Ca2+ responses to thyrotropin-releasing hormone and angiotensin II: the role of plasma membrane integrity and effect of G11alpha protein overexpression on homologous and heterologous desensitization

The molecular mechanisms involved in GPCR-initiated signaling cascades where the two receptors share the same signaling cascade, such as thyrotropin-releasing hormone (TRH) and angiotensin II (ANG II), are still far from being understood. Here, we analyzed hormone-induced Ca(2+) responses and the process of desensitization in HEK-293 cells, which express endogenous ANG II receptors. These cells were transfected to express exogenously high levels of TRH receptors (clone E2) or both TRH receptors and G(11)alpha protein (clone E2M11). We observed that the characteristics of the Ca(2+) response, as well as the process of desensitization, were both strongly dependent on receptor number and G(11)alpha protein level. Whereas treatment of E2 cells with TRH or ANG II led to significant desensitization of the Ca(2+) response to subsequent addition of either hormone, the response was not desensitized in E2M11 cells expressing high levels of G(11)alpha. In addition, stimulation of both cell lines with THR elicited a clear heterologous desensitization to subsequent stimulation with ANG II. On the other hand, ANG II did not affect a subsequent response to TRH. ANG II-mediated signal transduction was strongly dependent on plasma membrane integrity modified by cholesterol depletion, but signaling through TRH receptors was altered only slightly under these conditions. It may be concluded that the level of expression of G-protein-coupled receptors and their cognate G-proteins strongly influences not only the magnitude of the Ca(2+) response but also the process of desensitization and resistance to subsequent hormone addition.     

A comparison of BRCT domains involved in nonhomologous end-joining: introducing the solution structure of the BRCT domain of polymerase lambda

Three of the four family X polymerases, DNA polymerase lambda, DNA polymerase mu, and TdT, have been associated with repair of double-strand DNA breaks by nonhomologous end-joining. Their involvement in this DNA repair process requires an N-terminal BRCT domain that mediates interaction with other protein factors required for recognition and binding of broken DNA ends. Here we present the NMR solution structure of the BRCT domain of DNA polymerase lambda, completing the structural portrait for this family of enzymes. Analysis of the overall fold of the polymerase lambda BRCT domain reveals structural similarity to the BRCT domains of polymerase mu and TdT, yet highlights some key sequence and structural differences that may account for important differences in the biological activities of these enzymes and their roles in nonhomologous end-joining. Mutagenesis studies indicate that the conserved Arg57 residue of Pol lambda plays a more critical role for binding to the XRCC4-Ligase IV complex than its structural homolog in Pol mu, Arg43. In contrast, the hydrophobic Leu60 residue of Pol lambda contributes less significantly to binding than the structurally homologous Phe46 residue of Pol mu. A third leucine residue involved in the binding and activity of Pol mu, is nonconservatively replaced by a glutamine in Pol lambda (Gln64) and, based on binding and activity data, is apparently unimportant for Pol lambda interactions with the NHEJ complex. In conclusion, both the structure of the Pol lambda BRCT domain and its mode of interaction with the other components of the NHEJ complex significantly differ from the two previously studied homologs, Pol mu and TdT.     

The functions and possible significance of Kremen as the gatekeeper of Wnt signalling in development and pathology

Kremen (Krm) was originally discovered as a novel transmembrane protein containing the kringle domain. Both Krm1 (the first identified Krm) and its relative Krm2 were later identified to be the high-affinity receptors for Dickkopf (Dkk), the inhibitor of Wnt/beta-catenin signalling. The formation of a ternary complex composed of Krm, Dkk, and Lrp5/6 (the coreceptor of Wnt) inhibits Wnt/beta-catenin signalling. In Xenopus gastrula embryos, Wnt/beta-catenin signalling regulates anterior-posterior patterning, with low-signalling in anterior regions. Inhibition of Krm1/2 induces embryonic head defects. Together with anterior localization of Krms and Dkks, the inhibition of Wnt signalling by Dkk-Krm action seems to allow anterior embryonic development. During mammalian development, krm1 mRNA expression is low in the early stages, but gradually and continuously increases with developmental progression and differentiation. In contrast with the wide, strong expression of krm1 mRNA in mature tissues, expression of krm1 is diminished in a variety of human tumor cells. Since stem cells and undifferentiated cells rely on Wnt/beta-catenin signalling for maintenance in a low differentiation state, the physiological shutdown of Wnt/beta-catenin signalling by Dkk-Krm is likely to set cells on a divergent path toward differentiation. In tumour cells, a deficit of Krm may increase the susceptibility to tumourigenic transformation. Both positive and negative regulation of Wnt/beta-catenin signalling definitively contributes to diverse developmental and physiological processes, including cell-fate determination, tissue patterning and stem cell regulation. Krm is quite significant in these processes as the gatekeeper of the Wnt/beta-catenin signalling pathway.     

Identification of Residues Surrounding the Active Site of Type A Botulinum Neurotoxin Important for Substrate Recognition and Catalytic Activity

Type A botulinum neurotoxin is one of the most lethal of the seven serotypes and is increasingly used as a therapeutic agent in neuromuscular dysfunctions. Its toxic function is related to zinc-endopeptidase activity of the N-terminal light chain (LC) on synaptosome-associated protein-25 kDa (SNAP-25) of the SNARE complex. To understand the determinants of substrate specificity and assist the development of strategies for effective inhibitors, we used site-directed mutagenesis to investigate the effects of 13 polar residues of the LC on substrate binding and catalysis. Selection of the residues for mutation was based on a computational analysis of the three-dimensional structure of the LC modeled with a 17-residue substrate fragment of SNAP-25. Steady-state kinetic parameters for proteolysis of the substrate fragment were determined for a set of 16 single mutants. Of the mutated residues non-conserved among the serotypes, replacement of Arg-230 and Asp-369 by polar or apolar residues resulted in drastic lowering of the catalytic rate constant (k _cat), but had less effect on substrate affinity (K _m). Substitution of Arg-230 with Lys decreased the catalytic efficiency (k _cat/K _m) by 50-fold, whereas replacement by Leu yielded an inactive protein. Removal of the electrostatic charge at Asp-369 by mutation to Asn resulted in 140-fold decrease in k _cat/K _m. Replacement of other variable residues surrounding the catalytic cleft (Glu-54, Glu-63, Asn-66, Asp-130, Asn-161, Glu-163, Glu-170, Glu-256), had only marginal effect on decreasing the catalytic efficiency, but unexpectedly the substitution of Lys-165 with Leu resulted in fourfold increase in k _cat/K _m. For comparison purposes, two conserved residues Arg-362 and Tyr-365 were investigated with substitutions of Leu and Phe, respectively, and their catalytic efficiency decreased 140- and 10-fold, respectively, whereas substitution of the tyrosine ring with Asn abolished activity. The altered catalytic efficiencies of the mutants were not due to any significant changes in secondary or tertiary structures, or in zinc content and thermal stability. We suggest that, despite the large minimal substrate size for catalysis, only a few non-conserved residues surrounding the active site are important to render the LC competent for catalysis or provide conformational selection of the substrate.     

The biochemical basis of antimicrobial responses in Manduca sexta

Innate immunity is essential for the wellbeing of vertebrates and invertebrates. Key components of this defense system include pattern recognition receptors that bind to infectious agents, extra-and intra-cellular proteins that relay signals, as well as molecules and cells that eliminate pathogens. We have been studying the defense mechanisms in a biochemical model insect, Manduca sexta. In this insect, hemolin, peptidoglycan recognition proteins, β-1,3-glucan recognition proteins and C-type lectins detect microbial surface molecules and induce immune responses such as phagocytosis, nodulation, encapsulation, melanization and production of antimicrobial peptides. Some of these responses are mediated by extracellular serine proteinase pathways. The proteolytic activation of prophenoloxidase （proPO） yields active phenoloxidase （PO） which catalyzes the formation of quinones and melanin for wound healing and microbe killing. M. sexta hemolymph proteinase 14 （HP 14） precursor interacts with peptidoglycan or β-1,3-glucan, autoactivates, and leads to the activation of other HPs including HP21 and proPO-activating proteinases （PAPs）. PAP-1, -2 and -3 cut proPO to generate active PO in the presence of two serine proteinase homologs. Inhibition of the proteinases by serpins and association of the proteinase homologs with bacteria ensure a localized defense reaction. M. sexta HP1, HP6, HP8, HP17 and other proteinases may also participate in proPO activation or processing of spatzle and plasmatocyte spreading peptide.     

Bioinformatic identification of genes encoding C1q-domain containing proteins in zebrafish

C1q is the first subcomponent of classical pathway in the complement system and a major link between innate and acquired immunities. The globular (gC1q) domain similar with C1q was also found in many non-complement C1q-domain-containing (C1qDC) proteins which have similar crystal structure to that of the multifunctional tumor necrosis factor (TNF) ligand family, and also have diverse functions. In this study, we identified a total of 52 independent gene sequences encoding C1q-domain-containing proteins through comprehensive searches of zebrafish genome, cDNA and EST databases. In comparison to 31 orthologous genes in human and different numbers in other species, a significant selective pressure was suggested during vertebrate evolution. Domain organization of C1q-domain-containing (C1qDC) proteins mainly includes a leading signal peptide, a collagen-like region of variable length, and a C-terminal C1q domain. There are 11 highly conserved residues within the C1q domain, among which 2 are invariant within the zebrafish gene set. A more extensive database searches also revealed homologous C1qDC proteins in other vertebrates, invertebrates and even bacterium, but no homologous sequences for encoding C1qDC proteins were found in many species that have a more recent evolutionary history with zebrafish. Therefore, further studies on C1q-domain-containing genes among different species will help us understand evolutionary mechanism of innate and acquired immunities.     

NMR assignment of the C-terminal actin-binding domain of talin

Talin is a large dimeric 270 kDa adapter protein which binds the cytoplasmic face of a subset of integrin β-subunits and couples them to the actin cytoskeleton. Here we report the near complete ¹⁵N, ¹³C and ¹H chemical shift assignments for the C-terminal actin-binding domain.     

Complete resonance assignment of the first and second apple domains of MIC4 from Toxoplasma gondii, using a new NMRView-based assignment aid

Microneme protein 4 is involved in cell binding by the important parasite Toxoplasma gondii. We present here the backbone and side-chain assignments of the first two apple domains together with a new graphical aid for their assignment using NMRView. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Setup and dosimetry for exposing anaesthetised pigs in vivo to 900 MHz GSM mobile phone fields

The aim of this study was a dosimetrical analysis of the setup used in the exposure of the heads of domestic pigs to GSM-modulated radio frequency electromagnetic fields (RF-EMF) at 900 MHz. The heads of pigs were irradiated with a half wave dipole using three different exposure routines; short bursts of 1-3 s at two different exposure levels and a continuous 10-min exposure. The electroencephalogram (EEG) was registered continuously during the exposures to search for RF-EMF originated changes. The dosimetry was based on simulations with the anatomical heterogeneous numerical model of the pig head. The simulation results were validated by experimental measurements with the exposure dipole and a homogeneous liquid phantom resembling the pig head. The specific absorption rate (SAR), defined as a maximum average over 10 g tissue mass (SAR(10g)), was 7.3 W/kg for the first set of short bursts and 31 W/kg for the second set of short bursts. The SAR(10g) in the continuous 10-min exposure was 31 W/kg. The estimated uncertainty for the dosimetry was +/-25% (K = 2).