Giotto Biotech Srl
Giotto Biotech Srl
Takis P.G.,University of Ioannina |
Takis P.G.,GIOTTO Biotech S.r.l. |
Papavasileiou K.D.,Greek National Center For Scientific Research |
Peristeras L.D.,Greek National Center For Scientific Research |
And 3 more authors.
Physical Chemistry Chemical Physics | Year: 2017
Dimethyl sulfoxide (DMSO) has a significant, multi-faceted role in medicine, pharmacy, and biology as well as in biophysical chemistry and catalysis. Its physical properties and impact on biomolecular structures still attract major scientific interest, especially the interactions of DMSO with biomolecular functional groups. In the present study, we shed light on the "isolated" carboxylic (-COOH) and amide (-NH) interactions in neat DMSO via1H NMR studies along with extensive theoretical approaches, i.e. molecular dynamics (MD) simulations, density functional theory (DFT), and ab initio calculations, applied on model compounds (i.e. acetic and benzoic acid, ethyl acetamidocyanoacetate). Both experimental and theoretical results show excellent agreement, thereby permitting the calculation of the association constants between the studied compounds and DMSO molecules. Our coupled MD simulations, DFT and ab initio calculations, and NMR spectroscopy results indicated that complex formation is entropically driven and DMSO molecules undergo multiple strong interactions with the studied molecules, particularly with the -COOH groups. The combined experimental and theoretical techniques unraveled the interactions of DMSO with the most abundant functional groups of peptides (i.e. peptide bonds, side chain and terminal carboxyl groups) in high detail, providing significant insights on the underlying thermodynamics driving these interactions. Moreover, the developed methodology for the analysis of the simulation results could serve as a template for future thermodynamic and kinetic studies of similar systems. © the Owner Societies 2017.
Agency: European Commission | Branch: H2020 | Program: MSCA-ITN-ETN | Phase: MSCA-ITN-2015-ETN | Award Amount: 3.84M | Year: 2016
The development of effective novel drugs - especially for rare and neglected diseases - is one of the biggest challenges of the upcoming decades, as illustrated by the recent Ebola outbreak. Moreover, European innovation in new drug registrations is dramatically falling behind compared to the US and Asia. The principal aim of the AEGIS ITN is to implement the first comprehensive, intersectoral cross-disciplinary and structured curriculum for doctoral students in the European Research Area by establishing a unique training platform for the next generation of European researchers in early drug discovery. A significant added value is provided through networking with key European pharmaceutical companies. A key research aim of AEGIS is improving the efficiency and success of early stage drug development by combining innovative methods and techniques to tackle difficult but promising targets (i.e. protein-protein interactions), as potentially valuable drug targets are often neglected due to the high risk associated with their validation. The consortium joins leading academic and industry researchers in an open innovation environment for innovative drug development in Europe. It is supported by several additional partners and stakeholders in the field. Integrated training of the fellows takes place at the host institute and by secondments, research schools and individual training within the AEGIS network. The scientific training includes complementary skills, management, intellectual property rights, fund raising, communication and career planning. AEGIS will improve the availability of a highly skilled workforce for European industries and research, greatly enhance the employability and the career perspectives of young researchers for academia as well as for industry, and will be the seed of a sustainable development in innovative drug discovery, in particular for rare and neglected diseases.
Agency: European Commission | Branch: FP7 | Program: CP-TP | Phase: KBBE.2012.2.2-01 | Award Amount: 7.92M | Year: 2013
Scientific understanding of the role and mechanisms of bioactives is fragmented. Research often addresses the theoretical possibility of health improvement effects rather than their real, practical use for everyday diets. Bioactives cannot be considered as discrete chemical compounds and research must focus on bioactive-enriched foods (BEF), if consumer demands for foods delivering appropriate health and wellbeing benefits are to be fulfilled. PATHWAY, a pan-European interdisciplinary team of 16 life/social scientists and 10 high tech/ food processing SMEs, uniquely addresses the role and mechanisms of action of 3 bioactives (docosahexaenoic acid, -glucan, anthocyanins, chosen for known/claimed effectiveness in reducing some risk factors of Metabolic Syndrome (MS), enriching 3 different widely-consumed food matrices (dairy-, bakery-, egg products). Critical evaluation of bioactive-food matrix interactions and determining the extent of synergies between the 3 bioactives are key elements of PATHWAY. PATHWAY will determine the impact of BEF on physiologically-relevant MS (a risk factor for many diseases) endpoints and deliver a better understanding of the role and mechanisms of action of the 3 bioactives and BEF. Parallel in vitro/in vivo studies will enable selection of robust biomarkers by advanced omics techniques. Deliverables will include BEF and generic protocols, best practices and guidelines for planning dietary interventions, and guidance to SMEs for producing health-promoting BEF and for submitting convincing health claim dossiers to EFSA; the latter will be greatly facilitated by one SME partner who has submitted 3 successful dossiers. PATHWAY guidelines will be generic and will apply to a wide range of bioactives and BEF. Impact will be optimised across Europe by targeted dissemination to industry (especially SME), consumer and S&T stakeholders. Young people will be trained in a stimulating interdisciplinary, trans-sectoral environment.
Luchinat E.,University of Florence |
Barbieri L.,University of Florence |
Rubino J.T.,University of Florence |
Kozyreva T.,Giotto Biotech S.r.l. |
And 2 more authors.
Nature Communications | Year: 2014
Mutations in the superoxide dismutase 1 (SOD1) gene are related to familial cases of amyotrophic lateral sclerosis (fALS). Here we exploit in-cell NMR to characterize the protein folding and maturation of a series of fALS-linked SOD1 mutants in human cells and to obtain insight into their behaviour in the cellular context, at the molecular level. The effect of various mutations on SOD1 maturation are investigated by changing the availability of metal ions in the cells, and by coexpressing the copper chaperone for SOD1, hCCS. We observe for most of the mutants the occurrence of an unstructured SOD1 species, unable to bind zinc. This species may be a common precursor of potentially toxic oligomeric species, that are associated with fALS. Coexpression of hCCS in the presence of copper restores the correct maturation of the SOD1 mutants and prevents the formation of the unstructured species, confirming that hCCS also acts as a molecular chaperone. © 2014 Macmillan Publishers Limited. All rights reserved.
Agency: European Commission | Branch: FP7 | Program: MC-ITN | Phase: FP7-PEOPLE-2012-ITN | Award Amount: 3.47M | Year: 2013
A network combining 9 academic research groups and 4 collaborating industrial companies is proposed to train the next generation of PhD students and post-doctoral researchers, in developing and applying novel experimental and theoretical methods in the NMR spectroscopy of systems containing paramagnetic metals. The assembled team, with researchers distributed throughout the EU, will investigate a variety of important problems in chemistry and biology including catalysts, battery materials, metalloproteins and large protein-protein assemblies. The researchers will be trained to attack key problems that prevent the widespread usage of NMR spectroscopy as applied to paramagnetic materials, and to develop new methods to improve significantly the structural and electronic information that can be obtained from these systems. Three experimental and theoretical work programs are proposed, which build on, but also move significantly beyond the recent advances in pNMR, many of which have originated from members of this network: i) developing experimental approaches for obtaining NMR spectra from challenging paramagnetic molecules and materials, ii) extending the fundamental theoretical understanding of pNMR parameters, and facilitating their quantum-chemical implementations in first-principles software; iii) attacking relevant chemical and biological problems, with novel techniques to determine structure (e.g., of insoluble proteins and disordered battery electrode materials), dynamics and reactivity around metal centres, and exploring interactions between, e.g., biomolecules, catalytic centres and supports. Integral to the research-based training programme is the series of workshops, practical training courses, international conferences, and outreach actions, located at the different sites. These will i) train the young researchers of the network in the basics of pNMR and ii) disseminate the results of the network to the larger NMR community and to the general public.
PubMed | University of Vienna, University of Florence and Giotto Biotech S.R.L.
Type: Journal Article | Journal: Chemistry (Weinheim an der Bergstrasse, Germany) | Year: 2016
Among protein immobilization strategies, encapsulation in bioinspired silica is increasingly popular. Encapsulation offers high yields and the solid support is created through a protein-catalyzed polycondensation reaction that occurs under mild conditions. An integrated strategy is reported for the characterization of both the protein and bioinspired silica scaffold generated by the encapsulation of enzymes with an external silica-forming promoter or with the promoter expressed as a fusion to the enzyme. This strategy is applied to the catalytic domain of matrix metalloproteinase12. Analysis reveals that the structure of the protein encapsulated by either method is not significantly altered with respect to the native form. The structural features of silica obtained by either strategy are also similar, but differ from those obtained by other approaches. In case of the covalently linked R5-enzyme construct, immobilization yields are higher. Encapsulation through a fusion protein, therefore, appears to be the method of choice.
PubMed | University of Florence and Giotto Biotech S.R.L.
Type: | Journal: Scientific reports | Year: 2016
Proton-detection in solid-state NMR, enabled by high magnetic fields (>18 T) and fast magic angle spinning (>50kHz), allows for the acquisition of traditional (1)H-(15)N experiments on systems that are too big to be observed in solution. Among those, proteins entrapped in a bioinspired silica matrix are an attractive target that is receiving a large share of attention. We demonstrate that (1)H-detected SSNMR provides a novel approach to the rapid assessment of structural integrity in proteins entrapped in bioinspired silica.
PubMed | University of Florence, Florida Atlantic University, Max Planck Institute of Molecular Cell Biology and Genetics and Giotto Biotech S.R.L.
Type: | Journal: Scientific reports | Year: 2016
Cell surface proteolysis is an integral yet poorly understood physiological process. The present study has examined how the pericellular collagenase membrane-type 1 matrix metalloproteinase (MT1-MMP) and membrane-mimicking environments interplay in substrate binding and processing. NMR derived structural models indicate that MT1-MMP transiently associates with bicelles and cells through distinct residues in blades III and IV of its hemopexin-like domain, while binding of collagen-like triple-helices occurs within blades I and II of this domain. Examination of simultaneous membrane interaction and triple-helix binding revealed a possible regulation of proteolysis due to steric effects of the membrane. At bicelle concentrations of 1%, enzymatic activity towards triple-helices was increased 1.5-fold. A single mutation in the putative membrane interaction region of MT1-MMP (Ser466Pro) resulted in lower enzyme activation by bicelles. An initial structural framework has thus been developed to define the role(s) of cell membranes in modulating proteolysis.
PubMed | University of Florence and Giotto Biotech S.R.L.
Type: Journal Article | Journal: Journal of biomolecular NMR | Year: 2016
In-cell NMR provides structural and functional information on proteins directly inside living cells. At present, the high costs of the labeled media for mammalian cells represent a limiting factor for the development of this methodology. Here we report a protocol to prepare a homemade growth medium from Spirulina platensis autolysate, suitable to express uniformly labeled proteins inside mammalian cells at a reduced cost-per-sample. The human proteins SOD1 and Mia40 were overexpressed in human cells grown in (15)N-enriched S. platensis algal-derived medium, and high quality in-cell NMR spectra were obtained.
Agency: European Commission | Branch: FP7 | Program: MC-ITN | Phase: FP7-PEOPLE-2010-ITN | Award Amount: 3.43M | Year: 2010
Recent evidence shows that a large share of proteins gain functional advantages by remaining natively unstructured, either completely or partially, thus challenging well-established concepts in structural biology. In order to characterize the highly dynamical nature of such intrinsically disordered proteins, and follow their (possible) reorganization by interacting with partners, new integrated multidisciplinary approaches combining a variety of experimental and computational techniques are needed. Our proposal is focused on NMR spectroscopy, since this technique offers an arsenal of effective tools for the study of the structural and dynamical properties of disordered states of proteins at atomic resolution in systems as complex as whole cells. In order to achieve the full potential of this approach, however, current methods should be further developed and properly interfaced with other complementary techniques. The purpose of our network is thus to establish a framework to train a new generation of young researchers and help them develop the necessary skills to successfully respond to the challenges associated with the elucidation of the functional role of intrinsically disordered protein states. If achieved, this goal will provide us with a more quantitative understanding of the biochemical processes at the basis of life and have a significant impact in biomedical research and in the design of new drugs.