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1.
Molecules ; 28(3)2023 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-36771001

RESUMEN

Persistence and degradation are important factors in determining the safe use of such synthetic products, and numerous studies have been addressed to develop pesticide remediation methods aimed at ameliorating these features. In this frame, the use of different cyclodextrins (CDs) molecules has attracted considerable attention due to their well-known non-toxic nature, limited environmental impact, and capability to reduce the environmental and health risks of pesticides. CDs appear to be a valuable tool for the elimination of pesticides from polluted areas as well as for better pesticide formulations that positively influence their hydrolysis or degradation. The present work investigates the interaction between ß-cyclodextrins and three commonly used pesticides (i.e., chlorpropham, monuron, and propanil) both in solution and in the solid state by means of UV-Vis, FT-IR, and X-ray powder diffractometry. We show that such interactions result in all three cases in the formation of inclusion complexes with a 1:1 stoichiometry and binding constants (Kb) of 369.9 M-1 for chlorpropham, 292.3 M-1 for monuron, and 298.3 M-1 for propanil. We also report the energy-minimized structures in silico for each complex. Our data expand and complement the available literature data in indicating CDs as a low-cost and very effective tool capable of modulating the properties that determine the environmental fate of pesticides.


Asunto(s)
Ciclodextrinas , Plaguicidas , Propanil , beta-Ciclodextrinas , Plaguicidas/análisis , Clorprofam , Espectroscopía Infrarroja por Transformada de Fourier , beta-Ciclodextrinas/química , Ciclodextrinas/química , Solubilidad
2.
Int J Mol Sci ; 23(19)2022 Sep 20.
Artículo en Inglés | MEDLINE | ID: mdl-36232306

RESUMEN

A strict interplay is known to involve copper and zinc in many cellular processes. For this reason, the results of copper's interaction with zinc binding proteins are of great interest. For instance, copper interferences with the DNA-binding activity of zinc finger proteins are associated with the development of a variety of diseases. The biological impact of copper depends on the chemical properties of its two common oxidation states (Cu(I) and Cu(II)). In this framework, following the attention addressed to unveil the effect of metal ion replacement in zinc fingers and in zinc-containing proteins, we explore the effects of the Zn(II) to Cu(I) or Cu(II) replacement in the prokaryotic zinc finger domain. The prokaryotic zinc finger protein Ros, involved in the horizontal transfer of genes from A. tumefaciens to a host plant infected by it, belongs to a family of proteins, namely Ros/MucR, whose members have been recognized in different bacteria symbionts and pathogens of mammals and plants. Interestingly, the amino acids of the coordination sphere are poorly conserved in most of these proteins, although their sequence identity can be very high. In fact, some members of this family of proteins do not bind zinc or any other metal, but assume a 3D structure similar to that of Ros with the residues replacing the zinc ligands, forming a network of hydrogen bonds and hydrophobic interactions that surrogates the Zn-coordinating role. These peculiar features of the Ros ZF domain prompted us to study the metal ion replacement with ions that have different electronic configuration and ionic radius. The protein was intensely studied as a perfectly suited model of a metal-binding protein to study the effects of the metal ion replacement; it appeared to tolerate the Zn to Cd substitution, but not the replacement of the wildtype metal by Ni(II), Pb(II) and Hg(II). The structural characterization reported here gives a high-resolution description of the interaction of copper with Ros, demonstrating that copper, in both oxidation states, binds the protein, but the replacement does not give rise to a functional domain.


Asunto(s)
Mercurio , Zinc , Aminoácidos , Cadmio , Cobre/química , ADN/metabolismo , Iones , Plomo , Proteínas , Zinc/metabolismo , Dedos de Zinc
3.
Int J Mol Sci ; 23(19)2022 Sep 20.
Artículo en Inglés | MEDLINE | ID: mdl-36232339

RESUMEN

The crucial role of integrin in pathological processes such as tumor progression and metastasis formation has inspired intense efforts to design novel pharmaceutical agents modulating integrin functions in order to provide new tools for potential therapies. In the past decade, we have investigated the biological proprieties of the chimeric peptide RGDechi, containing a cyclic RGD motif linked to an echistatin C-terminal fragment, able to specifically recognize αvß3 without cross reacting with αvß5 and αIIbß3 integrin. Additionally, we have demonstrated using two RGDechi-derived peptides, called RGDechi1-14 and ψRGDechi, that chemical modifications introduced in the C-terminal part of the peptide alter or abolish the binding to the αvß3 integrin. Here, to shed light on the structural and dynamical determinants involved in the integrin recognition mechanism, we investigate the effects of the chemical modifications by exploring the conformational space sampled by RGDechi1-14 and ψRGDechi using an integrated natural-abundance NMR/MD approach. Our data demonstrate that the flexibility of the RGD-containing cycle is driven by the echistatin C-terminal region of the RGDechi peptide through a coupling mechanism between the N- and C-terminal regions.


Asunto(s)
Integrina alfaVbeta3 , Péptidos , Integrina alfaVbeta3/metabolismo , Espectroscopía de Resonancia Magnética , Oligopéptidos/química , Péptidos/química , Preparaciones Farmacéuticas
4.
Molecules ; 27(20)2022 Oct 17.
Artículo en Inglés | MEDLINE | ID: mdl-36296575

RESUMEN

The N-capping region of an α-helix is a short N-terminal amino acid stretch that contributes to nucleate and stabilize the helical structure. In the VEGF mimetic helical peptide QK, the N-capping region was previously demonstrated to be a key factor of QK helical folding. In this paper, we explored the effect of the chiral inversion of the N-capping sequence on QK folding, performing conformational analysis in solution by circular dichroism and NMR spectroscopy. The effect of such a modification on QK stability in serum and the proliferative effect were also evaluated.


Asunto(s)
Aminoácidos , Factor A de Crecimiento Endotelial Vascular , Secuencia de Aminoácidos , Péptidos/química , Dicroismo Circular , Conformación Proteica
5.
Bioorg Chem ; 116: 105379, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34563997

RESUMEN

The analysis of the forces governing helix formation and stability in peptides and proteins has attracted considerable interest in order to shed light on folding mechanism. We analyzed the role of hydrophobic interaction, steric hindrance and chain length on i, i + 3 position in QK peptide, a VEGF mimetic helical peptide. We focused on position 10 of QK, occupied by a leucine, as previous studies highlighted the key role of the Leu7-Leu10 interaction in modulating the helix formation and inducing an unusual thermodynamic stability. Leu10 has been replaced by hydrophobic amino acids with different side-chain length, hydrophobicity and steric hindrance. Ten peptides were, hence, synthesized and analyzed combining circular dichroism, calorimetry and NMR spectroscopy. We found that helical content and thermal stability of peptide QK changed when Leu10 was replaced. Interestingly, we observed that the changes in the helical content and thermal stability were not always correlated and they depend on the type of interaction (strength and geometry) that could be established between Leu7 and the residue in position 10.


Asunto(s)
Péptidos/química , Factores de Crecimiento Endotelial Vascular/química , Interacciones Hidrofóbicas e Hidrofílicas , Conformación Proteica
6.
J Pept Sci ; 26(2): e3231, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31749266

RESUMEN

The binding process of A9 peptide toward HER2-DIVMP, a synthetic model of the receptor domain IV, was studied by using the surface plasmon resonance (SPR) technique, with the aim of validating it as a fast and reliable screening method for selecting peptide ligands specifically targeting a domain of their target. To investigate the structural basis of A9 binding to the model of HER2-DIVMP, multiple ligand-based nuclear magnetic resonance (NMR) methods were applied. The use of saturation transfer difference (STD) and WaterLOGSY NMR experiments identified key residues in the peptide for the receptor binding. Moreover, the bound conformation of the A9 peptide was obtained using transferred nuclear Overhauser effect spectroscopy (trNOESY) experiments. The NMR data revealed an extended binding surface that confirms an in silico model previously reported. These structural findings could provide good starting points for future lead structures optimization specific for the receptor target.


Asunto(s)
Péptidos/química , Péptidos/farmacología , Receptor ErbB-2/metabolismo , Espectroscopía de Resonancia Magnética , Unión Proteica , Dominios Proteicos , Receptor ErbB-2/química , Resonancia por Plasmón de Superficie
7.
Int J Mol Sci ; 21(21)2020 Nov 05.
Artículo en Inglés | MEDLINE | ID: mdl-33167398

RESUMEN

The structural effects of zinc replacement by xenobiotic metal ions have been widely studied in several eukaryotic and prokaryotic zinc-finger-containing proteins. The prokaryotic zinc finger, that presents a bigger ßßßαα domain with a larger hydrophobic core with respect to its eukaryotic counterpart, represents a valuable model protein to study metal ion interaction with metallo-proteins. Several studies have been conducted on Ros87, the DNA binding domain of the prokaryotic zinc finger Ros, and have demonstrated that the domain appears to structurally tolerate Ni(II), albeit with important structural perturbations, but not Pb(II) and Hg(II), and it is in vitro functional when the zinc ion is replaced by Cd(II). We have previously shown that Ros87 unfolding is a two-step process in which a zinc binding intermediate converts to the native structure thorough a delicate downhill folding transition. Here, we explore the folding/unfolding behaviour of Ros87 coordinated to Co(II), Ni(II) or Cd(II), by UV-Vis, CD, DSC and NMR techniques. Interestingly, we show how the substitution of the native metal ion results in complete different folding scenarios. We found a two-state unfolding mechanism for Cd-Ros87 whose metal affinity Kd is comparable to the one obtained for the native Zn-Ros87, and a more complex mechanism for Co-Ros87 and Ni-Ros87, that show higher Kd values. Our data outline the complex cross-correlation between the protein-metal ion equilibrium and the folding mechanism proposing such an interplay as a key factor in the proper metal ion selection by a specific metallo-protein.


Asunto(s)
Cadmio/química , Cobalto/química , Níquel/química , Pliegue de Proteína/efectos de los fármacos , Proteínas Represoras , Zinc/química , Agrobacterium tumefaciens , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión/efectos de los fármacos , Cadmio/metabolismo , Cadmio/farmacología , Cobalto/metabolismo , Cobalto/farmacología , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Níquel/metabolismo , Níquel/farmacología , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Espectrofotometría Ultravioleta , Termodinámica , Zinc/metabolismo , Dedos de Zinc
8.
Int J Mol Sci ; 21(19)2020 Sep 29.
Artículo en Inglés | MEDLINE | ID: mdl-33003385

RESUMEN

The present study provides new evidence that cationic porphyrins may be considered as tunable platforms to interfere with the structural "key code" present on the 20S proteasome α-rings and, by consequence, with its catalytic activity. Here, we describe the functional and conformational effects on the 20S proteasome induced by the cooperative binding of the tri-cationic 5-(phenyl)-10,15,20-(tri N-methyl-4-pyridyl) porphyrin (Tris-T4). Our integrated kinetic, NMR, and in silico analysis allowed us to disclose a complex effect on the 20S catalytic activity depending on substrate/porphyrin concentration. The analysis of the kinetic data shows that Tris-T4 shifts the relative populations of the multiple interconverting 20S proteasome conformations leading to an increase in substrate hydrolysis by an allosteric pathway. Based on our Tris-T4/h20S interaction model, Tris-T4 is able to affect gating dynamics and substrate hydrolysis by binding to an array of negatively charged and hydrophobic residues present on the protein surface involved in the 20S molecular activation by the regulatory proteins (RPs). Accordingly, despite the fact that Tris-T4 also binds to the α3ΔN mutant, allosteric modulation is not observed since the molecular mechanism connecting gate dynamics with substrate hydrolysis is impaired. We envisage that the dynamic view of the 20S conformational equilibria, activated through cooperative Tris-T4 binding, may work as a simplified model for a better understanding of the intricate network of 20S conformational/functional states that may be mobilized by exogenous ligands, paving the way for the development of a new generation of proteasome allosteric modulators.


Asunto(s)
Regulación Alostérica/genética , Cationes/metabolismo , Porfirinas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Catálisis , Cationes/farmacología , Citoplasma/genética , Humanos , Cinética , Resonancia Magnética Nuclear Biomolecular , Porfirinas/farmacología , Complejo de la Endopetidasa Proteasomal/genética , Unión Proteica/efectos de los fármacos
9.
Molecules ; 25(18)2020 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-32961684

RESUMEN

Recently, the research community has become increasingly concerned with the receptor αvß5, a member of the well-known integrin family. Different ongoing studies have evidenced that αvß5 integrin regulates not only physiological processes but also a wide array of pathological events, suggesting the receptor as a valuable biomarker to specifically target for therapeutic/diagnostic purposes. Remarkably, in some tumors the involvement of the receptor in cell proliferation, tumor dissemination and angiogenesis is well-documented. In this scenario, the availability of a selective αvß5 antagonist without 'off-target' protein effects may improve survival rate in patients with highly aggressive tumors, such as hepatocellular carcinoma. We recently reported a cyclic peptide, RGDechi15D, obtained by structure-activity studies. To our knowledge it represents the first peptide-based molecule reported in the literature able to specifically bind αvß5 integrin and not cross react with αvß3. Here we demonstrated the ability of the peptide to diminish both adhesion and invasion of HepG2 cells, an in vitro model system for hepatocellular carcinoma, to reduce the cell proliferation through an apoptotic process, and to interfere with the PI3K pathway. The peptide, also decreases the formation of new vessels in endothelial cells. Taken together these results indicate that the peptide can be considered a promising molecule with properties suited to be assessed in the future for its validation as a selective therapeutic/diagnostic weapon in hepatocarcinoma.


Asunto(s)
Péptidos/metabolismo , Receptores de Vitronectina/metabolismo , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Hep G2 , Humanos , Oligopéptidos/química , Péptidos/química , Péptidos/farmacología , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Vitronectina/antagonistas & inhibidores
10.
Inorg Chem ; 58(2): 1067-1080, 2019 Jan 22.
Artículo en Inglés | MEDLINE | ID: mdl-30596504

RESUMEN

Zinc ion binding is a principal event in the achievement of the correct fold in classical zinc finger domains since the motif is largely unfolded in the absence of metal. In the case of a prokaryotic zinc finger, the larger ßßßαα domain contributes to the folding mechanism with a larger hydrophobic core. For these reasons, following the great amount of attention devoted to unveiling the effect of xenobiotic metal ion replacement in zinc fingers and in zinc-containing proteins in general, the prokaryotic zinc finger domain appears to be an interesting model for studying metal ion interaction with metalloproteins. Here, we explore the binding of Ni(II), Hg(II), and Pb(II) to Ros87, the DNA binding domain of the prokaryotic zinc finger protein Ros. We measured Ros87-metal ion dissociation constants and monitored the effects on the structure and function of the domain. Interestingly, we found that the protein folds in the presence of Ni(II) with important structural perturbations, while in the presence of Pb(II) and Hg(II) it does not appear to be significantly folded. Accordingly, an overall strong reduction in the DNA binding capability is observed for all of the examined proteins. Our data integrate and complement the information collected in the past few years concerning the functional and structural effects of metal ion substitution in classical zinc fingers in order to contribute to a better comprehension of the toxicity of these metals in biological systems.


Asunto(s)
Plomo/química , Mercurio/química , Metaloproteínas/química , Níquel/química , Sitios de Unión , Modelos Moleculares , Dedos de Zinc
11.
J Pept Sci ; 25(5): e3166, 2019 May.
Artículo en Inglés | MEDLINE | ID: mdl-30884005

RESUMEN

Integrins are heterodimeric cell-surface proteins that play important roles during developmental and pathological processes. Diverse human pathologies involve integrin adhesion including thrombotic diseases, inflammation, tumour progression, fibrosis, and infectious diseases. Although in the past decade, novel integrin-inhibitor drugs have been developed for integrin-based medical applications, the structural determinants modulating integrin-ligands recognition mechanisms are still poorly understood, reducing the number of integrin subtype exclusive antagonists. In this scenario, we have very recently showed, by means of chemical and biological assays, that a chimeric peptide (named RGDechi), containing a cyclic RGD motif linked to an echistatin C-terminal fragment, is able to interact with the components of integrin family with variable affinities, the highest for αv ß3. Here, in order to understand the mechanistic details driving the molecular recognition mechanism of αv ß3 by RGDechi, we have performed a detailed structural and dynamics characterization of the free peptide by natural abundance nuclear magnetic resonance (NMR) spectroscopy. Our data indicate that RGDechi presents in solution an heterogeneous conformational ensemble characterized by a more constrained and rigid pentacyclic ring and a largely unstructured acyclic region. Moreover, we propose that the molecular recognition of αv ß3 integrin by RGDechi occurs by a combination of conformational selection and induced fit mechanisms. Finally, our study indicates that a detailed NMR characterization, by means of natural abundance 15 N and 13 C, of a mostly unstructured bioactive peptide may provide the molecular basis to get essential structural insights into the binding mechanism to the biological partner.


Asunto(s)
Oligopéptidos/química , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Temperatura
12.
Brain ; 141(5): 1300-1319, 2018 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-29490009

RESUMEN

Genetic modifications during development of paediatric groups 3 and 4 medulloblastoma are responsible for their highly metastatic properties and poor patient survival rates. PRUNE1 is highly expressed in metastatic medulloblastoma group 3, which is characterized by TGF-ß signalling activation, c-MYC amplification, and OTX2 expression. We describe the process of activation of the PRUNE1 signalling pathway that includes its binding to NME1, TGF-ß activation, OTX2 upregulation, SNAIL (SNAI1) upregulation, and PTEN inhibition. The newly identified small molecule pyrimido-pyrimidine derivative AA7.1 enhances PRUNE1 degradation, inhibits this activation network, and augments PTEN expression. Both AA7.1 and a competitive permeable peptide that impairs PRUNE1/NME1 complex formation, impair tumour growth and metastatic dissemination in orthotopic xenograft models with a metastatic medulloblastoma group 3 cell line (D425-Med cells). Using whole exome sequencing technology in metastatic medulloblastoma primary tumour cells, we also define 23 common 'non-synonymous homozygous' deleterious gene variants as part of the protein molecular network of relevance for metastatic processes. This PRUNE1/TGF-ß/OTX2/PTEN axis, together with the medulloblastoma-driver mutations, is of relevance for future rational and targeted therapies for metastatic medulloblastoma group 3.10.1093/brain/awy039_video1awy039media15742053534001.


Asunto(s)
Proteínas Portadoras/metabolismo , Neoplasias Cerebelosas/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Meduloblastoma/metabolismo , Metástasis de la Neoplasia/fisiopatología , Fosfohidrolasa PTEN/metabolismo , Adolescente , Animales , Proteínas Portadoras/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Neoplasias Cerebelosas/patología , Niño , Preescolar , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes , Humanos , Lactante , Masculino , Meduloblastoma/patología , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Metástasis de la Neoplasia/genética , Fosfohidrolasa PTEN/genética , Monoéster Fosfórico Hidrolasas , Pirimidinonas/química , Pirimidinonas/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factores de Transcripción de la Familia Snail/metabolismo , Factor de Crecimiento Transformador beta/metabolismo
13.
Biochem J ; 475(14): 2377-2393, 2018 07 31.
Artículo en Inglés | MEDLINE | ID: mdl-29891613

RESUMEN

The complex formation between the proteins apoptosis-inducing factor (AIF) and cyclophilin A (CypA) following oxidative stress in neuronal cells has been suggested as a main target for reverting ischemia-stroke damage. Recently, a peptide encompassing AIF residues 370-394 has been developed to target the AIF-binding site on CypA, to prevent the association between the two proteins and suppress glutamate-induced cell death in neuronal cells. Using a combined approach based on NMR spectroscopy, synthesis and in vitro testing of all Ala-scan mutants of the peptide and molecular docking/molecular dynamics, we have generated a detailed model of the AIF (370-394)/CypA complex. The model suggests us that the central region of the peptide spanning residues V374-K384 mostly interacts with the protein and that for efficient complex inhibition and preservation of CypA activity, it is bent around amino acids F46-G75 of the protein. The model is consistent with experimental data also from previous works and supports the concept that the peptide does not interfere with other CypA activities unrelated to AIF activation; therefore, it may serve as an ideal template for generating future non-peptidic antagonists.


Asunto(s)
Factor Inductor de la Apoptosis/química , Ciclofilina A/química , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Péptidos/química , Humanos , Espectroscopía de Resonancia Magnética
14.
Chemistry ; 24(44): 11461-11466, 2018 Aug 06.
Artículo en Inglés | MEDLINE | ID: mdl-29799174

RESUMEN

QK peptide is a vascular endothelial growth factor (VEGF)-mimetic molecule with significant proangiogenic activity. In particular, QK is able to bind and activate VEGF receptors (VEGFRs) to stimulate a functional response in endothelial cells. To characterize the peptide bioactivity and its molecular recognition properties, a detailed picture of the interaction between peptide QK and VEGF receptors is reported. By combining NMR spectroscopy studies in solution on the purified receptor and in the presence of intact endothelial cells, a molecular description of the binding interaction between peptide QK and VEGFR2 in the cellular context is obtained. These results reveal useful insights into the peptide biological mechanism, which opens the way to further optimization of this class of VEGF-mimicking peptides.


Asunto(s)
Materiales Biomiméticos/química , Péptidos/química , Receptores de Factores de Crecimiento Endotelial Vascular/química , Factor A de Crecimiento Endotelial Vascular/química , Células Endoteliales , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Unión Proteica , Conformación Proteica
15.
Org Biomol Chem ; 16(5): 787-795, 2018 01 31.
Artículo en Inglés | MEDLINE | ID: mdl-29319097

RESUMEN

Molecular tools to stabilize the ß-hairpin conformation are needed as ß-hairpin peptides are useful molecules for pharmaceutical, biological and materials applications. We explored the use of a "triazole bridge", a covalent link between two ß-hairpin strands obtained through Cu-catalyzed alkyne-azide cycloaddition, combined with an aromatic-aromatic interaction. Highly conformationally stable peptides were identified by NMR screening of a small collection of cyclic peptides based on the Trpzip2 scaffold. The characteristic Trp-Trp interaction of Trpzip2 was replaced by a diagonal triazole bridge of variable length. NMR and CD analyses showed that triazole and indole rings could favorably interact to stabilize a ß-hairpin conformation. The conformational stabilization depends on the length of the triazole bridge and the reciprocal position between the aromatic rings. Combining aromatic interactions and the covalent inter-strand triazole bridge is a useful strategy to obtain peptides with a high ß-hairpin content.


Asunto(s)
Péptidos/química , Triazoles/química , Triptófano/química , Secuencia de Aminoácidos , Catálisis , Cobre/química , Reacción de Cicloadición , Péptidos/síntesis química , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/química , Conformación Proteica en Lámina beta , Estabilidad Proteica , Termodinámica , Triazoles/síntesis química , Triptófano/síntesis química
16.
Biochim Biophys Acta Gen Subj ; 1861(9): 2155-2164, 2017 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-28625421

RESUMEN

BACKGROUND: The peptide VLL-28, identified in the sequence of an archaeal protein, the transcription factor Stf76 from Sulfolobus islandicus, was previously identified and characterized as an antimicrobial peptide, possessing a broad-spectrum antibacterial activity. METHODS: Through a combined approach of NMR and Circular Dichroism spectroscopy, Dynamic Light Scattering, confocal microscopy and cell viability assays, the interaction of VLL-28 with the membranes of both parental and malignant cell lines has been characterized and peptide mechanism of action has been studied. RESULTS: It is here demonstrated that VLL-28 selectively exerts cytotoxic activity against murine and human tumor cells. By means of structural methodologies, VLL-28 interaction with the membranes has been proven and the binding residues have been identified. Confocal microscopy data show that VLL-28 is internalized only into tumor cells. Finally, it is shown that cell death is mainly caused by a time-dependent activation of apoptotic pathways. CONCLUSIONS: VLL-28, deriving from the archaeal kingdom, is here found to be endowed with selective cytotoxic activity towards both murine and human cancer cells and consequently can be classified as an ACP. GENERAL SIGNIFICANCE: VLL-28 represents the first ACP identified in an archaeal microorganism, exerting a trans-kingdom activity.


Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Antineoplásicos/farmacología , Sulfolobus/química , Animales , Péptidos Catiónicos Antimicrobianos/química , Antineoplásicos/química , Células 3T3 BALB , Muerte Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Dicroismo Circular , Humanos , Ratones , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica
17.
Chemistry ; 22(49): 17767-17775, 2016 Dec 05.
Artículo en Inglés | MEDLINE | ID: mdl-27759905

RESUMEN

Many biochemical pathways involving nerve growth factor (NGF), a neurotrophin with copper(II) binding abilities, are regulated by the ubiquitin (Ub) proteasome system. However, whether NGF binds Ub and the role played by copper(II) ions in modulating their interactions have not yet been investigated. Herein NMR spectroscopy, circular dichroism, ESI-MS, and titration calorimetry are employed to characterize the interactions of NGF with Ub. NGF1-14 , which is a short model peptide encompassing the first 14 N-terminal residues of NGF, binds the copper-binding regions of Ub (KD =8.6 10-5 m). Moreover, the peptide undergoes a random coil-polyproline type II helix structural conversion upon binding to Ub. Notably, copper(II) ions inhibit NGF1-14 /Ub interactions. Further experiments performed with the full-length NGF confirmed the existence of a copper(II)-dependent association between Ub and NGF and indicated that the N-terminal domain of NGF was a valuable paradigm that recapitulated many traits of the full-length protein.


Asunto(s)
Cobre/química , Factor de Crecimiento Nervioso/química , Péptidos/química , Ubiquitina/química , Dicroismo Circular , Humanos , Iones , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Unión Proteica
18.
Chemistry ; 22(2): 681-93, 2016 Jan 11.
Artículo en Inglés | MEDLINE | ID: mdl-26548575

RESUMEN

The critical role of integrins in tumor progression and metastasis has stimulated intense efforts to identify pharmacological agents that can modulate integrin function. In recent years, αv ß3 and αv ß5 integrin antagonists were demonstrated to be effective in blocking tumor progression. RGDechi-hCit, a chimeric peptide containing a cyclic RGD motif linked to an echistatin C-terminal fragment, is able to recognize selectively αv ß3 integrin both in vitro and in vivo. High-resolution molecular details of the selective αv ß3 recognition of the peptide are certainly required, nonetheless RGDechi-hCit internalization limited the use of classical in cell NMR experiments. To overcome such limitations, we used WM266 isolated cellular membranes to accomplish a detailed NMR interaction study that, combined with a computational analysis, provides significant structural insights into αv ß3 molecular recognition by RGDechi-hCit. Remarkably, on the basis of the identified molecular determinants, we design a RGDechi-hCit mutant that is selective for αv ß5 integrin.


Asunto(s)
Membrana Celular/química , Integrina alfaVbeta3/química , Espectroscopía de Resonancia Magnética , Oligopéptidos/química , Péptidos/química , Receptores de Vitronectina/química , Membrana Celular/metabolismo , Computadores Moleculares , Integrina alfaVbeta3/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Ligandos , Péptidos/metabolismo , Receptores de Vitronectina/metabolismo
19.
Nucleic Acids Res ; 42(9): 5993-6011, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24682827

RESUMEN

The hybrid plasmid-virus pSSVx from Sulfolobus islandicus presents an open reading frame encoding a 76 amino acid protein, namely Stf76, that does not show significant sequence homology with any protein with known 3D structure. The recombinant protein recognizes specifically two DNA-binding sites located in its own promoter, thus suggesting an auto-regulated role of its expression. Circular dichroism, spectrofluorimetric, light scattering and isothermal titration calorimetry experiments indicated a 2:1 molar ratio (protein:DNA) upon binding to the DNA target containing a single site. Furthermore, the solution structure of Stf76, determined by nuclear magnetic resonance (NMR) using chemical shift Rosetta software, has shown that the protein assumes a winged helix-turn-helix fold. NMR chemical shift perturbation analysis has been performed for the identification of the residues responsible for DNA interaction. In addition, a model of the Stf76-DNA complex has been built using as template a structurally related homolog.


Asunto(s)
Fuselloviridae/química , Proteínas Virales/química , Factores de Transcripción Winged-Helix/química , Secuencia de Aminoácidos , Secuencia de Bases , Dicroismo Circular , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Soluciones , Sulfolobus/virología
20.
Molecules ; 21(12)2016 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-27916966

RESUMEN

Cyclodextrins are natural macrocyclic oligosaccharides able to form inclusion complexes with a wide variety of guests, affecting their physicochemical and pharmaceutical properties. In order to obtain an improvement of the bioavailability and solubility of 5-fluorouracil, a pyrimidine analogue used as chemotherapeutic agent in the treatment of the colon, liver, and stomac cancers, the drug was complexed with alpha- and beta-cyclodextrin. The inclusion complexes were prepared in the solid state by kneading method and characterized by Fourier transform-infrared (FT-IR) spectroscopy and X-ray powder diffractometry. In solution, the 1:1 stoichiometry for all the inclusion complexes was established by the Job plot method and the binding constants were determined at different pHs by UV-VIS titration. Furthermore, the cytotoxic activity of 5-fluorouracil and its complexation products were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on MCF-7 (breast cancer cell line), Hep G2 (hepatocyte carcinoma cell line), Caco-2 (colon adenocarcinoma cell line), and A-549 (alveolar basal epithelial carcinoma cell line). The results showed that both inclusion complexes increased the 5-fluorouracil capability of inhibiting cell growth. In particular, 5-fluorouracil complexed with beta-cyclodextrin had the highest cytotoxic activity on MCF-7; with alpha-cyclodextrin the highest cytotoxic activity was observed on A-549. The IC50 values were equal to 31 and 73 µM at 72 h, respectively. Our results underline the possibility of using these inclusion complexes in pharmaceutical formulations for improving 5-fluorouracil therapeutic efficacy.


Asunto(s)
Fluorouracilo , Neoplasias/tratamiento farmacológico , alfa-Ciclodextrinas , beta-Ciclodextrinas , Células CACO-2 , Fluorouracilo/química , Fluorouracilo/farmacología , Células Hep G2 , Humanos , Células MCF-7 , alfa-Ciclodextrinas/química , alfa-Ciclodextrinas/farmacología , beta-Ciclodextrinas/química , beta-Ciclodextrinas/farmacología
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