Light-sensitive coupling of rhodopsin and melanopsin to G(i/o) and G(q) signal transduction in Caenorhabditis elegans

Activation of G-protein-coupled receptors (GPCRs) initiates signal transduction cascades that affect many physiological responses. The worm Caenorhabditis elegans expresses >1000 of these receptors along with their cognate heterotrimeric G proteins. Here, we report properties of 9-cis-retinal regenerated bovine opsin [(b)isoRho] and human melanopsin [(h)Mo], two light-activated, heterologously expressed GPCRs in the nervous system of C. elegans with various genetically engineered alterations. Profound transient photoactivation of G(i/o) signaling by (b)isoRho led to a sudden and transient loss of worm motility dependent on cyclic adenosine monophosphate, whereas transient photoactivation of G(q) signaling by (h)Mo enhanced worm locomotion dependent on phospholipase Cβ. These transgenic C. elegans models provide a unique way to study the consequences of G(i/o) and G(q) signaling in vivo with temporal and spatial precision and, by analogy, their relationship to human neuromotor function.     

Heterologous expression of functional G-protein-coupled receptors in Caenorhabditis elegans

New strategies for expression, purification, functional characterization, and structural determination of membrane-spanning G-protein-coupled receptors (GPCRs) are constantly being developed because of their importance to human health. Here, we report a Caenorhabditis elegans heterologous expression system able to produce milligram amounts of functional native and engineered GPCRs. Both bovine opsin [(b)opsin] and human adenosine A(2A) subtype receptor [(h)A(2A)R] expressed in neurons or muscles of C. elegans were localized to cell membranes. Worms expressing these GPCRs manifested changes in motor behavior in response to light and ligands, respectively. With a newly devised protocol, 0.6-1 mg of purified homogenous 9-cis-retinal-bound bovine isorhodopsin [(b)isoRho] and ligand-bound (h)A(2A)R were obtained from C. elegans from one 10-L fermentation at low cost. Purified recombinant (b)isoRho exhibited its signature absorbance spectrum and activated its cognate G-protein transducin in vitro at a rate similar to native rhodopsin (Rho) obtained from bovine retina. Generally high expression levels of 11 native and mutant GPCRs demonstrated the potential of this C. elegans system to produce milligram quantities of high-quality GPCRs and possibly other membrane proteins suitable for detailed characterization.     

Sequence, structure and functional diversity of PD-(D/E)XK phosphodiesterase superfamily

Proteins belonging to PD-(D/E)XK phosphodiesterases constitute a functionally diverse superfamily with representatives involved in replication, restriction, DNA repair and tRNA–intron splicing. Their malfunction in humans triggers severe diseases, such as Fanconi anemia and Xeroderma pigmentosum. To date there have been several attempts to identify and classify new PD-(D/E)KK phosphodiesterases using remote homology detection methods. Such efforts are complicated, because the superfamily exhibits extreme sequence and structural divergence. Using advanced homology detection methods supported with superfamily-wide domain architecture and horizontal gene transfer analyses, we provide a comprehensive reclassification of proteins containing a PD-(D/E)XK domain. The PD-(D/E)XK phosphodiesterases span over 21 900 proteins, which can be classified into 121 groups of various families. Eleven of them, including DUF4420, DUF3883, DUF4263, COG5482, COG1395, Tsp45I, HaeII, Eco47II, ScaI, HpaII and Replic_Relax, are newly assigned to the PD-(D/E)XK superfamily. Some groups of PD-(D/E)XK proteins are present in all domains of life, whereas others occur within small numbers of organisms. We observed multiple horizontal gene transfers even between human pathogenic bacteria or from Prokaryota to Eukaryota. Uncommon domain arrangements greatly elaborate the PD-(D/E)XK world. These include domain architectures suggesting regulatory roles in Eukaryotes, like stress sensing and cell-cycle regulation. Our results may inspire further experimental studies aimed at identification of exact biological functions, specific substrates and molecular mechanisms of reactions performed by these highly diverse proteins.     

Lecithin:retinol acyltransferase is critical for cellular uptake of vitamin A from serum retinol-binding protein

Vitamin A (all-trans-retinol) must be adequately distributed within the mammalian body to produce visual chromophore in the eyes and all-trans-retinoic acid in other tissues. Vitamin A is transported in the blood bound to retinol-binding protein (holo-RBP), and its target cells express an RBP receptor encoded by the Stra6 (stimulated by retinoic acid 6) gene. Here we show in mice that cellular uptake of vitamin A from holo-RBP depends on functional coupling of STRA6 with intracellular lecithin:retinol acyltransferase (LRAT). Thus, vitamin A uptake from recombinant holo-RBP exhibited by wild type mice was impaired in Lrat(-/-) mice. We further provide evidence that vitamin A uptake is regulated by all-trans-retinoic acid in non-ocular tissues of mice. When in excess, vitamin A was rapidly taken up and converted to its inert ester form in peripheral tissues, such as lung, whereas in vitamin A deficiency, ocular retinoid uptake was favored. Finally, we show that the drug fenretinide, used clinically to presumably lower blood RBP levels and thus decrease circulating retinol, targets the functional coupling of STRA6 and LRAT to increase cellular vitamin A uptake in peripheral tissues. These studies provide mechanistic insights into how vitamin A is distributed to peripheral tissues in a regulated manner and identify LRAT as a critical component of this process.     

Post-translational modifications of the serotonin type 4 receptor heterologously expressed in mouse rod cells

G-protein-coupled serotonin receptor type 4 (5-HT(4)R) is a pharmacological target implicated in a variety of gastrointestinal and nervous system disorders. As for many other integral membrane proteins, structural and functional studies of this receptor could be facilitated by its heterologous overexpression in eukaryotic systems that can perform appropriate post-translational modifications (PTMs) on the protein. We previously reported the development of an expression system that employs rhodopsin's biosynthetic machinery in rod cells of the retina to express heterologous G-protein-coupled receptors (GPCRs) in a pharmacologically functional form. In this study, we analyzed the glycosylation, phosphorylation, and palmitoylation of 5-HT(4)R heterologously expressed in rod cells of transgenic mice. We found that the glycosylation pattern in 5-HT(4)R was more complex than in murine and bovine rhodopsin. Moreover, overexpression of this exogenous GPCR in rod cells also affected the glycosylation pattern of coexisting native rhodopsin. These results highlight not only the occurrence of heterogeneous PTMs on transgenic proteins but also the complications that non-native PTMs can cause in the structural and functional characterization of both endogenous and heterologous protein targets.     

Analysis of mercury levels in historical bone material from syphilitic subjects--pilot studies (short report)

The aim of the present work is to study the remains of seven individuals with typical symptoms of tertiary syphilis in terms of mercury content in bones, thereby verifying whether they were subjected to treatment and, if they were, how long their organisms were exposed to toxic mercury fumes. Mercury was used, mainly in the Middle Ages and in the early modern period, as a preventive measure in case of individuals suffering from syphilis, a venereal disease, and also leprosy. Syphilitic patients treated this way should demonstrate increased concentration of mercury in their bones. The skeletons studied in the present work originate from various archaeological sites in southern and north-central Poland. The analyses concerned individuals with diagnosed syphilis as well as healthy individuals who constituted the control group. The analyses were performed by the LA-ICP-MS technique, with the use of laser Nd: YAG, Macro, 266 nm, New Wave, USA, coupled with Spectrometer Elan DRC-e Perkin Elmer, USA. The content analysis of the studied bone material revealed with high probability that the contact method of mercurial treatment was used only in the case of two women from north-central Poland, deceased at the turn of the 15th century at the earliest.     

Association study of interleukin-4 polymorphisms with paranoid schizophrenia in the Polish population: a critical approach

Changes in immunological system are one of dysfunctions reported in schizophrenia. Some changes based on an imbalance between Th1 and Th2 cytokines results from cytokine gene polymorphisms. Interleukin-4 gene (IL4) is considered as a potential candidate gene in schizophrenia association studies. The aim of the current case-control study was to examine whether the -590C/T (rs2243250) and -33C/T (rs2070874) IL4 gene polymorphisms are implicated in paranoid schizophrenia development in the Polish population. Genotyping of polymorphisms was performed by using PCR-RFLP technique. The genotypes and alleles distribution of both SNPs were analysed in patients (n = 182) and healthy individuals constituted the control group (n = 215). The connection between some clinical variables and studied polymorphisms has been examined as well. We did not revealed any association between the -590C/T and -33C/T polymorphisms and paranoid schizophrenia. In case of both SNPs the homozygous TT genotype was extremely rare. Both polymorphic sites of the IL4 gene were found to be in a very strong linkage disequilibrium. However we did not identify a haplotype predispose to paranoid schizophrenia. No associations were also observed between the clinical course and psychopathology of the disease and the genotypes of both analysed polymorphisms. Our results suggest that the polymorphisms -590C/T in IL4 gene promoter region and -33C/T in the 5'-UTR are not involved in the pathophysiology of paranoid schizophrenia in Polish residents.     

Structural and functional interaction of (±)-2-(N-tert-butylamino)-3′-iodo-4′-azidopropiophenone,a photoreactive bupropion derivative,with nicotinic acetylcholine receptors

The pharmacological properties of (±)-2-(N-tert-butylamino)-3′-iodo-4′-azidopropiophenone [(±)-SADU-3-72], a photoreactive analog of bupropion (BP), were characterized at different muscle nicotinic acetylcholine receptors (AChRs) by functional and structural approaches. Ca²⁺ influx results indicate that (±)-SADU-3-72 is 17- and 6-fold more potent than BP in inhibiting human (h) embryonic (hα1β1γδ) and adult (hα1β1εδ) muscle AChRs, respectively. (±)-SADU-3-72 binds with high affinity to the [³H]TCP site within the resting or desensitized Torpedo AChR ion channel, whereas BP has higher affinity for desensitized AChRs. Molecular docking results indicate that both SADU-3-72 enantiomers interact with the valine (position 13′) and serine (position 6′) rings. However, an additional domain, between the outer (position 20′) and valine rings, is observed in Torpedo AChR ion channels. Our results indicate that the azido group of (±)-SADU-3-72 may enhance its interaction with polar groups and the formation of hydrogen bonds at AChRs, thus supporting the observed higher potency and affinity of (±)-SADU-3-72 compared to BP. Collectively our results are consistent with a model where BP/SADU-3-72 and TCP bind to overlapping sites within the lumen of muscle AChR ion channels. Based on these results, we believe that (±)-SADU-3-72 is a promising photoprobe for mapping the BP binding site, especially within the resting AChR ion channel.     

The role of leukotrienes in the pathogenesis of systemic sclerosis

Systemic sclerosis (SSc, scleroderma) is an autoimmune disease characterized by widespread vascular injury and progressive fibrosis of the skin and internal organs. SSc-related involvement of the lungs, heart, kidneys and/or the gastrointestinal system accounts for the increased mortality of scleroderma patients. Despite the progress which has recently been made in this field, the treatment of SSc is still unsatisfactory due to the low efficacy and/or high toxicity of available therapies. Leukotrienes are a family of lipid mediators synthesized from arachidonic acid in a process mediated by 5-lipoxygenase; they include leukotriene B4 and a group of cysteinyl leukotrienes: C4, D4, and E4. Leukotrienes play an important role in the regulation of all the processes vital to the pathogenesis of SSc, namely inflammation, vascular function and connective tissue remodeling. The available data suggests that an excessive synthesis of leukotrienes may contribute to the development and progression of SSc. Accordingly, blockade of leukotriene pathways appears to be a new, promising target for the treatment of SSc.