RESUMEN
The majority of tight junction (TJ) proteins restrict the paracellular permeation of solutes via their extracellular loops (ECLs). Tricellulin tightens tricellular TJs (tTJs) and regulates bicellular TJ (bTJ) proteins. We demonstrate that the addition of recombinantly produced extracellular loop 2 (ECL2) of tricellulin opens cellular barriers. The peptidomimetic trictide, a synthetic peptide derived from tricellulin ECL2, increases the passage of ions, as well as of small and larger molecules up to 10 kDa, between 16 and 30 h after application to human epithelial colorectal adenocarcinoma cell line 2. Tricellulin and lipolysis-stimulated lipoprotein receptor relocate from tTJs toward bTJs, while the TJ proteins claudin-1 and occludin redistribute from bTJs to the cytosol. Analyzing the opening of the tricellular sealing tube by the peptidomimetic using super-resolution stimulated-emission depletion microscopy revealed a tricellulin-free area at the tricellular region. Cis-interactions (as measured by fluorescence resonance energy transfer) of tricellulin-tricellulin (tTJs), tricellulin-claudin-1, tricellulin-marvelD3, and occludin-occludin (bTJs) were strongly affected by trictide treatment. Circular dichroism spectroscopy and molecular modeling suggest that trictide adopts a ß-sheet structure, resulting in a peculiar interaction surface for its binding to tricellulin. In conclusion, trictide is a novel and promising tool for overcoming cellular barriers at bTJs and tTJs with the potential to transiently improve drug delivery.
Asunto(s)
Células Epiteliales/efectos de los fármacos , Proteína 2 con Dominio MARVEL/farmacología , Proteínas de Uniones Estrechas/metabolismo , Uniones Estrechas/metabolismo , Línea Celular Tumoral , Células Epiteliales/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Humanos , Dominios y Motivos de Interacción de Proteínas , Receptores de LDL/metabolismoRESUMEN
The blood-brain barrier (BBB) formed by the microvascular endothelium limits cerebral drug delivery. The paraendothelial cleft is sealed by tight junctions (TJs) with a major contribution from claudin-5, which we selected as a target to modulate BBB permeability. For this purpose, drug-enhancer peptides were designed based on the first extracellular loop (ECL) of claudin-5 to allow transient BBB permeabilization. Peptidomimetics (C5C2 and derivatives, nanomolar affinity to claudin-5) size-selectively (≤40 kDa) and reversibly (12-48 h) increased the permeability of brain endothelial and claudin-5-transfected epithelial cell monolayers. Upon peptide uptake, the number of TJ strand particles diminished, claudin-5 was downregulated and redistributed from cell-cell contacts to the cytosol, and the cell shape was altered. Cellular permeability of doxorubicin (cytostatic drug, 580 Da) was enhanced after peptide administration. Mouse studies (3.5 µmol/kg i.v.) confirmed that, for both C5C2 and a d-amino acid derivative, brain uptake of Gd-diethylene-triamine penta-acetic acid (547 Da) was enhanced within 4 h of treatment. On the basis of our functional data, circular dichroism measurements, molecular modeling, and docking experiments, we suggest an association model between ß-sheets flanked by α-helices, formed by claudin-5 ECLs, and the peptides. In conclusion, we identified claudin-5 peptidomimetics that improve drug delivery through endothelial and epithelial barriers expressing claudin-5.
Asunto(s)
Barrera Hematoencefálica/efectos de los fármacos , Claudina-5/farmacología , Células Endoteliales/efectos de los fármacos , Peptidomiméticos/farmacología , Animales , Antibióticos Antineoplásicos/administración & dosificación , Antibióticos Antineoplásicos/farmacocinética , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/ultraestructura , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Línea Celular , Células Cultivadas , Dicroismo Circular , Claudina-5/química , Claudina-5/farmacocinética , Doxorrubicina/administración & dosificación , Doxorrubicina/farmacocinética , Sistemas de Liberación de Medicamentos/métodos , Células Endoteliales/metabolismo , Células Endoteliales/ultraestructura , Gadolinio DTPA/administración & dosificación , Gadolinio DTPA/farmacocinética , Células HEK293 , Humanos , Ratones Endogámicos C57BL , Microscopía Confocal , Microscopía Electrónica/métodos , Modelos Moleculares , Peptidomiméticos/química , Peptidomiméticos/farmacocinética , Permeabilidad/efectos de los fármacos , Conformación Proteica , Ratas , Rodaminas/administración & dosificación , Rodaminas/farmacocinética , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Uniones Estrechas/ultraestructura , Imagen de Lapso de Tiempo/métodosRESUMEN
In epithelial/endothelial barriers, claudins form tight junctions, seal the paracellular cleft, and limit the uptake of solutes and drugs. The peptidomimetic C1C2 from the C-terminal half of claudin-1's first extracellular loop increases drug delivery through epithelial claudin-1 barriers. However, its molecular and structural mode of action remains unknown. In the present study, >100 µM C1C2 caused paracellular opening of various barriers with different claudin compositions, ranging from epithelial to endothelial cells, preferentially modulating claudin-1 and claudin-5. After 6 h incubation, C1C2 reversibly increased the permeability to molecules of different sizes; this was accompanied by redistribution of claudins and occludin from junctions to cytosol. Internalization of C1C2 in epithelial cells depended on claudin-1 expression and clathrin pathway, whereby most C1C2 was retained in recyclosomes >2 h. In freeze-fracture electron microscopy, C1C2 changed claudin-1 tight junction strands to a more parallel arrangement and claudin-5 strands from E-face to P-face association - drastic and novel effects. In conclusion, C1C2 is largely recycled in the presence of a claudin, which explains the delayed onset of barrier and junction loss, the high peptide concentration required and the long-lasting effect. Epithelial/endothelial barriers are specifically modulated via claudin-1/claudin-5, which can be targeted to improve drug delivery.
Asunto(s)
Permeabilidad de la Membrana Celular/fisiología , Células Endoteliales/metabolismo , Células Epiteliales/metabolismo , Peptidomiméticos/metabolismo , Proteínas de Uniones Estrechas/metabolismo , Uniones Estrechas/metabolismo , Células CACO-2 , Células HEK293 , HumanosRESUMEN
UNLABELLED: The paracellular cleft within epithelia/endothelia is sealed by tight junction (TJ) proteins. Their extracellular loops (ECLs) are assumed to control paracellular permeability and are targets of pathogenes. We demonstrated that claudin-1 is crucial for paracellular tightening. Its ECL1 is essential for the sealing and contains two cysteines conserved throughout all claudins. AIMS: We prove the hypothesis that this cysteine motif forms a redox-sensitive intramolecular disulfide bridge and, hence, the claudin-1-ECL1 constitutes a functional structure which is associated to ECLs of this and other TJ proteins. RESULTS: The structure and function of claudin-1-ECL1 was elucidated by investigating sequences of this ECL as synthetic peptides, C1C2, and as recombinant proteins, and exhibited a ß-sheet binding surface flanked by an α-helix. These sequences bound to different claudins, their ECL1, and peptides with nanomolar binding constants. C-terminally truncated C1C2 (-4aaC) opened cellular barriers and the perineurium. Recombinant ECL1 formed oligomers, and bound to claudin-1 expressing cells. Oligomerization and claudin association were abolished by reducing agents, indicating intraloop disulfide bridging and redox sensitivity. INNOVATION: The structural and functional model based on our in vitro and in vivo investigations suggested that claudin-1-ECL1 constitutes a functional and ECL-binding ß-sheet, stabilized by a shielded and redox-sensitive disulfide bond. CONCLUSION: Since the ß-sheet represents a consensus sequence of claudins and further junctional proteins, a general structural feature is implied. Therefore, our model is of general relevance for the TJ assembly in normal and pathological conditions. C1C2-4aaC is a new drug enhancer that is used to improve pharmacological treatment through tissue barriers.
Asunto(s)
Claudina-1/química , Claudina-1/metabolismo , Animales , Western Blotting , Línea Celular , Electroforesis en Gel de Poliacrilamida , Humanos , Inmunohistoquímica , Inmunoprecipitación , Oxidación-Reducción , Estructura Secundaria de Proteína , Ratas , Ratas Wistar , Uniones Estrechas/metabolismoRESUMEN
The blood-nerve barrier consists of the perineurium and endoneurial vessels. The perineurial barrier is composed of a basal membrane and a layer of perineurial cells sealed by tight junction proteins preventing e.g. application of analgesics for selective regional pain control. One of the barrier-sealing proteins in the blood-nerve barrier is claudin-1. Therefore, the claudin-1-peptidomimetics (C1C2), derived from the first extracellular loop (ECL1) on claudin-1 was developed. In this study, we further evaluated the expression of tight junction proteins in the perineurium in Wistar rats and characterized the specificity, in vivo applicability, mechanism of action as well as the biocompatibility of C1C2. In the perineurium, claudin-19, tricellulin and ZO-1, but no claudin-2, 3, 8 and -11 were expressed. C1C2 specifically bound to the ECL1 of claudin-1 and fluorescent 5,6-carboxytetramethylrhodamine-C1C2 was rapidly internalized. Opening the perineurium with C1C2 reduced the mRNA and protein expression of claudin-1 and increased small and macromolecule permeability into the peripheral nerve. Application of C1C2 facilitated regional analgesia using µ-opioid receptor agonists like DAMGO or morphine without motor impairment in naïve rats as well as rats with hind paw inflammation. In contrast the control peptide C2C2 derived from ECL1 on claudin-2 did neither open the barrier nor facilitated opioid-mediated regional analgesia. C1C2 delivery was well tolerated and caused no morphological and functional nerve damage. C1C2 effects could be reversed by interference with the wnt-signal-transduction pathway, specifically the homeobox transcription factor cdx2, using a glycogen-synthase-kinase-3 inhibitor. In summary, we describe the composition of and a pathway to open the perineurial barrier employing a peptide to deliver hydrophilic substances to the peripheral nerve.
Asunto(s)
Claudina-1/química , Claudina-1/farmacología , Péptidos/química , Péptidos/farmacología , Nervios Periféricos/efectos de los fármacos , Permeabilidad/efectos de los fármacos , Secuencia de Aminoácidos , Analgesia , Analgésicos Opioides/administración & dosificación , Analgésicos Opioides/farmacología , Animales , Línea Celular , Claudina-1/metabolismo , Humanos , Masculino , Datos de Secuencia Molecular , Nervios Periféricos/metabolismo , Ratas , Ratas Wistar , Nervio Ciático/efectos de los fármacos , Nervio Ciático/metabolismo , Uniones Estrechas/metabolismoRESUMEN
Claudin proteins are involved in the paracellular tightening of epithelia and endothelia. Their internalization, which can be modulated by extracellular stimuli, for example, proinflammatory cytokines, is a prerequisite for the regulation of the paracellular barrier to allow, for instance, cell migration or drug delivery. The internalization of peptide sequences of claudins is completely unknown. Here, we studied the internalization of two peptides, TAMRA-claudin-1 and TAMRA-claudin-5, derivatives of the extracellular loop of claudin-1 and -5, respectively, in either epithelial or endothelial cells. The cellular uptake of the claudin-1 peptide follows the clathrin-mediated endocytosis as indicated by inhibitors and respective tracers for colocalization. In addition, macropinocytosis and caveolae-mediated endocytosis of the peptide was observed. In contrast, the claudin-5 peptide is mainly internalized via the caveolae-mediated endocytosis evidenced by the colocalization with respective tracers and vesicle markers, whereas the nonselective macropinocytosis seems to be involved in a less effective manner. In conclusion, the assumption is supported that claudin peptides can be internalized by specific and nonspecific pathways.
Asunto(s)
Caveolas/metabolismo , Clatrina/metabolismo , Claudinas/metabolismo , Endocitosis/fisiología , Péptidos/metabolismo , Uniones Estrechas/metabolismo , Caveolas/fisiología , Técnicas de Cultivo de Célula , Clatrina/fisiología , Técnica del Anticuerpo Fluorescente , Células HEK293 , HumanosRESUMEN
The paracellular flux of solutes through tissue barriers is limited by transmembrane tight junction proteins. Within the family of tight junction proteins, claudin-1 seems to be a key protein for tightness formation and integrity. In the peripheral nervous system, the nerve fibers are surrounded with a barrier formed by the perineurium which expresses claudin-1. To enhance the access of hydrophilic pharmaceutical agents via the paracellular route, a claudin-1 specific modulator was developed. For this purpose, we designed and investigated the claudin-1 derived peptide C1C2. It transiently increased the paracellular permeability for ions and high and low molecular weight compounds through a cellular barrier model. Structural studies revealed a ß-sheet potential for the functionality of the peptide. Perineurial injection of C1C2 in rats facilitated the effect of hydrophilic antinociceptive agents and raised mechanical nociceptive thresholds. The mechanism is related to the internalization of C1C2 and to a vesicle-like distribution within the cells. The peptide mainly colocalized with intracellular claudin-1. C1C2 decreased membrane-localized claudin-1 of cells in culture and in vivo in the perineurium of rats after perineurial injection. In conclusion, a novel tool was developed to improve the delivery of pharmaceutical agents through the perineurial barrier by transient modulation of claudin-1.