Structure and membrane-targeting of a Bordetella pertussis effector N-terminal domain

BteA, a 69-kDa cytotoxic protein, is a type III secretion system (T3SS) effector in the classical Bordetella, the etiological agents of pertussis and related mammalian respiratory diseases. Like other cytotoxicity-mediating effectors, BteA uses its multifunctional N-terminal domain to target phosphatidylinositol (PI)-rich microdomains in the host membrane. Despite their structural similarity, T3SS effectors exhibit a variable range of membrane interaction modes, and currently only limited structural information is available for the BteA membrane-targeting domain and the molecular mechanisms underlying its function. Employing a synergistic combination of structural methods, here we determine the structure of this functional domain and uncover key molecular determinants mediating its interaction with membranes. Residues 29–121 of BteA form an elongated four-helix bundle packed against two shorter perpendicular helices, the second of which caps the domain in a critical ‘tip motif’. A flexible region preceding the BteA helical bundle contains the characteristic β-motif required for binding its cognate chaperone BtcA. We show that BteA targets PI(4,5)P₂-containing lipoprotein nanodiscs and binds a soluble PI(4,5)P₂ analog via an extensive positively charged surface spanning its first two helices, and that this interaction is weaker for PI(3,5)P₂ and abolished for PI(4)P. We confirmed this model of membrane-targeting by observation of BteA-induced changes in the structure of PI(4,5)P₂-containing phospholipid bilayers using small-angle X-ray scattering (SAXS). We also extended these results to a larger BteA domain (residues 1–287), confirming its interaction with bilayers using calorimetry, fluorescence and SAXS methods. This novel view of the structural underpinnings of membrane targeting by BteA is an important step towards a comprehensive understanding of cytotoxicity in Bordetella, as well as interactions of a broad range of pathogens with their respective hosts.     

Taste bud form and distribution on lips and in the oropharyngeal cavity of cardinal fish species (Apogonidae, Teleostei), with remarks on their dentition

The oral dentition and type and number of taste buds (TB) on the lips and in the oropharyngeal cavity were compared by means of SEM in 11 species of cardinal fishes (Apogonidae) belonging to five genera. The occurrence of a dense cover of skin papillae on the lips of some species (e.g., Apogon frenatus), as well as differences in structure of vomer, tongue, and palatinum, expose additional morphological characters important for clarification of the taxonomy of this group of fishes. Differences are also revealed in the type of dentition, such as on the vomer and epi-hypopharyngeal bones. Strong and dense dentition of the anterior part of the oral cavity and a high number of TB on this site in species feeding on larger prey (e.g., Cheilodipterus spp) is compared to the relatively feeble jaw armor and richness of TB on the more pharyngeal site in species feeding on smaller prey (e.g., Apogon angustatus, A. frenatus). In addition to the three types of TB (Types I-III) previously described from various teleost fish, a fourth type (Type IV), comprising very small buds, was found in some cardinal fish (Apogon angustatus, A. frenatus). The various TB are distributed from the lips to the pharyngeal bones, on the breathing valves, tongue, palatinum, and pharyngeal bones; their number and type on the various sites differ in the different species. In all species studied the Types I and II TB, elevated above the surrounding epithelium, dominated the lips and anterior part of mouth, while Types III and IV, which end apically at the level with the epithelium, dominated the more posterior pharyngeal region. The highest number of TB, around 24,600, were found in Fowleria variegata, a typical nocturnal species, and the lowest in the diurnal and crepuscular Apogon cyanosoma (1,660) and Cheilodipterus quinquestriatus (2,400). Differences are also revealed in the type of dentition, such as on the vomer and epi-hypopharyngeal bones. The number of TB increased with growth of the fishes. The differences in the total number of TB and their distribution in the oropharyngeal cavity in the various species indicates possible different mechanisms of foraging and food-recognition.     

Recruitment of ectodermal attachment cells via an EGFR-dependent mechanism during the organogenesis of Drosophila proprioceptors

Drosophila proprioceptors (chordotonal organs) are structured as a linear array of four lineage-related cells: a neuron, a glial cell, and two accessory cells, called cap and ligament, between which the neuron is stretched. To function properly as stretch receptors, chordotonal organs must be stably anchored at both edges. The cap cells are anchored to the cuticle through specialized lineage-related attachment cells. However, the mechanism by which the ligament cells at the other edge of the organ attach is not known. Here, we report the identification of specialized attachment cells that anchor the ligament cells of pentascolopidial chordotonal organs (lch5) to the cuticle. The ligament attachment cells are recruited by the approaching ligament cells upon reaching their attachment site, through an EGFR-dependent mechanism. Molecular characterization of lch5 attachment cells demonstrated that they share significant properties with Drosophila tendon cells and with mammalian proprioceptive organs.     

Selectivity and promiscuity of the first and second PDZ domains of PSD-95 and synapse-associated protein 102

PDZ domains typically interact with the very carboxyl terminus of their binding partners. Type 1 PDZ domains usually require valine, leucine, or isoleucine at the very COOH-terminal (P(0)) position, and serine or threonine 2 residues upstream at P(-2). We quantitatively defined the contributions of carboxyl-terminal residues to binding selectivity of the prototypic interactions of the PDZ domains of postsynaptic density protein 95 (PSD-95) and its homolog synapse-associated protein 90 (SAP102) with the NR2b subunit of the N-methyl-d-aspartate-type glutamate receptor. Our studies indicate that all of the last five residues of NR2b contribute to the binding selectivity. Prominent were a requirement for glutamate or glutamine at P(-3) and for valine at P(0) for high affinity binding and a preference for threonine over serine at P(-2), in the context of the last 11 residues of the NR2b COOH terminus. This analysis predicts a COOH-terminal (E/Q)(S/T)XV consensus sequence for the strongest binding to the first two PDZ domains of PSD-95 and SAP102. A search of the human genome sequences for proteins with a COOH-terminal (E/Q)(S/T)XV motif yielded 50 proteins, many of which have not been previously identified as PSD-95 or SAP102 binding partners. Two of these proteins, brain-specific angiogenesis inhibitor 1 and protein kinase Calpha, co-immunoprecipitated with PSD-95 and SAP102 from rat brain extracts.     

Novel indolo[2,1-b]quinazoline analogues as cytostatic agents: synthesis,biological evaluation and structure-activity relationship

In our endeavor to design and synthesize novel anticancer agents, a new series of indoloquinazoline compounds were prepared and tested initially for anticancer activity in vitro against a panel of human cancer cell lines. Most of these compounds exhibited cytotoxic activity in in vitro screens. Compounds were selected and further evaluated using a modified Hollow Fiber Assay for their preliminary in vivo activity against 12 cell lines implanted in the subcutaneous and intraperitoneal compartments in mice. The results indicate that these compounds may constitute a new class of anticancer agents.     

Noise-induced divisive gain control in neuron models

A recent computational study of gain control via shunting inhibition has shown that the slope of the frequency-versus-input (f-I) characteristic of a neuron can be decreased by increasing the noise associated with the inhibitory input (Neural Comput. 13, 227-248). This novel noise-induced divisive gain control relies on the concommittant increase of the noise variance with the mean of the total inhibitory conductance. Here we investigate this effect using different neuronal models. The effect is shown to occur in the standard leaky integrate-and-fire (LIF) model with additive Gaussian white noise, and in the LIF with multiplicative noise acting on the inhibitory conductance. The noisy scaling of input currents is also shown to occur in the one-dimensional theta-neuron model, which has firing dynamics, as well as a large scale compartmental model of a pyramidal cell in the electrosensory lateral line lobe of a weakly electric fish. In this latter case, both the inhibition and the excitatory input have Poisson statistics; noise-induced divisive inhibition is thus seen in f-I curves for which the noise increases along with the input I. We discuss how the variation of the noise intensity along with inputs is constrained by the physiological context and the class of model used, and further provide a comparison of the divisive effect across models.     

Elg1, an alternative subunit of the RFC clamp loader,preferentially interacts with SUMOylated PCNA

Replication‐factor C (RFC) is a protein complex that loads the processivity clamp PCNA onto DNA. Elg1 is a conserved protein with homology to the largest subunit of RFC, but its function remained enigmatic. Here, we show that yeast Elg1 interacts physically and genetically with PCNA, in a manner that depends on PCNA modification, and exhibits preferential affinity for SUMOylated PCNA. This interaction is mediated by three small ubiquitin‐like modifier (SUMO)‐interacting motifs and a PCNA‐interacting protein box close to the N‐terminus of Elg1. These motifs are important for the ability of Elg1 to maintain genomic stability. SUMOylated PCNA is known to recruit the helicase Srs2, and in the absence of Elg1, Srs2 and SUMOylated PCNA accumulate on chromatin. Strains carrying mutations in both ELG1 and SRS2 exhibit a synthetic fitness defect that depends on PCNA modification. Our results underscore the importance of Elg1, Srs2 and SUMOylated PCNA in the maintenance of genomic stability.