4 pags., 5 figs., 1 tab. ; Herein we report for the first time the site-selective C-H bond arylation of tryptophan and tryptophan residues in proteins in aqueous media at room temperature by using a PdNP bionanohybrid as a heterogeneous catalyst. The reaction proceeds using a stable aryldiazonium salt without a base. ; This work was supported by the Spanish Government the Spanish National Research Council (CSIC), and the Ministry of Education, Youth and Sports of the Community of Madrid and the European Social Fund (PEJD-2017PRE/SAL-3762). The authors thank the European Cooperation in Science and Technology (COST) program under CA15106 grant (CHAOS: CH Activation in Organic Synthesis). The authors also thank Irene Orera and Giuseppe Lattanzio from Proteomics Unit of Servicios Cientıfico Tecnicos del CIBA (IACS-Universidad de Zaragoza), ProteoRed ISCIII member, for mass spectra analysis.
12 pags., 8 figs., 4 tabs. ; Mussel adhesives proteins have been extensively studied as a promising alternative in bioadhesives due to their ability to provide durable anchoring under diferent surfaces in seawater. These charasteristics have been attributed to the presence of the reduced catechol form, 3,4-dihydroxyphenylalanine (DOPA) of its structure. However, its practical applications have been limited due to drawbacks with natural extraction. Here, a novel method have been described for site-specifc chemical incorporation of l-3,4-dihydroxyphenylalanine methyl ester (l-DOPAME) into proteins, in particular Candida antartica fraction B (CAL-B) lipase. Two strategies were followed, direct conjugation of DOPA at the C-terminus on the surface of the protein, and protein conjugation with tailor-made glycopolymers (DOPA-hyaluronic acid (HA) polymers) at the N-terminus. In all cases, the characterization of the new DOPA-proteins was carried out using circular dichroism, fuorescence or mass spectrometry. ; An improvement in the activity (in some cases more than 2 times) or the thermostability of CAL-B (with a half live 4 fold greater in some cases) was found by the incorporation of DOPA molecules. These DOPA-proteins showed excellent underwater covalent adhesive ability on amino functionalized surfaces in aqueous media compared to other modifed [e.g. tyrosine modifed (TYR)] CAL-B proteins. At pH 8.5, CALB-DOPA proteins were completely adsorbed after 90 min of incubation, whereas about 10% of CALB-HA or CALB-TYR proteins were adsorbed at the same time. Native CAL-B adsorption was not observed. These results suggest a potential application of these DOPA-proteins as bioglues or bioadhesives for practical underwater applications. ; s This work was supported by the Spanish National Research Council (CSIC), Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III, and European Union (ERDF/ ESF, 'Investing in your future') (PI15/00663 and PI18/00349 to OA); Diputación General de Aragón (Translation Research group in Digestive Pathology Group—E25_17R to OA); and Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd). The authors thank the COST Action CA15216 (ENBA) for COST meeting support. We also thank Dr. Martinez from Novozymes and Proteomics Facility of Servicios Científco Técnicos del CIBA (IACS-Universidad de Zaragoza), ProteoRed ISCIII, Zaragoza, Spain. ; Peer reviewed
Research in solid-gas heterogeneous catalytic processes is typically aimed toward optimization of catalyst composition to achieve a higher conversion and, especially, a higher selectivity. However, even with the most selective catalysts, an upper limit is found: Above a certain temperature, gas-phase reactions become important and their effects cannot be neglected. Here, we apply a microwave field to a catalyst-support ensemble capable of direct microwave heating (MWH). We have taken extra precautions to ensure that (i) the solid phase is free from significant hot spots and (ii) an accurate estimation of both solid and gas temperatures is obtained. MWH allows operating with a catalyst that is significantly hotter than the surrounding gas, achieving a high conversion on the catalyst while reducing undesired homogeneous reactions. We demonstrate the concept with the CO 2 -mediated oxidative dehydrogenation of isobutane, but it can be applied to any system with significant undesired homogeneous contributions. ; Financial support from the European Research Council Advanced Grant (HECTOR267626), the Regional Government of Aragon (DGA), and the MINECO and FEDER (project CTQ2015-65226) is acknowledged. ; Peer reviewed
18 pags., 8 figs., 6 tabs. -- This article belongs to the Special Issue The Amazing World of IDPs in Human Diseases II ; Intrinsic disorder plays an important functional role in proteins. Disordered regions are linked to posttranslational modifications, conformational switching, extra/intracellular trafficking, and allosteric control, among other phenomena. Disorder provides proteins with enhanced plasticity, resulting in a dynamic protein conformational/functional landscape, with well-structured and disordered regions displaying reciprocal, interdependent features. Although lacking well-defined conformation, disordered regions may affect the intrinsic stability and functional properties of ordered regions. MeCP2, methyl-CpG binding protein 2, is a multifunctional transcriptional regulator associated with neuronal development and maturation. MeCP2 multidomain structure makes it a prototype for multidomain, multifunctional, intrinsically disordered proteins (IDP). The methyl-binding domain (MBD) is one of the key domains in MeCP2, responsible for DNA recognition. It has been reported previously that the two disordered domains flanking MBD, the N-terminal domain (NTD) and the intervening domain (ID), increase the intrinsic stability of MBD against thermal denaturation. In order to prove unequivocally this stabilization effect, ruling out any artifactual result from monitoring the unfolding MBD with a local fluorescence probe (the single tryptophan in MBD) or from driving the protein unfolding by temperature, we have studied the MBD stability by differential scanning calorimetry (reporting on the global unfolding process) and chemical denaturation (altering intramolecular interactions by a different mechanism compared to thermal denaturation). ; This research was funded by the Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) (BFU2016-78232-P to A.V.C.; BES-2017-080739 to D.O.A.); Miguel Servet Program from Instituto de Salud Carlos III (CPII13/00017 to O.A.); Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III and European Union (ERDF/ESF, "Investing in your future") (PI15/00663 and PI18/00349 to O.A.); Diputación General de Aragón (Protein Targets and Bioactive Compounds Group E45_20R to A.V.C. and Digestive Pathology Group B25_20R to O.A.); and the Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd).
19 pags., 8 figs., 4 tabs. -- This article belongs to the Special Issue The Amazing World of IDPs in Human Diseases ; Methyl-CpG binding protein 2 (MeCP2) is a transcriptional regulator and a chromatin-binding protein involved in neuronal development and maturation. Loss-of-function mutations in MeCP2 result in Rett syndrome (RTT), a neurodevelopmental disorder that is the main cause of mental retardation in females. MeCP2 is an intrinsically disordered protein (IDP) constituted by six domains. Two domains are the main responsible elements for DNA binding (methyl-CpG binding domain, MBD) and recruitment of gene transcription/silencing machinery (transcription repressor domain, TRD). These two domains concentrate most of the RTT-associated mutations. R106W and R133C are associated with severe and mild RTT phenotype, respectively. We have performed a comprehensive characterization of the structural and functional impact of these substitutions at molecular level. Because we have previously shown that the MBD-flanking disordered domains (N-terminal domain, NTD, and intervening domain, ID) exert a considerable influence on the structural and functional features of the MBD (Claveria-Gimeno, R. et al. Sci Rep. 2017, 7, 41635), here we report the biophysical study of the influence of the protein scaffold on the structural and functional effect induced by these two RTT-associated mutations. These results represent an example of how a given mutation may show different effects (sometimes opposing effects) depending on the molecular context. ; This research was funded by the Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) (BFU2016-78232-P to A.V.C.; BES-2017-080739 to D.O.A.); Miguel Servet Program from Instituto de Salud Carlos III (CPII13/00017 to O.A.); Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III and European Union (ERDF/ESF, "Investing in your future") (PI15/00663 and PI18/00349 to O.A.); Diputación General de Aragón (Protein Targets and Bioactive Compounds Group E45_17R to A.V.C. and Digestive Pathology Group B25_17R to O.A.); and the Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd). ; Peer reviewed
22 pags., 4 figs., 2 tabs. -- This article belongs to the Special Issue The Amazing World of IDPs in Human Diseases ; Several carrier proteins are involved in protein transport from the cytoplasm to the nucleus in eukaryotic cells. One of those is importin α, of which there are several human isoforms; among them, importin α3 (Impα3) has a high flexibility. The protein NUPR1, a nuclear protein involved in the cell-stress response and cell cycle regulation, is an intrinsically disordered protein (IDP) that has a nuclear localization sequence (NLS) to allow for nuclear translocation. NUPR1 does localize through the whole cell. In this work, we studied the affinity of the isolated wild-type NLS region (residues 54–74) of NUPR1 towards Impα3 and several mutants of the NLS region by using several biophysical techniques and molecular docking approaches. The NLS region of NUPR1 interacted with Impα3, opening the way to model the nuclear translocation of disordered proteins. All the isolated NLS peptides were disordered. They bound to Impα3 with low micromolar affinity (1.7–27 μM). Binding was hampered by removal of either Lys65 or Lys69 residues, indicating that positive charges were important; furthermore, binding decreased when Thr68 was phosphorylated. The peptide phosphorylated at Thr68, as well as four phospho-mimetic peptides (all containing the Thr68Glu mutation), showed the presence of a sequential NN(i,i + 1) nuclear Overhauser effect (NOE) in the 2D-1H-NMR (two-dimensional–proton NMR) spectra, indicating the presence of turn-like conformations. Thus, the phosphorylation of Thr68 modulates the binding of NUPR1 to Impα3 by a conformational, entropy-driven switch from a random-coil conformation to a turn-like structure. ; This research was funded by the Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) (RTI2018-097991-B-I00 to J.L.N. and BFU2016-78232-P to A.V.C.); La Ligue Contre le Cancer, INCa, Canceropole PACA, and INSERM to J.L.I.; Miguel Servet Program from Instituto de Salud Carlos III (CPII13/00017 to OA); Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III and European Union (ERDF/ESF, "Investing in your future") (PI15/00663 and PI18/00349 to O.A.); Diputación General de Aragón (Protein Targets and Bioactive Compounds Group E45_17R to A.V.C. and Digestive Pathology Group B25_17R to O.A.); and the Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd). The NMR equipment used in this work was funded by the Generalitat Valenciana and cofinanced with ERDF funds (OP ERDF of Comunitat Valenciana 2014-2020). ; Peer reviewed
16 pags., 9 figs. ; Lactic acid is one of the key biobased chemical building blocks, given its readily availability from sugars through fermentation and facile conversion into a range of important chemical intermediates and polymers. Herein, well-defined rubbery polymers derived from butyl lactate solvent were successfully prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization of the corresponding monomeric acrylic derivative. Good control over molecular weight and molecular weight distribution was achieved in bulk using either monofunctional or bifunctional trithiocarbonate-type chain transfer agents. Subsequently, poly(butyl lactate acrylate), with a relative low Tg (-20 °C), good thermal stability (5% wt. loss at 340 °C) and low toxicity was evaluated as a sustainable middle block in all-acrylic ABA copolymers using isosorbide and vanillin-derived glassy polyacrylates as representative end blocks. Thermal, morphological and mechanical properties of copolymers containing hard segment contents of <20 wt% were evaluated to demonstrate the suitability of rubbery poly(alkyl lactate) building blocks for developing functional sustainable materials. Noteworthy, 180° peel adhesion measurements showed that the synthesized biosourced all-acrylic ABA copolymers possess competitive performance when compared with commercial pressure-sensitive tapes. ; This research was funded by Spanish Ministerio de Ciencia, Innovación y Universidades through project MAT2017-82669-R (to G.L. and J.C.R.), Serra Hunter Programme of the Government of Catalonia (to G.L.), University Rovira i Virgili (DL003536 grant to N.B.), Miguel Servet Program from Instituto de Salud Carlos III (CPII13/00017 to O.A.), Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III, and European Union (ERDF/ESF, 'Investing in your future') (PI18/00349 to O.A.), Diputación General de Aragón (Predoctoral Research Contract 2019 to A.J.-A., 'Digestive Pathology Group' B25_20R to O.A.), and Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd). I.L. and L.V. gratefully acknowledge support from the European Regional Development Fund (No. MOBTT21), the Estonia-Russia Cross Border Cooperation Programme (ER30) and the Baltic Research Programme in Estonia (EEA grant no. EMP426)
20 pags, 5 figs, 7 tabs ; Risk population screening programs are instrumental for advancing cancer management and reducing economic costs of therapeutic interventions and the burden of the disease, as well as increasing the survival rate and improving the quality of life for cancer patients. Lung cancer, with high incidence and mortality rates, is not excluded from this situation. The success of screening programs relies on many factors, with some of them being the appropriate definition of the risk population and the implementation of detection techniques with an optimal discrimination power and strong patient adherence. Liquid biopsy based on serum or plasma detection of circulating tumor cells or DNA/RNA is increasingly employed nowadays, but certain limitations constrain its wide application. In this work, we present a new implementation of thermal liquid biopsy (TLB) for lung cancer patients. TLB provides a prediction score based on the ability to detect plasma/serum proteome alterations through calorimetric thermograms that strongly correlates with the presence of lung cancer disease (91% accuracy rate, 90% sensitivity, 92% specificity, diagnostic odds ratio 104). TLB is a quick, minimally-invasive, low-risk technique that can be applied in clinical practice for evidencing lung cancer, and it can be used in screening and monitoring actions. ; This research was funded by Miguel Servet Program from Instituto de Salud Carlos III (CPII13/00017 to OA); Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III, and European Union (ERDF/ESF, 'Investing in your future') (PI15/00663 and PI18/00349 to OA, and PI17/01109 to AL); Spanish Ministry of Economy and Competitiveness (BFU2016-78232-P to AVC); Diputación General de Aragón (Protein Targets and Bioactive Compounds Group – E45_17R to AVC, and Digestive Pathology Group – E25_17R to OA and AL); and Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd). ; Peer reviewed
Rafael Claveria-Gimeno,1–3 Sonia Vega,3 Valeria Grazu,4 Jesús M de la Fuente,4–6 Angel Lanas,2,8–10 Adrian Velazquez-Campoy,2,3,7 Olga Abian1–3,8 1Instituto Aragonés de Ciencias de la Salud (IACS), Zaragoza, Spain; 2IIS Aragón, Zaragoza, Spain; 3Institute of Biocomputation and Physics of Complex Systems (BIFI), Joint Unit IQFR-CSIC-BIFI, Universidad de Zaragoza, Zaragoza, Spain; 4Instituto de Nanociencia de Aragon (INA), Universidad de Zaragoza, Zaragoza, Spain; 5Instituto de Ciencia de Materiales de Aragón (ICMA), CSIC-Universidad de Zaragoza, Zaragoza, Spain; 6Institute NanoBiomedicine and Engineering, Shanghai Jiao Tong University, Shanghai, People's Republic of China; 7Fundacion ARAID, Government of Aragon, Spain; 8Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain; 9Servicio de Aparato Digestivo, Hospital Clínico Universitario Lozano Blesa, Zaragoza, Spain; 10Department of Medicine, University of Zaragoza, Zaragoza, Spain Abstract: In vitro primary screening for identifying bioactive compounds (inhibitors, activators or pharmacological chaperones) against a protein target results in the discovery of lead compounds that must be tested in cell-based efficacy secondary screenings. Very often lead compounds do not succeed because of an apparent low potency in cell assays, despite an excellent performance in primary screening. Primary and secondary screenings differ significantly according to the conditions and challenges the compounds must overcome in order to interact with their intended target. Cellular internalization and intracellular metabolism are some of the difficulties the compounds must confront and different strategies can be envisaged for minimizing that problem. Using a novel screening procedure we have identified 15 compounds inhibiting the hepatitis C NS3 protease in an allosteric fashion. After characterizing biophysically the interaction with the target, some of the compounds were not able to inhibit viral replication in cell assays. In order to overcome this obstacle and potentially improve cellular internalization three of these compounds were complexed with γ-cyclodextrin. Two of them showed a five- and 16-fold activity increase, compared to their activity when delivered as free compounds in solution (while γ-cyclodextrin did not show antiviral activity by itself). The most remarkable result came from a third compound that showed no antiviral activity in cell assays when delivered free in solution, but its γ-cyclodextrin complex exhibited a 50% effective concentration of 5 µM. Thus, the antiviral activity of these compounds can be significantly improved, even completely rescued, using γ-cyclodextrin as carrier molecule. Keywords: primary and secondary screenings, drug activity, antiviral compounds, cyclodextrins, vehiculization, drug delivery, hepatitis C, NS3 protease, virus replicon system
3 pags, 1 fig ; Intrinsically disordered proteins (IDPs) do not have a well-defined structure, but they have key biological tasks in cancer development. By using the disordered cancer-related protein NUPR1 as a proof-of-concept, we have developed a new multidisciplinary approach to tackle drug-design against IDPs, using it to repurpose drugs for treating pancreatic adenocarcinoma (PDAC). ; This work in the authors' laboratories was supported by [La Ligue Contre le Cancer], INCa, [Canceropole PACA] and [INSERM] to JLI and LP; [Miguel Servet Program from Instituto de Salud Carlos III] under grant [CPII13/00017] to OA; Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III, and European Union (ERDF/ESF, 'Investing in your future') under grants [PI15/00663 and PI18/00349] to OA; Ministerio de Economia, Industria y Competitividad, Gobierno de Espana under grants [BFU2016-78232-P to AVC, CTQ2015-64445-R to JLN]; [Diputacion General de Aragon] under grants [Protein Targets and Bioactive Compound Group to AVC, and Digestive Pathology Group to OA]; [Centro de Investigacion Biomedica en Red en Enfermedades Hepaticas y Digestivas] (CIBERehd) toOAand AVC; [Programme XUGUANGQI] to YX and JLI; and [National Natural Science Foundation of China (81502920), the Fundamental Research Funds for the Central Universities] under grant [106112017CDJQJ468823] to YX. The Fundacion Alfonso Martin-Escudero and Fondation de France supported to PSC. ; Peer reviewed
Inhibiting the main protease 3CLpro is the most common strategy in the search for antiviral drugs to fight the infection from SARS-CoV-2. We report that the natural compound eugenol is able to hamper in vitro the enzymatic activity of 3CLpro, the SARS-CoV-2 main protease, with an inhibition constant in the sub-micromolar range (Ki = 0.81 μM). Two phenylpropene analogs were also tested: the same effect was observed for estragole with a lower potency (Ki = 4.1 μM), whereas anethole was less active. The binding efficiency index of these compounds is remarkably favorable due also to their small molecular mass (MW < 165 Da). We envision that nanomolar inhibition of 3CLpro is widely accessible within the chemical space of simple natural compounds ; This work was supported by Fundación hna (grant to A.V.-C. and O.A.); Miguel Servet Program from Instituto de Salud Carlos III (CPII13/00017 to O.A.); Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III, and European Union (ERDF/ESF, 'Investing in your future') (PI18/00349 to O.A. and a FIS Research Contract to L.C.-L.); Spanish Ministry of Economy and Competitiveness (BFU2016-78232-P to A.V.-C.); Spanish Ministry of Science, Innovation and Universities (FPI Predoctoral Research Contract BES-2017-080739 to D.O.-A.); Diputación General de Aragón (Predoctoral Research Contract 2019 to A.J.-A., 'Protein Targets and Bioactive Compounds Group' E45_20R to A.V.-C., 'Digestive Pathology Group' B25_20R to O.A.); Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd). ; Peer reviewed
24 pags., 8 figs., 3 tabs. -- This article belongs to the Special Issue Dendrimers and Dendritic Materials against Infectious Diseases ; The use of nanocarriers has been revealed as a valid strategy to facilitate drug bioavailability, and this allows for expanding the drug libraries for the treatment of certain diseases such as viral diseases. In the case of Hepatitis C, the compounds iopanoic acid and 3,3′,5-triiodothyroacetic acid (or tiratricol) were identified in a primary screening as bioactive allosteric inhibitors of viral NS3 protease, but they did not exhibit accurate activity inhibiting viral replication in cell-based assays. In this work, dendritic nanocarriers are proposed due to their unique properties as drug delivery systems to rescue the bioactivity of these two drugs. Specifically, four different amphiphilic Janus dendrimers synthesized by combining 2,2′-bis(hydroxymethyl)propionic acid (bis-MPA) and 2,2′-bis(glyciloxy)propionic acid (bis-GMPA) functionalized with either hydrophilic or lipophilic moieties at their periphery were used to entrap iopanoic acid and tiratricol. Interestingly, differences were found in the loading efficiencies depending on the dendrimer design, which also led to morphological changes of the resulting dendrimer aggregates. The most remarkable results consist of the increased water solubility of the bioactive compounds within the dendrimers and the improved antiviral activity of some of the dendrimer/drug aggregates, considerably improving antiviral activity in comparison to the free drugs. Moreover, imaging studies have been developed in order to elucidate the mechanism of cellular internalization. ; This research was funded by the Ministerio de Ciencia e Innovación, Spain, which included FEDER funds (PGC2018-097583-B-I00, PGC2018-093761-B-C31, MSA grant BES-2016-078774, AL grant FPU12/05210 and R.C.-G. grant FPU13/3870) Gobierno de Aragón-FSE (E47_20R); Miguel Servet Program from Instituto deSalud Carlos III (CPII13/00017 to OA); Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III and European Union (ERDF/ESF, "Investing in your future") (PI15/00663 and PI18/00349 to O.A.); Diputación General de Aragón (Digestive Pathology Group B25_17R to O.A.); and Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd). ; Peer reviewed
The pandemic, due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has stimulated the search for antivirals to tackle COVID-19 infection. Molecules with known pharmacokinetics and already approved for human use have been demonstrated or predicted to be suitable to be used either directly or as a base for a scaffold-based drug design. Among these substances, quercetin is known to be a potent in vitro inhibitor of 3CLpro, the SARS-CoV-2 main protease. However, its low in vivo bioavailability calls for modifications to its molecular structure. In this work, this issue is addressed by using rutin, a natural flavonoid that is the most common glycosylated conjugate of quercetin, as a model. Combining experimental (spectroscopy and calorimetry) and simulation techniques (docking and molecular dynamics simulations), we demonstrate that the sugar adduct does not hamper rutin binding to 3CLpro, and the conjugated compound preserves a high potency (inhibition constant in the low micromolar range, Ki = 11 μM). Although showing a disruption of the pseudo-symmetry in the chemical structure, a larger steric volume and molecular weight, and a higher solubility compared to quercetin, rutin is able to associate in the active site of 3CLpro, interacting with the catalytic dyad (His41/Cys145). The overall results have implications in the drug-design of quercetin analogs, and possibly other antivirals, to target the catalytic site of the SARS-CoV-2 3CLpro ; This work was supported by Fundación hna (to A.V.C. and O.A.); Miguel Servet Program from Instituto de Salud Carlos III (CPII13/00017 to O.A.); Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III, and European Union (ERDF/ESF, 'Investing in your future') (PI18/00349 to O.A. and a FIS Research Contract to L.C.-L.); Spanish Ministry of Economy and Competitiveness (BFU2016-78232-P to A.V.-C. and SAF2017-83265-R to H.T.R.); Spanish Ministry of Science, Innovation and Universities (FPI Predoctoral Research Contract BES-2017-080739 to D.O.-A.); the Spanish National Research Council (CSIC, project 202020E079); Diputación General de Aragón (Predoctoral Research Contract 2019 to A.J.-A., 'Protein Targets and Bioactive Compounds Group' E45_20R to A.V.-C., 'Digestive Pathology Group' B25_20R to O.A.); and Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd). ; Peer reviewed
18 pags., 6 figs., 1 tab. -- Open Access funded by Creative Commons Atribution Licence 4.0 ; Coordinated conformational transitions in oligomeric enzymatic complexes modulate function in response to substrates and play a crucial role in enzyme inhibition and activation. Caseinolytic protease (ClpP) is a tetradecameric complex, which has emerged as a drug target against multiple pathogenic bacteria. Activation of different ClpPs by inhibitors has been independently reported from drug development efforts, but no rationale for inhibitor-induced activation has been hitherto proposed. Using an integrated approach that includes x-ray crystallography, solid- and solution-state nuclear magnetic resonance, molecular dynamics simulations, and isothermal titration calorimetry, we show that the proteasome inhibitor bortezomib binds to the ClpP active-site serine, mimicking a peptide substrate, and induces a concerted allosteric activation of the complex. The bortezomib-activated conformation also exhibits a higher affinity for its cognate unfoldase ClpX. We propose a universal allosteric mechanism, where substrate binding to a single subunit locks ClpP into an active conformation optimized for chaperone association and protein processive degradation. ; This work used the platforms of the Grenoble Instruct center (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) with support from INSTRUCT ("Innovative EM/NMR approach for the characterization of the drug target ClpP APPID: 301"), FRISBI (ANR-10-INSB-05-02), and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). We thank the ESRF for beamtime at ID30A and ID23-1. Funding: This work was supported by Spanish Ministerio de Economia y Competitividad (BFU2016-78232-P) and Instituto de Salud Carlos III co-funded by European Union (PI15/00663 and PI18/00349, ERDF/ ESF, "Investing in your future"). This work was financially supported by the European Research Council (ERC-Stg-2012-311318 to P.S.). J.F. is supported by an EMBO long-term post-doctoral fellowship (ALTF441-2017).
22 pags., 14 figs., 9 tabs. 1 sch. -- This article belongs to the Special Issue Nanostructures Applied to Drug Delivery and Diagnosis ; (1) Background: Biophysical techniques applied to serum samples characterization could promote the development of new diagnostic tools. Fluorescence spectroscopy has been previously applied to biological samples from cancer patients and differences from healthy individuals were observed. Dendronized hyperbranched polymers (DHP) based on bis(hydroxymethyl)propionic acid (bis-MPA) were developed in our group and their potential biomedical applications explored. (2) Methods: A total of 94 serum samples from diagnosed cancer patients and healthy individuals were studied (20 pancreatic ductal adenocarcinoma, 25 blood donor, 24 ovarian cancer, and 25 benign ovarian cyst samples). (3) Results: Fluorescence spectra of serum samples (fluorescence liquid biopsy, FLB) in the presence and the absence of DHP-bMPA were recorded and two parameters from the signal curves obtained. A secondary parameter, the fluorescence spectrum score (FSscore), was calculated, and the diagnostic model assessed. For pancreatic ductal adenocarcinoma (PDAC) and ovarian cancer, the classification performance was improved when including DHP-bMPA, achieving high values of statistical sensitivity and specificity (over 85% for both pathologies). (4) Conclusions: We have applied FLB as a quick, simple, and minimally invasive promising technique in cancer diagnosis. The classification performance of the diagnostic method was further improved by using DHP-bMPA, which interacted differentially with serum samples from healthy and diseased subjects. These preliminary results set the basis for a larger study and move FLB closer to its clinical application, providing useful information for the oncologist during patient diagnosis. ; This research was funded by the Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) (BFU2016-78232-P to A.V.C.); Projects funded by Instituto de Salud Carlos III and co-funded by European Union (ESF, "Investing in your future"): "PI15/00663 (FIS project to O.A.)", "PI18/00349 (FIS project to O.A.)", "FI19/00146 (PFIS contract for SHD)"; Gobierno de Aragón-ESF (Predoctoral Research Contract FEDER 2014-2020 "Construyendo Europa desde Aragón") to V.M.-V., Protein Targets and Bioactive Compounds Group E45_17R to A.V.C., Digestive Pathology Group B25_17R to S.H.-D., O.A. and CLIP group E47_20R, to V.M.-V. and T.S.); and the Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd)
