Up-regulation of GABAB Receptor Signaling by Constitutive Assembly with the K+ Channel Tetramerization Domain-containing Protein 12 (KCTD12)

GABA_B receptors are the G-protein coupled receptors (GPCRs) for GABA, the main inhibitory neurotransmitter in the central nervous system. Native GABA_B receptors comprise principle and auxiliary subunits that regulate receptor properties in distinct ways. The principle subunits GABA_B1a, GABA_B1b, and GABA_B2 form fully functional heteromeric GABA_B(1a,2) and GABA_B(1b,2) receptors. Principal subunits regulate forward trafficking of the receptors from the endoplasmic reticulum to the plasma membrane and control receptor distribution to axons and dendrites. The auxiliary subunits KCTD8, -12, -12b, and -16 are cytosolic proteins that influence agonist potency and G-protein signaling of GABA_B(1a,2) and GABA_B(1b,2) receptors. Here, we used transfected cells to study assembly, surface trafficking, and internalization of GABA_B receptors in the presence of the KCTD12 subunit. Using bimolecular fluorescence complementation and metabolic labeling, we show that GABA_B receptors associate with KCTD12 while they reside in the endoplasmic reticulum. Glycosylation experiments support that association with KCTD12 does not influence maturation of the receptor complex. Immunoprecipitation and bioluminescence resonance energy transfer experiments demonstrate that KCTD12 remains associated with the receptor during receptor activity and receptor internalization from the cell surface. We further show that KCTD12 reduces constitutive receptor internalization and thereby increases the magnitude of receptor signaling at the cell surface. Accordingly, knock-out or knockdown of KCTD12 in cultured hippocampal neurons reduces the magnitude of the GABA_B receptor-mediated K⁺ current response. In summary, our experiments support that the up-regulation of functional GABA_B receptors at the neuronal plasma membrane is an additional physiological role of the auxiliary subunit KCTD12.     

p87 and p101 Subunits Are Distinct Regulators Determining Class IB Phosphoinositide 3-Kinase (PI3K) Specificity

Class I_B phosphoinositide 3-kinase γ (PI3Kγ) comprises a single catalytic p110γ subunit, which binds to two non-catalytic subunits, p87 or p101, and controls a plethora of fundamental cellular responses. The non-catalytic subunits are assumed to be redundant adaptors for Gβγ enabling G-protein-coupled receptor-mediated regulation of PI3Kγ. Growing experimental data provide contradictory evidence. To elucidate the roles of the non-catalytic subunits in determining the specificity of PI3Kγ, we tested the impact of p87 and p101 in heterodimeric p87-p110γ and p101-p110γ complexes on the modulation of PI3Kγ activity in vitro and in living cells. RT-PCR, biochemical, and imaging data provide four lines of evidence: (i) specific expression patterns of p87 and p101, (ii) up-regulation of p101, providing the basis to consider p87 as a protein forming a constitutively and p101 as a protein forming an inducibly expressed PI3Kγ, (iii) differences in basal and stimulated enzymatic activities, and (iv) differences in complex stability, all indicating apparent diversity within class I_B PI3Kγ. In conclusion, expression and activities of PI3Kγ are modified differently by p87 and p101 in vitro and in living cells, arguing for specific regulatory roles of the non-catalytic subunits in the differentiation of PI3Kγ signaling pathways.     

Development and characterization of a compensating wheat-Thinopyrum intermedium Robertsonian translocation with Sr44 resistance to stem rust (Ug99)

The emergence of the highly virulent Ug99 race complex of the stem rust fungus (Puccinia graminis Pers. f. sp. tritici Eriks. and Henn.) threatens wheat (Triticum aestivum L.) production worldwide. One of the effective genes against the Ug99 race complex is Sr44, which was derived from Thinopyrum intermedium (Host) Barkworth and D.R. Dewey and mapped to the short arm of 7J (designated 7J#1S) present in the noncompensating T7DS-7J#1L?7J#1S translocation. Noncompensating wheat-alien translocations are known to cause genomic duplications and deficiencies leading to poor agronomic performance, precluding their direct use in wheat improvement. The present study was initiated to produce compensating wheat-Th. intermedium Robertsonian translocations with Sr44 resistance. One compensating RobT was identified consisting of the wheat 7DL arm translocated to the Th. intermedium 7J#1S arm resulting in T7DL?7J#1S. The T7DL?7J#1S stock was designated as TA5657. The 7DL?7J#1S stock carries Sr44 and has resistance to the Ug99 race complex. This compensating RobT with Sr44 resistance may be useful in wheat improvement. In addition, we identified an unnamed stem rust resistance gene located on the 7J#1L arm that confers resistance not only to Ug99, but also to race TRTTF, which is virulent to Sr44. However, the action of the second gene can be modified by the presence of suppressors in the recipient wheat cultivars.     

Genome wide sequence analysis grants unbiased definition of species boundaries in “Candidatus Phytoplasma”

The phytoplasmas are currently named using the Candidatus category, as the inability to grow them in vitro prevented (i) the performance of tests, such as DNA-DNA hybridization, that are regarded as necessary to establish species boundaries, and (ii) the deposition of type strains in culture collections. The recent accession to complete or nearly complete genome sequence information disclosed the opportunity to apply to the uncultivable phytoplasmas the same taxonomic approaches used for other bacteria. In this work, the genomes of 14 strains, belonging to the 16SrI, 16SrIII, 16SrV and 16SrX groups, including the species “Ca. P. asteris”, “Ca. P. mali”, “Ca. P. pyri”, “Ca. P. pruni”, and “Ca. P. australiense” were analyzed along with Acholeplasma laidlawi, to determine their taxonomic relatedness. Average nucleotide index (ANIm), tetranucleotide signature frequency correlation index (Tetra), and multilocus sequence analysis of 107 shared genes using both phylogenetic inference of concatenated (DNA and amino acid) sequences and consensus networks, were carried out. The results were in large agreement with the previously established 16S rDNA based classification schemes. Moreover, the taxonomic relationships within the 16SrI, 16SrIII and 16SrX groups, that represent clusters of strains whose relatedness could not be determined by 16SrDNA analysis, could be comparatively evaluated with non-subjective criteria. “Ca. P. mali” and “Ca. P. pyri” were found to meet the genome characteristics for the retention into two different, yet strictly related species; representatives of subgroups 16SrI-A and 16SrI-B were also found to meet the standards used in other bacteria to distinguish separate species; the genomes of the strains belonging to 16SrIII were found more closely related, suggesting that their subdivision into Candidatus species should be approached with caution.     

Homooligomeric dA·dU and dA·dT Sequences in Parallel and Antiparallel Strand Orientation: Consequence of the 5-methyl Groups on Stability,Structure and Interaction with the Minor Groove Binding Drug HOECHST 33258

Abstract

Oligodeoxyribonucleotides containing dA·dU base combinations were shown to form parallel stranded DNA. CD spectra and hyperchromicity profiles provide evidence that the structure is very similar to that of a related parallel stranded dA·oligomer. Thermal denaturation studies show that these parallel dAdU sequences are significantly less stable than their dA·analogues in either antiparallel or parallel stranded orientations. The stabilizing effect of the 5- methyl group is similar for parallel and antiparallel sequences. The minor groove binding drug Hoechst 33258 binds with similar affinity to APS dA·and APS dA·dU sequences. However, binding to the PS dA·hairpin is significantly impaired as a consequence of the different groove dimensions and the presence of thymine methyl groups at the binding site. This results in an 8.6 kJmoF reduced free energy of binding for the PS dA·sequence. Replacement of the bulky methyl group with a hydrogen (ie. T -> U) results in significantly stronger Hoechst 33258 binding to the parallel dA·dU sequences with a penalty of only 4.1 kJmol^?1. Our data demonstrate that although Hoechst 33258 detects the altered groove, it is still able to bind a PS duplex containing dA·dU base pairs with high affinity, despite the large structural differences from its regular binding site in APS DNA.     

Towards an Understanding of DNA Recognition by the Methyl-CpG Binding Domain 1

Abstract

CpG methylation determines a variety of biological functions of DNA. The methylation signal is interpreted by proteins containing a methyl-CpG binding domain (MBDs). Based on the NMR structure of MBD1 complexed with methylated DNA we analysed the recognition mode by means of molecular dynamics simulations.

As the protein is monomeric and recognizes a symmetrically methylated CpG step, the recognition mode is an asymmetric one. We find that the two methyl groups do not contribute equally to the binding energy. One methyl group is associated with the major part of the binding energy and the other one nearly does not contribute at all. The contribution of the two cytosine methyl groups to binding energy is calculated to be ?3.6 kcal/mol. This implies a contribution of greater than two orders of magnitude to the binding constant. The conserved amino acid Asp32 is known to be essential for DNA binding by MBD1, but so far no direct contact with DNA has been observed. We detected a direct DNA base contact to Asp32. This could be the main reason for the importance of this amino acid. MBD contacts DNA exclusively in the major groove, the minor groove is reserved for histone contacts. We found a deformation of the minor groove shape due to complexation by MBD1, which indicates an information transfer between the major and the minor groove.     

The Involvement of Mitochondrial Amidoxime Reducing Components 1 and 2 and Mitochondrial Cytochrome b5 in N-Reductive Metabolism in Human Cells

The mitochondrial amidoxime reducing component mARC is a recently discovered molybdenum enzyme in mammals. mARC is not active as a standalone protein, but together with the electron transport proteins NADH-cytochrome b₅ reductase (CYB5R) and cytochrome b₅ (CYB5), it catalyzes the reduction of N-hydroxylated compounds such as amidoximes. The mARC-containing enzyme system is therefore considered to be responsible for the activation of amidoxime prodrugs. All hitherto analyzed mammalian genomes code for two mARC genes (also referred to as MOSC1 and MOSC2), which share high sequence similarities. By RNAi experiments in two different human cell lines, we demonstrate for the first time that both mARC proteins are capable of reducing N-hydroxylated substrates in cell metabolism. The extent of involvement is highly dependent on the expression level of the particular mARC protein. Furthermore, the mitochondrial isoform of CYB5 (CYB5B) is clearly identified as an essential component of the mARC-containing N-reductase system in human cells. The participation of the microsomal isoform (CYB5A) in N-reduction could be excluded by siRNA-mediated down-regulation in HEK-293 cells and knock-out in mice. Using heme-free apo-CYB5, the contribution of mitochondrial CYB5 to N-reductive catalysis was proven to strictly depend on heme. Finally, we created recombinant CYB5B variants corresponding to four nonsynonymous single nucleotide polymorphisms (SNPs). Investigated mutations of the heme protein seemed to have no significant impact on N-reductive activity of the reconstituted enzyme system.