G protein-coupled receptor signalling and cross-talk: achieving rapidity and specificity

Activation of a given type of G protein-coupled receptor (GPCR) triggers a limited set of signalling events in a very rapid and specific manner. The classical paradigm of GPCR signalling was rather linear and sequential. Emerging evidence, however, has revealed that this is only a part of the complex signalling mediated by GPCR. Propagation of GPCR signalling involves cross-regulation of many but specific pathways, including cross-talks between different GPCRs as well as with other signalling pathways. Moreover, it is increasingly apparent that GPCRs can activate both heterotrimeric G protein-dependent and G protein-independent signalling pathways. In this review, we discuss how the signallings initiated by GPCRs achieve rapidity as well as specificity, and how the GPCRs can cross-regulate other specific signalling pathways at the same time. New concepts regarding GPCR signalling have been arising to address this issue, which include multiprotein signalling complex and signalling compartment in microdomain concepts that enable close colocalization or even contact among the proteins engaged in the specific signal transduction. The final outcome of a stimulation of GPCR will thus be the sum of its own specific set of intracellular signalling pathways it regulates.     

Genomic analysis of the aromatic catabolic pathways from Pseudomonas putida KT2440

Analysis of the catabolic potential of Pseudomonas putida KT2440 against a wide range of natural aromatic compounds and sequence comparisons with the entire genome of this microorganism predicted the existence of at least four main pathways for the catabolism of central aromatic intermediates, that is, the protocatechuate (pca genes) and catechol (cat genes) branches of the beta-ketoadipate pathway, the homogentisate pathway (hmg/fah/mai genes) and the phenylacetate pathway (pha genes). Two additional gene clusters that might be involved in the catabolism of N-heterocyclic aromatic compounds (nic cluster) and in a central meta-cleavage pathway (pcm genes) were also identified. Furthermore, the genes encoding the peripheral pathways for the catabolism of p-hydroxybenzoate (pob), benzoate (ben), quinate (qui), phenylpropenoid compounds (fcs, ech, vdh, cal, van, acd and acs), phenylalanine and tyrosine (phh, hpd) and n-phenylalkanoic acids (fad) were mapped in the chromosome of P. putida KT2440. Although a repetitive extragenic palindromic (REP) element is usually associated with the gene clusters, a supraoperonic clustering of catabolic genes that channel different aromatic compounds into a common central pathway (catabolic island) was not observed in P. putida KT2440. The global view on the mineralization of aromatic compounds by P. putida KT2440 will facilitate the rational manipulation of this strain for improving biodegradation/biotransformation processes, and reveals this bacterium as a useful model system for studying biochemical, genetic, evolutionary and ecological aspects of the catabolism of aromatic compounds.     

Invertase in Immobilized Cells of Eschscholtzia Californica

Cell suspension culture of Eschscholtzia californica Cham. were permeabilized by Tween 20 or 80, and immobilized by glutaraldehyde. The highest invertase activity was at pH 4.5 and temperature 50 °C. The hydrolysis of the substrate was linear for 5 h reaching 60 % conversion. The cells had high invertase activity and a good stability, and in long-term storage they showed convenient physico-mechanical properties.     

An unusual orientation for Tyr75 in the active site of the aspartic proteinase from Saccharomyces cerevisiae

The structures of the native Saccharomyces cerevisiae proteinase A have been solved by molecular replacement in the monoclinic and trigonal crystal forms and refined at 2.6-2.7A resolution. These structures agree overall with those of other uninhibited aspartic proteinases. However, an unusual orientation for the side chain of Tyr75, a conserved residue on the flexible "flap" that covers the active site and is important for the activity of these enzymes, was found in the trigonal crystals. A similar conformation of Tyr75 occupying the S1 substrate-binding pocket was previously reported only for chymosin (where it was interpreted as representing a "self-inhibited" state of the enzyme), but for no other aspartic proteinases. Since this orientation of Tyr75 has now been seen in the structures of two members of the family of aspartic proteinases, it might indicate that the placement of that residue in the S1 substrate-binding pocket might have some functional significance, analogous to what was seen for self-inhibited structures of serine proteinases.     

The Pco proteins are involved in periplasmic copper handling in Escherichia coli

The interactions between the plasmid-borne copper resistance determinant, pco, and the main copper export system in Escherichia coli have been investigated and no direct interaction has been found. The PcoE and PcoC proteins are periplasmic and PcoC binds one Cu ion per protein molecule. PcoA is also periplasmic and can substitute for the chromosomally encoded CueO protein. The pco determinant is proposed to exert its effect through periplasmic handling of excess copper ions and to increase the level of resistance to copper ions above that conferred by copA alone.     

Comparative analysis of expressed sequence tags from Sesamum indicum and Arabidopsis thaliana developing seeds

Sesame (Sesamum indicum) is an important oilseed crop which produces seeds with 50% oil that have a distinct flavor and contains antioxidant lignans. Because sesame lignans are known to have antioxidant and health-protecting properties, metabolic pathways for lignans have been of interest in developing sesame seeds. As an initial approach to identify genes involved in accumulation of storage products and in the biosynthesis of antioxidant lignans, 3328 expressed sequence tags (ESTs) were obtained from a cDNA library of immature seeds 5-25 days old. ESTs were clustered and analyzed by the BLASTX or FASTAX program against the GenBank NR and Arabidopsis proteome databases. To compare gene expression profiles during development of green and non-green seeds, a comparative analysis was carried out between developing sesame and Arabidopsis seed ESTs. Analyses of these two seed EST sets have helped to identify similar and different gene expression profiles during seed development, and to identify a large number of sesame seed-specific genes. In particular, we have identified EST candidates for genes possibly involved in biosynthesis of sesame lignans, sesamin and sesamolin, and also suggest a possible metabolic pathway for the generation of cofactors required for synthesis of storage lipid in non-green oilseeds. Seed-specific expression of several candidate genes has been confirmed by northern blot analysis.     

Crystal structure of the BAFF-BAFF-R complex and its implications for receptor activation

B-cell activating factor (BAFF) is a key regulator of B-lymphocyte development. Its biological role is mediated by the specific receptors BCMA, TACI and BAFF-R. We have determined the crystal structure of the extracellular domain of BAFF-R bound to BAFF at a resolution of 3.3 A. The cysteine-rich domain (CRD) of the BAFF-R extracellular domain adopts a beta-hairpin structure and binds to the virus-like BAFF cage in a 1:1 molar ratio. The conserved DxL motif of BAFF-R is located on the tip of the beta-turn and is indispensable in the binding of BAFF. The crystal structure shows that a unique dimeric contact occurs between the BAFF-R monomers in the virus-like cage complex. The extracellular domain of TACI contains two CRDs, both of which contain the DxL motif. Modeling of TACI-BAFF complex suggests that both CDRs simultaneously interact with the BAFF dimer in the virus-like cage.     

Detection of substance P and its mRNA in human blast cells in childhood lymphoblastic leukaemia using immunocytochemistry and in situ hybridisation

The study focused on determining the expression of substance P (SP) in neoplastic cells of childhood acute lymphoblastic leukaemia (ALL) at the levels of its mRNA and the protein production. The study group comprised 44 children treated for ALL in the Department of Paediatric Haematology and Oncology, Karol Marcinkowski University of Medical Sciences in Poznań, in the years 1999-2001. Bone marrow smears were obtained by needle biopsy. Expression of SP was examined by immunocytochemistry with specific antibody against human SP and by in situ hybridisation with anti-mRNA 5'-biotinylated probe. The results of the study demonstrated that SP could be detected in the cytoplasm of lymphoblasts (mean percentage of 81.8% for immunocytochemical and 84.5% for in situ hybridisation technique) in leukaemias of the common and T-cell types. SP was absent from blasts in B-cell leukaemia and from normal haematopoietic, cells in children of the control group. The results show that lymphoblasts of common and T-cell origin acquire the capability to synthesise SP after their neoplastic transformation in childhood acute leukaemia. SP may be involved in auto- and paracrine mechanisms capable of inducing hyperplasia of the neoplastic cells.