BBA - General Subjects (v.1810, #6)

Characterization of the L683P mutation of SLC26A9 in Xenopus oocytes by Martine Avella; Franck Borgese; Jordi Ehrenfeld (277-283).
In the present study, we characterized a STAS-domain amino acid mutation of SLC26A9 having a significant impact on ion transport. We focused on the sole conserved L- leucine residue of the STAS domain identified among SLC26 members. We therefore characterized the L683P mutation of SLC26A9 in Xenopus oocytes by monitoring the protein functional expression (two-electrode technique for voltage-clamp analysis) and its presence at the cell membrane (surface protein biotinylation technique). This mutation was found to reduce Cl transport through SLC26A9 as well as the positive interaction exerted by SLC26A9 on CFTR ion transport activity. The origin of this effect is discussed in the light of the presence of the SLC26A9–L683P mutant at the plasma membrane.► In the present study, we characterized a STAS-domain amino acid mutation of SLC26A9 having a significant impact on ion transport. ► This mutation was found to reduce Cl transport through SLC26A9 as well as the positive interaction exerted by SLC26A9 on CFTR ion transport activity. ► This study strengthens the role of the STAS domain in channel function as accounted for its interaction or stabilization of CFTR. ► SLC26A9 mutations in the STAS domain may be associated with some “cystic fibrosis-like” patient phenotypes that do not imply a CFTR gene defect.
Keywords: Cl channel; Xenopus oocytes; SLC26A9–L683P; CFTR;

A T/C polymorphism in the GPX4 3′UTR affects the selenoprotein expression pattern and cell viability in transfected Caco-2 cells by Hannah Gautrey; Fergus Nicol; Alan A. Sneddon; Judith Hall; John Hesketh (284-291).
Synthesis of selenoproteins such as glutathione peroxidases (GPx) requires a specific tRNA and a stem-loop structure in the 3′untranslated region (3′UTR) of the mRNA. A common single nucleotide polymorphism occurs in the GPX4 gene in a region corresponding to the 3′UTR.The two variant 3′UTR sequences were linked to sequences from a selenoprotein reporter gene (iodothyronine deiodinase) and expressed in Caco-2 cells. Clones expressing comparable levels of deiodinase (assessed by real-time PCR) were selected and their response to tert-butyl hydroperoxide assessed by cell viability and measurement of reactive oxygen species. Selenoprotein expression was assessed by real-time PCR, enzyme activity and immunoassay.When selenium supply was low, cells overexpressing the C variant 3′UTR showed lower viability after oxidative challenge, increased levels of reactive oxygen species and lower GPx activity and SelH mRNA expression compared to cells overexpressing the T variant. After selenium supplementation, cell viability and GPx4 expression were higher in the cells overexpressing the C variant. Expression of transgenes incorporating the T/C variant GPX4 (rs713041) sequences in Caco-2 cells leads to alterations in both cell viability after an oxidative challenge and selenoprotein expression. This suggests that the two variants compete differently in the selenoprotein hierarchy.The data provide evidence that the T/C variant GPX4 (rs713041) alters the pattern of selenoprotein synthesis if selenium intake is low. Further work is required to assess the impact on disease susceptibility.► Clones over-expressed a GPX4 3′UTR transgene with either T or C variant of rs713041. ► T/C polymorphism modulated effect of over-expression on selenoprotein hierarchy. ► T/C polymorphism modulated effect of transgene on sensitivity to oxidative challenge.
Keywords: Selenium; Glutathione peroxidase 4; Oxidative stress; 3′untranslated region; Selenoprotein hierarchy; Transfection;

G protein-coupled receptors fused to a Gα-subunit are functionally similar to their unfused counterparts. They offer an intriguing view into the nature of the receptor–G protein complex, but their usefulness depends upon the stability of the fusion.Fusion proteins of the M2 muscarinic receptor and the α-subunit of Gi1 were expressed in CHO and Sf9 cells, extracted in digitonin–cholate, and examined for their binding properties and their electrophoretic mobility on western blots.Receptor fused to native αi1 underwent proteolysis near the point of fusion to release a fragment with the mobility of αi1. The cleavage was prevented by truncation of the α-subunit at position 18. Binding of the agonist oxotremorine-M to the stable fusion protein from Sf9 cells was biphasic, and guanylylimidodiphosphate promoted an apparent interconversion of sites from higher to lower affinity. With receptor from CHO cells, the apparent capacity for N-[3H]methylscopolamine was 60% of that for [3H]quinuclidinylbenzilate; binding at saturating concentrations of the latter was inhibited in a noncompetitive manner at low concentrations of unlabeled N-methylscopolamine.A stable fusion protein of the M2 receptor and truncated αi1 resembles the native receptor–G protein complex with respect to the guanyl nucleotide-sensitive binding of agonists and the noncompetitive binding of antagonists.Release of the α-subunit is likely to occur with other such fusion proteins, rendering the data ambiguous or misleading. The properties of a chemically stable fusion protein support the notion that signaling proceeds via a stable multimeric complex of receptor and G protein.► Fusion proteins of the M2 muscarinic receptor and Gαi1 undergo proteolysis near the point of fusion. ► The cleavage was prevented by deletion of the first 17 amino acids from the N-terminus of Gαi1. ► The stable adduct from Sf9 cells exhibits GMP-PNP-sensitive heterogeneity toward agonists. ► Signaling therefore proceeds via a stable complex of two or more receptors and G proteins. ► Cleavage may be common to fusion proteins, thereby rendering the data ambiguous or misleading.
Keywords: Cooperativity; Fusion protein; G protein-coupled receptor; Gα-subunit; Oligomers; Radioligand binding studies;

Quantitative modeling of the self-assembly of DNA tiles leading either to defined end-products or distribution of biopolymers is of practical importance for biotechnology and synthetic biology.The combinatorial process describing tile assembly was implemented into a generic algorithm allowing quantitative description of the population of significant species accumulating during the reaction course. Experimental formation and characterization by optical and electrophoresis approaches of copolymers resulting from the self-assembly of a limited number of half-complementary tiles were used to define and validate generic rules allowing definition of model parameters.Factors controlling the structure and the dynamic of the oligomer population were evidenced for assemblies leading or not to defined end-products. Primary parameters were experimentally determined using rapid mixing experiments. Adjustment of simulations to experimental profiles allowed definition of generic rules for calculation of secondary parameters that take into account macro- and microenvironment of individual hybridization steps. In the case of copolymers, accurate simulation of experimental profiles was achieved for formation of linear assemblies.Overall length of species and structure of the DNA regions flanking the hybridization sites are critical parameters for which calculation rules were defined. The computational approach quantitatively predicted the parameters affecting time-course and distribution of accumulating products for different experimental designs.The computational and parameter evaluation procedures designed for the assembly of DNA tiles into large 1D-structures are more generally applicable for the construction of non-DNA polymers by extremities-specific recognition of molecular blocks.Display Omitted► Open and converging combinatorial assembly of DNA tiles was modeled. ► Parameters affecting time-course and distribution of end-products are predicted. ► We defined rules allowing estimation of model parameters from experimental data. ► Dependence of hybridization rates on tile structure and length is characterized. ► Model is expected to facilitate design of gene and combinatorial library synthesis.
Keywords: Combinatorial assembly; Deoxyribonucleic acid tile; System biology; Modeling; Kinetic control; Gene synthesis;

Analogs of 5-(substituted benzylidene)hydantoin as inhibitors of tyrosinase and melanin formation by Young Mi Ha; Jin-Ah Kim; Yun Jung Park; Daeui Park; Ji Min Kim; Ki Wung Chung; Eun Kyeong Lee; Ji Young Park; Ji Yeon Lee; Hye Jin Lee; Jeong Hyun Yoon; Hyung Ryong Moon; Hae Young Chung (612-619).
Many tyrosinase inhibitors find application in cosmetics and pharmaceutical products for the prevention of the overproduction of melanin in the epidermis. A series of 5-(substituted benzylidene)hydantoin derivatives 2a2k were prepared, and their inhibitory activities toward tyrosinase and melanin formation were evaluated.The structures of the compounds were established using 1H and 13C NMR spectroscopy and mass spectral analyses. All the synthesized compounds were evaluated for their mushroom tyrosinase inhibition activity.The best results were obtained for compound 2e which possessed hydroxyl group at R2 and methoxy group at R3, respectively. We predicted the tertiary structure of tyrosinase, simulated its docking with compound 2e and confirmed that this compound interacts strongly with mushroom tyrosinase residues as a competitive tyrosinase inhibitor. In addition, we found that 2e inhibited melanin production and tyrosinase activity in B16 cells.Compound 2e could be considered as a promising candidate for preclinical drug development in skin hyperpigmentation applications.This study will enhance understanding of the mechanism of tyrosinase inhibition and will contribute to the development of effective drugs for use hyperpigmentation.Computational structure prediction for mushroom tyrosinase and docking simulation with compound 2e. Display Omitted► A series of 5-(substituted benzylidene)hydantoin derivatives were synthesized. ► We examine the inhibitory activities toward melanin formation of the compounds. ► Increasing information will contribute to the development of effective drugs.
Keywords: 5-(substituted benzylidene)hydantoin derivatives; Tyrosinase inhibitory activity; Docking study with mushroom tyrosinase; Antimelanogenesis;

CoPK32 is a novel stress-responsive protein kinase in the mushroom Coprinopsis cinerea by Keisuke Kaneko; Yasunori Sugiyama; Yusuke Yamada; Noriyuki Sueyoshi; Akira Watanabe; Yasuhiko Asada; Atsuhiko Ishida; Isamu Kameshita (620-629).
In a previous study, we conducted an expression cloning screen of a cDNA library prepared from Coprinopsis cinerea mycelia using Multi-PK antibodies and detected a wide variety of Ser/Thr protein kinases. One of the isolated clones, CMZ032, was found to encode a putative Ser/Thr protein kinase designated CoPK32. In the present study, we investigated the biochemical properties and physiological significance of CoPK32.CoPK32 was expressed in Escherichia coli, and its biochemical properties were examined. The effects of high osmotic stresses on the growth of C. cinerea and on the endogenous CoPK32 activity in mycelia were also examined.CoPK32 showed autophosphorylation activity and effectively phosphorylated exogenous protein substrates. CoPK32S, a splice variant that was 18 amino acids shorter than CoPK32, showed much lower protein kinase activity than CoPK32. The catalytic properties of CoPK32 deletion mutants suggested that the C-terminal region of CoPK32 was important for the kinase activity and recognition of substrates. CoPK32 was highly expressed in the actively growing region of the mycelial colony. When mycelia were stimulated by high osmotic stresses, endogenous CoPK32 was markedly activated and the mycelial growth was severely inhibited. The activation of CoPK32 activity by high osmotic stresses was abrogated by SB202190 or SB239063 as well-known inhibitors of p38 mitogen-activated protein kinase.CoPK32 is involved in the stress response pathway in mycelia of C. cinerea in response to environmental stresses.In C. cinerea, protein kinases such as CoPK32 play important roles in signal transduction pathways involved in stress responses.► CoPK32 is a MAPKAPK homologue in Coprinopsis cinerea. ► C-terminal region of CoPK32 is involved in substrate recognition. ► CoPK32 is predominantly expressed in actively growing mycelial colony. ► CoPK32 is significantly activated by high osmotic stress.
Keywords: Basidiomycetes; Coprinopsis cinerea (Coprinus cinereus); Mushroom; Osmotic stress; Protein kinase; Stress response;

Inositol lipid metabolism in mycobacteria: Biosynthesis and regulatory mechanisms by Yasu S. Morita; Takeshi Fukuda; Chubert B.C. Sena; Yoshiki Yamaryo-Botte; Malcolm J. McConville; Taroh Kinoshita (630-641).
The genus Mycobacterium includes a number of medically important pathogens. The cell walls of these bacteria have many unique features, including the abundance of various inositol lipids, such as phosphatidylinositol mannosides (PIMs), lipomannan (LM), and lipoarabinomannan (LAM). The biosynthesis of these lipids is believed to be prime drug targets, and has been clarified in detail over the past several years.Here we summarize our current understanding of the inositol lipid metabolism in mycobacteria. We will highlight unsolved issues and future directions especially in the context of metabolic regulation.Inositol is a building block of phosphatidylinositol (PI), which is further elaborated to become PIMs, LM and LAM. d-myo-inositol 3-phosphate is an intermediate of the de novo inositol synthesis, but it is also the starting substrate for mycothiol synthesis. Controlling the level of d-myo-inositol 3-phosphate appears to be important for maintaining the steady state levels of mycothiol and inositol lipids. Several additional control mechanisms must exist to control the complex biosynthetic pathways of PI, PIMs, LM and LAM. These may include regulatory proteins such as a lipoprotein LpqW, and spatial separation of enzymes, such as the amphipathic PimA mannosyltransferase and later enzymes in the PIMs/LM biosynthetic pathway. Finally, we discuss mechanisms that underlie control of LM/LAM glycan polymer elongation.Mycobacteria have evolved a complex network of inositol metabolism. Clarifying its metabolism will not only provide better understanding of bacterial pathogenesis, but also understanding of the evolution and general functions of inositol lipids in nature.► Mycobacteria include many medically important pathogens. ► Biosynthesis of inositol lipids is complex and requires regulatory mechanisms. ► Controlling the level of d-myo-inositol 3-phosphate appears to be important. ► Regulatory proteins and spatial separations control inositol lipid biosynthesis. ► Control mechanisms exist for polymer elongation of lipomannan and lipoarabinomannan.
Keywords: Mycobacterium; Phosphatidylinositol mannoside; Lipomannan; Lipoarabinomannan; Inositol; Metabolic regulation;