ABSTRACT
Tryptophan hydroxylases catalyze the first and rate-limiting step in the biosynthesis of serotonin, a well-known neurotransmitter that plays an important role in multiple physiological functions. A reduction of serotonin levels, especially in the brain, can cause dysregulation leading to depression or insomnia. In contrast, overproduction of peripheral serotonin is associated with symptoms like carcinoid syndrome and pulmonary arterial hypertension. Recently, we developed a class of TPH inhibitors based on xanthine-benzimidazoles, characterized by a tripartite-binding mode spanning the binding sites of the cosubstrate pterin and the substrate tryptophan and by chelation of the catalytic iron ion. Herein, we describe the structure-based development of a second generation of xanthine-imidiazopyridines and -imidazothiazoles designed to inhibit TPH1 in the periphery while preventing the interaction with TPH2 in the brain. Lead compound 32 (TPT-004) shows superior pharmacokinetic and pharmacodynamic properties as well as efficacy in preclinical models of peripheral serotonin attenuation and colorectal tumor growth.
Subject(s)
Tryptophan Hydroxylase , Tryptophan , Tryptophan/metabolism , Xanthine , Serotonin/metabolismABSTRACT
Claudins regulate paracellular permeability, contribute to epithelial polarization and are dysregulated during inflammation and carcinogenesis. Variants of the claudin-binding domain of Clostridium perfringens enterotoxin (cCPE) are highly sensitive protein ligands for generic detection of a broad spectrum of claudins. Here, we investigated the preferential binding of YFP- or GST-cCPE fusion proteins to non-junctional claudin molecules. Plate reader assays, flow cytometry and microscopy were used to assess the binding of YFP- or GST-cCPE to non-junctional claudins in multiple in vitro and ex vivo models of human and rat gastrointestinal epithelia and to monitor formation of a tight junction barrier. Furthermore, YFP-cCPE was used to probe expression, polar localization and dysregulation of claudins in patient-derived organoids generated from gastric dysplasia and gastric cancer. Live-cell imaging and immunocytochemistry revealed cell polarity and presence of tight junctions in glandular organoids (originating from intestinal-type gastric cancer and gastric dysplasia) and, in contrast, a disrupted diffusion barrier for granular organoids (originating from discohesive tumor areas). In sum, we report the use of cCPE fusion proteins as molecular probes to specifically and efficiently detect claudin expression, localization and tight junction dysregulation in cell lines, tissue explants and patient-derived organoids of the gastrointestinal tract.
ABSTRACT
Glioblastoma (GBM) heterogeneity, aggressiveness and infiltrative growth drastically limit success of current standard of care drugs and efficacy of various new therapeutic approaches. There is a need for new therapies and models reflecting the complex biology of these tumors to analyze the molecular mechanisms of tumor formation and resistance, as well as to identify new therapeutic targets. We established and screened a panel of 26 patient-derived subcutaneous (s.c.) xenograft (PDX) GBM models on immunodeficient mice, of which 15 were also established as orthotopic models. Sensitivity toward a drug panel, selected for their different modes of action, was determined. Best treatment responses were observed for standard of care temozolomide, irinotecan and bevacizumab. Matching orthotopic models frequently show reduced sensitivity, as the blood-brain barrier limits crossing of the drugs to the GBM. Molecular characterization of 23 PDX identified all of them as IDH-wt (R132) with frequent mutations in EGFR, TP53, FAT1, and within the PI3K/Akt/mTOR pathway. Their expression profiles resemble proposed molecular GBM subtypes mesenchymal, proneural and classical, with pronounced clustering for gene sets related to angiogenesis and MAPK signaling. Subsequent gene set enrichment analysis identified hallmark gene sets of hypoxia and mTORC1 signaling as enriched in temozolomide resistant PDX. In models sensitive for mTOR inhibitor everolimus, hypoxia-related gene sets reactive oxygen species pathway and angiogenesis were enriched. Our results highlight how our platform of s.c. GBM PDX can reflect the complex, heterogeneous biology of GBM. Combined with transcriptome analyses, it is a valuable tool in identification of molecular signatures correlating with monitored responses. Available matching orthotopic PDX models can be used to assess the impact of the tumor microenvironment and blood-brain barrier on efficacy. Our GBM PDX panel therefore represents a valuable platform for screening regarding molecular markers and pharmacologically active drugs, as well as optimizing delivery of active drugs to the tumor.
ABSTRACT
Drug delivery to the brain is limited for most pharmaceuticals by the blood-brain barrier (BBB) where claudin-5 dominates the paraendothelial tightening. For circumventing the BBB, we identified the compound M01 as a claudin-5 interaction inhibitor. M01 causes transient permeabilisation of the BBB depending on the concentration of small molecules in different cell culture models within 3 to 48 h. In mice, brain uptake of fluorescein peaked within the first 3 h after M01 injection and normalised within 48 h. Compared to the cytostatic paclitaxel alone, M01 improved delivery of paclitaxel to mouse brain and reduced orthotopic glioblastoma growth. Results on interactions of M01 with claudin-5 were incorporated into a binding model which suggests association of its aromatic parts with highly conserved residues of the extracellular domain of claudin-5 and adjacent transmembrane segments. Our results indicate the following mode of action: M01 preferentially binds to the extracellular claudin-5 domain, which weakens trans-interactions between adhering cells. Further decrease in membranous claudin-5 levels due to internalization and transcriptional downregulation enables the paracellular passage of small molecules. In summary, the first small molecule is introduced here as a drug enhancer, which specifically permeabilises the BBB for a sufficient interval for allowing neuropharmaceuticals to enter the brain.
Subject(s)
Blood-Brain Barrier , Pharmaceutical Preparations , Animals , Blood-Brain Barrier/metabolism , Brain/metabolism , Claudin-5/metabolism , Mice , Tight Junctions/metabolismABSTRACT
The outcome of stroke is greatly influenced by the state of the blood-brain barrier (BBB). The BBB endothelium is sealed paracellularly by tight junction (TJ) proteins, i.e., claudins (Cldns) and the redox regulator occludin. Functions of Cldn3 and occludin at the BBB are largely unknown, particularly after stroke. We address the effects of Cldn3 deficiency and stress factors on the BBB and its TJs. Cldn3 tightened the BBB for small molecules and ions, limited endothelial endocytosis, strengthened the TJ structure and controlled Cldn1 expression. After middle cerebral artery occlusion (MCAO) and 3-h reperfusion or hypoxia of isolated brain capillaries, Cldn1, Cldn3 and occludin were downregulated. In Cldn3 knockout mice (C3KO), the reduction in Cldn1 was even greater and TJ ultrastructure was impaired; 48 h after MCAO of wt mice, infarct volumes were enlarged and edema developed, but endothelial TJs were preserved. In contrast, junctional localization of Cldn5 and occludin, TJ density, swelling and infarction size were reduced in affected brain areas of C3KO. Taken together, Cldn3 and occludin protect TJs in stroke, and this keeps the BBB intact. However, functional Cldn3, Cldn3-regulated TJ proteins and occludin promote edema and infarction, which suggests that TJ modulation could improve the outcome of stroke.
Subject(s)
Blood-Brain Barrier/physiopathology , Brain Ischemia/physiopathology , Edema/physiopathology , Stroke/physiopathology , Animals , Humans , Male , Mice , Tight Junctions/metabolismABSTRACT
Improving diagnostic imaging and therapy by targeted compound delivery to pathological areas and across biological barriers is of urgent need. A lipopeptide, P-CrA-A2, composed of a highly cationic peptide sequence (A2), an N-terminally attached palmitoyl chain (P) and cryptophane molecule (CrA) for preferred uptake into blood-brain barrier (BBB) capillary endothelial cells, was generated. CrA allows reversible binding of Xe for NMR detection with hyperpolarized nuclei. The lipopeptide forms size-optimized micelles with a diameter of about 11 nm at low micromolar concentration. Their high local CrA payload has a strong and switchable impact on the bulk magnetization through Hyper-CEST detection. Covalent fixation of CrA does not impede micelle formation and does not hamper its host functionality but simplifies Xe access to hosts for inducing saturation transfer. Xe Hyper-CEST magnetic resonance imaging (MRI) allows for distinguishing BBB endothelial cells from control aortic endothelial cells, and the small micelle volume with a sevenfold improved CrA-loading density compared to liposomal carriers allows preferred cell labelling with a minimally invasive volume (≈16â¯000-fold more efficient than 19 F cell labelling). Thus, these nanoscopic particles combine selectivity for human brain capillary endothelial cells with great sensitivity of Xe Hyper-CEST MRI and might be a potential MRI tool in brain diagnostics.
Subject(s)
Cytological Techniques/methods , Lipopeptides , Magnetic Resonance Imaging/methods , Micelles , Aorta/cytology , Blood-Brain Barrier/cytology , Cells, Cultured , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , Humans , Lipopeptides/chemistry , Lipopeptides/metabolism , Nanoparticles/chemistry , Nanoparticles/metabolism , Xenon/chemistryABSTRACT
At the blood-brain barrier (BBB), claudin (Cldn)-5 is thought to be the dominant tight junction (TJ) protein, with minor contributions from Cldn3 and -12, and occludin. However, the BBB appears ultrastructurally normal in Cldn5 knock-out mice, suggesting that further Cldns and/or TJ-associated marvel proteins (TAMPs) are involved. Microdissected human and murine brain capillaries, quickly frozen to recapitulate the in vivo situation, showed high transcript expression of Cldn5, -11, -12, and -25, and occludin, but also abundant levels of Cldn1 and -27 in man. Protein levels were quantified by a novel epitope dilution assay and confirmed the respective mRNA data. In contrast to the in vivo situation, Cldn5 dominates BBB expression in vitro, since all other TJ proteins are at comparably low levels or are not expressed. Cldn11 was highly abundant in vivo and contributed to paracellular tightness by homophilic oligomerization, but almost disappeared in vitro. Cldn25, also found at high levels, neither tightened the paracellular barrier nor interconnected opposing cells, but contributed to proper TJ strand morphology. Pathological conditions (in vivo ischemia and in vitro hypoxia) down-regulated Cldn1, -3, and -12, and occludin in cerebral capillaries, which was paralleled by up-regulation of Cldn5 after middle cerebral artery occlusion in rats. Cldn1 expression increased after Cldn5 knock-down. In conclusion, this complete Cldn/TAMP profile demonstrates the presence of up to a dozen TJ proteins in brain capillaries. Mouse and human share a similar and complex TJ profile in vivo, but this complexity is widely lost under in vitro conditions.
Subject(s)
Blood-Brain Barrier , Claudin-5/genetics , Tight Junction Proteins/genetics , Tight Junctions/metabolism , Adult , Animals , Brain/blood supply , Brain/metabolism , Cells, Cultured , Claudin-5/metabolism , Female , Gene Expression , HEK293 Cells , Humans , Male , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Middle Aged , Protein Isoforms/genetics , Protein Isoforms/metabolism , Tight Junction Proteins/metabolism , Tight Junctions/ultrastructureABSTRACT
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.
Subject(s)
Epithelial Cells/drug effects , MARVEL Domain Containing 2 Protein/pharmacology , Tight Junction Proteins/metabolism , Tight Junctions/metabolism , Cell Line, Tumor , Epithelial Cells/metabolism , Fluorescence Resonance Energy Transfer , Humans , Protein Interaction Domains and Motifs , Receptors, LDL/metabolismABSTRACT
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.
Subject(s)
Blood-Brain Barrier/drug effects , Claudin-5/pharmacology , Endothelial Cells/drug effects , Peptidomimetics/pharmacology , Animals , Antibiotics, Antineoplastic/administration & dosage , Antibiotics, Antineoplastic/pharmacokinetics , Blood-Brain Barrier/metabolism , Blood-Brain Barrier/ultrastructure , Brain/drug effects , Brain/metabolism , Cell Line , Cells, Cultured , Circular Dichroism , Claudin-5/chemistry , Claudin-5/pharmacokinetics , Doxorubicin/administration & dosage , Doxorubicin/pharmacokinetics , Drug Delivery Systems/methods , Endothelial Cells/metabolism , Endothelial Cells/ultrastructure , Gadolinium DTPA/administration & dosage , Gadolinium DTPA/pharmacokinetics , HEK293 Cells , Humans , Mice, Inbred C57BL , Microscopy, Confocal , Microscopy, Electron/methods , Models, Molecular , Peptidomimetics/chemistry , Peptidomimetics/pharmacokinetics , Permeability/drug effects , Protein Conformation , Rats , Rhodamines/administration & dosage , Rhodamines/pharmacokinetics , Tight Junctions/drug effects , Tight Junctions/metabolism , Tight Junctions/ultrastructure , Time-Lapse Imaging/methodsABSTRACT
UNLABELLED: Tight junctions (TJs) seal paracellular clefts in epithelia/endothelia and form tissue barriers for proper organ function. TJ-associated marvel proteins (TAMPs; tricellulin, occludin, marvelD3) are thought to be relevant to regulation. Under normal conditions, tricellulin tightens tricellular junctions against macromolecules. Traces of tricellulin occur in bicellular junctions. AIMS: As pathological disturbances have not been analyzed, the structure and function of human tricellulin, including potentially redox-sensitive Cys sites, were investigated under reducing/oxidizing conditions at 3- and 2-cell contacts. RESULTS: Ischemia, hypoxia, and reductants redistributed tricellulin from 3- to 2-cell contacts. The extracellular loop 2 (ECL2; conserved Cys321, Cys335) trans-oligomerized between three opposing cells. Substitutions of these residues caused bicellular localization. Cys362 in transmembrane domain 4 contributed to bicellular heterophilic cis-interactions along the cell membrane with claudin-1 and marvelD3, while Cys395 in the cytosolic C-terminal tail promoted homophilic tricellullar cis-interactions. The Cys sites included in homo-/heterophilic bi-/tricellular cis-/trans-interactions contributed to cell barrier tightness for small/large molecules. INNOVATION: Tricellulin forms TJs via trans- and cis-association in 3-cell contacts, as demonstrated electron and quantified fluorescence microscopically; it tightens 3- and 2-cell contacts. Tricellulin's ECL2 specifically seals 3-cell contacts redox dependently; a structural model is proposed. CONCLUSIONS: TAMP ECL2 and claudins' ECL1 share functionally and structurally similar features involved in homo-/heterophilic tightening of cell-cell contacts. Tricellulin is a specific redox sensor and sealing element at 3-cell contacts and may compensate as a redox mediator for occludin loss at 2-cell contacts in vivo and in vitro. Molecular interaction mechanisms were proposed that contribute to tricellulin's function. In conclusion, tricellulin is a junctional redox regulator for ischemia-related alterations.
Subject(s)
Cysteine/metabolism , Ischemia/metabolism , Kidney/blood supply , MARVEL Domain Containing 2 Protein/metabolism , Occludin/metabolism , Tight Junctions/metabolism , Animals , Binding Sites , Cell Hypoxia , Cell Membrane Permeability , Dogs , Epithelial Cells/physiology , HEK293 Cells , Humans , Ischemia/pathology , Kidney/metabolism , Kidney/pathology , MARVEL Domain Containing 2 Protein/chemistry , Madin Darby Canine Kidney Cells , Male , Mice, Inbred C57BL , Oxidation-Reduction , Oxidative Stress , Protein Folding , Protein Interaction Domains and Motifs , Protein TransportABSTRACT
The blood-brain barrier (BBB) is formed by microvascular endothelial cells sealed by tetraspanning tight junction (TJ) proteins, such as claudins and TAMPs (TJ-associated marvel proteins, occludin and tricellulin). Claudins are the major components of the TJs. At the BBB, claudin-5 dominates the TJs by preventing the paracellular permeation of small molecules. On the other hand, TAMPs regulate the structure and function of the TJs; tricellulin may tighten the barrier for large molecules. This review aims at integrating and summarizing the most relevant and recent work on how the BBB is influenced by claudin-1, -3, -5, -12 and the TAMPs occludin and tricellulin, all of which are four-transmembrane TJ proteins. The exact functions of claudin-1, -3, -12 and TAMPs at this barrier still need to be elucidated.
Subject(s)
Blood-Brain Barrier/cytology , Blood-Brain Barrier/physiology , Membrane Proteins/metabolism , Tight Junctions/physiology , Animals , Biological Transport , Endothelial Cells/metabolism , Humans , Tight Junctions/ultrastructureABSTRACT
A limitation in the uptake of many drugs is the restricted permeation through tissue barriers. There are two general ways to cross barriers formed by cell layers: by transcytosis or by diffusion through the intercellular space. In the latter, tight junctions (TJs) play the decisive role in the regulation of the barrier permeability. Thus, transient modulation of TJs is a potent strategy to improve drug delivery. There have been extensive studies on surfactant-like absorption enhancers. One of the most effective enhancers found is sodium caprate. However, this modulates TJs in an unspecific fashion. A novel approach would be the specific modulation of TJ-associated marvel proteins and claudins, which are the main structural components of the TJs. Recent studies have identified synthetic peptidomimetics and RNA interference techniques to downregulate the expression of targeted TJ proteins. This review summarizes current progress and discusses the impact on TJs' barrier function.
ABSTRACT
BACKGROUND: Claudins, a family of protein localized in tight junctions, are essential for the control of paracellular permeation in epithelia and endothelia. The interaction of several claudins with Clostridium perfringens enterotoxin (CPE) has been exploited for an affinity-based enrichment of CPE-binding claudins from lysates of normal rat cholangiocytes. RESULTS: Immunoblotting and mass spectrometry (MS) experiments demonstrate strong enrichment of the CPE-binding claudins -3, -4 and -7, indicating specific association with glutathione-S-transferase (GST)-CPE(116-319) fusion protein. In parallel, the co-elution of (non-CPE-binding) claudin-1 and claudin-5 was observed. The complete set of co-enriched proteins was identified by MS after electrophoretic separation. Relative mass spectrometric protein quantification with stable isotope labeling with amino acids in cell culture (SILAC) made it possible to discriminate specific binding from non-specific association to GST and/or matrix material. CONCLUSION: CPE(116-319) provides an efficient tool for single step enrichment of different claudins from cell lysates. Numerous proteins were shown to be co-enriched with the CPE-binding claudins, but there are no indications (except for claudins -1 and -5) for an association with tight junctions.
Subject(s)
Clostridium perfringens/metabolism , Enterotoxins/metabolism , Membrane Proteins/isolation & purification , Tight Junctions/metabolism , Animals , Cell Line , Chromatography, Affinity , Mass Spectrometry , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Protein Binding , RatsABSTRACT
Most claudins are tight junction (TJ)-forming proteins. However, their interaction on the molecular level remains unresolved. It is hypothesized that the extracellular loops specify these claudin functions. It is assumed that the first extracellular loop (ECL1) is critical for determining the paracellular tightness and the selective paracellular ion permeability, and that the second extracellular loop may cause narrowing of the paracellular cleft. Using a combination of site-directed mutagenesis and homology modeling for the second extracellular loop (ECL2) of claudin-5, we found several amino acids important for claudin folding and/or trans-interaction to claudins in neighboring cells. These sensitive residues are highly conserved within one group of claudins, whereas the corresponding positions in the remaining claudins show a large sequence variety. Further functional data and analysis of sequence similarity for all claudins has led to their differentiation into two groups, designated as classic claudins (1-10, 14, 15, 17, 19) and nonclassic claudins (11-13, 16, 18, 20-24). This also corresponds to conserved structural features at ECL1 for classic claudins. Based on this, we propose a hypothesis for different pore-forming claudins. Pore formation or tightness is supported by the spatial encounter of a surplus of repulsing or attracting amino acid types at ECL1. A pore is likely opened by repulsion of equally charged residues, while an encounter of unequally charged residues leads to tight interaction. These considerations may reveal the ECLs of claudins as decisive submolecular determinants that specify the function of a claudin.
Subject(s)
Membrane Proteins/chemistry , Membrane Proteins/metabolism , Amino Acid Sequence , Cells, Cultured , Claudin-5 , Humans , Membrane Proteins/genetics , Models, Biological , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Phylogeny , Protein Folding , Protein Structure, Tertiary , Sequence Alignment , Structure-Activity Relationship , Tight Junctions/metabolismABSTRACT
Clostridium perfringens enterotoxin (CPE) binds to the extracellular loop 2 of a subset of claudins, e.g. claudin-3. Here, the molecular mechanism of the CPE-claudin interaction was analyzed. Using peptide arrays, recombinant CPE-(116-319) bound to loop 2 peptides of mouse claudin-3, -6, -7, -9, and -14 but not of 1, 2, 4, 5, 8, 10-13, 15, 16, 18-20, and 22. Substitution peptide mapping identified the central motif (148)NPL(150)VP, supposed to represent a turn region in the loop 2, as essential for the interaction between CPE and murine claudin-3 peptides. CPE-binding assays with claudin-3 mutant-transfected HEK293 cells or lysates thereof demonstrated the involvement of Asn(148) and Leu(150) of full-length claudin-3 in the binding. CPE-(116-319) and CPE-(194-319) bound to HEK293 cells expressing claudin-3, whereas CPE-(116-319) bound to claudin-5-expressing HEK293 cells, also. This binding was inhibited by substitutions T151A and Q156E in claudin-5. In contrast, removal of the aromatic side chains in the loop 2 of claudin-3 and -5, involved in trans-interaction between claudins, increased the amount of CPE-(116-319) bound. These findings and molecular modeling indicate different molecular mechanisms of claudin-claudin trans-interaction and claudin-CPE interaction. Confocal microscopy showed that CPE-(116-319) and CPE-(194-319) bind to claudin-3 at the plasma membrane, outside cell-cell contacts. Together, these findings demonstrate that CPE binds to the hydrophobic turn and flanking polar residues in the loop 2 of claudin-3 outside tight junctions. The data can be used for the specific design of CPE-based modulators of tight junctions, to improve drug delivery, and as chemotherapeutics for tumors overexpressing claudins.
Subject(s)
Clostridium perfringens/metabolism , Enterotoxins/metabolism , Membrane Proteins/physiology , Amino Acid Motifs , Amino Acid Sequence , Animals , Caco-2 Cells , Cell Line , Claudin-3 , Humans , Membrane Proteins/metabolism , Mice , Molecular Sequence Data , Peptides/chemistry , Protein Binding , Surface Plasmon Resonance , Tight Junctions/metabolismABSTRACT
Claudins are tetraspan transmembrane proteins of tight junctions. They determine the barrier properties of this type of cell-cell contact existing between the plasma membranes of two neighbouring cells, such as occurring in endothelia or epithelia. Claudins can completely tighten the paracellular cleft for solutes, and they can form paracellular ion pores. It is assumed that the extracellular loops specify these claudin functions. It is hypothesised that the larger first extracellular loop is critical for determining the paracellular tightness and the selective ion permeability. The shorter second extracellular loop may cause narrowing of the paracellular cleft and have a holding function between the opposing cell membranes. Sequence analysis of claudins has led to differentiation into two groups, designated as classic claudins (1-10, 14, 15, 17, 19) and non-classic claudins (11-13, 16, 18, 20-24), according to their degree of sequence similarity. This is also reflected in the derived sequence-structure function relationships for extracellular loops 1 and 2. The concepts evolved from these findings and first tentative molecular models for homophilic interactions may explain the different functional contribution of the two extracellular loops at tight junctions.
Subject(s)
Membrane Proteins/chemistry , Membrane Proteins/physiology , Tight Junctions/physiology , Amino Acid Sequence , Animals , Humans , Membrane Proteins/genetics , Models, Molecular , Molecular Sequence Data , Molecular Structure , Multiprotein Complexes , Phylogeny , Protein Processing, Post-Translational , Sequence Homology, Amino AcidABSTRACT
Claudins are the critical transmembrane proteins in tight junctions. Claudin-5, for instance, prevents paracellular permeation of small molecules. However, the molecular interaction mechanism is unknown. Hence, the claudin-claudin interaction and tight junction strand formation were investigated using systematic single mutations. Claudin-5 mutants transfected into tight junction-free cells demonstrated that the extracellular loop 2 is involved in strand formation via trans-interaction, but not via polymerization, along the plasma membrane of one cell. Three phenotypes were obtained: the tight junction type (wild-type-like trans- and cis-interaction; the disjunction type (blocked trans-interaction); the intracellular type (disturbed folding). Combining site-directed mutagenesis, live-cell imaging-, electron microscopy-, and molecular modeling data led to an antiparallel homodimer homology model of the loop. These data for the first time explain how two claudins hold onto each other and constrict the paracellular space. The intermolecular interface includes aromatic (F147, Y148, Y158) and hydrophilic (Q156, E159) residues. The aromatic residues form a strong binding core between two loops from opposing cells. Since nearly all these residues are conserved in most claudins, our findings are of general relevance for all classical claudins. On the basis of the data we have established a novel molecular concept for tight junction formation.
Subject(s)
Membrane Proteins/metabolism , Tight Junctions , Amino Acid Substitution , Cell Line , Claudin-5 , Fluorescence Resonance Energy Transfer , Humans , Immunohistochemistry , Membrane Proteins/chemistry , Membrane Proteins/genetics , Microscopy, Electron, Transmission , Mutagenesis, Site-Directed , Subcellular Fractions/metabolismABSTRACT
Bone cells seeded directly on depots of bone morphogenetic protein-2 (BMP-2) increase alkaline phosphatase (ALP) expression. Heating of such BMP-2 depots to 100 degrees C augmented the intensity of this local ALP induction. To understand this unexpected finding, we investigated the effect of heat treatment on BMP-2 depots more closely. Using a novel bioassay based on ALP-induction of remote cells, we found that the amount of released bioactive BMP-2 from heat-treated depots decays within days and could be described by an exponential function. From this function, we expected that pre-incubation of BMP-2 depots in culture medium for 4 weeks renders them insufficient to induce ALP. However, preincubated, heat-treated depots still induced maximal ALP, unless treated with the selective BMP-2 inhibitor noggin. Furthermore, heat treatment of BMP-2 depots generated a layer of immunoreactive BMP-2 at the surface of the carrier. In contrast, BMP-2 was washed off completely if heat treatment of adsorbed protein was omitted. Results show that heat treatment generates both a soluble pool of BMP-2 and a material-bound layer of BMP-2 in which the protein is protected against degradation. Therefore, heat treatment appears useful to locally immobilize BMP-2 on various implant surfaces.
Subject(s)
Bacterial Proteins/biosynthesis , Bone Morphogenetic Proteins , Bone and Bones/enzymology , Coated Materials, Biocompatible , Endopeptidases/biosynthesis , Prostheses and Implants , Transforming Growth Factor beta , Bone Morphogenetic Protein 2 , Bone Morphogenetic Proteins/chemistry , Bone and Bones/cytology , Cell Culture Techniques/methods , Cell Line , Delayed-Action Preparations/chemistry , Hot Temperature , Humans , Time Factors , Transforming Growth Factor beta/chemistryABSTRACT
The transcription factor RovA of Yersinia pseudotuberculosis and analogous proteins in other Enterobacteriaceae activate the expression of virulence genes that play a crucial role in stress adaptation and pathogenesis. In this study, we demonstrate that the RovA protein forms dimers independent of DNA binding, stimulates RNA polymerase, most likely via its C-terminal domain, and counteracts transcriptional repression by the histone-like protein H-NS. As the molecular function of the RovA family is largely uncharacterized, random mutagenesis and terminal deletions were used to identify functionally important domains. Our analysis showed that a winged-helix motif in the center of the molecule is essential and directly involved in DNA binding. Terminal deletions and amino acid changes within both termini also abrogate RovA activation and DNA-binding functions, most likely due to their implication in dimer formation. Finally, we show that the last four amino acids of RovA are crucial for activation of gene transcription. Successive deletions of these residues result in a continuous loss of RovA activity. Their removal reduced the capacity of RovA to activate RNA polymerase and abolished transcription of RovA-activated promoters in the presence of H-NS, although dimerization and DNA binding functions were retained. Our structural model implies that the final amino acids of RovA play a role in protein-protein interactions, adjusting RovA activity.