RESUMEN
The possible link between hIAPP accumulation and ß-cell death in diabetic patients has inspired numerous studies focusing on amyloid structures and aggregation pathways of this hormone. Recent studies have reported on the importance of early oligomeric intermediates, the many roles of their interactions with lipid membrane, pH, insulin, and zinc on the mechanism of aggregation of hIAPP. The challenges posed by the transient nature of amyloid oligomers, their structural heterogeneity, and the complex nature of their interaction with lipid membranes have resulted in the development of a wide range of biophysical and chemical approaches to characterize the aggregation process. While the cellular processes and factors activating hIAPP-mediated cytotoxicity are still not clear, it has recently been suggested that its impaired turnover and cellular processing by proteasome and autophagy may contribute significantly toward toxic hIAPP accumulation and, eventually, ß-cell death. Therefore, studies focusing on the restoration of hIAPP proteostasis may represent a promising arena for the design of effective therapies. In this review we discuss the current knowledge of the structures and pathology associated with hIAPP self-assembly and point out the opportunities for therapy that a detailed biochemical, biophysical, and cellular understanding of its aggregation may unveil.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Proteostasis , Humanos , Polipéptido Amiloide de los Islotes Pancreáticos/química , Factores de RiesgoRESUMEN
Dipyridamole is currently used as a medication that inhibits blood clot formation and it is also investigated in the context of neurodegenerative and other amyloid related diseases. Here, we propose this molecule as a new diagnostic tool to follow the aggregation properties of three different amyloidogenic proteins tested (insulin, amylin and amyloid ß peptide 1-40). Results show that dipyridamole is sensitive to early stage amyloid formation undetected by thioflavin T, giving a different response for the aggregation of the three different proteins. In addition, we show that dipyridamole is also able to enhance ubiquitin chain growth, paving the way to its potential application as therapeutic agent in neurodegenerative diseases.
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Péptidos beta-Amiloides , Proteínas Amiloidogénicas , Amiloide , Dipiridamol , Polipéptido Amiloide de los Islotes PancreáticosRESUMEN
A reduced proteasome activity tiles excessive amyloid growth during the progress of protein conformational diseases (PCDs). Hence, the development of safe and effective proteasome enhancers represents an attractive target for the therapeutic treatment of these chronic disorders. Here we analyze two natural diastereoisomers belonging to the family of flavonolignans, Sil A and Sil B, by evaluating their capacity to increase proteasome activity. Enzyme assays carried out on yeast 20S (y20S) proteasome and in parallel on a permanently "open gate" mutant (α3ΔN) evidenced that Sil B is a more efficient 20S activator than Sil A. Conversely, in the case of human 20S proteasome (h20S) a higher affinity and more efficient activation is observed for Sil A. Driven by experimental data, computational studies further demonstrated that the taxifolin group of both diastereoisomers plays a crucial role in their anchoring to the α5/α6 groove of the outer α-ring. However, due to the different stereochemistry at C-7" and C-8" of ring D, only Sil A was able to reproduce the interactions responsible for h20S proteasome activation induced by their cognate regulatory particles. The provided silybins/h20S interaction models allowed us to rationalize their different ability to activate the peptidase activities of h20S and y20S. Our results provide structural details concerning the important role played by stereospecific interactions in driving Sil A and Sil B binding to the 20S proteasome and may support future rational design of proteasome enhancers.
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Complejo de la Endopetidasa Proteasomal , Saccharomyces cerevisiae , Citoplasma/metabolismo , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Conformación Proteica , SilibinaRESUMEN
In the last two decades, the amyloid hypothesis, i.e., the abnormal accumulation of toxic Aß assemblies in the brain, has been considered the mainstream concept sustaining research in Alzheimer's Disease (AD). However, the course of cognitive decline and AD development better correlates with tau accumulation rather than amyloid peptide deposition. Moreover, all clinical trials of amyloid-targeting drug candidates have been unsuccessful, implicitly suggesting that the amyloid hypothesis needs significant amendments. Accumulating evidence supports the existence of a series of potentially dangerous relationships between Aß oligomeric species and tau protein in AD. However, the molecular determinants underlying pathogenic Aß/tau cross interactions are not fully understood. Here, we discuss the common features of Aß and tau molecules, with special emphasis on: (i) the critical role played by metal dyshomeostasis in promoting both Aß and tau aggregation and oxidative stress, in AD; (ii) the effects of lipid membranes on Aß and tau (co)-aggregation at the membrane interface; (iii) the potential of small peptide-based inhibitors of Aß and tau misfolding as therapeutic tools in AD. Although the molecular mechanism underlying the direct Aß/tau interaction remains largely unknown, the arguments discussed in this review may help reinforcing the current view of a synergistic Aß/tau molecular crosstalk in AD and stimulate further research to mechanism elucidation and next-generation AD therapeutics.
Asunto(s)
Enfermedad de Alzheimer , Amiloidosis , Enfermedad de Alzheimer/metabolismo , Amiloide , Péptidos beta-Amiloides/metabolismo , Proteínas Amiloidogénicas , Humanos , Iones , Lípidos/uso terapéutico , Metales , Proteínas tau/metabolismoRESUMEN
Prodrugs are ingenious derivatives of therapeutic agents designed to improve the pharmacokinetic profile of the drug. Here, we report an efficient and regioselective solid phase approach for obtaining new prodrugs of 9â³-silybins conjugated with 3'-ribonucleotide units (uridine and adenosine) as pro-moieties. Uridine and adenosine conjugates were obtained in good yields (41-50%), beginning with silibinin and its diastereomers (silybin A and silybin B), using a NovaSyn® support functionalized with an ad hoc linker, which allowed selective detachment of only the desired products. As expected, the solubility of both uridine and adenosine conjugates was higher than that of the parental natural product (5 mg/mL and 3 mg/mL for uridine and adenosine, respectively). Our investigations revealed that uridine conjugates were quickly cleaved by RNase A, releasing silybin drugs, even at low enzyme concentrations. No toxic effects were found for any ribonucleotide conjugate on differentiated neuroblastoma SH-SY5Y cells when tested at increasing concentrations. All results strongly encourage further investigations of uridine-silybin prodrugs as potential therapeutic agents for both oral and intravenous administration. The present synthetic approach represents a valuable strategy to the future design of new prodrugs with modified nucleoside pro-moieties to modulate the pharmacokinetics of silybins or different natural products with strong pharmacological activities but poor bioavailability.
Asunto(s)
Adenosina/química , Profármacos/síntesis química , Silibina/síntesis química , Técnicas de Síntesis en Fase Sólida , Uridina/química , Humanos , Estructura Molecular , Profármacos/química , Silibina/química , Solubilidad , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
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.
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Péptidos/química , Factores de Crecimiento Endotelial Vascular/química , Interacciones Hidrofóbicas e Hidrofílicas , Conformación ProteicaRESUMEN
Protein misfolding and subsequent self-association are complex, intertwined processes, resulting in development of a heterogeneous population of aggregates closely related to many chronic pathological conditions including Type 2 Diabetes Mellitus and Alzheimer's disease. To address this issue, here, we develop a theoretical model in the general framework of linear stability analysis. According to this model, self-assemblies of peptides with pronounced conformational flexibility may become, under particular conditions, unstable and spontaneously evolve toward an alternating array of partially ordered and disordered monomers. The predictions of the theory were verified by atomistic molecular dynamics (MD) simulations of islet amyloid polypeptide (IAPP) used as a paradigm of aggregation-prone polypeptides (proteins). Simulations of dimeric, tetrameric, and hexameric human-IAPP self-assemblies at physiological electrolyte concentration reveal an alternating distribution of the smallest domains (of the order of the peptide mean length) formed by partially ordered (mainly ß-strands) and disordered (turns and coil) arrays. Periodicity disappears upon weakening of the inter-peptide binding, a result in line with the predictions of the theory. To further probe the general validity of our hypothesis, we extended the simulations to other peptides, the Aß(1-40) amyloid peptide, and the ovine prion peptide as well as to other proteins (SOD1 dimer) that do not belong to the broad class of intrinsically disordered proteins. In all cases, the oligomeric aggregates show an alternate distribution of partially ordered and disordered monomers. We also carried out Surface Enhanced Raman Scattering (SERS) measurements of hIAPP as an experimental validation of both the theory and in silico simulations.
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Polipéptido Amiloide de los Islotes Pancreáticos/química , Desnaturalización Proteica , Pliegue de Proteína , Coloides/química , Simulación por Computador , Electrólitos , Humanos , Cinética , Modelos Teóricos , Simulación de Dinámica Molecular , Péptidos/química , Multimerización de Proteína , Estructura Secundaria de Proteína , Reproducibilidad de los Resultados , Solventes , Espectrometría Raman , TermodinámicaRESUMEN
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 ZincRESUMEN
Copper plays an important role as a regulator in many pathologies involving the angiogenesis process. In cancerogenesis, tumor progression, and angiogenic diseases, copper homeostasis is altered. Although many details in the pathways involved are still unknown, some copper-specific ligands have been successfully used as therapeutic agents. Copper-binding peptides able to modulate angiogenesis represent a possible way to value new drugs. We previously reported that a fragment (VEGF73-101) of vascular endothelial growth factor (VEGF165), a potent angiogenic, induced an apoptotic effect on human umbilical vein endothelial cells. The aim of this study was to investigate the putative copper ionophoric activity of VEGF73-101, as well as establish a relationship between the structure of the peptide fragment and the cytotoxic activity in the presence of copper(II) ions. Here, we studied the stoichiometry and the conformation of the VEGF73-101/Cu(II) complexes and some of its mutated peptides by electrospray ionization mass spectrometry and circular dichroism spectroscopy. Furthermore, we evaluated the effect of all peptides in the absence and presence of copper ions by cell viability and cytofuorimetric assays. The obtained results suggest that VEGF73-101 could be considered an interesting candidate in the development of new molecules with ionophoric properties as agents in antiangiogenic therapeutic approaches.
Asunto(s)
Apoptosis , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Secuencia de Aminoácidos , Apoptosis/efectos de los fármacos , Apoptosis/genética , Permeabilidad de la Membrana Celular , Supervivencia Celular/efectos de los fármacos , Quelantes/farmacología , Cobre/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Neovascularización Fisiológica/efectos de los fármacos , Neovascularización Fisiológica/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Unión Proteica , Conformación Proteica , Espectrometría de Masa por Ionización de Electrospray , Análisis Espectral , Relación Estructura-Actividad , Factor A de Crecimiento Endotelial Vascular/química , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
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.
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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 fármacosRESUMEN
Insulin-degrading enzyme (IDE) is a ubiquitous zinc peptidase of the inverzincin family, which has been initially discovered as the enzyme responsible for insulin catabolism; therefore, its involvement in the onset of diabetes has been largely investigated. However, further studies on IDE unraveled its ability to degrade several other polypeptides, such as ß-amyloid, amylin, and glucagon, envisaging the possible implication of IDE dys-regulation in the "aggregopathies" and, in particular, in neurodegenerative diseases. Over the last decade, a novel scenario on IDE biology has emerged, pointing out a multi-functional role of this enzyme in several basic cellular processes. In particular, latest advances indicate that IDE behaves as a heat shock protein and modulates the ubiquitin-proteasome system, suggesting a major implication in proteins turnover and cell homeostasis. In addition, recent observations have highlighted that the regulation of glucose metabolism by IDE is not merely based on its largely proposed role in the degradation of insulin in vivo. There is increasing evidence that improper IDE function, regulation, or trafficking might contribute to the etiology of metabolic diseases. In addition, the enzymatic activity of IDE is affected by metals levels, thus suggesting a role also in the metal homeostasis (metallostasis), which is thought to be tightly linked to the malfunction of the "quality control" machinery of the cell. Focusing on the physiological role of IDE, we will address a comprehensive vision of the very complex scenario in which IDE takes part, outlining its crucial role in interconnecting several relevant cellular processes.
Asunto(s)
Insulisina/metabolismo , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/patología , Animales , Diabetes Mellitus Tipo 2/enzimología , Diabetes Mellitus Tipo 2/patología , Humanos , Insulisina/fisiología , Agregación Patológica de Proteínas/enzimología , Agregación Patológica de Proteínas/patología , Conformación ProteicaRESUMEN
The interaction of insulin-degrading enzyme (IDE) with the main intracellular proteasome assemblies (i.e, 30S, 26S and 20S) was analyzed by enzymatic activity, mass spectrometry and native gel electrophoresis. IDE was mainly detected in association with assemblies with at least one free 20S end and biochemical investigations suggest that IDE competes with the 19S in vitro. IDE directly binds the 20S and affects its proteolytic activities in a bimodal fashion, very similar in human and yeast 20S, inhibiting at (IDE) ≤ 30 nM and activating at (IDE) ≥ 30 nM. Only an activating effect is observed in a yeast mutant locked in the "open" conformation (i.e., the α-3ΔN 20S), envisaging a possible role of IDE as modulator of the 20S "open"-"closed" allosteric equilibrium. Protein-protein docking in silico proposes that the interaction between IDE and the 20S could involve the C-term helix of the 20S α-3 subunit which regulates the gate opening of the 20S.
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Insulisina/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Regulación Alostérica , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Células HEK293 , Humanos , Insulisina/química , Cinética , Simulación del Acoplamiento Molecular , Electroforesis en Gel de Poliacrilamida Nativa , Complejo de la Endopetidasa Proteasomal/química , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Espectrometría de Masas en Tándem , Levaduras/metabolismoRESUMEN
Our knowledge of the molecular events underlying type 2 diabetes mellitus-a protein conformational disease characterized by the aggregation of islet amyloid polypeptide (IAPP) in pancreatic ß cells-is limited. However, amyloid-mediated membrane damage is known to play a key role in IAPP cytotoxicity, and therefore the effects of lipid composition on modulating IAPP-membrane interactions have been the focus of intense research. In particular, membrane cholesterol content varies with aging and consequently with adverse environmental factors such as diet and lifestyle, but its role in the development of the disease is controversial. In this study, we employ a combination of experimental techniques and in silico molecular simulations to shed light on the role of cholesterol in IAPP aggregation and the related membrane disruption. We show that if anionic POPC/POPS vesicles are used as model membranes, cholesterol has a negligible effect on the kinetics of IAPP fibril growth on the surface of the bilayer. In addition, cholesterol inhibits membrane damage by amyloid-induced poration on membranes, but enhances leakage through fiber growth on the membrane surface. Conversely, if 1:2 DOPC/DPPC raft-like model membranes are used, cholesterol accelerates fiber growth. Next, it enhances pore formation and suppresses fiber growth on the membrane surface, leading to leakage. Our results highlight a twofold effect of cholesterol on the amyloidogenicity of IAPP and help explain its debated role in type 2 diabetes mellitus.
Asunto(s)
Membrana Celular/metabolismo , Colesterol/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Humanos , Polipéptido Amiloide de los Islotes Pancreáticos/química , Cinética , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Microdominios de Membrana/química , Microdominios de Membrana/metabolismo , Simulación de Dinámica Molecular , Fosfatidilcolinas/metabolismo , Porosidad , Unión Proteica , Estructura Secundaria de Proteína , Propiedades de SuperficieRESUMEN
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 typeâ 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.
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Cobre/química , Factor de Crecimiento Nervioso/química , Péptidos/química , Ubiquitina/química , Dicroismo Circular , Humanos , Iones , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Unión ProteicaRESUMEN
Imidazoline ligands in I2-type binding sites in the brain alter monoamine turnover and release. One example of an I2 binding site characterized by binding studies, kinetics, and crystal structure has been described in monoamine oxidase B (MAO B). MAO A also binds imidazolines but has a different active site structure. Docking and molecular dynamics were used to explore how 2-(2-benzofuranyl)-2-imidazoline hydrochloride (2-BFI) binds to MAO A and to explain why tranylcypromine increases tight binding to MAO B. The energy for 2-BFI binding to MAO A was comparable to that for tranylcypromine-modified MAO B, but the location of 2-BFI in the MAO A could be anywhere in the monopartite substrate cavity. Binding to the tranylcypromine-modified MAO B was with high affinity and in the entrance cavity as in the crystal structure, but the energies of interaction with the native MAO B were less favorable. Molecular dynamics revealed that the entrance cavity of MAO B after tranylcypromine modification is both smaller and less flexible. This change in the presence of tranylcypromine may be responsible for the greater affinity of tranylcypromine-modified MAO B for imidazoline ligands.
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Imidazolinas/metabolismo , Monoaminooxidasa/metabolismo , Sitios de Unión , Humanos , Imidazolinas/química , Simulación de Dinámica MolecularRESUMEN
Novel conjugated G-quadruplex-forming d(TG3AG) oligonucleotides, linked to hydrophobic groups through phosphodiester bonds at 5'-end, have been synthesized as potential anti-HIV aptamers, via a fully automated, online phosphoramidite-based solid-phase strategy. Conjugated quadruplexes showed pronounced anti-HIV activity with some preference for HIV-1, with inhibitory activity invariably in the low micromolar range. The CD and DSC monitored thermal denaturation studies on the resulting quadruplexes, indicated the insertion of lipophilic residue at the 5'-end, conferring always improved stability to the quadruplex complex (20<ΔTm<40°C). The data suggest no direct functional relationship between the thermal stability and anti-HIV activity of the folded conjugated G-quartets. It would appear that the nature of the residue at 5' end of the d(TG3AG) quadruplexes plays an important role in the thermodynamic stabilization but a minor influence on the anti-HIV activity. Moreover, a detailed CD and DSC analyses indicate a monophasic behaviour for sequences I and V, while for ODNs (II-IV) clearly show that these quadruplex structures deviate from simple two-state melting, supporting the hypothesis that intermediate states along the dissociation pathway may exist.
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Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , G-Cuádruplex , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/metabolismo , Aptámeros de Nucleótidos/química , Rastreo Diferencial de Calorimetría , Células Cultivadas/virología , Dicroismo Circular , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Proteína gp120 de Envoltorio del VIH/metabolismo , Proteína gp41 de Envoltorio del VIH/metabolismo , Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH-1/efectos de los fármacos , VIH-1/patogenicidad , VIH-2/efectos de los fármacos , VIH-2/patogenicidad , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Oligonucleótidos/química , Oligonucleótidos/farmacología , Albúmina Sérica/metabolismo , Técnicas de Síntesis en Fase Sólida , Relación Estructura-Actividad , Resonancia por Plasmón de Superficie , TermodinámicaRESUMEN
We investigate the therapeutic potential of Aloin A and Aloin B, two natural compounds derived from Aloe vera leaves, focusing on their neuroprotective and anticancer properties. The structural differences between these two epimers suggest that they may exhibit distinct pharmacological properties. Our investigations revealed that both epimers are not stable in aqueous solution and tend to degrade rapidly, with their concentration decreasing by over 50% within approximately 12 h. These results underscore the importance of addressing issues such as the need for encapsulation into effective drug delivery systems to enhance stability. ThT fluorescence experiments showed that neither compound was able to inhibit Aß amyloid aggregation, indicating that other mechanisms may be responsible for their neuroprotective effects. Next, an equimolar mixture of Aloin A and Aloin B demonstrated an ability to inhibit proteasome in tube tests, which is suggestive of potential anticancer properties, in accordance with antiproliferative effects observed in neuroblastoma SH-SY5Y and HeLa cell lines. Higher water stability and increased antiproliferative activity were observed by encapsulation in carbon dot nanoparticles, suggesting a promising potential for further in vivo studies.
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Emodina , Fármacos Neuroprotectores , Humanos , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/química , Emodina/farmacología , Emodina/análogos & derivados , Emodina/química , Células HeLa , Línea Celular Tumoral , Estabilidad de Medicamentos , Antineoplásicos/farmacología , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Péptidos beta-Amiloides/metabolismo , Nanopartículas/química , Aloe/química , Complejo de la Endopetidasa Proteasomal/metabolismoRESUMEN
Disruption of the integrity of the plasma membrane by amyloidogenic proteins is linked to the pathogenesis of a number of common age-related diseases. Although accumulating evidence suggests that adverse environmental stressors such as unbalanced levels of metal ions may trigger amyloid-mediated membrane damage, many features of the molecular mechanisms underlying these events are unknown. Using human islet amyloid polypeptide (hIAPP, aka amylin), an amyloidogenic peptide associated with ß-cell death in type 2 diabetes, we demonstrate that the presence of Ca(2+) ions inhibits membrane damage occurring immediately after the interaction of freshly dissolved hIAPP with the membrane, but significantly enhances fiber-dependent membrane disruption. In particular, dye leakage, quartz crystal microbalance, atomic force microscopy, and NMR experiments show that Ca(2+) ions promote a shallow membrane insertion of hIAPP, which leads to the removal of lipids from the bilayer through a detergent-like mechanism triggered by fiber growth. Because both types of membrane-damage mechanisms are common to amyloid toxicity by most amyloidogenic proteins, it is likely that unregulated ion homeostasis, amyloid aggregation, and membrane disruption are all parts of a self-perpetuating cycle that fuels amyloid cytotoxicity.
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Amiloide/metabolismo , Calcio/metabolismo , Membrana Celular/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Animales , Cationes , Colorantes , Detergentes/farmacología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas/efectos de los fármacos , Membrana Dobles de Lípidos/química , Microscopía de Fuerza Atómica , Fosfatidilcolinas/química , Fosfatidilgliceroles/química , Fosfatidilserinas/química , Tecnicas de Microbalanza del Cristal de Cuarzo , RatasRESUMEN
In the funneled landscape, proteins fold to their native states through a stochastic process in which the free energy decreases spontaneously and unfolded, transition, native, and possible intermediate states correspond to local minima or saddle points. Atomic description of the folding pathway appears therefore to be essential for a deep comprehension of the folding mechanism. In metallo-proteins, characterization of the folding pathways becomes even more complex, and therefore, despite their fundamental role in critical biological processes, little is known about their folding and assembly. The study of the mechanisms through which a cofactor influences the protein folding/unfolding reaction has been the rationale of the present study aimed at contributing to the search for cofactors' general roles in protein folding reactions. In particular, we have investigated the folding pathway of two homologous proteins, Ros87, which contains a prokaryotic zinc finger domain, and Ml452-151, lacking the zinc ion. Using a combination of CD, DSC and NMR techniques, we determined the thermodynamics and the structural features, at an atomic level, of the thermal unfolding of Ros87 and compared them to the behavior of Ml452-151. Our results, also corroborated by NMR (1)H/(2)H exchange measurements, show that the presence of the structural Zn(II) in Ros87 implies a switch from the Ml452-151 fully cooperative to a two-step unfolding process in which the intermediate converts to the native state through a downhill barrierless transition. This observation, which has never been reported for any metal ion so far, may have a significant role in the understanding of the protein misfolding associated with the presence of metal ions, as observed in neurodegenerative diseases.