RESUMO
Medicinal chemistry is constantly searching for new approaches to develop more effective and targeted therapeutic molecules. The design of peptidomimetics is a promising emerging strategy that is aimed at developing peptides that mimic or modulate the biological activity of proteins. Among these, stapled peptides stand out for their unique ability to stabilize highly frequent helical motifs, but they have failed to be systematically reported. Here, we exploit chemically diverse helix-inducing i, i + 4 constraints-lactam, hydrocarbon, triazole, double triazole and thioether-on two distinct short sequences derived from the N-terminal peptidase domain of hACE2 upon structural characterization and in silico alanine scan. Our overall objective was to provide a sequence-independent comparison of α-helix-inducing staples using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. We identified a 9-mer lactam stapled peptide derived from the hACE2 sequence (His34-Gln42) capable of reaching its maximal helicity of 55% with antiviral activity in bioreporter- and pseudovirus-based inhibition assays. To the best of our knowledge, this study is the first comprehensive investigation comparing several cyclization methods with the goal of generating stapled peptides and correlating their secondary structures with PPI inhibitions using a highly topical model system (i.e., the interaction of SARS-CoV-2 Spike RBD with hACE2).
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Ciclização , Lactamas , Peptídeos/farmacologia , TriazóisRESUMO
Pepducins are cell-penetrating, membrane-tethered lipopeptides designed to target the intracellular region of a G protein-coupled receptor (GPCR) in order to allosterically modulate the receptor's signaling output. In this proof-of-concept study, we explored the pain-relief potential of a pepducin series derived from the first intracellular loop of neurotensin receptor type 1 (NTS1), a class A GPCR that mediates many of the effects of the neurotensin (NT) tridecapeptide, including hypothermia, hypotension and analgesia. We used BRET-based biosensors to determine the pepducins' ability to engage G protein signaling pathways associated with NTS1 activation. We observed partial Gαq and Gα13 activation at a 10 µM concentration, indicating that these pepducins may act as allosteric agonists of NTS1. Additionally, we used surface plasmon resonance (SPR) as a label-free assay to monitor pepducin-induced responses in CHO-K1 cells stably expressing hNTS1. This whole-cell integrated assay enabled us to subdivide our pepducin series into three profile response groups. In order to determine the pepducins' antinociceptive potential, we then screened the series in an acute pain model (tail-flick test) by measuring tail withdrawal latencies to a thermal nociceptive stimulus, following intrathecal (i.t.) pepducin administration (275 nmol/kg). We further evaluated promising pepducins in a tonic pain model (formalin test), as well as in neuropathic (Chronic Constriction Injury) and inflammatory (Complete Freund's Adjuvant) chronic pain models. We report one pepducin, PP-001, that consistently reduced rat nociceptive behaviors, even in chronic pain paradigms. Finally, we designed a TAMRA-tagged version of PP-001 and found by confocal microscopy that the pepducin reached the rat dorsal root ganglia post i.t. injection, thus potentially modulating the activity of NTS1 at this location to produce its analgesic effect. Altogether, these results suggest that NTS1-derived pepducins may represent a promising strategy in pain-relief.
Assuntos
Analgésicos/uso terapêutico , Peptídeos Penetradores de Células/uso terapêutico , Lipopeptídeos/uso terapêutico , Dor/tratamento farmacológico , Receptores de Neurotensina , Analgésicos/farmacologia , Animais , Células CHO , Peptídeos Penetradores de Células/farmacologia , Cricetulus , Proteínas de Ligação ao GTP/metabolismo , Gânglios Espinais/metabolismo , Lipopeptídeos/farmacologia , Masculino , Dor/genética , Dor/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Thaxtomin A (TA) is a natural cellulose biosynthesis inhibitor (CBI) synthesized by the potato common scab-causing pathogen Streptomyces scabies. Inhibition of cellulose synthesis by TA compromises cell wall organization and integrity, leading to the induction of an atypical program of cell death (PCD). These processes may facilitate S. scabies entry into plant tissues. To study the mechanisms that regulate the induction of cell death in response to inhibition of cellulose synthesis, we used Arabidopsis thaliana cell suspension cultures treated with two structurally different CBIs, TA and the herbicide isoxaben (IXB). RESULTS: The induction of cell death by TA and IXB was abrogated following pretreatment with the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) and the natural auxin indole-3-acetic acid (IAA). The addition of auxin efflux inhibitors also inhibited the CBI-mediated induction of PCD. This effect may be due to intracellular accumulation of auxin. Auxin has a wide range of effects in plant cells, including a role in the control of cell wall composition and rigidity to facilitate cell elongation. Using Atomic Force Microscopy (AFM)-based force spectroscopy, we found that inhibition of cellulose synthesis by TA and IXB in suspension-cultured cells decreased cell wall stiffness to a level slightly different than that caused by auxin. However, the cell wall stiffness in cells pretreated with auxin prior to CBI treatment was equivalent to that of cells treated with auxin only. CONCLUSIONS: Addition of auxin to Arabidopsis cell suspension cultures prevented the TA- and IXB-mediated induction of cell death. Cell survival was also stimulated by inhibition of polar auxin transport during CBI-treatment. Inhibition of cellulose synthesis perturbed cell wall mechanical properties of Arabidopsis cells. Auxin treatment alone or with CBI also decreased cell wall stiffness, showing that the mechanical properties of the cell wall perturbed by CBIs were not restored by auxin. However, since auxin's effects on the cell wall stiffness apparently overrode those induced by CBIs, we suggest that auxin may limit the impact of CBIs by restoring its own transport and/or by stabilizing the plasma membrane - cell wall - cytoskeleton continuum.
Assuntos
Apoptose/efeitos dos fármacos , Arabidopsis/fisiologia , Benzamidas/farmacologia , Celulose/biossíntese , Ácidos Indolacéticos/metabolismo , Indóis/farmacologia , Piperazinas/farmacologia , Reguladores de Crescimento de Plantas/metabolismo , Arabidopsis/efeitos dos fármacos , Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Células Cultivadas , Celulose/antagonistas & inibidores , Herbicidas/farmacologiaRESUMO
Angiotensin II (AngII) type 1 receptor (AT1R) is a G protein-coupled receptor known for its role in numerous physiological processes and its implication in many vascular diseases. Its functions are mediated through G protein dependent and independent signaling pathways. AT1R has several endogenous peptidic agonists, all derived from angiotensinogen, as well as several synthetic ligands known to elicit biased signaling responses. Here, surface plasmon resonance (SPR) was used as a cell-based and label-free technique to quantify, in real time, the response of HEK293 cells stably expressing the human AT1R. The goal was to take advantage of the integrative nature of this assay to identify specific signaling pathways in the features of the response profiles generated by numerous endogenous and synthetic ligands of AT1R. First, we assessed the contributions of Gq, G12/13, Gi, Gßγ,â¯ERK1/2 and ß-arrestins pathways in the cellular responses measured by SPR where Gq, G12/Rho/ROCK together with ß-arrestins and ERK1/2 were found to play significant roles. More specifically, we established a major role for G12 in the early events of the AT1R-dependent response, which was followed by a robust ERK1/2 component associated to the later phase of the signal. Interestingly, endogenous AT1R ligands (AngII, AngIII and AngIV) exhibited distinct responses signatures with a significant increase of the ERK1/2-like components for both AngIII and AngIV, which points toward possibly distinct physiological roles for the later. We also tested AT1R biased ligands, all of which affected both the early and later events. Our results support SPR-based integrative cellular assays as a powerful approach to delineate the contribution of specific signaling pathways for a given cell response and reveal response differences associated with ligands with distinct pharmacological properties.
Assuntos
Angiotensina II/análogos & derivados , Angiotensina II/farmacologia , Proteínas de Ligação ao GTP/fisiologia , Receptor Tipo 1 de Angiotensina/fisiologia , Células HEK293 , Humanos , RNA Interferente Pequeno/genética , Transdução de Sinais , Ressonância de Plasmônio de SuperfícieRESUMO
Evanescent field based biosensing systems such as surface plasmon resonance (SPR), diffraction gratings, or metal-clad waveguides (MCWGs) are powerful tools for label-free real-time monitoring of signaling activity of living cells exposed to hormones, pharmacological agents, and toxins. In particular, MCWG-based imaging is well suited for studying relatively thick objects such as cells due to its greater depth of penetration into the sensing medium, compared to SPR. Label-free methods, however, provide only indirect measurements in that the measured signal arises from local changes in material properties rather than from specific biomolecular targets. In the case of cells, the situation is especially complex as the measured label-free signal may result from a combination of very diverse sources: morphological changes, intra-cellular reorganization, cascaded molecular events, protein expression etc. Consequently, deconvolving the contributions of specific sources to a particular cell response profile can be challenging. In the following, we present a cell imaging platform that combines two distinct sensing modalities, namely label-free MCWG imaging and label-based surface enhanced fluorescence (SEF), designed to facilitate the identification of the underlying molecular and structural contributions to the label-free MCWG images. We demonstrate the bimodal capabilities of this imaging platform in experiments designed to visualize actin cytoskeleton organization in vascular smooth muscle cells. We then monitored the real-time response of HEK293 cells expressing the Angiotensin 1 receptor (AT1R), when stimulated by the receptor agonist Angiotensin II (AngII). The analysis of the simultaneous label-free signal obtained by MCWG and the intracellular calcium signal resulting form AT1R activation, measured by SEF, allows relating label-free signal features to specific markers of receptor activation. Our results show that the intracellular calcium levels normally observed following AT1R activation are not required for the initial burst of cellular activity observed in the MCWG signal but rather indicates signaling activity involving the intracellular kinase ROCK.
Assuntos
Citoesqueleto de Actina/metabolismo , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Transdução de Sinais/fisiologia , Animais , Fluorescência , Corantes Fluorescentes/química , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Ratos , Receptor Tipo 1 de Angiotensina/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
Evanescent-field based methods such as surface plasmon resonance (SPR) have been used very effectively for label-free imaging of microscopic biological material in close proximity to a sensing surface. However, the shallow probing depth of SPR (typically less than ~200 nm) can be problematic when imaging relatively thick biological objects such as cells or bacteria. In this paper, we demonstrate how metal-clad waveguides (MCWG) can be used to achieve deeper probing depth compared to SPR while maintaining good imaging spatial resolution. Comparative numerical simulations of imaging spatial resolution versus probing depth are shown for a number of common SPR, long-range SPR, and MCWG configurations, demonstrating that MCWG offer the best compromise between resolution and depth for imaging thick biological objects. Experimental results of synthetic target and live cell imaging are shown that validate the numerical simulations and demonstrate the capabilities of the method.
RESUMO
Advanced glycation end-products (AGE) and the receptor for AGE (RAGE) have been linked to numerous diabetic vascular complications. RAGE activation promotes a self-sustaining state of chronic inflammation and has been shown to induce apoptosis in various cell types. Although previous studies in vascular smooth muscle cells (VSMC) showed that RAGE activation increases vascular calcification and interferes with their contractile phenotype, little is known on the potential of RAGE to induce apoptosis in VSMC. Using a combination of apoptotic assays, we showed that RAGE stimulation with its ligand CML-HSA promotes apoptosis of VSMC. The formation of stress granules and the increase in the level of the associated protein HuR point toward RAGE-dependent endoplasmic reticulum (ER) stress, which is proposed as a key contributor of RAGE-induced apoptosis in VSMC as it has been shown to promote cell death via numerous mechanisms, including up-regulation of caspase-9. Chronic NF-κB activation and modulation of Bcl-2 homologs are also suspected to contribute to RAGE-dependent apoptosis in VSMC. With the goal of reducing RAGE signaling and its detrimental impact on VSMC, we designed a RAGE antagonist (iRAGE) derived from the primary amino acid sequence of HSA. The resulting CML peptide was selected for the high glycation frequency of the primary sequence in the native protein in vivo. Pretreatment with iRAGE blocked 69.6% of the increase in NF-κB signaling caused by RAGE activation with CML-HSA after 48h. Preincubation with iRAGE was successful in reducing RAGE-induced apoptosis, as seen through enhanced cell survival by SPR and reduced PARP cleavage. Activation of executioner caspases was 63.5% lower in cells treated with iRAGE before stimulation with CML-HSA. To our knowledge, iRAGE is the first antagonist shown to block AGE-RAGE interaction and we propose the molecule as an initial candidate for drug discovery.
Assuntos
Lisina/análogos & derivados , Peptídeos/farmacologia , Receptor para Produtos Finais de Glicação Avançada/antagonistas & inibidores , Albumina Sérica/farmacologia , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Produtos Finais de Glicação Avançada/metabolismo , Lisina/química , Lisina/farmacologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Albumina Sérica/químicaRESUMO
In high-resolution surface plasmon (SPR) imaging, lateral resolution is limited along the direction of plasmon propagation by the longitudinal decay length. Though SPR systems can achieve sub-micrometer resolution, the decay length causes a degradation in the images in the direction of plasmon propagation akin to a blurring artifact, with ringing along resonant to nonresonant transition edges. We present a method to significantly reduce this effect based on combining images of a sample acquired with distinct guided-mode propagation directions. As SPR is a special case of the class of optical structures known as metal-clad waveguides (MCWG) that are also affected by the decay length, this work is broadly applicable.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Metais , Microscopia/métodos , Ressonância de Plasmônio de Superfície/métodos , Impedância ElétricaRESUMO
Protein glycation is a ubiquitous process involved in vascular complications observed in diabetes. Glyoxal (GO), an intracellular reactive oxoaldehyde that is one of the most potent glycation agents, readily reacts with amines present on proteins to produce the lysine-derived adduct carboxymethyllysine, which is a prevalent advanced glycation end-product (AGE). Our group previously showed that cell exposure to GO leads to an alteration in the cell contractile activity that could occur as a result of the glycation of various proteins regulating the cell contractile machinery. Here, we measured the extent of glycation on three functionally distinct proteins known to participate in cell contraction and cytoskeletal organization-Rho-kinase (ROCK), actin, and gelsolin (GSN)-using an assay based on the reaction of the cell membrane-permeable fluorescent probe carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), which reacts with primary amine groups of proteins. By combining CFDA-SE fluorescence and Western blot detection, we observed (following GO incubation) increased glycation of actin and ROCK as well as an increased interaction between actin and GSN as observed by co-immunoprecipitation. Thus, we conclude that the use of the fluorescent probe CFDA-SE offers an interesting alternative to perform a comparative analysis of the extent of intracellular protein glycation in live cells.
Assuntos
Fluoresceínas/metabolismo , Corantes Fluorescentes/metabolismo , Glicosilação , Succinimidas/metabolismo , Actinas/análise , Actinas/metabolismo , Western Blotting , Linhagem Celular , Fluoresceínas/análise , Corantes Fluorescentes/análise , Gelsolina/análise , Gelsolina/metabolismo , Glioxal/metabolismo , Humanos , Microscopia de Fluorescência , Succinimidas/análise , Quinases Associadas a rho/análise , Quinases Associadas a rho/metabolismoRESUMO
In this study, we used a combination of traditional signaling investigation approaches, bioluminescence resonance energy transfer (BRET) biosensors, and the label-free approach surface plasmon resonance (SPR) spectroscopy to monitor the signaling cascades of the µ-opioid receptor (MOP). In human embryonic kidney cells stably expressing a Flag-tagged version of human MOP, we compared the signals triggered by the noninternalizing and internalizing MOP agonists morphine and DAMGO (Tyr-D-Ala-Gly-N-methyl-Phe-Gly-ol), respectively. We studied three major and well described components of MOP signaling: receptor internalization, G protein coupling, and activation of extracellular signal-regulated kinase ERK1/ERK2. Our results show that morphine and DAMGO display different profiles of receptor internalization and a similar ability to trigger the phosphorylation of ERK1/ERK2. Our SPR analyses revealed that morphine and DAMGO evoke similar SPR signatures and that Gαi, cAMP-dependent pathways, and ERK1/ERK2 have key roles in morphine- and DAMGO-mediated signaling. Most interestingly, we found that the so-called MOP neutral antagonists CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2)), naloxone, and naltrexone behave like partial agonists. Even more intriguing, BRET experiments indicate that CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2)) induces similar conformational changes as naltrexone at the Gαi-ßγ interface, whereas it appears as an inverse agonist based on its SPR response thus indicating distinct signaling mechanisms for the two ligands. Taken together, our results support the usefulness of label-free methods such as SPR to study whole-cell responses and signaling cascades triggered by G protein-coupled receptors and complement the conventional approaches by revealing cellular responses that would have been otherwise undetectable.
Assuntos
Receptores Opioides mu/metabolismo , Transdução de Sinais/fisiologia , Linhagem Celular , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases/genética , Fosforilação/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Receptores Opioides mu/agonistas , Receptores Opioides mu/antagonistas & inibidoresRESUMO
STIM1 plays a crucial role in Ca(2+) homeostasis, particularly in replenishing the intracellular Ca(2+) store following its depletion. In cardiomyocytes, the Ca(2+) content of the sarcoplasmic reticulum must be tightly controlled to sustain contractile activity. The presence of STIM1 in cardiomyocytes suggests that it may play a role in regulating the contraction of cardiomyocytes. The aim of the present study was to determine how STIM1 participates in the regulation of cardiac contractility. Atomic force microscopy revealed that knocking down STIM1 disrupts the contractility of cardiomyocyte-derived HL-1 cells. Ca(2+) imaging also revealed that knocking down STIM1 causes irregular spontaneous Ca(2+) oscillations in HL-1 cells. Action potential recordings further showed that knocking down STIM1 induces early and delayed afterdepolarizations. Knocking down STIM1 increased the peak amplitude and current density of T-type voltage-dependent Ca(2+) channels (T-VDCC) and shifted the activation curve toward more negative membrane potentials in HL-1 cells. Biotinylation assays revealed that knocking down STIM1 increased T-VDCC surface expression and co-immunoprecipitation assays suggested that STIM1 directly regulates T-VDCC activity. Thus, STIM1 is a negative regulator of T-VDCC activity and maintains a constant cardiac rhythm by preventing a Ca(2+) overload that elicits arrhythmogenic events.
Assuntos
Canais de Cálcio Tipo T/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Glicoproteínas de Membrana/metabolismo , Contração Muscular , Miócitos Cardíacos/metabolismo , Taquicardia , Animais , Western Blotting , Canais de Cálcio , Células Cultivadas , Eletrofisiologia , Imunoprecipitação , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/genética , Camundongos , Miócitos Cardíacos/citologia , Molécula 1 de Interação EstromalRESUMO
Compounds beyond the rule-of-five are generating interest as they expand the molecular toolbox for modulating targets previously considered "undruggable". Macrocyclic peptides are an efficient class of molecules for modulating protein-protein interactions. However, predicting their permeability is difficult as they differ from small molecules. Although constrained by macrocyclization, they generally retain some conformational flexibility associated with an enhanced ability to cross biological membranes. In this study, we investigated the relationship between the structure of semi-peptidic macrocycles and their membrane permeability through structural modifications. Based on a scaffold of four amino acids and a linker, we synthesized 56 macrocycles incorporating modifications in either stereochemistry, N-methylation, or lipophilicity and assessed their passive permeability using the parallel artificial membrane permeability assay (PAMPA). Our results show that some semi-peptidic macrocycles have adequate passive permeability even with properties outside the Lipinski rule of five. We found that N-methylation in position 2 and the addition of lipophilic groups to the side chain of tyrosine led to an improvement in permeability with a decrease in tPSA and 3D-PSA. This enhancement could be attributed to the shielding effect of the lipophilic group on some regions of the macrocycle, which in turn, facilitates a favorable macrocycle conformation for permeability, suggesting some degree of chameleonic behavior.
Assuntos
Aminoácidos , Peptídeos , Peptídeos/química , Conformação Molecular , Permeabilidade , TirosinaRESUMO
Apoptosis can be routinely characterized using biomolecular markers such as in the TUNEL and the annexin V assays or by using fluorescent caspase substrates. Apoptosis can also be semi-quantitatively characterized using microscopy, which targets morphological features such as cell rounding, nuclear condensation and fragmentation as well as cell membrane blebbing. This label-free approach provides a limited resolution for the evolution of these events in time and relies heavily on subjective identification of the morphological features. Here we propose a label-free assay based on surface plasmon resonance (SPR) detection of minute morphology changes occurring as a result of apoptosis induction in an endothelial cell model (EA.hy926). At first, annexin V assays confirmed that our cellular model was responsive to TRAIL over a 12-hour period. Then, we show that SPR allows accurate monitoring of apoptosis by measuring (1) the duration of the latency period during which the apoptotic signal is integrated by the initiator caspases and transmitted to the executioner caspases, (2) the rate of the execution phase in which death substrates are cleaved and morphological changes occur, and (3) the total extent of apoptosis. Using these parameters, we characterized the responses obtained with TRAIL (EA.hy926, HeLa, AD-293) and the anti-Fas antibody (HeLa) for the extrinsic pathways and UV exposure (HeLa) for the intrinsic pathways. By comparing the SPR time-course of apoptosis with phase contrast micrographs, we demonstrate that the cell morphological hallmarks of apoptosis are the major contributors to the SPR signal. Altogether, our results validate the use of SPR as an accurate label-free assay for the real-time monitoring of apoptosis-triggered cell morphological changes.
Assuntos
Apoptose , Forma Celular , Ressonância de Plasmônio de Superfície , Anexina A5/metabolismo , Apoptose/efeitos dos fármacos , Células Cultivadas , Humanos , Ligante Indutor de Apoptose Relacionado a TNF/farmacologiaRESUMO
The extracellular matrix (ECM) is an interconnected macromolecular scaffold occupying the space between cells. Amongst other functions, the ECM provides structural support to tissues and serves as a microenvironmental niche that conveys regulatory signals to cells. Cell-matrix adhesions, which link the ECM to the cytoskeleton, are dynamic multi-protein complexes containing surface receptors and intracellular effectors that control various downstream pathways. In skeletal muscle, the most abundant tissue of the body, each individual muscle fiber and its associated muscle stem cells (MuSCs) are surrounded by a layer of ECM referred to as the basal lamina. The core scaffold of the basal lamina consists of self-assembling polymeric laminins and a network of collagens that tether proteoglycans, which provide lateral crosslinking, establish collateral associations with cell surface receptors, and serve as a sink and reservoir for growth factors. Skeletal muscle also contains the fibrillar collagenous interstitial ECM that plays an important role in determining tissue elasticity, connects the basal laminae to each other, and contains matrix secreting mesenchymal fibroblast-like cell types and blood vessels. During skeletal muscle regeneration fibroblast-like cell populations expand and contribute to the transitional fibronectin-rich regenerative matrix that instructs angiogenesis and MuSC function. Here, we provide a comprehensive overview of the role of the skeletal muscle ECM in health and disease and outline its role in orchestrating tissue regeneration and MuSC function.
RESUMO
Label-free biosensing, such as with surface plasmon resonance (SPR), is a highly efficient method for monitoring the responses of living cells exposed to pharmacological agents and biochemical stimuli in vitro. Conventional cell culture protocols used in cell-based biosensing generally provide little direct control over cell morphologies and phenotypes. Surface micropatterning techniques have been exploited for the controlled immobilization and establishment of well-defined cell morphologies and phenotypes. In this article, surface adhesion micropatterns are used to control the adhesion of endothelial cells within adjacent hexagonal microstructures to promote the emergence of a well-controlled and standardized cell layer phenotype onto SPR sensor surfaces. We show that the formation of cell-cell junctions can be controlled by tuning the inter-cellular spacing in groups of 3 neighbouring cells. Fluorescence microscopy was used to confirm the formation of vascular endothelium cadherin junctions, a structural marker of a functional endothelium. In order to confirm the functionality of the proposed model, the response to thrombin, a modulator of endothelium integrity, was monitored by surface plasmon resonance imaging (SPRI). Experiments demonstrate the potential of the proposed model as a primary biological signal transducer for SPRI-based analysis, with potential applications in cell biology, pharmacology and diagnostic.
Assuntos
Técnicas Biossensoriais , Técnicas Biossensoriais/métodos , Células Endoteliais , Endotélio , Ressonância de Plasmônio de Superfície/métodos , TrombinaRESUMO
Interaction between hyaluronic acid (HA) present at the surface of tubular epithelial cells and calcium oxalate monohydrate (COM) crystals is thought to play an important role in kidney stone formation. AFM-based force spectroscopy, where HA is covalently attached to AFM-probes, was used to quantify the interaction between HA and the surfaces of COM crystals. The work of adhesion of the HA-probe as well as the rupture force of single HA molecules were quantified in order to understand the molecular regulation of HA binding to COM crystals. Our results reveal that HA adsorbs to the crystal surface in physiological conditions. We also observed increased adhesion when the pH is lowered to a value that increases the risk of kidney stone formation. HA adhesion to the COM crystal surface can be suppressed by citrate, a physiological inhibitor of stone retention currently used in the treatment and prevention of kidney stone formation. Interestingly, we also observed preferential binding of HA onto the [100] face versus the [010] face, suggesting a major contribution of the [100] faces in the crystal retention process at the surface of tubular epithelial cells and the promotion of stone formation. Our results clearly establish a direct role for the glycosaminoglycan HA present at the surface of kidney tubular epithelium in the process of COM crystal retention.
Assuntos
Oxalato de Cálcio/química , Ácido Cítrico/química , Ácido Hialurônico/química , Concentração de Íons de Hidrogênio , Microscopia de Força AtômicaRESUMO
We report on the use of tris(hydroxymethyl)phosphine-alanine (THPAL) functionalized gold nanoparticles as a multivalent cross-linking agent to assemble collagen fibrils into a mesh-like structure. Atomic force microscopy (AFM) was used for characterization of the structure after adsorption onto an atomically flat mica substrate, revealing a mesh-like construct in which the collagen fibrils and the gold nanoparticles interact to form interconnected nodes measuring from 100 to 500 nm. As expected, the density of the collagen mesh can be increased with a higher initial concentration of gold nanoparticles. The maximum thickness of the meshes (â¼ 20 nm) obtained through cross-sectional height measurements confirms that the adsorbed structure consists of a single layer of collagen fibrils/gold nanoparticles assembled in two-dimensions. We propose that the capability of gold nanoparticles functionalized with the THPAL to bind to several collagen fibrils combined with the large persistence length of the fibrils, which was reported to be in the hundreds of nanometer range, are determinant factors for the preferential 2D growth of the mesh in solution.
Assuntos
Alanina/química , Colágeno/química , Reagentes de Ligações Cruzadas/química , Ouro/química , Nanopartículas Metálicas/química , Fosfinas/química , Microscopia de Força Atômica/métodosRESUMO
Incorporating small modifications to peptidic macrocycles can have a major influence on their properties. For instance, N-methylation has been shown to impact permeability. A better understanding of the relationship between permeability and structure is of key importance as peptidic drugs are often associated with unfavorable pharmacokinetic profiles. Starting from a semipeptidic macrocycle backbone composed of a tripeptide tethered head-to-tail with an alkyl linker, we investigated two small changes: peptide-to-peptoid substitution and various methyl placements on the nonpeptidic linker. Implementing these changes in parallel, we created a collection of 36 compounds. Their permeability was then assessed in parallel artificial membrane permeability assay (PAMPA) and Caco-2 assays. Our results show a systematic improvement in permeability associated with one peptoid position in the cycle, while the influence of methyl substitution varies on a case-by-case basis. Using a combination of molecular dynamics simulations and NMR measurements, we offer hypotheses to explain such behavior.
Assuntos
Compostos Macrocíclicos/química , Peptidomiméticos/química , Células CACO-2 , Permeabilidade da Membrana Celular/efeitos dos fármacos , Desenho de Fármacos , Humanos , Ligação de Hidrogênio , Compostos Macrocíclicos/metabolismo , Compostos Macrocíclicos/farmacologia , Espectroscopia de Ressonância Magnética , Metilação , Conformação Molecular , Simulação de Dinâmica Molecular , Peptidomiméticos/metabolismo , Peptidomiméticos/farmacologiaRESUMO
The morphological and functional integrity of the microcirculation is compromised in many cardiovascular diseases such as hypertension, diabetes, stroke, and sepsis. Angiotensin converting enzyme inhibitors (ACEi), which are known to favor bradykinin (BK) bioactivity by reducing its metabolism, may have a positive impact on preventing the microvascular structural rarefaction that occurs in these diseases. Our study was designed to test the hypothesis that BK, via B2 receptors (B2R), protects the viability of the microvascular endothelium exposed to the necrotic and apoptotic cell death inducers H(2)O(2) and LPS independently of hemodynamics. Expression (RT-PCR and radioligand binding) and functional (calcium mobilization with fura-2AM, and p42/p44MAPK and Akt phosphorylation assays) experiments revealed the presence of functional B2R in pig cerebral microvascular endothelial cells (pCMVEC). In vitro results showed that the cytocidal effects of H(2)O(2) and LPS on pCMVEC were significantly decreased by a BK pretreatment (MTT and crystal violet tests, annexin-V staining/FACS analysis), which was countered by the B2R antagonist HOE 140. BK treatment coincided with enhanced expression of the cytoprotective proteins COX-2, Bcl-2, and (Cu/Zn)SOD. Ex vivo assays on rat brain explants showed that BK impeded (by approximately 40%) H(2)O(2)-induced microvascular degeneration (lectin-FITC staining). The present study proposes a novel role for BK in microvascular endothelial protection, which may be pertinent to the complex mechanism of action of ACEi explaining their long-term beneficial effects in maintaining vascular integrity.
Assuntos
Bradicinina/farmacologia , Encéfalo/patologia , Citoproteção/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Microvasos/patologia , Substâncias Protetoras/farmacologia , Animais , Encéfalo/irrigação sanguínea , Morte Celular/efeitos dos fármacos , Células Endoteliais/enzimologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/farmacologia , Lipopolissacarídeos/farmacologia , Microvasos/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor B2 da Bradicinina/genética , Receptor B2 da Bradicinina/metabolismo , Sus scrofaRESUMO
Physiological processes, occurring as a result of specific receptor stimulation, are generally assessed via molecular biology techniques and microscopic approaches with the involvement of specific molecular markers. The recent progress in experimental approaches, allowing the mechanical characterization of individual biological entities, now makes it possible to address cellular processes occurring in individual cells as a result of their stimulation by hormones. Here, we demonstrate that the atomic force microscope (AFM) can be used to mechanically probe individual cells following the activation of the angiotensin-1 receptor, a receptor well known for its role in cell homeostasis regulation. Our goal is to demonstrate that the measurement of cantilever deflection can be used to quantify in real time the mechanical and morphological cell activity associated with the activation of the receptor. By combining the AFM with time-lapse sequences of phase-contrast and confocal micrographs, we show that the angiotensin-1 receptor stimulation with 100 nM angiotensin II produces an actin-dependent contractile response with an amplitude of 262 +/- 52 nm. We validated the mechanical origin of the responses by measuring the elastic modulus of the cell from indentation experiments performed at 30-s intervals. Additionally, nanoscaled height fluctuations of the cell membrane occurring after the initial contraction response could be attributed to an increased actin cytoskeleton activity and remodeling detected by confocal microscopy. Finally, by using inhibitors for specific elements of the angiotensin-1 receptor signaling pathways, we demonstrate that AFM real-time height monitoring allows a read out of the molecular processes responsible for the cell mechanical response.