Paralogous chemoreceptors mediate chemotaxis towards protein amino acids and the non‐protein amino acid gamma‐aminobutyrate (GABA)

The paralogous receptors PctA, PctB and PctC of Pseudomonas aeruginosa were reported to mediate chemotaxis to amino acids, intermediates of amino acid metabolism and chlorinated hydrocarbons. We show that the recombinant ligand binding regions (LBRs) of PctA, PctB and PctC bind 17, 5 and 2 l ‐amino acids respectively. In addition, PctC‐LBR recognized GABA but not any other structurally related compound. l ‐Gln, one of the three amino acids that is not recognized by PctA‐LBR, was the most tightly binding ligand to PctB suggesting that PctB has evolved to mediate chemotaxis primarily towards l ‐Gln. Bacteria were efficiently attracted to l ‐Gln and GABA, but mutation of pctB and pctC, respectively, abolished chemoattraction. The physiological relevance of taxis towards GABA is proposed to reside in an interaction with plants. LBRs were predicted to adopt double PDC (P hoQ/D cuS/C itA) like structures and site‐directed mutagenesis studies showed that ligands bind to the membrane‐distal module. Analytical ultracentrifugation studies have shown that PctA‐LBR and PctB‐LBR are monomeric in the absence and presence of ligands, which is in contrast to the enterobacterial receptors that require sensor domain dimers for ligand recognition.     

Structural conservation of Notch receptors and ligands

The Notch signalling pathway is an evolutionarily conserved cell-to-cell communication system utilized multiple times and in many tissues during development. The outcome of an interaction between Notch and its ligands is highly influenced by factors both extrinsic and intrinsic to Notch expressing cells, suggesting that Notch functions either directly or in parallel with other signalling systems to regulate cellular differentiation events. Protein domains common to all ligands and receptors of this system suggest conserved functional properties that likely relate to regulatory mechanisms for Notch signalling. Within this review, the known functional properties of these domains are analyzed with respect to their contributions to ligand/receptor interactions and Notch signalling.     

Spatial organization of transmembrane receptor signalling

The spatial organization of transmembrane receptors is a critical step in signal transduction and receptor trafficking in cells. Transmembrane receptors engage in lateral homotypic and heterotypic cis‐interactions as well as intercellular trans‐interactions that result in the formation of signalling foci for the initiation of different signalling networks. Several aspects of ligand‐induced receptor clustering and association with signalling proteins are also influenced by the lipid composition of membranes. Thus, lipid microdomains have a function in tuning the activity of many transmembrane receptors by positively or negatively affecting receptor clustering and signal transduction. We review the current knowledge about the functions of clustering of transmembrane receptors and lipid–protein interactions important for the spatial organization of signalling at the membrane.     

Lateral density of receptor arrays in the membrane plane influences sensitivity of the E. coli chemotaxis response

In chemotactic bacteria, transmembrane chemoreceptors, CheA and CheW form the core signalling complex of the chemotaxis sensory apparatus. These complexes are organized in extended arrays in the cytoplasmic membrane that allow bacteria to respond to changes in concentration of extracellular ligands via a cooperative, allosteric response that leads to substantial amplification of the signal induced by ligand binding. Here, we have combined cryo-electron tomographic studies of the 3D spatial architecture of chemoreceptor arrays in intact E. coli cells with computational modelling to develop a predictive model for the cooperativity and sensitivity of the chemotaxis response. The predictions were tested experimentally using fluorescence resonance energy transfer (FRET) microscopy. Our results demonstrate that changes in lateral packing densities of the partially ordered, spatially extended chemoreceptor arrays can modulate the bacterial chemotaxis response, and that information about the molecular organization of the arrays derived by cryo-electron tomography of intact cells can be translated into testable, predictive computational models of the chemotaxis response.     

Adapt locally and act globally: strategy to maintain high chemoreceptor sensitivity in complex environments

In bacterial chemotaxis, several types of ligand‐specific receptors form mixed clusters, wherein receptor–receptor interactions lead to signal amplification and integration. However, it remains unclear how a mixed receptor cluster adapts to individual stimuli and whether it can differentiate between different types of ligands. Here, we combine theoretical modeling with experiments to reveal the adaptation dynamics of the mixed chemoreceptor cluster in Escherichia coli. We show that adaptation occurs locally and is ligand‐specific: only the receptor that binds the external ligand changes its methylation level when the system adapts, whereas other types of receptors change methylation levels transiently. Permanent methylation crosstalk occurs when the system fails to adapt accurately. This local adaptation mechanism enables cells to differentiate individual stimuli by encoding them into the methylation levels of corresponding types of chemoreceptors. It tunes each receptor to its most responsive state to maintain high sensitivity in complex environments and prevents saturation of the cluster by one signal.     

Ionotropic and metabotropic mechanisms in chemoreception: ‘chance or design'?

Chemosensory receptors convert an enormous diversity of chemical signals from the external world into a common language of electrical activity in the brain. Mammals and insects use several families of transmembrane receptor proteins to recognize distinct classes of volatile and non‐volatile chemicals that are produced by conspecifics or other environmental sources. A comparison of the signalling mechanisms of mammalian and insect receptors has revealed an unexpected functional distinction: mammals rely almost exclusively on metabotropic ligand‐binding receptors, which use second messenger signalling cascades to indirectly activate ion channels, whereas insects use ionotropic receptors, which are gated directly by chemical stimuli, thereby leading to neuronal depolarization. In this review, we consider possible reasons for this dichotomy, taking into account biophysical, cell biological, ecological and evolutionary influences on how information is extracted from chemosensory cues by these animal classes.     

Deficiency in mTORC1‐controlled C/EBPβ‐mRNA translation improves metabolic health in mice

The mammalian target of rapamycin complex 1 (mTORC1) is a central regulator of physiological adaptations in response to changes in nutrient supply. Major downstream targets of mTORC1 signalling are the mRNA translation regulators p70 ribosomal protein S6 kinase 1 (S6K1p70) and the 4E‐binding proteins (4E‐BPs). However, little is known about vertebrate mRNAs that are specifically controlled by mTORC1 signalling and are engaged in regulating mTORC1‐associated physiology. Here, we show that translation of the CCAAT/enhancer binding protein beta (C/EBPβ) mRNA into the C/EBPβ‐LIP isoform is suppressed in response to mTORC1 inhibition either through pharmacological treatment or through calorie restriction. Our data indicate that the function of 4E‐BPs is required for suppression of LIP. Intriguingly, mice lacking the cis‐regulatory upstream open reading frame (uORF) in the C/EBPβ‐mRNA, which is required for mTORC1‐stimulated translation into C/EBPβ‐LIP, display an improved metabolic phenotype with features also found under calorie restriction. Thus, our data suggest that translational adjustment of C/EBPβ‐isoform expression is one of the key processes that direct metabolic adaptation in response to changes in mTORC1 activity.     

Chemoreceptors in signalling complexes: shifted conformation and asymmetric coupling

Bacterial chemotaxis is mediated by signalling complexes of chemoreceptors, histidine kinase CheA and coupling protein CheW. Interactions in complexes profoundly affect the kinase. We investigated effects of these interactions on chemoreceptors by comparing receptors alone and in complexes. Assays of initial rates of methylation indicated that signalling complexes shifted receptor conformation towards the methylation-on, higher-ligand-affinity, kinase-off state, tuning receptors for greater sensitivity. In contrast, transmembrane and conformational signalling within chemoreceptors was essentially unaltered, consistent with other evidence identifying receptor dimers as the fundamental units of such signalling. In signalling complexes, coupling of ligand binding to kinase activity is cooperative and the dynamic range of kinase control expanded > 100-fold by receptor adaptational modification. We observed no cooperativity in influence of ligand on receptor conformation, only on kinase activity. However, receptor modification generated increased dynamic range in a stepwise fashion, partly in coupling ligand to receptor conformation and partly in coupling receptor conformation to kinase activity. Thus, receptors and kinase were not equivalently affected by interactions in signalling complexes or by ligand binding and adaptational modification, indicating asymmetrical coupling between them. This has implications for mechanisms of precise adaptation. Coupling might vary, providing a previously unappreciated locus for sensory control.     

Atomic force microscopy study of the antigen‐antibody binding force on patient cancer cells based on ROR1 fluorescence recognition

Knowledge of drug–target interaction is critical to our understanding of drug action and can help design better drugs. Due to the lack of adequate single‐molecule techniques, the information of individual interactions between ligand‐receptors is scarce until the advent of atomic force microscopy (AFM) that can be used to directly measure the individual ligand‐receptor forces under near‐physiological conditions by linking ligands onto the surface of the AFM tip and then obtaining force curves on cells. Most of the current AFM single‐molecule force spectroscopy experiments were performed on cells grown in vitro (cell lines) that are quite different from the human cells in vivo. From the view of clinical practice, investigating the drug–target interactions directly on the patient cancer cells will bring more valuable knowledge that may potentially serve as an important parameter in personalized treatment. Here, we demonstrate the capability of AFM to measure the binding force between target (CD20) and drug (rituximab, an anti‐CD20 monoclonal antibody targeted drug) directly on lymphoma patient cancer cells under the assistance of ROR1 fluorescence recognition. ROR1 is a receptor expressed on some B‐cell lymphomas but not on normal cells. First, B‐cell lymphoma Raji cells (a cell line) were used for ROR1 fluorescence labeling and subsequent measurement of CD20‐rituximab binding force. The results showed that Raji cells expressed ROR1, and the labeling of ROR1 did not influence the measurement of CD20‐rituximab binding force. Then the established experimental procedures were performed on the pathological samples prepared from the bone marrow of a follicular lymphoma patient. Cancer cells were recognized by ROR1 fluorescence. Under the guidance of fluorescence, with the use of a rituximab‐conjugated tip, the cellular topography was visualized by using AFM imaging and the CD20‐Rituximab binding force was measured by single‐molecule force spectroscopy. Copyright © 2013 John Wiley & Sons, Ltd.     

Effect of SXWS/WSXWS peptides on chemotaxis and adhesion of the macrophage‐like cell line J774

WSXWS motif is a conserved amino acid sequence that is present in type I cytokine receptors. This motif that can be found both in the ligand binding chains and signal transducer molecule of the receptors with different amino acids at the position “X” plays a role in the receptor folding, ligand binding and signal transduction as well. Structural analysis proved that WSEWS motif of IL‐6R is located in a highly accessible location in the protein. Structural properties and chemotaxis of a tetrapeptide library with SXWS sequence, where X was the 19 proteinogenic amino acids except cystein were systematically studied earlier. It has been proved that C‐terminal amidation and the identity of amino acid X had a pronounced influence on the chemotactic properties but less of the structure of the peptides. Here, we present our findings on the effect of a tetrapeptide and a pentapeptide library with the sequence of SXWS and WSXWS on the chemotaxis and adhesion of J774 murine macrophage cell line. We studied the effect of the presence/absence of N‐terminal tryptophan and the different amino acids at the X position on these physiological responses. Results indicated that amino acid X had a marked influence on chemotaxis, adhesion as well as on proliferation induced by (W)SXWS peptides. Elongation of SXWS sequence with a tryptophan at the N terminus also altered pronouncedly all the physiological responses of the cells studied. A good correlation could be observed between the chemotaxis and the proliferation and physicochemical parameters of the amino acid X. Copyright © 2015 John Wiley & Sons, Ltd.     

Stimulus response coupling in bacterial chemotaxis: receptor dimers in signalling arrays

In the Escherichia coli chemotaxis system, a family of chemoreceptors in the cytoplasmic membrane binds stimulatory ligands and regulates the activity of an associated histidine kinase CheA to modulate swimming behaviour and thereby cause a net migration towards attractants and away from repellents. The chemoreceptors themselves have been shown to be predominantly dimeric, but in the presence of the kinase CheA plus an adapter protein, CheW, much higher order structures have been observed. Recent results indicate that transmembrane signalling occurs within receptor clusters rather than through isolated dimers. We propose that the mechanism involves receptor arrays where binding of ligands at the outside surface of the membrane affects lateral packing interactions that cause perturbations in the organization of the signalling array at the opposing surface of the membrane. Results with receptor chimeras as well as findings with tyrosine kinase receptors suggest that this mechanism may represent a common theme in membrane receptor function.     

Adaptational modification and ligand occupancy have opposite effects on positioning of the transmembrane signalling helix of a chemoreceptor

Sensory systems adapt to persistent stimulation. In the transmembrane receptors of bacterial chemotaxis, adaptation is mediated by methylation at specific glutamyl residues in the cytoplasmic domain. Methylation counteracts effects of ligand binding on functional activities of that domain. Both ligand binding and adaptational modification are thought to act through conformational changes. As characterized for Escherichia coli chemoreceptors, a mechanistically crucial feature of the ligand-induced conformational change is piston sliding towards the cytoplasm of a signalling helix in the periplasmic/transmembrane domain. Adaptational modification could counteract this signalling movement by blocking its influence on the cytoplasmic domain or by reversing it. To investigate, we characterized effects of adaptational modification on the position of the signalling helix in chemoreceptor Trg using rates of disulphide formation between introduced cysteines. We utilized an intact cell procedure in which receptors were in their native, functional state. In vivo rates of disulphide formation between diagnostic cysteine pairs spanning a signalling helix interface changed as a function of adaptational modification. Strikingly, those changes were opposite those caused by ligand occupancy for each diagnostic pair tested. This suggests that adaptational modification resets the receptor complex to its null state by reversal of the conformational change generated by ligand binding.     

An overview of odorant-binding protein functions in insect peripheral olfactory reception

Insect olfactory perception involves many aspects of insect life, and can directly or indirectly evoke either individual or group behaviors. Insect olfactory receptors and odorant-binding proteins (OBPs) are considered to be crucial to insect-specific and -sensitive olfaction. Although the mechanisms of interaction between OBPs or OBP/ligand complex with olfactory receptors are still not well understood, it has been shown that many OBPs contribute to insect olfactory perception at various levels. Some of these are numerous and divergent members in OBP family; expression in the olfactory organ at high concentration; a variety of combinational patterns between different OBPs and ligands, but exclusive affinity for one OBP to specific binding ligands; complicated interactions between OBP/ligand complex and transmembrane proteins (olfactory receptors or sensory neuron membrane proteins). First, we review OBPs' ligand-binding property based on OBP structural research and ligand-binding test; then, we review current progress around the points cited above to show the role of such proteins in insect olfactory signal transmission; finally, we discuss applications based on insect OBP research.     

Spatial organization in bacterial chemotaxis

Spatial organization of signalling is not an exclusive property of eukaryotic cells. Despite the fact that bacterial signalling pathways are generally simpler than those in eukaryotes, there are several well‐documented examples of higher‐order intracellular signalling structures in bacteria. One of the most prominent and best‐characterized structures is formed by proteins that control bacterial chemotaxis. Signals in chemotaxis are processed by ordered arrays, or clusters, of receptors and associated proteins, which amplify and integrate chemotactic stimuli in a highly cooperative manner. Receptor clusters further serve to scaffold protein interactions, enhancing the efficiency and specificity of the pathway reactions and preventing the formation of signalling gradients through the cell body. Moreover, clustering can also ensure spatial separation of multiple chemotaxis systems in one bacterium. Assembly of receptor clusters appears to be a stochastic process, but bacteria evolved mechanisms to ensure optimal cluster distribution along the cell body for partitioning to daughter cells at division.     

Transmembrane interactions and the mechanisms of transport of proteins across membranes

We have made observations, by double fluorescence staining of the same cell, of the distributions of surface receptors, and of intracellular actin and myosin, on cultured normal fibroblasts and other flat cells, and on lymphocytes and other rounded cells. The binding of multivalent ligands (a lectin or specific antibodies) to a cell surface receptor on flat cells clusters the cell receptors into small patches, which line up directly over the actin- and myosin-containing stress fibers inside the cell. Similar ligands binding to rounded cells can cause their surface receptors to be collected into caps on the surface, and these caps are invariably found to be associated with concentrations of actin and myosin under the capped membrane. Although these ligand-induced surface phenomena appear to be different on flat and rounded cells, we propose that in both cases clusters of receptors become linked across the membrane to actin- and myosin-containing structures. In flat cells these structures are very long stress fibers; therefore, when clusters of receptors become linked to these fibers, the clusters are immobilized. In round cells, membrane-associated actin- and myosin-containing structures are apparently much less extensive than in flat cells; therefore, clusters of receptors linked to these structures are still mobile in the plane of the membrane. We suggest that in this case the clusters are then actively collected into a cap by an analogue of the muscle sliding filament mechanism. To explain the transmembrane linkage, we propose that actin is associated with the plasma membrane as a peripheral protein which is directly or indirectly bound to an integral protein (or proteins) X of the membrane. Individual molecules of any receptor are not bound to X, but after they are specifically clustered into patches, a patch of receptors then becomes bound to S and hence to actin/myosin. Patching and capping of specific receptors on rounded cells is often accompanied by a specific endocytosis of the ligand-receptor complexes. This represents one common transport mechanism of a protein (the ligand) across the plasma membrane. The possibility is discussed that this type of endocytosis is mediated by a transmembrane linkage of the clustered receptor to actin/myosin. Another mechanism of endocytosis involves the “coated pit” structures that are observed by electron microscopy of plasma membranes. The possible relationships between an actin/myosin and a coated pit mechanism of endocytosis are explored.     

Variable sizes of Escherichia coli chemoreceptor signaling teams

Like many sensory receptors, bacterial chemotaxis receptors form clusters. In bacteria, large‐scale clusters are subdivided into signaling teams that act as ‘antennas’ allowing detection of ligands with remarkable sensitivity. The range of sensitivity is greatly extended by adaptation of receptors to changes in concentrations through covalent modification. However, surprisingly little is known about the sizes of receptor signaling teams. Here, we combine measurements of the signaling response, obtained from in vivo fluorescence resonance energy transfer, with the statistical method of principal component analysis, to quantify the size of signaling teams within the framework of the previously successful Monod–Wyman–Changeux model. We find that size of signaling teams increases 2‐ to 3‐fold with receptor modification, indicating an additional, previously unrecognized level of adaptation of the chemotaxis network. This variation of signaling‐team size shows that receptor cooperativity is dynamic and likely optimized for sensing noisy ligand concentrations.     

Competition between Delta and the Abruptex domain of Notch

Background  

Extracellular domains of the Notch family of signalling receptors contain many EGF repeat domains, as do their major ligands. Some EGF repeats are modified by O-fucosylation, and most have no identified role in ligand binding.     

Correlation between signal input and output in PctA and PctB amino acid chemoreceptor of Pseudomonas aeruginosa

The PctA and PctB chemoreceptors of Pseudomonas aeruginosa mediate chemotaxis toward amino acids. A general feature of signal transduction processes is that a signal input is converted into an output. We have generated chimeras combining the Tar signaling domain with either the PctA or PctB ligand binding domain (LBD). Escherichia coli harboring either PctA‐Tar or PctB‐Tar mediated chemotaxis toward amino acids. The responses of both chimeras were determined using fluorescence resonance energy transfer, and the derived EC₅₀ values are a measure of output. PctA‐Tar and PctB‐Tar responded to 19 and 11 L‐amino acids respectively. The EC₅₀ values of PctA‐Tar responses differed by more than three orders of magnitude, whereas PctB‐Tar responded preferentially to L‐Gln. The comparison of amino acid binding constants and the corresponding EC₅₀ values for both receptors revealed statistically significant correlations between inputs and outputs. PctA and PctB possess a double PDC (PhoQ‐DcuS‐CitA) LBD – a family of binding domain found in various other amino acid chemoreceptors. Similarly, various chemoreceptors share the preferential response to certain amino acids (e.g. L‐Cys, L‐Ser and L‐Thr) that we observed for PctA. Defining the specific inputs and outputs of these chemoreceptors is an important step toward better understanding of their physiological role.     

Structural and functional dissection of the interplay between lipid and Notch binding by human Notch ligands

Recent data have expanded our understanding of Notch signalling by identifying a C2 domain at the N‐terminus of Notch ligands, which has both lipid‐ and receptor‐binding properties. We present novel structures of human ligands Jagged2 and Delta‐like4 and human Notch2, together with functional assays, which suggest that ligand‐mediated coupling of membrane recognition and Notch binding is likely to be critical in establishing the optimal context for Notch signalling. Comparisons between the Jagged and Delta family show a huge diversity in the structures of the loops at the apex of the C2 domain implicated in membrane recognition and Jagged1 missense mutations, which affect these loops and are associated with extrahepatic biliary atresia, lead to a loss of membrane recognition, but do not alter Notch binding. Taken together, these data suggest that C2 domain binding to membranes is an important element in tuning ligand‐dependent Notch signalling in different physiological contexts.     

Delineation of the cell-extrinsic apoptosis pathway in the zebrafish

The mammalian extrinsic apoptosis pathway is triggered by Fas ligand (FasL) and Apo2 ligand/tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL). Ligand binding to cognate receptors activates initiator caspases directly in a death-inducing signaling complex. In Drosophila, TNF ligand binding activates initiator caspases indirectly, through JNK. We characterized the extrinsic pathway in zebrafish to determine how it operates in a nonmammalian vertebrate. We identified homologs of FasL and Apo2L/TRAIL, their receptors, and other components of the cell death machinery. Studies with three Apo2L/TRAIL homologs demonstrated that they bind the receptors zHDR (previously linked to hematopoiesis) and ovarian TNFR (zOTR). Ectopic expression of these ligands during embryogenesis induced apoptosis in erythroblasts and notochord cells. Inhibition of zHDR, zOTR, the adaptor zFADD, or caspase-8-like proteases blocked ligand-induced apoptosis, as did antiapoptotic Bcl-2 family members. Thus, the extrinsic apoptosis pathway in zebrafish closely resembles its mammalian counterpart and cooperates with the intrinsic pathway to trigger tissue-specific apoptosis during embryogenesis in response to ectopic Apo2L/TRAIL expression.