RESUMEN
The in-depth studies over the years on the defence barriers by tomato plants have shown that the Systemin peptide controls the response to a wealth of environmental stress agents. This multifaceted stress reaction seems to be related to the intrinsic disorder of its precursor protein, Prosystemin (ProSys). Since latest findings show that ProSys has biological functions besides Systemin sequence, here we wanted to assess if this precursor includes peptide motifs able to trigger stress-related pathways. Candidate peptides were identified in silico and synthesized to test their capacity to trigger defence responses in tomato plants against different biotic stressors. Our results demonstrated that ProSys harbours several repeat motifs which triggered plant immune reactions against pathogens and pest insects. Three of these peptides were detected by mass spectrometry in plants expressing ProSys, demonstrating their effective presence in vivo. These experimental data shed light on unrecognized functions of ProSys, mediated by multiple biologically active sequences which may partly account for the capacity of ProSys to induce defense responses to different stress agents.
Asunto(s)
Péptidos , Proteínas de Plantas , Péptidos/metabolismo , Proteínas de Plantas/metabolismoRESUMEN
Aliphatic sulfonamides are an interesting class of carbonic anhydrase inhibitors (CAIs) proven to be effective for several carbonic anhydrase (CA) isoforms involved in pathologic states. Here we report the crystallographic structures of hCA II in complex with two aliphatic sulfonamides incorporating coumarin rings, which showed a good inhibition and selectivity for this isoform. Although these two molecules have a very similar chemical structure, differing only in the substitution of the two aliphatic hydrogen atoms with two fluorine atoms, they adopt a significantly different binding mode within the enzyme active site. Theoretical binding free energy calculations, performed to rationalize these data, showed that a delicate balance of electrostatic and steric effects modulate the protein-ligand interactions. Data presented here can be fruitfully used for the rational design of novel and effective isozyme-specific inhibitor molecules.
RESUMEN
Carbonic anhydrases (CAs) are ubiquitous enzymes that catalyze the reversible carbon dioxide hydration reaction. Among the eight different CA classes existing in nature, the α-class is the largest one being present in animals, bacteria, protozoa, fungi, and photosynthetic organisms. Although many studies have been reported on these enzymes, few functional, biochemical, and structural data are currently available on α-CAs isolated from photosynthetic organisms. Here, we give an overview of the most recent literature on the topic. In higher plants, these enzymes are engaged in both supplying CO2 at the Rubisco and determining proton concentration in PSII membranes, while in algae and cyanobacteria they are involved in carbon-concentrating mechanism (CCM), photosynthetic reactions and in detecting or signaling changes in the CO2 level in the environment. Crystal structures are only available for three algal α-CAs, thus not allowing to associate specific structural features to cellular localizations or physiological roles. Therefore, further studies on α-CAs from photosynthetic organisms are strongly needed to provide insights into their structure-function relationship.
Asunto(s)
Anhidrasas Carbónicas , Animales , Dióxido de Carbono , Anhidrasas Carbónicas/metabolismo , Fotosíntesis/fisiología , Plantas/metabolismo , Protones , Ribulosa-Bifosfato CarboxilasaRESUMEN
Melioidosis is a severe disease caused by the highly pathogenic gram-negative bacterium Burkholderia pseudomallei. Several studies have highlighted the broad resistance of this pathogen to many antibiotics and pointed out the pivotal importance of improving the pharmacological arsenal against it. Since γ-carbonic anhydrases (γ-CAs) have been recently introduced as potential and novel antibacterial drug targets, in this paper, we report a detailed characterization of BpsγCA, a γ-CA from B. pseudomallei by a multidisciplinary approach. In particular, the enzyme was recombinantly produced and biochemically characterized. Its catalytic activity at different pH values was measured, the crystal structure was determined and theoretical pKa calculations were carried out. Results provided a snapshot of the enzyme active site and dissected the role of residues involved in the catalytic mechanism and ligand recognition. These findings are an important starting point for developing new anti-melioidosis drugs targeting BpsγCA.
RESUMEN
Prosystemin is a 200-amino acid precursor expressed in Solanaceae plants which releases at the C-terminal part a peptidic hormone called Systemin in response to wounding and herbivore attack. We recently showed that Prosystemin is not only a mere scaffold of Systemin but, even when deprived of Systemin, is biologically active. These results, combined with recent discoveries that Prosystemin is an intrinsically disordered protein containing disordered regions within its sequence, prompted us to investigate the N-terminal portions of the precursor, which contribute to the greatest disorder within the sequence. To this aim, PS1-70 and PS1-120 were designed, produced, and structurally and functionally characterized. Both the fragments, which maintained their intrinsic disorder, were able to induce defense-related genes and to protect tomato plants against Botrytis cinerea and Spodoptera littoralis larvae. Intriguingly, the biological activity of each of the two N-terminal fragments and of Systemin is similar but not quite the same and does not show any toxicity on experimental non-targets considered. These regions account for different anti-stress activities conferred to tomato plants by their overexpression. The two N-terminal fragments identified in this study may represent new promising tools for sustainable crop protection.
RESUMEN
Intrinsically Disordered Proteins (IDPs) lack stable tertiary and secondary structures and are extensively distributed across eukaryotic cells, playing critical roles in cell signaling and regulation [...].
Asunto(s)
Proteínas Intrínsecamente Desordenadas , Humanos , Proteínas Intrínsecamente Desordenadas/química , Conformación Proteica , Estructura Secundaria de ProteínaRESUMEN
CDCA1 is a very peculiar member of the Carbonic Anhydrase (CA) family. It has been the first enzyme to show an efficient utilization of Cd(II) ions in Nature and a unique adaptation capability to live on the surface ocean. Indeed, in this environment, which is extremely depleted in essential metal ions, CDCA1 can utilize Zn(II) or Cd(II) as catalytic metal to support the metabolic needs of fast growing diatoms. In this paper we demonstrate a further catalytic versatility of this enzyme by using a combination of X-ray crystallography, molecular dynamics simulations and enzymatic experiments. First we identified the CO2 binding site and the way in which this substrate travels from the environment to the enzyme active site. Then, starting from the observation of a structural similarity with the substrate entry route of CS2 hydrolase from Acidanius A1-3, we hypothesized and demonstrated that also CS2 is a substrate for CDCA1. This finding is new and unexpected since until now only few CS2 hydrolases have been characterized, and none of them is reported to have any CO2 hydratase action. The physiological implications of this supplementary catalytic activity still remain to be unveiled. We suggest here that it could represent another ability of diatoms expressing CDCA1 to adapt to the external environment. Indeed, the ability of this enzyme to convert CS2 could represent an alternative source of carbon acquisition for diatoms, in addition to CO2.
RESUMEN
It has been clearly established that some proteins or protein regions are devoid of any stable secondary and/or tertiary structure under physiological conditions, but still possess fundamental biological functions [...].
Asunto(s)
Proteínas Intrínsecamente Desordenadas/química , Citoesqueleto de Actina/metabolismo , Animales , Helicobacter pylori , Humanos , Proteínas Intrínsecamente Desordenadas/metabolismo , Modelos Moleculares , Conformación Proteica , Pliegue de Proteína , Proteínas/químicaRESUMEN
hCA IX is a multi-domain protein belonging to the family of hCAs which are ubiquitous zinc enzymes that catalyze the reversible hydration of CO2 to HCO3- and H+. hCA IX is a tumor-associated enzyme with a limited distribution in normal tissues, but over-expressed in many tumors, and is a promising drug target. Although many studies concerning the CA IX catalytic domain were performed, little is known about the proteoglycan-like (PG-like) domain of hCA IX which has been poorly investigated so far. Here we attempt to fill this gap by providing an overview on the functional, structural and therapeutic studies of the PG-like domain of hCA IX which represents a unique feature within the CA family. The main studies and recent advances concerning PG role in modulating hCA IX catalytic activity as well as in tumor spreading and migration are here reported. Special attention has been paid to the newly discovered disordered features of the PG domain which open new perspectives about its molecular mechanisms of action under physiological and pathological conditions, since disorder is likely involved in mediating interactions with partner proteins. The emerged disordered features of PG domain will be explored for putative diagnostic and therapeutic applications involving CA IX targeting in tumors.
Asunto(s)
Anhidrasa Carbónica IX/metabolismo , Dominio Catalítico , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteoglicanos/metabolismo , Bicarbonatos/metabolismo , Biocatálisis/efectos de los fármacos , Dióxido de Carbono/metabolismo , Anhidrasa Carbónica IX/química , Inhibidores de Anhidrasa Carbónica/farmacología , Inhibidores de Anhidrasa Carbónica/uso terapéutico , Concentración de Iones de Hidrógeno , Proteínas Intrínsecamente Desordenadas/química , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Neoplasias/metabolismo , Proteoglicanos/químicaRESUMEN
In the present work, structural features of the interaction between peptide nucleic acid (PNA)-based analogs of the tumor-suppressor microRNA-34a with both its binding sites on MYCN mRNA were investigated. In particular, the region from base 1 to 8 ("seed" region) of miR-34a was reproduced in the form of an 8-mer PNA fragment (tiny PNA), and binding to target 3'UTR MYCN mRNA, was studied by a seldom reported and detailed NMR characterization, providing evidence for the formation of anti-parallel duplexes with a well-organized structural core. The formation of PNA-3'UTR duplexes was also confirmed by Circular Dichroism, and their melting curves were measured by UV spectroscopy. Nevertheless, this study offered a valuable comparison between molecular dynamics predictions and experimental evidence, which showed great correlation. Preliminary uptake assays were carried out in Neuroblastoma Kelly cells, using short peptide conjugates as carriers and FITC fluorescent tag for subcellular localization. Moderate internalization was observed without the use of transfecting agents. The reported results corroborate the interest toward the design and development of chimeric PNA/RNA sequences as effective RNA-targeting agents.
RESUMEN
Carbonic anhydrases (CAs) are ubiquitous metallo-enzymes that catalyse the reversible hydration of carbon dioxide to bicarbonate and proton. In humans there are 15 isoforms among which only 12 are catalytically active. Since active human (h) CAs show different efficiency, the understanding of the molecular determinants affecting it is a matter of debate. Here we investigated, by a site-specific mutagenesis approach, residues modulating the catalytic features of one of the least investigated cytosolic isoform, i.e. hCA XIII. Results showed that residues assisting the formation of an ordered solvent network within the catalytic site as well as those forming a histidine cluster on the protein surface are important to guarantee an efficient proton transfer.
Asunto(s)
Biocatálisis , Anhidrasas Carbónicas/genética , Anhidrasas Carbónicas/metabolismo , Mutagénesis Sitio-Dirigida , Anhidrasas Carbónicas/química , Relación Dosis-Respuesta a Droga , Humanos , Modelos Moleculares , Estructura Molecular , Relación Estructura-ActividadRESUMEN
MicroRNAs are a ubiquitous class of non-coding RNAs able to regulate gene expression in diverse biological processes. Widespread miRNAs deregulation was reported in numerous diseases including cancer, with several miRNAs playing oncogenic and/or tumor suppressive role by targeting multiple mRNAs simultaneously. Based on these findings, miRNAs have emerged as promising therapeutic tools for cancer treatment. Herein, for the first time, peptide nucleic acids (PNAs) were studied to develop a new class of molecules able to target 3'UTR on MYCN mRNA without a fully complementary base pairing sequence (as miRNAs). For our proof of concept study we have selected as a model the miRNA-34a, which acts as a tumor suppressor in a number of cancers including neuroblastoma. In particular, miRNA-34a is a direct regulator of MYCN oncogene, whose overexpression is a prominent biomarker for the highly aggressive neuroblastoma phenotype. The design and synthesis of three PNA-based oligomers of different length was described, and their interaction with two binding sites on the target MYCN mRNA was investigated by molecular dynamics simulation, and spectroscopic techniques (CD, UV). Intake assay and confocal microscopy of PNA sequences were also carried out in vitro on neuroblastoma Kelly cells. Despite the presence of multiple mismatches, the PNA/RNA hetero duplexes retain very interesting features in terms of stability, affinity as well as of cellular uptake.
Asunto(s)
Proliferación Celular , MicroARNs/genética , Proteína Proto-Oncogénica N-Myc/antagonistas & inhibidores , Neuroblastoma/tratamiento farmacológico , Ácidos Nucleicos de Péptidos/farmacología , ARN Mensajero/antagonistas & inhibidores , Química Computacional , Humanos , Microscopía Confocal , Simulación de Dinámica Molecular , Proteína Proto-Oncogénica N-Myc/genética , Neuroblastoma/genética , Neuroblastoma/patología , Ácidos Nucleicos de Péptidos/síntesis química , ARN Mensajero/genética , Células Tumorales CultivadasRESUMEN
Recent studies identified the benzoxaborole moiety as a new zinc-binding group able to interact with carbonic anhydrase (CA) active site. Here, we report a structural analysis of benzoxaboroles containing urea/thiourea groups, showing that these molecules are very versatile since they can bind the enzyme assuming different binding conformations and coordination geometries of the catalytic zinc ion. In addition, theoretical calculations of binding free energy were performed highlighting the key role of specific residues for protein-inhibitor recognition. Overall, these data are very useful for the development of new inhibitors with higher selectivity and efficacy for various CAs.
Asunto(s)
Compuestos de Boro/farmacología , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/metabolismo , Compuestos de Boro/síntesis química , Compuestos de Boro/química , Inhibidores de Anhidrasa Carbónica/síntesis química , Inhibidores de Anhidrasa Carbónica/química , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Humanos , Modelos Moleculares , Conformación Molecular , Relación Estructura-ActividadRESUMEN
Although important progress has been achieved in understanding the catalytic mechanism of Carbonic Anhydrases, a detailed picture of all factors influencing the catalytic efficiency of the various human isoforms is still missing. In this paper we report a detailed structural study and theoretical pKa calculations on a hCA VII variant. The obtained data were compared with those already known for another thoroughly investigated cytosolic isoform, hCA II. Our structural studies show that in hCA VII the network of ordered water molecules, which connects the zinc bound solvent molecule to the proton shuttle His64, is altered compared to hCA II, causing a reduction of the catalytic efficiency. Theoretical calculations suggest that changes in solvent network are related to the difference in pKa of the proton shuttle in the two enzymes. The residue that plays a major role in determining the diverse pKa values of the proton shuttle is the one in position four, namely His for hCA II and Gly for hCA VII. This residue is located on the protein surface, outside of the active site cavity. These findings are in agreement with our previous studies that highlighted the importance of histidines on the protein surface of hCA II (among which His4) as crucial residues for the high catalytic efficiency of this isoform.
Asunto(s)
Anhidrasas Carbónicas/química , Dominio Catalítico , Protones , Sustitución de Aminoácidos , Anhidrasas Carbónicas/genética , Anhidrasas Carbónicas/metabolismo , Cristalografía por Rayos X , Humanos , Simulación de Dinámica Molecular , Unión Proteica , Zinc/farmacologíaRESUMEN
Human carbonic anhydrase IX (hCA IX) is a tumour-associated enzyme present in a limited number of normal tissues, but overexpressed in several malignant human tumours. It is a transmembrane protein, where the extracellular region consists of a greatly investigated catalytic CA domain and a much less investigated proteoglycan-like (PG) domain. Considering its important role in tumour biology, here, we report for the first time the full characterization of the PG domain, providing insights into its structural and functional features. In particular, this domain has been produced at high yields in bacterial cells and characterized by means of biochemical, biophysical and molecular dynamics studies. Results show that it belongs to the family of intrinsically disordered proteins, being globally unfolded with only some local residual polyproline II secondary structure. The observed conformational flexibility may have several important roles in tumour progression, facilitating interactions of hCA IX with partner proteins assisting tumour spreading and progression.
Asunto(s)
Antígenos de Neoplasias/química , Bioquímica/métodos , Biofisica/métodos , Anhidrasa Carbónica IX/química , Simulación de Dinámica Molecular , Secuencia de Aminoácidos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Sitios de Unión/genética , Anhidrasa Carbónica IX/genética , Anhidrasa Carbónica IX/metabolismo , Dominio Catalítico , Dicroismo Circular , Progresión de la Enfermedad , Humanos , Espectroscopía de Resonancia Magnética , Neoplasias/enzimología , Neoplasias/patología , Conformación Proteica , Proteoglicanos/metabolismo , Relación Estructura-ActividadRESUMEN
HER2 transmembrane receptor is an important target in immunotherapy treatment of breast and gastroesophageal cancer. Molecular imaging of HER2 expression may provide essential prognostic and predictive information concerning disseminated cancer and aid in selection of an optimal therapy. Radiolabeled low molecular weight peptide ligands are particularly attractive as probes for molecular imaging, since they reach and bind to the target and clear from non-target organs and blood stream faster than bulky antibodies. In this study, we evaluated a potential HER2-imaging probe, an A9 nonapeptide, derived from the trastuzumab-Fab portion. Its cellular uptake was investigated by mass spectrometry analysis of the cytoplasmic cellular extracts. Moreover, based on in-silico modeling, DTPA chelator was conjugated to N-terminus of A9. 111In-labeled A9 demonstrated nanomolar affinity to HER2-expressing BT474 cells and favorable biodistribution profile in NMRI mice. This study suggests that the peptide A9 represents a good lead candidate for development of molecular probe, to be used for imaging purposes and for the delivery of cytotoxic agents.
Asunto(s)
Neoplasias de la Mama/diagnóstico , Sondas Moleculares/metabolismo , Péptidos/metabolismo , Radioinmunodetección/métodos , Receptor ErbB-2/inmunología , Animales , Animales no Consanguíneos , Neoplasias de la Mama/patología , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos , Femenino , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Ratones , Sondas Moleculares/genética , Péptidos/genética , Unión Proteica , Trastuzumab/genéticaRESUMEN
Prosystemin, originally isolated from Lycopersicon esculentum, is a tomato pro-hormone of 200 aminoacid residues which releases a bioactive peptide of 18 aminoacids called Systemin. This signaling peptide is involved in the activation of defense genes in solanaceous plants in response to herbivore feeding damage. Using biochemical, biophysical and bioinformatics approaches we characterized Prosystemin, showing that it is an intrinsically disordered protein possessing a few secondary structure elements within the sequence. Plant treatment with recombinant Prosystemin promotes early and late plant defense genes, which limit the development and survival of Spodoptera littoralis larvae fed with treated plants.
Asunto(s)
Proteínas de Plantas/química , Proteínas de Plantas/farmacología , Solanum lycopersicum/metabolismo , Spodoptera/crecimiento & desarrollo , Animales , Dicroismo Circular , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/farmacología , Larva/efectos de los fármacos , Larva/crecimiento & desarrollo , Solanum lycopersicum/química , Solanum lycopersicum/genética , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Proteínas de Plantas/metabolismo , Estructura Secundaria de Proteína , Spodoptera/efectos de los fármacosRESUMEN
Sulphamate and sulphamide derivatives have been largely investigated as carbonic anhydrase inhibitors (CAIs) by means of different experimental techniques. However, the structural determinants responsible for their different binding mode to the enzyme active site were not clearly defined so far. In this paper, we report the X-ray crystal structure of hCA II in complex with a sulphamate inhibitor incorporating a nitroimidazole moiety. The comparison with the structure of hCA II in complex with its sulphamide analogue revealed that the two inhibitors adopt a completely different binding mode within the hCA II active site. Starting from these results, we performed a theoretical study on sulphamate and sulphamide derivatives, demonstrating that electrostatic interactions with residues within the enzyme active site play a key role in determining their binding conformation. These findings open new perspectives in the design of effective CAIs using the sulphamate and sulphamide zinc binding groups as lead compounds.
Asunto(s)
Anhidrasa Carbónica II/antagonistas & inhibidores , Inhibidores de Anhidrasa Carbónica/farmacología , Sulfonamidas/farmacología , Ácidos Sulfónicos/farmacología , Termodinámica , Sitios de Unión/efectos de los fármacos , Anhidrasa Carbónica II/metabolismo , Inhibidores de Anhidrasa Carbónica/síntesis química , Inhibidores de Anhidrasa Carbónica/química , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/química , Ácidos Sulfónicos/síntesis química , Ácidos Sulfónicos/químicaRESUMEN
Human Carbonic Anhydrase (hCA) IX is a membrane-associated member of the CA enzyme family, involved in solid tumor acidification. This enzyme is a marker of tumor hypoxia and a prognostic factor for several human cancers. In a recent paper, we showed that CA IX interacts with cullin-associated NEDD8-dissociated protein 1 (CAND1), a nuclear protein involved in gene transcription and assembly of SCF ubiquitin ligase complexes. A functional role for this interaction was also identified, since lower CA IX levels were observed in cells with decreased CAND1 expression via shRNA-mediated interference. In this paper, we describe the identification of the structural determinants responsible for the CA IX/CAND1 interaction by means of a multidisciplinary approach, consisting of binding assay measurements, molecular docking, and site-directed mutagenesis. These data open a novel scenario in the design of anticancer drugs targeting CA IX. Indeed, the knowledge of the structural determinants responsible for the CAND1/CA IX interaction provides the molecular basis to design molecules able to destabilize it. Due to the proposed function of CAND1 in stabilizing CA IX, these molecules could represent an efficient tool to lower the amount of CA IX in hypoxic cancer cells, thus limiting its action in survival and the metastatic spread of tumors.
Asunto(s)
Anhidrasa Carbónica IX/metabolismo , Factores de Transcripción/metabolismo , Anhidrasa Carbónica IX/química , Inhibidores de Anhidrasa Carbónica , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Estabilidad Proteica , Factores de Transcripción/químicaRESUMEN
Carbonic anhydrases (CAs) III and VII are two cytosolic isoforms of the α-CA family which catalyze the physiological reaction of carbon dioxide hydration to bicarbonate and proton. Despite these two enzymes share a 49% sequence identity and present a very similar three-dimensional structure, they show profound differences when comparing the specific activity for CO2 hydration reaction, with CA VII being much more active than CA III. Recently, CA III and CA VII have been proposed to play a new role as scavenger enzymes in cells where oxidative damage occurs. Here, we will examine functional and structural features of these two isoforms giving insights into their newly proposed protective role against oxidative stress.