Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 31
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Biophys J ; 122(24): 4730-4747, 2023 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-37978804

RESUMO

Vascular endothelial cells (ECs) form a semipermeable barrier separating vascular contents from the interstitium, thereby regulating the movement of water and molecular solutes across small intercellular gaps, which are continuously forming and closing. Under inflammatory conditions, however, larger EC gaps form resulting in increased vascular leakiness to circulating fluid, proteins, and cells, which results in organ edema and dysfunction responsible for key pathophysiologic findings in numerous inflammatory disorders. In this study, we extend our earlier work examining the biophysical properties of EC gap formation and now address the role of lamellipodia, thin sheet-like membrane projections from the leading edge, in modulating EC spatial-specific contractile properties and gap closure. Micropillars, fabricated by soft lithography, were utilized to form reproducible paracellular gaps in human lung ECs. Using time-lapse imaging via optical microscopy, rates of EC gap closure and motility were measured with and without EC stimulation with the barrier-enhancing sphingolipid, sphingosine-1-phosphate. Peripheral ruffle formation was ubiquitous during gap closure. Kymographs were generated to quantitatively compare the lamellipodia dynamics of sphingosine-1-phosphate-stimulated and -unstimulated ECs. Utilizing atomic force microscopy, we characterized the viscoelastic behavior of EC lamellipodia. Our results indicate decreased stiffness and increased liquid-like behavior of expanding lamellipodia compared with regions away from the cellular edge (lamella and cell body) during EC gap closure, results in sync with the rapid kinetics of protrusion/retraction motion. We hypothesize this dissipative EC behavior during gap closure is linked to actomyosin cytoskeletal rearrangement and decreased cross-linking during lamellipodia expansion. In summary, these studies of the kinetic and mechanical properties of EC lamellipodia and ruffles at gap boundaries yield insights into the mechanisms of vascular barrier restoration and potentially a model system for examining the druggability of lamellipodial protein targets to enhance vascular barrier integrity.


Assuntos
Células Endoteliais , Pseudópodes , Humanos , Pseudópodes/metabolismo , Lisofosfolipídeos/metabolismo , Citoesqueleto/metabolismo , Endotélio Vascular/metabolismo , Células Cultivadas
2.
Langmuir ; 38(1): 100-111, 2022 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-34968052

RESUMO

Polymerization enhances the stability of a planar supported lipid bilayer (PSLB) but it also changes its chemical and mechanical properties, attenuates lipid diffusion, and may affect the activity of integral membrane proteins. Mixed bilayers composed of fluid lipids and poly(lipids) may provide an appropriate combination of polymeric stability coupled with the fluidity and elasticity needed to maintain the bioactivity of reconstituted receptors. Previously (Langmuir, 2019, 35, 12483-12491) we showed that binary mixtures of the polymerizable lipid bis-SorbPC and the fluid lipid DPhPC form phase-segregated PSLBs composed of nanoscale fluid and poly(lipid) domains. Here we used atomic force microscopy (AFM) to compare the nanoscale mechanical properties of these binary PSLBs with single-component PSLBs. The elastic (Young's) modulus, area compressibility modulus, and bending modulus of bis-SorbPC PSLBs increased upon polymerization. Before polymerization, breakthrough events at forces below 5 nN were observed, but after polymerization, the AFM tip could not penetrate the PSLB up to an applied force of 20 nN. These results are attributed to the polymeric network in poly(bis-SorbPC), which increases the bilayer stiffness and resists compression and bending. In binary DPhPC/poly(bis-SorbPC) PSLBs, the DPhPC domains are less stiff, more compressible, and are less resistant to rupture and bending compared to pure DPhPC bilayers. These differences are attributed to bis-SorbPC monomers and oligomers present in DPhPC domains that disrupt the packing of DPhPC molecules. In contrast, the poly(bis-SorbPC) domains are stiffer and less compressible relative to pure PSLBs; this difference is attributed to DPhPC filling the nm-scale pores in the polymerized domains that are created during bis-SorbPC polymerization. Thus, incomplete phase segregation increases the stability of poly(bis-SorbPC) but has the opposite, detrimental effect for DPhPC. Overall, these results provide guidance for the design of partially polymerized bilayers for technological uses.


Assuntos
Bicamadas Lipídicas , Polímeros , Difusão , Microscopia de Força Atômica , Polimerização
3.
Cell Mol Bioeng ; 14(6): 597-612, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34900013

RESUMO

INTRODUCTION: Platelet activation by mechanical means such as shear stress exposure, is a vital driver of thrombotic risk in implantable blood-contacting devices used in the treatment of heart failure. Lipids are essential in platelets activation and have been studied following biochemical activation. However, little is known regarding lipid alterations occurring with mechanical shear-mediated platelet activation. METHODS: Here, we determined if shear-activation of platelets induced lipidome changes that differ from those associated with biochemically-mediated platelet activation. We performed high-resolution lipidomic analysis on purified platelets from four healthy human donors. For each donor, we compared the lipidome of platelets that were non-activated or activated by shear, ADP, or thrombin treatment. RESULTS: We found that shear activation altered cell-associated lipids and led to the release of lipids into the extracellular environment. Shear-activated platelets released 21 phospholipids and sphingomyelins at levels statistically higher than platelets activated by biochemical stimulation. CONCLUSIONS: We conclude that shear-mediated activation of platelets alters the basal platelet lipidome. Further, these alterations differ and are unique in comparison to the lipidome of biochemically activated platelets. Many of the released phospholipids contained an arachidonic acid tail or were phosphatidylserine lipids, which have known procoagulant properties. Our findings suggest that lipids released by shear-activated platelets may contribute to altered thrombosis in patients with implanted cardiovascular therapeutic devices. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-021-00692-x.

4.
EMBO Mol Med ; 10(1): 32-47, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29208638

RESUMO

Alzheimer's disease is a devastating neurodegenerative disease eventually leading to dementia. An effective treatment does not yet exist. Here we show that oral application of the compound anle138b restores hippocampal synaptic and transcriptional plasticity as well as spatial memory in a mouse model for Alzheimer's disease, when given orally before or after the onset of pathology. At the mechanistic level, we provide evidence that anle138b blocks the activity of conducting Aß pores without changing the membrane embedded Aß-oligomer structure. In conclusion, our data suggest that anle138b is a novel and promising compound to treat AD-related pathology that should be investigated further.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/metabolismo , Benzodioxóis/uso terapêutico , Hipocampo/efeitos dos fármacos , Pirazóis/uso terapêutico , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/genética , Animais , Benzodioxóis/farmacologia , Modelos Animais de Doenças , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasticidade Neuronal/efeitos dos fármacos , Fenótipo , Pirazóis/farmacologia , Memória Espacial/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos
5.
Nat Photonics ; 11: 352-355, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29576804

RESUMO

Ultrasensitive nanomechanical instruments, including the atomic force microscope (AFM)1-4 and optical and magnetic tweezers5-8, have helped shed new light on the complex mechanical environments of biological processes. However, it is difficult to scale down the size of these instruments due to their feedback mechanisms9, which, if overcome, would enable high-density nanomechanical probing inside materials. A variety of molecular force probes including mechanophores10, quantum dots11, fluorescent pairs12,13 and molecular rotors14-16 have been designed to measure intracellular stresses; however, fluorescence-based techniques can have short operating times due to photo-instability and it is still challenging to quantify the forces with high spatial and mechanical resolution. Here, we develop a compact nanofibre optic force transducer (NOFT) that utilizes strong near-field plasmon-dielectric interactions to measure local forces with a sensitivity of <200 fN. The NOFT system is tested by monitoring bacterial motion and heart-cell beating as well as detecting infrasound power in solution.

6.
Methods Mol Biol ; 1345: 251-68, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26453217

RESUMO

Aggregation can be studied by a range of methods, experimental and computational. Aggregates form in solution, across solid surfaces, and on and in the membrane, where they may assemble into unregulated leaking ion channels. Experimental probes of ion channel conformations and dynamics are challenging. Atomistic molecular dynamics (MD) simulations are capable of providing insight into structural details of amyloid ion channels in the membrane at a resolution not achievable experimentally. Since data suggest that late stage Alzheimer's disease involves formation of toxic ion channels, MD simulations have been used aiming to gain insight into the channel shapes, morphologies, pore dimensions, conformational heterogeneity, and activity. These can be exploited for drug discovery. Here we describe computational methods to model amyloid ion channels containing the ß-sheet motif at atomic scale and to calculate toxic pore activity in the membrane.


Assuntos
Doença de Alzheimer/genética , Peptídeos beta-Amiloides/química , Biologia Computacional/métodos , Canais Iônicos/química , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Humanos , Canais Iônicos/metabolismo , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/química , Estrutura Secundária de Proteína
7.
Biochemistry ; 53(47): 7407-14, 2014 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-25365227

RESUMO

Connexin proteins form hexameric assemblies known as hemichannels. When docked to form gap junction (GJ) channels, hemichannels play a critical role in cell-cell communication and cellular homeostasis, but often are functional entities on their own in unapposed cell membranes. Defects in the Connexin26 (Cx26) gene are the major cause of hereditary deafness arising from dysfunctional hemichannels in the cochlea. Structural studies of Cx26 hemichannels properly trafficked and inserted in plasma membranes, including their clustering that forms a plaque-like feature in whole gap junctions, are limited. We used atomic force microscopy (AFM) to study the surface topography of Cx26 hemichannels using two different membrane preparations. Rat Cx26 containing appended carboxy terminal V5 and hexahistidine tags were expressed in baculovirus/Sf9 cell systems. The expressed Cx26 proteins form hemichannels in situ in Sf9 cells that were then purified either as (1) Sf9 membrane fragments containing Cx26 hemichannels or (2) solubilized hemichannels. The latter were subsequently reconstituted in liposomes. AFM images of purified membrane fragments showed clusters of protein macromolecular structures in the membrane that at higher magnification corresponded to Cx26 hemichannels. Hemichannels reconstituted into DOPC bilayers displayed two populations of channel heights likely resulting from differences in orientations of inserted hemichannels. Hemichannels in the protein rich portions of purified membranes also showed a reduced channel height above the bilayer compared to membranes with reconstituted hemichannels perhaps due to reduced AFM probe access to the lipid bilayer. These preparations of purified membranes enriched for connexin hemichannels that have been properly trafficked and inserted in membranes provide a platform for high-resolution AFM imaging of the structure, interconnexon interactions, and cooperativity of properly trafficked and inserted noncrystalline connexin hemichannels.


Assuntos
Membrana Celular/metabolismo , Conexinas/química , Microscopia de Força Atômica , Fragmentos de Peptídeos/química , Animais , Conexina 26 , Conexinas/metabolismo , Detergentes/farmacologia , Bicamadas Lipídicas/metabolismo , Fragmentos de Peptídeos/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Ratos , Células Sf9 , Spodoptera
8.
Sci Rep ; 4: 4454, 2014 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-24663394

RESUMO

Biological membranes contain ion channels, which are nanoscale pores allowing controlled ionic transport and mediating key biological functions underlying normal/abnormal living. Synthetic membranes with defined pores are being developed to control various processes, including filtration of pollutants, charge transport for energy storage, and separation of fluids and molecules. Although ionic transport (currents) can be measured with single channel resolution, imaging their structure and ionic currents simultaneously is difficult. Atomic force microscopy enables high resolution imaging of nanoscale structures and can be modified to measure ionic currents simultaneously. Moreover, the ionic currents can also be used to image structures. A simple method for fabricating conducting AFM cantilevers to image pore structures at high resolution is reported. Tungsten microwires with nanoscale tips are insulated except at the apex. This allows simultaneous imaging via cantilever deflections in normal AFM force feedback mode as well as measuring localized ionic currents. These novel probes measure ionic currents as small as picoampere while providing nanoscale spatial resolution surface topography and is suitable for measuring ionic currents and conductance of biological ion channels.


Assuntos
Membrana Celular/ultraestrutura , Microscopia de Força Atômica , Nanoporos/ultraestrutura , Transporte Biológico , Canais Iônicos/ultraestrutura , Íons/química
9.
Sci Rep ; 4: 4424, 2014 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-24651823

RESUMO

Membrane ion channels regulate key cellular functions and their activity is dependent on their 3D structure. Atomic force microscopy (AFM) images 3D structure of membrane channels placed on a solid substrate. Solid substrate prevents molecular transport through ion channels thus hindering any direct structure-function relationship analysis. Here we designed a ~70 nm nanopore to suspend a membrane, allowing fluidic access to both sides. We used these nanopores with AFM and total internal reflection fluorescence microscopy (TIRFM) for high resolution imaging and molecular transport measurement. Significantly, membranes over the nanopore were stable for repeated AFM imaging. We studied structure-activity relationship of gap junction hemichannels reconstituted in lipid bilayers. Individual hemichannels in the membrane overlying the nanopore were resolved and transport of hemichannel-permeant LY dye was visualized when the hemichannel was opened by lowering calcium in the medium. This integrated technique will allow direct structure-permeability relationship of many ion channels and receptors.


Assuntos
Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Bicamadas Lipídicas/metabolismo , Animais , Transporte Biológico , Cálcio/metabolismo , Cálcio/farmacologia , Conexina 43/isolamento & purificação , Conexina 43/ultraestrutura , Fibroblastos/química , Corantes Fluorescentes/metabolismo , Junções Comunicantes/química , Junções Comunicantes/efeitos dos fármacos , Isoquinolinas/metabolismo , Bicamadas Lipídicas/química , Microscopia de Força Atômica , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Permeabilidade , Porosidade , Ratos , Relação Estrutura-Atividade
10.
Chem Soc Rev ; 43(19): 6750-64, 2014 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-24566672

RESUMO

Aggregation of disordered amyloidogenic peptides into oligomers is the causative agent of amyloid-related diseases. In solution, disordered protein states are characterized by heterogeneous ensembles. Among these, ß-rich conformers self-assemble via a conformational selection mechanism to form energetically-favored cross-ß structures, regardless of their precise sequences. These disordered peptides can also penetrate the membrane, and electrophysiological data indicate that they form ion-conducting channels. Based on these and additional data, including imaging and molecular dynamics simulations of a range of amyloid peptides, Alzheimer's amyloid-ß (Aß) peptide, its disease-related variants with point mutations and N-terminal truncated species, other amyloidogenic peptides, as well as a cytolytic peptide and a synthetic gel-forming peptide, we suggest that disordered amyloidogenic peptides can also present a common motif in the membrane. The motif consists of curved, moon-like ß-rich oligomers associated into annular organizations. The motif is favored in the lipid bilayer since it permits hydrophobic side chains to face and interact with the membrane and the charged/polar residues to face the solvated channel pores. Such channels are toxic since their pores allow uncontrolled leakage of ions into/out of the cell, destabilizing cellular ionic homeostasis. Here we detail Aß, whose aggregation is associated with Alzheimer's disease (AD) and for which there are the most abundant data. AD is a protein misfolding disease characterized by a build-up of Aß peptide as senile plaques, neurodegeneration, and memory loss. Excessively produced Aß peptides may directly induce cellular toxicity, even without the involvement of membrane receptors through Aß peptide-plasma membrane interactions.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Membrana Celular/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Membrana Celular/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Simulação de Dinâmica Molecular , Estrutura Terciária de Proteína
11.
J Phys Chem B ; 117(39): 11518-29, 2013 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-24000923

RESUMO

An unusual ΔE693 mutation in the amyloid precursor protein (APP) producing a ß-amyloid (Aß) peptide lacking glutamic acid at position 22 (Glu22) was recently discovered, and dabbed the Osaka mutant (ΔE22). Previously, several point mutations in the Aß peptide involving Glu22 substitutions were identified and implicated in the early onset of familial Alzheimer's disease (FAD). Despite the absence of Glu22, the Osaka mutant is also associated with FAD, showing a recessive inheritance in families affected by the disease. To see whether this aggregation-prone Aß mutant could directly relate to the Aß ion channel-mediated mechanism as observed for the wild type (WT) Aß peptide in AD pathology, we modeled Osaka mutant ß-barrels in a lipid bilayer. Using molecular dynamics (MD) simulations, two conformer ΔE22 barrels with the U-shaped monomer conformation derived from NMR-based WT Aß fibrils were simulated in explicit lipid environment. Here, we show that the ΔE22 barrels obtain the lipid-relaxed ß-sheet channel topology, indistinguishable from the WT Aß1-42 barrels, as do the outer and pore dimensions of octadecameric (18-mer) ΔE22 barrels. Although the ΔE22 barrels lose the cationic binding site in the pore which is normally provided by the negatively charged Glu22 side chains, the mutant pores gain a new cationic binding site by Glu11 at the lower bilayer leaflet, and exhibit ion fluctuations similar to the WT barrels. Of particular interest, this deletion mutant suggests that toxic WT Aß1-42 would preferentially adopt a less C-terminal turn similar to that observed for Aß17-42, and explains why the solid state NMR data for Aß1-40 point to a more C-terminal turn conformation. The observed ΔE22 barrels conformational preferences also suggest an explanation for the lower neurotoxicity in rat primary neurons as compared to WT Aß1-42.


Assuntos
Peptídeos beta-Amiloides/química , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/genética , Fragmentos de Peptídeos/química , Deleção de Sequência , Doença de Alzheimer/genética , Sequência de Aminoácidos , Amiloide/química , Sítios de Ligação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Fosfatidilcolinas/química , Conformação Proteica , Estrutura Secundária de Proteína
12.
Phys Chem Chem Phys ; 15(23): 8868-77, 2013 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-23450150

RESUMO

The current paradigm in the amyloid hypothesis brands small ß-amyloid (Aß) oligomers as the toxic species in Alzheimer's disease (AD). These oligomers are fibril-like; contain ß-sheet structure, and present exposed hydrophobic surface. Oligomers with this motif are capable of penetrating the cell membrane, gathering to form toxic ion channels. Current agents suppressing precursor Aß cleavage have only met partial success; and to date, those targeting the peptides and their assemblies in the aqueous environment of the extracellular space largely fail in clinical trials. One possible reason is failure to reach membrane-embedded targets of disease-'infected' cells. Here we provide an overview, point to the need to account for the lipid environment when aiming to prevent the formation of toxic channels, and propose a combination therapy to target the species spectrum.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Amiloide/antagonistas & inibidores , Amiloide/metabolismo , Descoberta de Drogas , Amiloide/química , Animais , Humanos , Modelos Moleculares
13.
Nanomedicine ; 9(7): 875-84, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23523769

RESUMO

In this study we employ atomic force microscopy, supported by finite element analysis and fluorescence microscopy, to characterize the elastic properties accompanying cytoskeletal structural rearrangements of lung microvascular endothelial cells in response to barrier altering stimuli. Statistical analysis of elasticity data obtained from multiple cells demonstrates a heterogeneous cellular elastic response to barrier-enhancing and barrier-disrupting agents; sphingosine 1-phosphate (S1P) and thrombin, respectively. A small but detectable (10%) increase in the average elastic modulus of all cells is observed for S1P, which is accompanied by a corresponding significant decrease in cell thickness. Variable effects of thrombin on these parameters were observed. To account for possible substrate effects in our elasticity analysis, we analyzed only the low-force sections of the force-displacement curves and utilized a finite-thickness correction to the Hertzian model. Our finite element analysis results substantiate this approach. The heterogeneous elastic behavior correlates with differential cytoskeletal rearrangements observed with fluorescence microscopy. FROM THE CLINICAL EDITOR: This team of investigators employed atomic force microscopy coupled with finite element analysis and fluorescence microscopy to characterize the elastic properties accompanying cytoskeletal structural rearrangements of lung microvascular endothelial cells in response to barrier altering stimuli, demonstrating the validity of their approach.


Assuntos
Elasticidade/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Pulmão/citologia , Lisofosfolipídeos/farmacologia , Microvasos/citologia , Esfingosina/análogos & derivados , Trombina/farmacologia , Actinas/metabolismo , Linhagem Celular , Citoesqueleto , Células Endoteliais/citologia , Análise de Elementos Finitos , Humanos , Modelos Biológicos , Esfingosina/farmacologia , Estresse Mecânico
14.
J Chem Theory Comput ; 9(1): 822-833, 2013 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-23316126

RESUMO

Amyloid-ß (Aß) oligomers destabilize cellular ionic homeostasis, mediating Alzheimer's disease (AD). It is still unclear whether the mechanism (i) is mediated by cell surface receptors; (ii) is direct, with Aß oligomers interacting with membrane lipids; or (iii) both mechanisms take place. Recent studies indicate that Aß oligomers may act by either of the last two. Little is known about the oligomers' structures and how they spontaneously insert into the membrane. Using explicit solvent molecular dynamics (MD) simulations, we show that fibril-like Aß(17-42) (p3) oligomer is capable of penetrating the membrane. Insertion is similar to that observed for protegrin-1 (PG-1), a cytolytic ß-sheet-rich antimicrobial peptide (AMP). Both Aß and PG-1 favor the amphipathic interface of the lipid bilayer in the early stage of interaction with the membrane. U-shaped Aß oligomers are observed in solution and in the membrane, suggesting that the preformed seeds can be shared by amyloid fibrils in the growth phase and membrane toxicity. Here we provide sequential events in possible Aß oligomer membrane-insertion pathways. We speculate that for the U-shaped motif, a trimer is the minimal oligomer size to insert effectively. We propose that monomers and dimers may insert in (apparently on-pathway) aggregation-intermediate ß-hairpin state, and may (or may not) convert to a U-shape in the bilayer. Together with earlier observations, our results point to a non-specific, broadly heterogeneous landscape of membrane-inserting oligomer conformations, pathways, and membrane-mediated toxicity of ß-rich oligomers.

15.
Biochemistry ; 51(14): 3031-8, 2012 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-22413858

RESUMO

Alzheimer's disease (AD) is a misfolded protein disease characterized by the accumulation of ß-amyloid (Aß) peptide as senile plaques, progressive neurodegeneration, and memory loss. Recent evidence suggests that AD pathology is linked to the destabilization of cellular ionic homeostasis mediated by toxic pores made of Aß peptides. Understanding the exact nature by which these pores conduct electrical and molecular signals could aid in identifying potential therapeutic targets for the prevention and treatment of AD. Here using atomic force microscopy (AFM) and molecular dynamics (MD) simulations, we compared the imaged pore structures with models to predict channel conformations as a function of amino acid sequence. Site-specific amino acid (AA) substitutions in the wild-type Aß(1-42) peptide yield information regarding the location and significance of individual AA residues to its characteristic structure-activity relationship. We selected two AAs that our MD simulation predicted to inhibit or permit pore conductance. The substitution of Phe19 with Pro has previously been shown to eliminate conductance in the planar lipid bilayer system. Our MD simulations predict a channel-like shape with a collapsed pore, which is supported by the AFM channel images. We suggest that proline, a known ß-sheet breaker, creates a kink in the center of the pore and prevents conductance via blockage. This residue may be a viable target for drug development studies aiming to inhibit Aß from inducing ionic destabilization toxicity. The substitution of Phe20 with Cys exhibits pore structures indistinguishable from the wild type in AFM images. MD simulations predict site 20 to face the solvated pore. Overall, the mutations support the previously predicted ß-sheet-based channel structure.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , Amiloide , Peptídeos beta-Amiloides/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Relação Estrutura-Atividade
16.
J Phys Chem B ; 116(5): 1728-35, 2012 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-22217000

RESUMO

Alzheimer's disease (AD) is a protein misfolding disease characterized by a buildup of ß-amyloid (Aß) peptide as senile plaques, uncontrolled neurodegeneration, and memory loss. AD pathology is linked to the destabilization of cellular ionic homeostasis and involves Aß peptide-plasma membrane interactions. In principle, there are two possible ways through which disturbance of the ionic homeostasis can take place: directly, where the Aß peptide either inserts into the membrane and creates ion-conductive pores or destabilizes the membrane organization, or, indirectly, where the Aß peptide interacts with existing cell membrane receptors. To distinguish between these two possible types of Aß-membrane interactions, we took advantage of the biochemical tenet that ligand-receptor interactions are stereospecific; L-amino acid peptides, but not their D-counterparts, bind to cell membrane receptors. However, with respect to the ion channel-mediated mechanism, like L-amino acids, D-amino acid peptides will also form ion channel-like structures. Using atomic force microscopy (AFM), we imaged the structures of both D- and L-enantiomers of the full length Aß(1-42) when reconstituted in lipid bilayers. AFM imaging shows that both L- and D-Aß isomers form similar channel-like structures. Molecular dynamics (MD) simulations support the AFM imaged 3D structures. Previously, we have shown that D-Aß(1-42) channels conduct ions similarly to their L- counterparts. Taken together, our results support the direct mechanism of Aß ion channel-mediated destabilization of ionic homeostasis rather than the indirect mechanism through Aß interaction with membrane receptors.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/ultraestrutura , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/ultraestrutura , Amiloide/química , Amiloide/metabolismo , Amiloide/ultraestrutura , Peptídeos beta-Amiloides/metabolismo , Humanos , Isomerismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Microscopia de Força Atômica , Fragmentos de Peptídeos/metabolismo
17.
Soft Matter ; 7(11): 5267-5273, 2011 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-21918653

RESUMO

Amyloid-ß (Aß) peptides are thought to be involved in neurodegenerative diseases such as Alzheimer's disease and Down's syndrome. They form a large number of polymorphic structures, including heterogeneous ionic pores in membranes as well as different types of fibrillar and globular structures on surfaces and in solution. Understanding the origin of these structures and the factors that influence their occurrence is of great biomedical interest because of the possible relationship between structure and pathogenicity. Here, we use atomic force microscopy (AFM) and molecular dynamics (MD) simulations to demonstrate that at room temperature a truncated Aß peptide which is generated in vivo and shown to be toxic in vitro forms fibrillar structures on hydrophobic graphite surfaces, but not on hydrophilic mica or lipid bilayers. Our results suggest that the toxic pores and fibrillar polymorphic organizations can be explained in terms of the U-shaped ß-strand-turn-ß-strand structural motif observed for full length Aß and other amyloids, as well as the physicochemical properties at the interfaces. The interactions of the hydrophobic, truncated Aß with its environment illustrate that the universal amyloid motif can provide a link between the pore and fibrillar structures and indicate that surfaces with different physicochemical properties can shift the polymorphic landscape toward other conformational states.

18.
Biophys J ; 100(7): 1775-83, 2011 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-21463591

RESUMO

Protegrin-1 (PG-1) is an 18 residues long, cysteine-rich ß-sheet antimicrobial peptide (AMP). PG-1 induces strong cytotoxic activities on cell membrane and acts as a potent antibiotic agent. Earlier we reported that its cytotoxicity is mediated by its channel-forming ability. In this study, we have examined the amyloidogenic fibril formation properties of PG-1 in comparison with a well-defined amyloid, the amyloid-ß (Aß(1-42)) peptide. We have used atomic force microscopy (AFM) and thioflavin-T staining to investigate the kinetics of PG-1 fibrils growth and molecular dynamics simulations to elucidate the underlying mechanism. AFM images of PG-1 on a highly hydrophilic surface (mica) show fibrils with morphological similarities to Aß(1-42) fibrils. Real-time AFM imaging of fibril growth suggests that PG-1 fibril growth follows a relatively fast kinetics compared to the Aß(1-42) fibrils. The AFM results are in close agreement with results from thioflavin-T staining data. Furthermore, the results indicate that PG-1 forms fibrils in solution. Significantly, in contrast, we do not detect fibrillar structures of PG-1 on an anionic lipid bilayer 2-dioleoyl-sn-glycero-3-phospho-L-serine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; only small PG-1 oligomers can be observed. Molecular dynamics simulations are able to identify the presence of these small oligomers on the membrane bilayer. Thus, our current results show that cytotoxic AMP PG-1 is amyloidogenic and capable of forming fibrils. Overall, comparing ß-rich AMPs and amyloids such as Aß, in addition to cytotoxicity and amyloidogenicity, they share a common structural motif, and are channel forming. These combined properties support a functional relationship between amyloidogenic peptides and ß-sheet-rich cytolytic AMPs, suggesting that amyloids channels may have an antimicrobial function.


Assuntos
Amiloide/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Adsorção , Silicatos de Alumínio/química , Amiloide/ultraestrutura , Peptídeos Catiônicos Antimicrobianos/química , Simulação por Computador , Cinética , Bicamadas Lipídicas , Microscopia de Força Atômica , Estrutura Secundária de Proteína , Fatores de Tempo
19.
J Mol Biol ; 404(5): 917-34, 2010 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-20970427

RESUMO

Emerging evidence supports the ion channel mechanism for Alzheimer's disease pathophysiology wherein small ß-amyloid (Aß) oligomers insert into the cell membrane, forming toxic ion channels and destabilizing the cellular ionic homeostasis. Solid-state NMR-based data of amyloid oligomers in solution indicate that they consist of a double-layered ß-sheets where each monomer folds into ß-strand-turn-ß-strand and the monomers are stacked atop each other. In the membrane, Aß peptides are proposed to be ß-type structures. Experimental structural data available from atomic force microscopy (AFM) imaging of Aß oligomers in membranes reveal heterogeneous channel morphologies. Previously, we modeled the channels in a non-tilted organization, parallel with the cross-membrane normal. Here, we modeled a ß-barrel-like organization. ß-Barrels are common in transmembrane toxin pores, typically consisting of a monomeric chain forming a pore, organized in a single-layered ß-sheet with antiparallel ß-strands and a right-handed twist. Our explicit solvent molecular dynamics simulations of a range of channel sizes and polymorphic turns and comparisons of these with AFM image dimensions support a ß-barrel channel organization. Different from the transmembrane ß-barrels where the monomers are folded into a circular ß-sheet with antiparallel ß-strands stabilized by the connecting loops, these Aß barrels consist of multimeric chains forming double ß-sheets with parallel ß-strands, where the strands of each monomer are connected by a turn. Although the Aß barrels adopt the right-handed ß-sheet twist, the barrels still break into heterogeneous, loosely attached subunits, in good agreement with AFM images and previous modeling. The subunits appear mobile, allowing unregulated, hence toxic, ion flux.


Assuntos
Peptídeos beta-Amiloides/química , Canais Iônicos/química , Animais , Humanos , Substâncias Macromoleculares/química , Microscopia de Força Atômica , Modelos Moleculares , Simulação de Dinâmica Molecular , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína
20.
Biophys J ; 98(11): 2644-52, 2010 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-20513409

RESUMO

Antimicrobial peptides (AMPs) are an emerging class of antibiotics for controlling health effects of antibiotic-resistant microbial strains. Protegrin-1 (PG-1) is a model antibiotic among beta-sheet AMPs. Antibiotic activity of AMPs involves cell membrane damage, yet their membrane interactions, their 3D membrane-associated structures and the mechanism underlying their ability to disrupt cell membrane are poorly understood. Using complementary approaches, including molecular dynamics simulations, atomic force microscopy (AFM) imaging, and planar lipid bilayer reconstitution, we provide computational and experimental evidence that PG-1, a beta-hairpin peptide, forms ion channels. Simulations indicate that PG-1 forms channel-like structures with loosely attached subunits when reconstituted in anionic lipid bilayers. AFM images show the presence of channel-like structures when PG-1 is reconstituted in dioleoylphosphatidylserine/palmitoyloleoyl phosphatidylethanolamine bilayers or added to preformed bilayers. Planar lipid bilayer electrical recordings show multiple single channel conductances that are consistent with the heterogeneous oligomeric channel structures seen in AFM images. PG-1 channel formation seems to be lipid-dependent: PG-1 does not easily show ion channel electrical activity in phosphatidylcholine membranes, but readily shows channel activity in membranes rich in phosphatidylethanolamine or phosphatidylserine. The combined results support a model wherein the beta-hairpin PG-1 peptide acts as an antibiotic by altering cell ionic homeostasis through ion channel formation in cell membranes.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Animais , Condutividade Elétrica , Bicamadas Lipídicas/química , Potenciais da Membrana , Microscopia de Força Atômica , Simulação de Dinâmica Molecular , Fosfatidiletanolaminas/química , Fosfatidilserinas/química , Probabilidade , Estrutura Secundária de Proteína , Suínos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...