RESUMO
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.
Assuntos
Peptídeos beta-Amiloides , Proteínas Amiloidogênicas , Amiloide , Dipiridamol , Polipeptídeo Amiloide das Ilhotas PancreáticasRESUMO
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.
Assuntos
Complexo de Endopeptidases do Proteassoma , Saccharomyces cerevisiae , Citoplasma/metabolismo , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Conformação Proteica , SilibinaRESUMO
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.
Assuntos
Cádmio/química , Cobalto/química , Níquel/química , Dobramento de Proteína/efeitos dos fármacos , Proteínas Repressoras , Zinco/química , Agrobacterium tumefaciens , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação/efeitos dos fármacos , Cádmio/metabolismo , Cádmio/farmacologia , Cobalto/metabolismo , Cobalto/farmacologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Níquel/metabolismo , Níquel/farmacologia , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Espectrofotometria Ultravioleta , Termodinâmica , Zinco/metabolismo , Dedos de ZincoRESUMO
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.
Assuntos
Apoptose , Células Endoteliais da Veia Umbilical Humana/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Sequência de Aminoácidos , Apoptose/efeitos dos fármacos , Apoptose/genética , Permeabilidade da Membrana Celular , Sobrevivência Celular/efeitos dos fármacos , Quelantes/farmacologia , Cobre/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Ligação Proteica , Conformação Proteica , Espectrometria de Massas por Ionização por Electrospray , Análise Espectral , Relação Estrutura-Atividade , Fator A de Crescimento do Endotélio Vascular/química , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Folding stability is a crucial feature of protein evolution and is essential for protein functions. Thus, the comprehension of protein folding mechanisms represents an important complement to protein structure and function, crucial to determine the structural basis of protein misfolding. In this context, thermal unfolding studies represent a useful tool to get a molecular description of the conformational transitions governing the folding/unfolding equilibrium of a given protein. Here, we report the thermal folding/unfolding pathway of VEGFR1D2, a member of the immunoglobulin superfamily by means of a high-resolution thermodynamic approach that combines differential scanning calorimetry with atomic-level unfolding monitored by NMR. We show how VEGFR1D2 folding is driven by an oxidatively induced disulfide pairing: the key event in the achievement of its functional structure is the formation of a small hydrophobic core that surrounds a disulfide bridge. Such a 'folding nucleus' induces the cooperative transition to the properly folded conformation supporting the hypothesis that a disulfide bond can act as a folding nucleus that eases the folding process.
Assuntos
Dobramento de Proteína , Proteínas , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Dissulfetos/química , Humanos , Desnaturação Proteica , TermodinâmicaRESUMO
Type 2 Diabetes is a major public health threat, and its prevalence is increasing worldwide. The abnormal accumulation of islet amyloid polypeptide (IAPP) in pancreatic ß-cells is associated with the onset of the disease. Therefore, the design of small molecules able to inhibit IAPP aggregation represents a promising strategy in the development of new therapies. Here we employ in vitro, biophysical, and computational methods to inspect the ability of Silybin A and Silybin B, two natural diastereoisomers extracted from milk thistle, to interfere with the toxic self-assembly of human IAPP (hIAPP). We show that Silybin B inhibits amyloid aggregation and protects INS-1 cells from hIAPP toxicity more than Silybin A. Molecular dynamics simulations revealed that the higher efficiency of Silybin B is ascribable to its interactions with precise hIAPP regions that are notoriously involved in hIAPP self-assembly i.e., the S20-S29 amyloidogenic core, H18, the N-terminal domain, and N35. These results highlight the importance of stereospecific ligand-peptide interactions in regulating amyloid aggregation and provide a blueprint for future studies aimed at designing Silybin derivatives with enhanced drug-like properties.
Assuntos
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Amiloide/química , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Silibina/farmacologiaRESUMO
n-Eicosane-melamine formaldehyde microcapsules of an average size of 1.1 µm and latent heat of fusion of 146.2 ± 5.3 J/g have been prepared. They have been characterized by scanning electron microscopy, FTIR spectroscopy, calorimetric techniques, and thermogravimetric analyses. Under processing conditions, the microcapsules apparently preserved their properties, also maintaining their n-eicosane loading and heat storage capacity under washing conditions (water with detergent at 60 °C). The microcapsules synthesis has been scaled up for the fabrication of functional films by extrusion. For that, polymer films containing 10 wt.% of microcapsules were prepared at a pilot plant level. In those films, even though a fraction of the n-eicosane loading was lost during the extrusion process, the microcapsules showed good compatibility within the polyamide. The percentage of PCM in the polyamide 6 films was estimated by TGA, verifying also the heat storage capacity predicted by DSC (2.6 ± 0.7 J/g).
RESUMO
Alzheimer's Diseases (AD) is characterized by the accumulation of amyloid deposits of Aß peptide in the brain. Besides genetic background, the presence of other diseases and an unhealthy lifestyle are known risk factors for AD development. Albeit accumulating clinical evidence suggests that an impaired lipid metabolism is related to Aß deposition, mechanistic insights on the link between amyloid fibril formation/clearance and aberrant lipid interactions are still unavailable. Recently, many studies have described the key role played by membrane bound Aß assemblies in neurotoxicity. Moreover, it has been suggested that a derangement of the ubiquitin proteasome pathway and autophagy is significantly correlated with toxic Aß aggregation and dysregulation of lipid levels. Thus, studies focusing on the role played by lipids in Aß aggregation and proteostasis could represent a promising area of investigation for the design of valuable treatments. In this review we examine current knowledge concerning the effects of lipids in Aß aggregation and degradation processes, focusing on the therapeutic opportunities that a comprehensive understanding of all biophysical, biochemical, and biological processes involved may disclose.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Lipídeos/química , Peptídeos beta-Amiloides/química , Animais , Homeostase , Humanos , Agregados Proteicos , Fatores de RiscoRESUMO
Alzheimer's disease (AD) is a multifactorial pathology that requires multifaceted agents able to address its peculiar nature. Increasing evidence has shown that aggregation of amyloid ß (Aß) and oxidative stress are strictly interconnected, and their modulation might have a positive and synergic effect in contrasting AD-related impairments. Herein, a new and efficient fragment-based approach towards tyrosol phosphodiester derivatives (TPDs) has been developed starting from suitable tyrosol building blocks and exploiting the well-established phosphoramidite chemistry. The antioxidant activity of new TPDs has been tested as well as their ability to inhibit Aß protein aggregation. In addition, their metal chelating ability has been evaluated as a possible strategy to develop new natural-based entities for the prevention or therapy of AD. Interestingly, TPDs containing a catechol moiety have demonstrated highly promising activity in inhibiting the aggregation of Aß40 and a strong ability to chelate biometals such as CuII and ZnII .
Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/antagonistas & inibidores , Quelantes/farmacologia , Complexos de Coordenação/farmacologia , Compostos Organofosforados/farmacologia , Álcool Feniletílico/análogos & derivados , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Quelantes/síntese química , Quelantes/química , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Compostos Organofosforados/química , Álcool Feniletílico/química , Álcool Feniletílico/farmacologia , Agregados Proteicos/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
The abnormal deposition of Aß amyloid deposits in the brain is a hallmark of Alzheimer's disease (AD). Based on this evidence, many current therapeutic approaches focus on the development of small molecules halting Aß aggregation. However, due to the temporary and elusive structures of amyloid assemblies, the rational design of aggregation inhibitors remains a challenging task. Here we combine ThT assays and MD simulations to study Aß aggregation in the presence of the natural compounds tyrosol (TY), 3-hydroxytyrosol (HDT), and 3-methoxytyrosol (homovanillyl alcohol - HVA). We show that albeit HDT is a potent inhibitor of amyloid growth, TY and HVA catalyze fibril formation. An inspection of MD simulations trajectories revealed that the different effects of these three molecules on Aß1-40 aggregation are ascribable to their capacity to arrange H-bonds network between the ligand (position C-3) and the peptide (Glu22). We believe that our results may contribute to the design of more effective and safe small molecules able to contrast pathogenic amyloid aggregation.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Catecóis/metabolismo , Fragmentos de Peptídeos/metabolismo , Álcool Feniletílico/análogos & derivados , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/química , Catálise , Ácido Homovanílico/farmacologia , Humanos , Ligação de Hidrogênio , Ligantes , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Álcool Feniletílico/química , Álcool Feniletílico/farmacologia , Ligação ProteicaRESUMO
Alzheimer's disease (AD) is linked to the abnormal accumulation of amyloid ß peptide (Aß) aggregates in the brain. Silybin B, a natural compound extracted from milk thistle (Silybum marianum), has been shown to significantly inhibit Aß aggregation in vitro and to exert neuroprotective properties in vivo. However, further explorations of silybin B's clinical potential are currently limited by three main factors: (a) poor solubility, (b) instability in blood serum, and (c) only partial knowledge of silybin's mechanism of action. Here, we address these three limitations. We demonstrate that conjugation of a trehalose moiety to silybin significantly increases both water solubility and stability in blood serum without significantly compromising its antiaggregation properties. Furthermore, using a combination of biophysical techniques with different spatial resolution, that is, TEM, ThT fluorescence, CD, and NMR spectroscopy, we profile the interactions of the trehalose conjugate with both Aß monomers and oligomers and evidence that silybin may shield the "toxic" surfaces formed by the N-terminal and central hydrophobic regions of Aß. Finally, comparative analysis with silybin A, a less active diastereoisomer of silybin B, revealed how even subtle differences in chemical structure may entail different effects on amyloid inhibition. The resulting insight on the mechanism of action of silybins as aggregation inhibitors is anticipated to facilitate the future investigation of silybin's therapeutic potential.
Assuntos
Peptídeos beta-Amiloides , Pró-Fármacos , Antioxidantes , Fragmentos de Peptídeos , Silibina , TrealoseRESUMO
An increasing number of human diseases has been shown to be linked to aggregation and amyloid formation by intrinsically disordered proteins (IDPs). Amylin, amyloid-ß, and α-synuclein are, indeed, involved in type-II diabetes, Alzheimer's, and Parkinson's, respectively. Despite the correlation of the toxicity of these proteins at early aggregation stages with membrane damage, the molecular events underlying the process is quite complex to understand. In this study, we demonstrate the crucial role of free lipids in the formation of lipid-protein complex, which enables an easy membrane insertion for amylin, amyloid-ß, and α-synuclein. Experimental results from a variety of biophysical methods and molecular dynamics results reveal that this common molecular pathway in membrane poration is shared by amyloidogenic (amylin, amyloid-ß, and α-synuclein) and nonamyloidogenic (rat IAPP, ß-synuclein) proteins. Based on these results, we propose a "lipid-chaperone" hypothesis as a unifying framework for protein-membrane poration.
Assuntos
Proteínas Intrinsicamente Desordenadas , Amiloide , Proteínas Amiloidogênicas , Animais , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Lipídeos , Ratos , alfa-SinucleínaRESUMO
Downhill folding has been defined as a unique thermodynamic process involving a conformations ensemble that progressively loses structure with the decrease of protein stability. Downhill folders are estimated to be rather rare in nature as they miss an energetically substantial folding barrier that can protect against aggregation and proteolysis. We have previously demonstrated that the prokaryotic zinc finger protein Ros87 shows a bipartite folding/unfolding process in which a metal binding intermediate converts to the native structure through a delicate barrier-less downhill transition. Significant variation in folding scenarios can be detected within protein families with high sequence identity and very similar folds and for the same sequence by varying conditions. For this reason, we here show, by means of DSC, CD and NMR, that also in different pH and ionic strength conditions Ros87 retains its partly downhill folding scenario demonstrating that, at least in metallo-proteins, the downhill mechanism can be found under a much wider range of conditions and coupled to other different transitions. We also show that mutations of Ros87 zinc coordination sphere produces a different folding scenario demonstrating that the organization of the metal ion core is determinant in the folding process of this family of proteins.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Dobramento de Proteína , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Desdobramento de Proteína , TermodinâmicaRESUMO
Mature microRNAs are short non-coding RNA sequences which upon incorporation into the RISC ribonucleoprotein complex, play a crucial role in regulation of gene expression. However, miRNAs can exist within the cell also as free molecules fulfilling their biological activity. Therefore, it is emerging that in addition to sequence even the structure adopted by mature miRNAs might play an important role to reach the target. Indeed, we analysed by several spectroscopic techniques the secondary structures of two artificial miRNAs selected by computational tool (miR-Synth) as best candidates to silence c-MET and EGFR genes and of two endogenous miRNAs (miR-15a and miR-15b) having the same seed region, but different biological activity. Our results demonstrate that both endogenous and artificial miRNAs can arrange in several 3D-structures which affect their activity and selectivity toward the targets.
Assuntos
MicroRNAs/química , MicroRNAs/genética , Sequência de Bases , Receptores ErbB/deficiência , Receptores ErbB/genética , Inativação Gênica , Conformação de Ácido Nucleico , Proteínas Proto-Oncogênicas c-met/deficiência , Proteínas Proto-Oncogênicas c-met/genética , Análise de Sequência de RNARESUMO
Proteasome malfunction parallels abnormal amyloid accumulation in Alzheimer's Disease (AD). Here we scrutinize a small library of pyrazolones by assaying their ability to enhance proteasome activity and protect neuronal cells from amyloid toxicity. Tube tests evidenced that aminopyrine and nifenazone behave as 20S proteasome activators. Enzyme assays carried out on an "open gate" mutant (α3ΔN) proteasome demonstrated that aminopyrine activates proteasome through binding the α-ring surfaces and influencing gating dynamics. Docking studies coupled with STD-NMR experiments showed that H-bonds and π-π stacking interactions between pyrazolones and the enzyme play a key role in bridging α1 to α2 and, alternatively, α5 to α6 subunits of the outer α-ring. Aminopyrine and nifenazone exhibit neurotrophic properties and protect differentiated human neuroblastoma SH-SY5Y cells from ß-amyloid (Aß) toxicity. ESI-MS studies confirmed that aminopyrine enhances Aß degradation by proteasome in a dose-dependent manner. Our results suggest that some pyrazolones and, in particular, aminopyrine are promising compounds for the development of proteasome activators for AD treatment.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/antagonistas & inibidores , Complexo de Endopeptidases do Proteassoma/metabolismo , Pirazolonas/farmacologia , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Complexo de Endopeptidases do Proteassoma/genética , Pirazolonas/química , Relação Estrutura-AtividadeRESUMO
Four specifically designed IDE mutants have been used to unveil the molecular basis of peptidase versus E1-like activity of the enzyme. We have found that physiological concentrations of copper(ii) ions inhibit the proteolytic activity of the enzyme towards small and large substrates but have no effect on the E1-like activity of the enzyme.