influence of the lipid environment in the receptor function. Besides, we aim to apply biophysical and single molecule tools to understand the dynamics of such complexes as well as to develop small molecules which target specific AMPAR complexes.

Herguedas´ group is part of the DGA group NEUROMOL, together with PIs Jose A. Carrodeguas, Nunilo Cremades, Javier García Nafría. Our focus is to study neuronal proteins -including ion channels, receptors and intrinsically disordered proteins- and their role in neuropathologies. We are also part of a the “Grupo de Acción BioF-DTE (Implementación y desarrollo de herramientas BIOFísicas en el estudio, Diagnóstico y Tratamiento de Enfermedades)” from the Campus Iberus, which aims to apply biophysical tools to develop novel strategies for the diagnosis and treatment of human diseases.

RELEVANT PUBLICATIONS

1. Architecture of the heteromeric GluA1/2 AMPA receptor in complex with the auxiliary subunit TARP y8. Herguedas B*; Watson JF, Ho H; Cais O; García-Nafría J; Greger H*. Science, 2019 (*corresponding author)
2. Druggability simulations of ionotropic glutamate receptors reveal a high-susceptibility binding site in the GluA3 AMPA receptor N-terminal domain. Lee* JY, Krieger J. *, Herguedas B. *, García-Nafría J. *, Dutta A., Shaikh S., Greger I.H., and Bahar I.. Structure, 2018. (*co-first author)
3. Structure and organization of heteromeric AMPA-type glutamate receptors Herguedas B*, García-Nafría J*, Cais O, Fernández-Leiro R, Krieger J, Ho H, Greger IH.. Science. 2016. (*co-first author)
4. The dynamic AMPA receptor extracellular region: a platform for synaptic protein interactionS. García-Nafría J, Herguedas B, Watson JF, Greger IH. s. J Physiol. Review. 2016.
5. Structural insights into the synthesis of FMN in prokaryotic organisms. Herguedas B, Lans I, Sebastián M, Hermoso JA, Martínez-Júlvez M, Medina M. Acta Crystallogr D Biol Crystallogr. 2015.
6. Mapping the interaction sites between AMPA receptors and TARPs reveals a role for the receptor N-terminal domain in channel gating. Cais O, Herguedas B, Krol K, Cull-Candy SG, Farrant M, Greger IH. Cell Reports, 2014.
7. A hydrogen bond network in the active site of Anabaena ferredoxin-NADP(+) reductase modulates its catalytic efficiency. Sánchez-Azqueta A, Herguedas B, Hurtado-Guerrero R, Hervás M, Navarro JA, Martínez-Júlvez M, Medina M. BBA bioenergetics. 2014.
8. Receptor heteromeric assembly-how it works and why it matters: the case of ionotropic glutamate receptors. Herguedas B, Krieger J, Greger IH. Prog Mol Biol Transl Sci. Review. 2013.
9. Oligomeric state in the crystal structure of modular FAD synthetase provides insights into its sequential catalysis in prokaryotes Herguedas B, Martínez-Júlvez M, Frago S, Medina M and Hermoso JA.. J Mol Biol 2010.
10. Crystallization and preliminary X-ray diffraction studies of FAD synthetase from Corynebacterium ammoniagenes. Herguedas B, Martínez-Júlvez M, Frago S, Medina M and Hermoso JA. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2009.
11. Flavodoxin: a compromise between efficiency and versatility in the electron transfer from Photosystem I to Ferredoxin-NADP(+) reductase. Goñi G, Herguedas B, Hervás M, Peregrina JR, De la Rosa MA, Gómez-Moreno C, Navarro JA, Hermoso JA, Martínez-Júlvez M, Medina M. BBA Bioenergetics. 2009.
12. Protein motifs involved in coenzyme interaction and enzymatic efficiency in anabaena ferredoxin-NADP+ reductase. Peregrina JR*, Herguedas B*, Hermoso JA, Martínez-Júlvez M, Medina M Biochemistry. 2009. (*co-first author)

MAIN RESEARCH PROJECTS

1. Structure and Dynamics of Calcium Permeable AMPA receptors. Agencia Estatal de Investigación. PID2019-106284GA-I00. (01/06/2020-31/05/2021). Principal Investigator.
2. Dotación Adicional Programa Ramón y Cajal. Agencia Estatal de Investigación. RYC2018-025720-I. 01/05/2020-30/06/2025. Principal Investigator.
3. AMPA Glutamate Receptors: the role of the extracellular domains in receptor assembly and allosteric regulation MRC Centenary Early Career Award. P.I. Beatriz Herguedas. 01/08/2012-30/09/2013. Principal Investigator.

COLLABORATORS

Ingo Greger (MRC Laboratory of Molecular Biology)
Javier García Nafría (Zaragoza)

Contact: bherguedas at unizar.es

Enzyme modulation & Reaction Mechanisms

Head of the Research Line:

Pedro Merino

Researchers:
Manuel Pedrón (contratado pre-doctoral Gobierno de Aragón)
Sara Orta (contratado pre-doctoral Gobierno de Aragón)
Sandra Pereira (contratado pre-doctoral FPI)
Ignacio Sanz (contratado pre-doctoral FPI)

Colaboradores:
Tomas Tejero Lopez (ISQCH)
Iñaki Delso (ISQCH)

SUMMARY

SUBLINE 1: Enzyme modulation and enzymatic mechanisms

Our group focus its activity on pursuing new insights in to our understanding of health related biological processes. With a wide experience on asymmetric organic synthesis and well-equipped laboratories in the Faculty of Sciences, the group, working in the field of Chemical Biology is interested in the design and synthesis of small molecules  including glycomimetics and nitrogenated compounds- acting as key modulators and/or inhibitors of target enzymes associated to specific biological functions. We are particularly interested in glycosyltransferases and transglycosylases among other enzymes. This activity is carried out by combining a series of multidisciplinary tools and techniques including in-house developed synthetic methodologies using metal- and organic catalysis, biocomputational approaches (docking and molecular dynamics) and advanced spectroscopic techniques (i.e. STD-NMR).

Figure 1. Glycosyltransferase GalNAc-T2: Molecular dynamics study and interactions with a designed ligand of the

Figure 2. Glycosyltransferase POFUT1: SN1-like catalytic mechanism with formation of an intimate ion pair in the transition state

SUBLINE 2: Organic Reactions Mechanisms.

The group is also interested in studying reaction mechanisms by using QM calculations and modern topological approaches such as ELF (electron localization function) and NCI (non-covalent interactions) analyses.

Figure 3. QM and topological studies of reaction mechanisms

The studies include elucidation of catalytic reactions considering both metal-catalyzed and organocatalyzed. In particular, we are dedicated to unravel the role of chiral phosphoric acids in organocatalytic reactions in which transient carbocations are developed.

RELEVANT PUBLICATIONS

1. Computational evidence of Glycosy Cations. Merino, P.; Delso, I.; Pereira, S.; Pedron, M.; Orta, S.; Tejero, T. Org. Biomol. Chem. 2021. doi:  10.1039/D0OB02373F
2. Enantio- and Diastereoselective Nucleophilic Addition of N-tert-Butyl Hydrazones to Isoquinolinium Ions through Anion-Binding Catalysis. Matador, E.; Iglesias-Sigüenza, J.; Monge, D.; Merino, P.; Fernández, R.; Lassaletta, J. M. Angew. Chem. Int. Ed. 2021,133, 5096-5101. doi:  10.1002/anie.202012861
3. Anomeric beta-triflate characterization enables the monitorization of glycosylation dynamics and suggests a non-canonical reinterpretation of the mechanism. Santana, A. G.; Montalvillo-Jiménez, L.; Díaz-Casado, L.; Corzana, F.; Jiménez-Osés, G.; Merino, P.; Cañada, F. J.; Jiménez-Barbero, J.; Gómez, A. M.; Asensio, J. L. J. Am. Chem. Soc. 2020, 142, 12501-12514. doi:  10.1021/jacs.0c05525
4. Enantioselective Synthesis of Tropanes through Brønsted Acid-Catalyzed Pseudotransannular Desymmetrization. Rodriguez, S.; Uria, U.; Reyes, E.; Carrillo, L.; Tejero, T.; Merino, P.; Vicario, J. L. Angew. Chem. Int. Ed. 2020, 132, 6846-6850. doi:  10.1002/anie.202000650
5. Dissecting the Structural and Chemical Determinants of the “Open-to-Closed” Motion in the Mannosyltransferase PimA from Mycobacteria. Unzueta, A. R.; Ghirardello, M.; Urresti, S.; Delso, I.; Giganti, D.; Anso-Miqueleiz, I.; Trastoy, B.; Comino, N.; Tersa, M.; D’Angelo, C.; Cifuente, J. O.; Marina, A.; Durana, A.; Chenal, A.; Svergun, D. I.; Alzari, P. M.; Albesa-Jové, D.; Merino, P.; Guerin, M. E. Biochemistry 2020, 59, 2934-2945. doi:  10.1021/acs.biochem.0c00376
6. Enantioselective Synthesis, DFT Calculations and Preliminary Antineoplastic Activity of Dibenzo 1-Azaspiro[4.5]decanes on Drug Resistant Leukemias. Mendes, J. A.; Merino, P.; Soler, T.; Salustiano, E. J.; Costa, P. R. R.; Yus, M.; Foubelo, F.; Buarque, C. D. J. Org. Chem. 2019, 84, 2219-2233. doi:  10.1021/acs.joc.8b03203
7. Sequential Metal-free Thermal 1,3-Dipolar Cycloaddition of Unactivated Azomethine Ylides. Selva, V.; Selva, E.; Merino, P.; Nájera, C.; Sansano, J. M. Org. Lett. 2018, 20, 3522-3526. doi:  10.1021/acs.orglett.8b01292
8. Catalytic Enantioselective Cloke-Wilson Rearrangement. Ortega, A.; Manzano, R.; Uria, U.; Carrillo, L.; Reyes, E.; Tejero, T.; Merino, P.; Vicario, J. L. Angew. Chem. Int. Ed. 2018, 57, 8225-8229. doi:  10.1002/anie.201804614
9. UDP-GlcNAc Analogs as Inhibitors of O-GlcNAc Transferase (OGT): Spectroscopic, Computational and Biological Studies. Ghirardello, M.; Perrone, D.; Chinaglia, N.; Sádaba, D.; Delso, I.; Tejero, T.; Marchesi, E.; Fogagnolo, M.; Rafie, K.; Aalten, D. M. F. v.; Merino, P. Chem. Eur. J. 2018, 24, 7264-7272. doi:  10.1002/chem.201801083
10. Inhibitors against Fungal Cell Wall-remodelling Enzymes. Delso, I.; Valero-Gonzalez, J.; Gomollón-Bel, F.; Castro-López, J.; Fang, W.; Navratilova, I.; Aalten, D. M. F. v.; Tejero, T.; Merino, P.; Hurtado-Guerrero, R. ChemMedChem 2018, 13, 128-132. doi:  10.1002/cmdc.201700720
11. (+)-Methyl (1R,2S)-2-{[4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl]methyl}-1-phenylcyclopropanecarboxylate [(+)-MR200] Derivatives as Potent and Selective Sigma Receptor Ligands: Stereochemistry and Pharmacological Properties. Amata, E.; Rescifina, A.; Prezzavento, O.; Arena, E.; Dichiara, M.; Pittalà, V.; Montilla-García, Á.; Punzo, F.; Merino, P.; Cobos, E. J.; Marrazzo, A. J. Med. Chem. 2018, 61, 372-384. doi:  10.1021/acs.jmedchem.7b01584
12. Regioselectivity Change in the Organocatalytic Enantioselective (3+2) Cycloaddition with Nitrones Through Cooperative H-Bonding Catalysis/Iminium Activation. Prieto, L.; Juste-Navarro, V.; Uria, U.; Delso, I.; Reyes, E.; Tejero, T.; Carrillo, L.; Merino, P.; Vicario, J. L. Chem. Eur. J. 2017, 23, 2764-2768. doi:  10.1002@chem.201605350
13. Racemic Hemiacetals as Oxygen-Centered Pronucleophiles Triggering Cascade 1,4-Addition/Michael Reaction through Dynamic Kinetic Resolution under Iminium catalysis. Reaction Development and Mechanistic Insights. Orue, A.; Uria, U.; Roca-López, D.; Delso, I.; Reyes, E.; Carrillo, L.; Merino, P.; Vicario, J. L. Chem. Sci. 2017, 8, 2904-2913. doi:  10.1039/C7SC00009J
14. The small molecule luteolin inhibits N-acetyl--galactosaminyltransferases and reduces mucin-type O-glycosylation of amyloid precursor protein Liu, F.; X, K.; Xu, Z.; Rivas, M. d. l.; Li, X.; Lu, J.; Delso, I.; Merino, P.; Hurtado-Guerrero, R.; Zhang, Y. Journal of Biological Chemistry 2017, 292, 21304-21319. doi:  10.1074/jbc.M117.814202.

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MAIN RESEARCH PROJECTS

1.- Rational Design and Stereoselective Synthesis of Glycomimetics. MINECO, 2014-2016 CTQ2013-44367-C2-1-P. 151.200 EURO. PI: Pedro Merino

2.- Rational Design of Glycomimetics Inhibitors of Glycosyltransferases. MINECO, 2017-2019 CTQ2016-76155-R. 172.800 EURO. PI: Pedro Merino

3. Estudios mecanísticos de reacciones de glicosilación y su aplicación al diseño de inhibidores de glicosiltransferasas. Ministerio de Ciencia, Innovacion y Universidades. PID2019-104090RB-100. 2020-2023. 163.350 EUROS PI: Pedro Merino Filella

COLABORATIONS

Dr. Loredana Maiuolo. Università della Calabria. Cosenza. Italy
Prof. Jose Luis Vicario. Universidad del Pais Vasco. Bilbao. Spain
Prof. Jose M.  Lassaaletta. IIQ-CSIC. Sevilla. Spain.
Dr. Francesca Cardona. Universitá di Firenze. Italy
Prof. Dan van Aalten. University of Dundee. United Kingdom.
Prof. Sonsoles Martin-Santamaría. CIB-CSIC. Madrid. Spain.
Prof. Juan Luis Asensio. IQO-CSIC, Madrid. Spain
Prof. Eric Oldfield. University of Illinois, USA
Dr. Francisco Corzana. Universidad de La Rioja. Spain
Dr. Marcelo Guerin. CIC-Biogune. Bilbao. Spain
Prof. Pedro Gois. Universidade de Coimbra. Portugal.
Dr. Ramon Hurtado. Universidad de Zaragoza. BIFI. Spain

CONTACT
pmerino@unizar.es
bioorganica@unizar.es
web: http://bioorganica.unizar.es