Solid state NMR sequential resonance assignments and conformational analysis of the 2×10.4 kDa dimeric form of the Bacillus subtilis protein Crh

Solid state NMR sample preparation and resonance assignments of the U-[¹³C,¹⁵N] 2×10.4 kDa dimeric form of the regulatory protein Crh in microcrystalline, PEG precipitated form are presented. Intra– and interresidue correlations using dipolar polarization transfer methods led to nearly complete sequential assignments of the protein, and to 88% of all ¹⁵N, ¹³C chemical shifts. For several residues, the resonance assignments differ significantly from those reported for the monomeric form analyzed by solution state NMR. Dihedral angles obtained from a TALOS-based statistical analysis suggest that the microcrystalline arrangement of Crh must be similar to the domain-swapped dimeric structure of a single crystal form recently solved using X-ray crystallography. For a limited number of protein residues, a remarkable doubling of the observed NMR resonances is observed indicative of local static or dynamic conformational disorder. Our study reports resonance assignments for the largest protein investigated by solid state NMR so far and describes the conformational dimeric variant of Crh with previously unknown chemical shifts.     

Characterization of two functional phosphoenolpyruvate/phosphate translocator (PPT) genes in Arabidopsis--AtPPT1 may be involved in the provision of signals for correct mesophyll development

The Arabidopsis thaliana chlorophyll a/b-binding protein underexpressed 1 (cue1) mutant shows a reticulate leaf phenotype and is defective in a plastidic phosphoenolpyruvate (PEP)/phosphate translocator (AtPPT1). A functional AtPPT1 providing plastids with PEP for the shikimate pathway is therefore essential for correct leaf development. The Arabidopsis genome contains a second PPT gene, AtPPT2. Both transporters share similar substrate specificities and are therefore able to transport PEP into plastids. The cue1 phenotype could partially be complemented by ectopic expression of AtPPT2 but obviously not by the endogeneous AtPPT2. Both genes are differentially expressed in most tissues: AtPPT1 is mainly expressed in the vasculature of leaves and roots, especially in xylem parenchyma cells, but not in leaf mesophyll cells, whereas AtPPT2 is expressed ubiquitously in leaves, but not in roots. The expression profiles are corroborated by tissue-specific transport data. As AtPPT1 expression is absent in mesophyll cells that are severely affected in the cue1 mutant, we propose that the vasculature-located AtPPT1 is involved in the generation of phenylpropanoid metabolism-derived signal molecules that trigger development in interveinal leaf regions. This signal probably originates from the root vasculature where only AtPPT1, but not AtPPT2, is present.     

Gene structure and expression of the 5'-(CGG)(n)-3'-binding protein (CGGBP1)

The human nuclear 5'-(CGG)(n)-3'-binding protein (CGGBP1) influences the expression of the fragile X mental retardation (FMR1) gene by specifically interacting with the 5'-(CGG)(n>5)-3' repeat in its 5' untranslated region. Here, we show that CGGBP1 binds to 5'-(CGG)(n)-3' repeats with n>or=5 and to interrupted repeats. The genomic and mRNA organization of the human and murine CGGBP1 genes was studied and the human gene was mapped to chromosome 3p. Due to alternative polyadenylation, mRNAs of 1.2 and 4.5 kb are transcribed at varying ratios in human and murine cells and in embryonic, fetal, and adult tissues. The human and the murine genes, including promoters and large parts of the untranslated regions, are highly conserved. A sequence of 235 nucleotides 5' upstream of CGGBP1 is essential for promoter activity in transfection experiments. Complete in vitro methylation inactivates the promoter, which is unmethylated in human cells as shown by bisulfite genomic sequencing.     

Hydrogen peroxide and ADP-ribose induce TRPM2-mediated calcium influx and cation currents in microglia

Microglial cells are the host macrophages in the central nervous system and respond to brain injury and various neurological diseases. In this process, microglial cells undergo multiple morphological and functional changes from the resting cell toward a fully activated, phagocyting tissue macrophage. In culture, bacterial lipopolysaccharide (LPS) is a frequently used tool to induce this activation. By using calcium-imaging and patch-clamp techniques, we investigated the effect of hydrogen peroxide (H₂O₂), which is released by macrophagic cells themselves, on the intracellular calcium concentration and ion currents in cultured rat microglia. Application of 0.1–5 mM H₂O₂ for several minutes induced small responses in untreated cells but a large calcium influx and cation current in LPS-treated cells. In both untreated and LPS-treated microglia, internal perfusion of ADP-ribose (ADPR) via the patch pipette elicited large cation currents. Both stimuli, H₂O₂ and ADPR, have been reported to activate the recently cloned nonselective cation channel TRPM2. RT-PCR analysis from cultured rat glial and neuronal cells confirmed a strong expression of TRPM2 in rat microglia but not in astrocytes and cerebellar granule cells. In situ hybridizations from mouse brain showed a distribution of TRPM2, which is compatible with the expression in microglial cells. In conclusion, we describe here a novel calcium influx pathway in microglia coupled to hydrogen peroxide and ADPR and provide evidence that this pathway involves TRPM2. The increased sensitivity to H₂O₂ in LPS-stimulated cells suggests a role for TRPM2 in the calcium signaling of activated microglia. nonselective cation channel; transient receptor potential channel; H₂O₂; activated microglia     

Arabidopsis trehalose-6-phosphate synthase 1 is essential for normal vegetative growth and transition to flowering

In resurrection plants and yeast, trehalose has a function in stress protection, but the absence of measurable amounts of trehalose in other plants precludes such a function. The identification of a trehalose biosynthetic pathway in angiosperms raises questions on the function of trehalose metabolism in nonresurrection plants. We previously identified a mutant in the Arabidopsis trehalose biosynthesis gene AtTPS1. Plants homozygous for the tps1 mutation do not develop mature seeds (Eastmond et al., 2002). AtTPS1 expression analysis and the spatial and temporal activity of its promoter suggest that this gene is active outside the seed-filling stage of development as well. A generally low expression is observed in all organs analyzed, peaking in metabolic sinks such as flower buds, ripening siliques, and young rosette leaves. The arrested tps1/tps1 embryonic state could be rescued using a dexamethasone-inducible AtTPS1 expression system enabling generation of homozygous mutant plants. When depleted in AtTPS1 expression, such mutant plants show reduced root growth, which is correlated with a reduced root meristematic region. Moreover, tps1/tps1 plants are retarded in growth and remain generative during their lifetime. Absence of Trehalose-6-Phosphate Synthase 1 in Arabidopsis plants precludes transition to flowering.     

Xylem sap protein composition is conserved among different plant species

Xylem sap from broccoli (Brassica oleracea L. cv. Calabrais), rape (Brassica napus L. cv. Drakkar), pumpkin (Cucurbita maxima Duch. cv. gelber Zentner) and cucumber (Cucumis sativus L. cv. Hoffmanns Giganta) was collected by root pressure exudation from the surface of cut stems of healthy, adult plants. Total protein concentrations were in the range of 100 g ml^–1. One-dimensional gel electrophoresis (SDS–PAGE) resulted in 10–20 visible protein bands in a molecular mass range from 10 to 100 kDa. The main bands were cut out, digested with trypsin, and analysed using tandem mass spectrometry. Fifty bands resulted in amino acid sequence information that was used to perform database similarity searches. Sequences from 30 bands showed high homology to proteins present in databases. Among them, we found mostly peroxidases, but could also identify the lectin-like xylem protein XSP30, a glycine-rich protein, serine proteases, an aspartyl protease family protein, chitinases, and a lipid transfer protein-like polypeptide. Sequence analysis predicted apoplastic secretion signals for all database entries similar to the partial xylem protein sequences. This and the lack of cross-reactivity with phloem protein-specific antibodies suggest that the proteins really originate from the xylem and do not result from phloem contamination. Most of the highly similar proteins probably function in repair and defence reactions. Some of the most abundant proteins (peroxidases, chitinases, serine proteases) were present in xylem exudate of all species analysed, often in more than one band. This indicates an important basic role of these proteins in maintaining xylem function.Abbreviations CHT Chitinase - 1D One-dimensional - GRP Glycine-rich protein - SP Serine protease - SSP Subtilisin-like serine protease - POX Peroxidase     

In situ identification of protein structural changes in prion-infected tissue

Transmissible spongiform encephalopathies (TSE) are fatal neurodegenerative disorders characterized by the conversion of the normal prion protein (PrP(C)) into aggregates of its pathological conformer (PrP(Sc)). The mechanism behind this structural conversion is unclear. We report the identification of disease-related protein structural differences directly within the tissue environment. Utilizing a synchrotron infrared (IR) light source, IR images of protein structure were obtained at a subcellular resolution, revealing regions of decreased alpha-helical content and elevated beta-sheet structure in and around infected neurons in the 263 K scrapie hamster model. PrP(Sc) immunostaining of the same tissue demonstrated that the elevated beta-sheet regions correspond to regions where the misfolded structure of PrP(Sc) is located. No evidence of these structural changes was observed in normal neurons.     

Formation of critical oligomers is a key event during conformational transition of recombinant syrian hamster prion protein

We have investigated the conformational transition and aggregation process of recombinant Syrian hamster prion protein (SHaPrP90-232) by Fourier transform infrared spectroscopy, circular dichroism spectroscopy, light scattering, and electron microscopy under equilibrium and kinetic conditions. SHaPrP90-232 showed an infrared absorbance spectrum typical of proteins with a predominant alpha-helical structure both at pH 7.0 and at pH 4.2 in the absence of guanidine hydrochloride. At pH 4.2 and destabilizing conditions (0.3-2 m guanidine hydrochloride), the secondary structure of SHaPrP90-232 was transformed to a strongly hydrogen-bonded, most probably intermolecularly arranged antiparallel beta-sheet structure as indicated by dominant amide I band components at 1620 and 1691 cm-1. Kinetic analysis of the transition process showed that the decrease in alpha-helical structures and the increase in beta-sheet structures occurred concomitantly according to a bimolecular reaction. However, the concentration dependence of the corresponding rate constant pointed to an apparent third order reaction. No beta-sheet structure was formed within the dead time (190 ms) of the infrared experiments. Light scattering measurements revealed that the structural transition of SHaPrP90-232 was accompanied by formation of oligomers, whose size was linearly dependent on protein concentration. Extrapolation to zero protein concentration yielded octamers as the smallest oligomers, which are considered as "critical oligomers." The small oligomers showed spherical and annular shapes in electron micrographs. Critical oligomers seem to play a key role during the transition and aggregation process of SHaPrP90-232. A new model for the structural transition and aggregation process of the prion protein is described.