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It has been brought to our attention that the affiliation of Dr. Jerzy Pieczykolan at the time when he was responsible for the work described in the paper [...].
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Compounds that recognize and strongly bind to molecular targets are one of the cornerstones of modern pharmaceutics. Work has been ongoing for the past 25 years on the therapeutic use of aptamers, nucleic acid molecules, whose three-dimensional structure is the result of interactions between complementary base pairs. The aptamers selection methods allow the oligonucleotides which bind the molecular target in its native environment to be quickly isolated from a large library of random oligonucleotides. The possibilities presented for aptamers in the field of targeted therapy require the application of effective carriers to counter the renal clearance effect and/or functional cargo to exert therapeutic action if the aptamer is only used as a targeting moiety. Lately, a material gaining ground in biomedical research is iron oxide particles, which exhibit a superparamagnetic characteristic at nanoscale levels. This allows the iron oxide nanoparticles to convert external magnetic energy into heat, a mechanism known as hyperthermy, and efficiently supports conventional oncological treatment. In this study, we describe an experimentally confirmed functional model of targeted anticancer hyperthermia therapy. Using the systematic evolution of ligands by exponential enrichment technique, we selected a DNA aptamer that specifically binds to the extracellular domain of recombinant fibroblast growth factor receptor type-1 (FGFR1) with a nanomolar dissociation constant. The chosen target plays an important role in many crucial cellular processes and is also considered a candidate protein that is involved in tumor initiation, survival and progression. Next, we combined the selected aptamer with iron oxide nanoparticles to produce aptamer superparamagnetic conjugates (ASCs). Finally, we found that targeted ASCs selectively destroy FGFR1-overexpressing human osteosarcoma cells U2OS upon magnetic field irradiation.
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Antineoplásicos/farmacología , Aptámeros de Nucleótidos/farmacología , Hipertermia Inducida/métodos , Nanopartículas de Magnetita/química , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Antineoplásicos/química , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/metabolismo , Avidina/química , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales/métodos , Óxido Ferrosoférrico/química , Humanos , Ligandos , Magnetoterapia/métodos , Terapia Molecular Dirigida , Neoplasias/terapia , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genéticaRESUMEN
Antibody-drug conjugates (ADCs) have recently emerged as efficient and selective cancer treatment therapeutics. Currently, alternative forms of drug carriers that can replace monoclonal antibodies are under intensive investigation. Here, a cytotoxic conjugate of an anti-HER2 (Human Epidermal Growth Factor Receptor 2) diaffibody with monomethyl-auristatin E (MMAE) is proposed as a potential anticancer therapeutic. The anti-HER2 diaffibody was based on the ZHER2:4 affibody amino acid sequence. The anti-HER2 diaffibody has been expressed as a His-tagged protein in E. coli and purified by Ni-nitrilotriacetyl (Ni-NTA) agarose chromatography. The molecule was properly folded, and the high affinity and specificity of its interaction with HER2 was confirmed by surface plasmon resonance (SPR) and flow cytometry, respectively. The (ZHER2:4)2DCS-MMAE conjugate was obtained by coupling the maleimide group linked with MMAE to cysteines, which were introduced in a drug conjugation sequence (DCS). Cytotoxicity of the conjugate was evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide MTT assay and the xCELLigence Real-Time Cell Analyzer. Our experiments demonstrated that the conjugate delivered auristatin E specifically to HER2-positive tumor cells, which finally led to their death. These results indicate that the cytotoxic diaffibody conjugate is a highly potent molecule for the treatment of various types of cancer overexpressing HER2 receptors.
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Aminobenzoatos/farmacología , Anticuerpos Monoclonales/farmacología , Inmunoconjugados/farmacología , Neoplasias/metabolismo , Oligopéptidos/farmacología , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/metabolismo , Aminobenzoatos/química , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Especificidad de Anticuerpos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Inmunoconjugados/química , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Oligopéptidos/química , Unión Proteica , Estabilidad Proteica , Termodinámica , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Targeted therapy is a method owing to its limited side effect profile, particularly in cancer treatment. Magnetic hyperthermia, which is induced by nanoparticles (NPs) conjugated with targeting agents, can be useful in combination with chemo- or radiotherapy. In this paper, we constructed dextran-coated ferric oxide NPs conjugated with specific anti-human epidermal growth factor receptor (HER2) aptamer and used them to induce magnetic hyperthermia in cultured cells. The specificity of the tagged NPs was determined by studying their effect relative to that of non-tagged NPs against two cell lines: human adenocarcinoma SK-BR3, overexpressing the HER2 receptor; and U-87 MG, a human glioblastoma epithelial cell line, not expressing HER2. In order to confirm the interaction of the tagged NPs with the cells we used, fluorescence microscopy and fluorescence-activated cell sorting analysis were performed. All of these experiments showed that the aptamer-tagged NPs were highly specific toward the HER2-expressing cells. In addition, a ninetyfold lower dose of the tagged NPs relative to that of the non-tagged NPs was needed to achieve ~50% cell killing by hyperthermia of the SK-BR3 cell line, while for the U-87 MG cells the viability level was close to 100%. These results show that targeted NPs can be applied at substantially lower doses than non-targeted ones to achieve similar effects of hyperthermia, which should greatly limit the side effects of treatment.
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Adenocarcinoma/genética , Adenocarcinoma/terapia , Aptámeros de Nucleótidos/genética , Aptámeros de Nucleótidos/uso terapéutico , Hipertermia Inducida/métodos , Nanopartículas de Magnetita/uso terapéutico , Receptor ErbB-2/genética , Adenocarcinoma/patología , Línea Celular Tumoral , Humanos , Nanopartículas de Magnetita/química , Nanopartículas de Magnetita/ultraestructura , Coloración y Etiquetado , Resultado del TratamientoRESUMEN
Aptamers emerged over 20 years ago as a class of nucleic acids able to recognize specific targets. Today, aptamer-related studies constitute a large and important field of biotechnology. Functional oligonucleotides have proved to be a versatile tool in biomedical research due to the ease of synthesis, a wide range of potentially recognized molecular targets and the simplicity of selection. Similarly to antibodies, aptamers can be used to detect or isolate specific molecules, as well as to act as targeting and therapeutic agents. In this review we present different approaches to aptamer application in nanobiotechnology, diagnostics and medicine.
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Aptámeros de Nucleótidos , Aptámeros de Péptidos , Biotecnología , Nanotecnología , Técnica SELEX de Producción de AptámerosRESUMEN
sGC (soluble guanylate cyclase) is the main mediator of NO signalling. Biochemical and physiological studies suggest that, besides NO, in vivo regulation of sGC involves direct interaction with other proteins. Using yeast two-hybrid screening, we identified that the multidomain LGN (Leu-Gly-Asn repeat-enriched protein) interacts with both α1 and ß1 sGC subunits. LGN and sGC co-localized in the cell cytoplasm, and the LGN-sGC complex was co-immunoprecipitated from cells expressing both proteins and from native tissues. Their interaction requires the N-terminal tetratricopeptide repeats of LGN, but does not require the N-terminal portions of α1 or ß1 sGC subunits. Overexpression of LGN decreases the activity of cellular sGC, whereas knockdown of LGN mRNA and protein correlated with increased sGC activity. Although purified LGN interacts directly with purified sGC, the inhibitory effect in vitro is observed only after supplementation of cell lysate to the reaction. Although resting sGC and sGC activated by the stimulator BAY41-2272 have very similar LGN-IC50 values to the NO-stimulated sGC, they have a much higher Hill coefficient, suggesting co-operative binding with respect to LGN in the low-activated state of sGC. AGS3 (activator of G-protein signalling 3), the closest LGN homologue, also inhibits sGC. The interaction of sGC with these scaffolding proteins may expand the cross-talk between NO/cGMP signalling and other cellular pathways and tailor sGC function to specific tissues or signals.
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Guanilato Ciclasa/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Óxido Nítrico/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal , Línea Celular Tumoral , GMP Cíclico/metabolismo , Guanilato Ciclasa/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Guanilil Ciclasa Soluble , TransfecciónRESUMEN
Plant pathogenesis-related (PR) proteins of class 10 are the only group among the 17 PR protein families that are intracellular and cytosolic. Sequence conservation and the wide distribution of PR-10 proteins throughout the plant kingdom are an indication of an indispensable function in plants, but their true biological role remains obscure. Crystal and solution structures for several homologues have shown a similar overall fold with a vast internal cavity which, together with structural similarities to the steroidogenic acute regulatory protein-related lipid transfer domain and cytokinin-specific binding proteins, strongly indicate a ligand-binding role for the PR-10 proteins. This article describes the structure of a complex between a classic PR-10 protein [Lupinus luteus (yellow lupine) PR-10 protein of subclass 2, LlPR-10.2B] and N,N'-diphenylurea, a synthetic cytokinin. Synthetic cytokinins have been shown in various bioassays to exhibit activity similar to that of natural cytokinins. The present 1.95 A resolution crystallographic model reveals four N,N'-diphenylurea molecules in the hydrophobic cavity of the protein and a degree of conformational changes accompanying ligand binding. The structural adaptability of LlPR-10.2B and its ability to bind different cytokinins suggest that this protein, and perhaps other PR-10 proteins as well, can act as a reservoir of cytokinin molecules in the aqueous environment of a plant cell.
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Citocininas/farmacología , Lupinus/química , Proteínas de Plantas/química , Secuencia de Aminoácidos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
We describe a detailed study of the RhoA-binding epitope of the GAP domain of Graf, including the determination of the thermodynamic and kinetic parameters of the interaction of wild-type domain, and of its 15 single-site mutants, with cognate GTPases. We show that residues important for the structural integrity of the Arg-finger loop are critical for binding Rho and for the catalytic activity of GAP, but GTPase selectivity appears to be modulated by a much more subtle interplay of electrostatic and hydrophobic interactions involving residues on the periphery of the main interface. The eight residues targeted in this study are involved in three distinct patches on the surface, two of which appear to interact with highly conserved regions of the GTPase, while the third plays a role in GTPase selectivity.
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Proteínas Activadoras de GTPasa/química , Proteínas Activadoras de GTPasa/metabolismo , Proteína de Unión al GTP rhoA/química , Proteína de Unión al GTP rhoA/metabolismo , Secuencia de Aminoácidos , Proteínas Activadoras de GTPasa/genética , Guanosina Trifosfato/metabolismo , Humanos , Datos de Secuencia Molecular , Alineación de Secuencia , Termodinámica , Proteína de Unión al GTP rhoA/genéticaRESUMEN
Soluble guanylyl cyclase (sGC), a key protein in the NO/cGMP signaling pathway, is an obligatory heterodimeric protein composed of one alpha- and one beta-subunit. The alpha(1)/beta(1) sGC heterodimer is the predominant form expressed in various tissues and is regarded as the major isoform mediating NO-dependent effects such as vasodilation. We have identified three new alpha(1) sGC protein variants generated by alternative splicing. The 363 residue N1-alpha(1) sGC splice variant contains the regulatory domain, but lacks the catalytic domain. The shorter N2-alpha(1) sGC maintains 126 N-terminal residues and gains an additional 17 unique residues. The C-alpha(1) sGC variant lacks 240 N-terminal amino acids, but maintains a part of the regulatory domain and the entire catalytic domain. Q-PCR of N1-alpha(1), N2-alpha(1) sGC mRNA levels together with RT-PCR analysis for C-alpha(1) sGC demonstrated that the expression of the alpha(1) sGC splice forms vary in different human tissues indicative of tissue-specific regulation. Functional analysis of the N1-alpha(1) sGC demonstrated that this protein has a dominant-negative effect on the activity of sGC when coexpressed with the alpha(1)/beta(1) heterodimer. The C-alpha(1) sGC variant heterodimerizes with the beta(1) subunit and produces a fully functional NO- and BAY41-2272-sensitive enzyme. We also found that despite identical susceptibility to inhibition by ODQ, intracellular levels of the 54-kDa C-alpha(1) band did not change in response to ODQ treatments, while the level of 83 kDa alpha(1) band was significantly affected by ODQ. These studies suggest that modulation of the level and diversity of splice forms may represent novel mechanisms modulating the function of sGC in different human tissues.
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Empalme Alternativo/fisiología , Inhibidores Enzimáticos/farmacología , Guanilato Ciclasa/metabolismo , Oxadiazoles/farmacología , Quinoxalinas/farmacología , Receptores Citoplasmáticos y Nucleares/metabolismo , Empalme Alternativo/efectos de los fármacos , Animales , Dominio Catalítico/fisiología , Línea Celular Tumoral , Guanilato Ciclasa/análisis , Guanilato Ciclasa/genética , Humanos , Especificidad de Órganos/fisiología , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Citoplasmáticos y Nucleares/análisis , Receptores Citoplasmáticos y Nucleares/genética , Guanilil Ciclasa Soluble , SpodopteraRESUMEN
A phage-displayed random 7-mer disulfide bridge-constrained peptide library was used to map the surface of the RhoA GTPase and to find peptides able to recognize RhoA switch regions. Several peptide sequences were selected after four rounds of enrichment, giving a high signal in ELISA against RhoA-GDP. A detailed analysis of one such selected peptide, called R2 (CWSFPGYAC), is reported. The RhoA-R2 interaction was investigated using fluorescence spectroscopy, chemical denaturation, and determination of the kinetics of nucleotide exchange and GTP hydrolysis in the presence of RhoA regulatory proteins. All measurements indicate that the affinity of the R2 peptide for RhoA is in the micromolar range and that R2 behaves as an inhibitor of: i) GDP binding to the apo form of RhoA (Mg2+-and nucleotide-free form of the GTPase), ii) nucleotide exchange stimulated by GEF (DH/PH tandem from PDZRhoGEF), and iii) GTP hydrolysis stimulated by the BH domain of GrafGAP protein.
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GTP Fosfohidrolasas/metabolismo , Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Péptidos/química , Péptidos/aislamiento & purificación , Secuencia de Aminoácidos , Bacteriófago M13/genética , GTP Fosfohidrolasas/antagonistas & inhibidores , Proteínas Activadoras de GTPasa/metabolismo , Guanosina Trifosfato/metabolismo , Biblioteca de Péptidos , Mapeo Peptídico/métodos , Péptidos/genética , Conformación Proteica , Proteínas Recombinantes/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
The cytosolic fraction of Vigna radiata contains a 17-kD protein that binds plant hormones from the cytokinin group, such as zeatin. Using recombinant protein and isothermal titration calorimetry as well as fluorescence measurements coupled with ligand displacement, we have reexamined the K(d) values and show them to range from approximately 10(-6) M (for 4PU30) to 10(-4) M (for zeatin) for 1:1 stoichiometry complexes. In addition, we have crystallized this cytokinin-specific binding protein (Vr CSBP) in complex with zeatin and refined the structure to 1.2 A resolution. Structurally, Vr CSBP is similar to plant pathogenesis-related class 10 (PR-10) proteins, despite low sequence identity (<20%). This unusual fold conservation reinforces the notion that classic PR-10 proteins have evolved to bind small-molecule ligands. The fold consists of an antiparallel beta-sheet wrapped around a C-terminal alpha-helix, with two short alpha-helices closing a cavity formed within the protein core. In each of the four independent CSBP molecules, there is a zeatin ligand located deep in the cavity with conserved conformation and protein-ligand interactions. In three cases, an additional zeatin molecule is found in variable orientation but with excellent definition in electron density, which plugs the entrance to the binding pocket, sealing the inner molecule from contact with bulk solvent.
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Proteínas Portadoras/metabolismo , Phaseolus/metabolismo , Proteínas de Plantas/metabolismo , Zeatina/metabolismo , Secuencia de Aminoácidos , Calorimetría , Proteínas Portadoras/química , Cristalografía por Rayos X , Citocininas , Ligandos , Datos de Secuencia Molecular , Proteínas de Plantas/química , Unión Proteica , Conformación Proteica , Homología de Secuencia de Aminoácido , Espectrometría de Fluorescencia , Zeatina/químicaRESUMEN
Proteases and their natural protein inhibitors are among the most intensively studied protein-protein complexes. There are about 30 structurally distinct inhibitor families that are able to block serine, cysteine, metallo- and aspartyl proteases. The mechanisms of inhibition can be related to the catalytic mechanism of protease action or include a mechanism-unrelated steric blockage of the active site or its neighborhood. The structural elements that are responsible for the inhibition most often include the N- or the C-terminus or exposed loop(s) either separately or in combination of several such elements. During complex formation, no major conformational changes are usually observed, but sometimes structural transitions of the inhibitor and enzyme occur. In many cases, convergent evolution, with respect to the inhibitors' parts that are responsible for the inhibition, can be inferred from comparisons of their structures or sequences, strongly suggesting that there are only limited ways to inhibit proteases by proteins.
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Evolución Molecular , Modelos Moleculares , Complejos Multiproteicos/metabolismo , Péptido Hidrolasas/metabolismo , Proteínas/antagonistas & inhibidores , Proteínas/metabolismo , Sitios de Unión/genética , Unión Competitiva , Catálisis , Péptido Hidrolasas/química , Péptido Hidrolasas/genética , Conformación ProteicaRESUMEN
Syntenin, a 33 kDa protein, interacts with several cell membrane receptors and with merlin, the product of the causal gene for neurofibromatosis type II. We report a crystal structure of the functional fragment of human syntenin containing two canonical PDZ domains, as well as binding studies for full-length syntenin, the PDZ tandem, and isolated PDZ domains. We show that the functional properties of syntenin are a result of independent interactions with target peptides, and that each domain is able to bind peptides belonging to two different classes: PDZ1 binds peptides from classes I and III, while PDZ2 interacts with classes I and II. The independent binding of merlin by PDZ1 and syndecan-4 by PDZ2 provides direct evidence for the coupling of syndecan-mediated signaling to actin regulation by merlin.
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Proteínas Portadoras/química , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/química , Estructura Terciaria de Proteína , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cristalografía por Rayos X , Citoesqueleto/metabolismo , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Neurofibromina 2/química , Neurofibromina 2/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica , Proteoglicanos/metabolismo , Alineación de Secuencia , Sindecanos , SinteninasRESUMEN
PDZ domains are ubiquitous protein interaction modules that play a key role in cellular signaling. Their binding specificity involves recognition of the carboxyl-terminus of various proteins, often belonging to receptor and ion channel families. PDZ domains also mediate more complicated molecular networks through PDZ-PDZ interactions, recognition of internal protein sequences or phosphatidylinositol moieties. The domains often form a tandem of multiple copies, but equally often such tandems or single PDZ domain occur in combination with other signaling domains (for example SH3, DH/PH, GUK, LIM, CaMK). Common occurrence of PDZ domains in Metazoans strongly suggests that their evolutionary appearance results from the complication of signaling mechanisms in multicellular organisms. Here, we focus on their structure, specificity and role in signaling pathways.
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Proteínas Portadoras/fisiología , Estructura Terciaria de Proteína/fisiología , Transducción de Señal/fisiología , Animales , Proteínas Portadoras/clasificación , Guanilato-Quinasas , Humanos , Ligandos , Nucleósido-Fosfato Quinasa/fisiología , Fosforilación , Proteína de Unión al Tracto de Polipirimidina/fisiología , Estructura Terciaria de Proteína/genéticaRESUMEN
Mitomycin C (MC) is a potent anticancer agent. Streptomyces lavendulae, which produces MC, protects itself from the lethal effects of the drug by expressing several resistance proteins. One of them (MRD) binds MC and functions as a drug exporter. We report the crystal structure of MRD and its complex with an MC metabolite, 1,2-cis-1-hydroxy-2,7-diaminomitosene, at 1.5 A resolution. The drug is sandwiched by pi-stacking interactions of His-38 and Trp-108. MRD is a dimer. The betaalphabetabetabeta fold of the MRD molecule is reminiscent of methylmalonyl-CoA epimerase, bleomycin resistance proteins, glyoxalase I, and extradiol dioxygenases. The location of the binding site is identical to the ones in evolutionarily related enzymes, suggesting that the protein may have been recruited from a different metabolic pathway.