Éva Klement

Phosphorylation of Phytochrome B Inhibits Light-Induced Signaling via Accelerated Dark Reversion in Arabidopsis

This work shows that the photoreceptor phytochrome B is phosphorylated in vivo and demonstrates that this posttranslational modification inhibits red light–induced photomorphogenesis under nonsaturating light conditions by accelerating light-independent inactivation and dark reversion of the biologically active phyB conformer. The photoreceptor phytochrome B (phyB) interconverts between the biologically active Pfr (λmax = 730 nm) and inactive Pr (λmax = 660 nm) forms in a red/far-red–dependent fashion and regulates, as molecular switch, many aspects of light-dependent development in Arabidopsis thaliana. phyB signaling is launched by the biologically active Pfr conformer and mediated by specific protein–protein interactions between phyB Pfr and its downstream regulatory partners, whereas conversion of Pfr to Pr terminates signaling. Here, we provide evidence that phyB is phosphorylated in planta at Ser-86 located in the N-terminal domain of the photoreceptor. Analysis of phyB-9 transgenic plants expressing phospho-mimic and nonphosphorylatable phyB–yellow fluorescent protein (YFP) fusions demonstrated that phosphorylation of Ser-86 negatively regulates all physiological responses tested. The Ser86Asp and Ser86Ala substitutions do not affect stability, photoconversion, and spectral properties of the photoreceptor, but light-independent relaxation of the phyBSer86Asp Pfr into Pr, also termed dark reversion, is strongly enhanced both in vivo and in vitro. Faster dark reversion attenuates red light–induced nuclear import and interaction of phyBSer86Asp-YFP Pfr with the negative regulator PHYTOCHROME INTERACTING FACTOR3 compared with phyB–green fluorescent protein. These data suggest that accelerated inactivation of the photoreceptor phyB via phosphorylation of Ser-86 represents a new paradigm for modulating phytochrome-controlled signaling.

Phosphorylation blocks the activity of tubulin polymerization promoting protein (TPPP): Identification of sites targeted by different kinases

Tubulin polymerization-promoting protein (TPPP), an unfolded brain-specific protein interacts with the tubulin/microtubule system in vitro and in vivo, and is enriched in human pathological brain inclusions. Here we show that TPPP induces tubulin self-assembly into intact frequently bundled microtubules, and that the phosphorylation of specific sites distinctly affects the function of TPPP. In vitro phosphorylation of wild type and the truncated form (Δ3-43TPPP) of human recombinant TPPP was performed by kinases involved in brain-specific processes. A stoichiometry of 2.9 ± 0.3, 2.2 ± 0.3, and 0.9 ± 0.1 mol P/mol protein with ERK2, cyclin-dependent kinase 5 (Cdk5), and cAMP-dependent protein kinase (PKA), respectively, was revealed for the full-length protein, and 0.4-0.5 mol P/mol protein was detected with all three kinases when the N-terminal tail was deleted. The phosphorylation sites Thr14, Ser18, Ser160 for Cdk5; Ser18, Ser160 for ERK2, and Ser32 for PKA were identified by mass spectrometry. These sites were consistent with the bioinformatic predictions. The three N-terminal sites were also found to be phosphorylated in vivo in TPPP isolated from bovine brain. Affinity binding experiments provided evidence for the direct interaction between TPPP and ERK2. The phosphorylation of TPPP by ERK2 or Cdk5, but not by PKA, perturbed the structural alterations induced by the interaction between TPPP and tubulin without affecting the binding affinity (Kd = 2.5-2.7 μm) or the stoichiometry (1 mol TPPP/mol tubulin) of the complex. The phosphorylation by ERK2 or Cdk5 resulted in the loss of microtubule-assembling activity of TPPP. The combination of our in vitro and in vivo data suggests that ERK2 can regulate TPPP activity via the phosphorylation of Thr14 and/or Ser18 in its unfolded N-terminal tail.

Enrichment of O-GlcNAc modified proteins by the periodate oxidation – hydrazide resin capture approach

A chemical derivatization approach has been developed for the enrichment of O-GlcNAc modified proteins. The procedure is based on the isolation technique used for N-glycoproteins with appropriate modifications because of the differences in the two types of glycosylation: a prolonged periodate oxidation is followed by hydrazide resin capture, on-resin proteolytic digestion, and release of the modified peptides by hydroxylamine. This enrichment strategy offers a fringe benefit in mass spectrometry analysis. Upon collisional activation, the presence of the open carbohydrate ring leads to characteristic fragmentation facilitating both glycopeptide identification and site assignment. The enrichment protocol was applied to the Drosophila proteasome complex previously described as O-GlcNAc modified. The O-GlcNAc modification was located on proteasome interacting proteins, deubiquitinating enzyme Faf (CG1945) and a ubiquitin-like domain containing protein (CG7546). Three other proteins were also found GlcNAc modified, a HSP70 homologue (CG2918), scribbled (CG5462) and the 205 kDa microtubule-associated protein (CG1483). Interestingly, in the HSP70 homologue the GlcNAc modification is attached to an asparagine residue of a N-glycosylation motif.

Active site closure facilitates juxtaposition of reactant atoms for initiation of catalysis by human dUTPase

Human dUTPase, essential for DNA integrity, is an important survival factor for cancer cells. We determined the crystal structure of the enzyme:α,β‐imino‐dUTP:Mg complex and performed equilibrium binding experiments in solution. Ordering of the C‐terminus upon the active site induces close juxtaposition of the incoming nucleophile attacker water oxygen and the α‐phosphorus of the substrate, decreasing their distance below the van der Waals limit. Complex interactions of the C‐terminus with both substrate and product were observed via a specifically designed tryptophan sensor, suitable for further detailed kinetic and ligand binding studies. Results explain the key functional role of the C‐terminus.

Flexible segments modulate co-folding of dUTPase and nucleocapsid proteins

The homotrimeric fusion protein nucleocapsid (NC)-dUTPase combines domains that participate in RNA/DNA folding, reverse transcription, and DNA repair in Mason-Pfizer monkey betaretrovirus infected cells. The structural organization of the fusion protein remained obscured by the N- and C-terminal flexible segments of dUTPase and the linker region connecting the two domains that are invisible in electron density maps. Small-angle X-ray scattering reveals that upon oligonucleotide binding the NC domains adopt the trimeric symmetry of dUTPase. High-resolution X-ray structures together with molecular modeling indicate that fusion with NC domains dramatically alters the conformation of the flexible C-terminus by perturbing the orientation of a critical β-strand. Consequently, the C-terminal segment is capable of double backing upon the active site of its own monomer and stabilized by non-covalent interactions formed with the N-terminal segment. This co-folding of the dUTPase terminal segments, not observable in other homologous enzymes, is due to the presence of the fused NC domain. Structural and genomic advantages of fusing the NC domain to a shortened dUTPase in betaretroviruses and the possible physiological consequences are envisaged.

Modular Broad-Host-Range Expression Vectors for Single-Protein and Protein Complex Purification

ABSTRACT A set of modular broad-host-range expression vectors with various affinity tags (six-His-tag, FLAG-tag, Strep-tag II, T7-tag) was created. The complete nucleotide sequences of the vectors are known, and these small vectors can be mobilized by conjugation. They are useful in the purification of proteins and protein complexes from gram-negative bacterial species. The plasmids were easily customized for Thiocapsa roseopersicina, Rhodobacter capsulatus, and Methylococcus capsulatus by inserting an appropriate promoter. These examples demonstrate the versatility and flexibility of the vectors. The constructs harbor the T7 promoter for easy overproduction of the desired protein in an appropriate Escherichia coli host. The vectors were useful in purifying different proteins from T. roseopersicina. The FLAG-tag-Strep-tag II combination was utilized for isolation of the HynL-HypC2 protein complex involved in hydrogenase maturation. These tools should be useful for protein purification and for studying protein-protein interactions in a range of bacterial species.

Electron-transfer subunits of the NiFe hydrogenases in Thiocapsa roseopersicina BBS.

Thiocapsa roseopersicina BBS contains at least three different active NiFe hydrogenases: two membrane‐bound enzymes and one apparently localized in the cytoplasm. In addition to the small and large structural subunits, additional proteins are usually associated with the NiFe hydrogenases, connecting their activity to other redox processes in the cells. The operon of the membrane‐associated hydrogenase, HynSL, has an unusual gene arrangement: between the genes coding for the large and small subunits, there are two open reading frames, namely isp1 and isp2. Isp1 is a b‐type haem‐containing transmembrane protein, whereas Isp2 displays marked sequence similarity to the heterodisulfide reductases. The other membrane‐bound (Hup) NiFe hydrogenase contains the hupC gene, which codes for a cytochrome b‐type protein that probably plays a role in electron transport. The operon of the NAD+‐reducing Hox hydrogenase contains a hoxE gene. In addition to the hydrogenase and diaphorase parts of the complex, the fifth HoxE subunit may serve as a third redox gate of this enzyme. The physiological functions of these putative electron‐mediating subunits were studied by disruption of their genes. The deletion of some accessory proteins dramatically reduced the in vivo activities of the hydrogenases, although they were fully active in vitro. The absence of HupC resulted in a decrease in HupSL activity in the membrane, but removal of the Isp1 and Isp2 proteins did not have any significant effect on the location of HynSL activity. Through the use of a tagged HoxE protein, the whole Hox hydrogenase pentamer could be purified as an intact complex.

Comparison of transcriptional and translational changes caused by long-term menadione exposure in Aspergillus nidulans

Under long-term oxidative stress caused by menadione sodium bisulfite, genome-wide transcriptional and proteome-wide translational changes were compared in Aspergillus nidulans vegetative cells. The comparison of proteomic and DNA microarray expression data demonstrated that global gene expression changes recorded with either flip-flop or dendrimer cDNA labeling techniques supported proteome changes moderately with 40% and 34% coincidence coefficients, respectively. Enzyme levels in the glycolytic pathway were alternating, which was a direct consequence of fluctuating gene expression patterns. Surprisingly, enzymes in the vitamin B2 and B6 biosynthetic pathways were repressed concomitantly with the repression of some protein folding chaperones and nuclear transport elements. Under long-term oxidative stress, the peroxide-detoxifying peroxiredoxins and cytochrome c peroxidase were replaced by thioredoxin reductase, a nitroreductase and a flavohemoprotein, and protein degradation became predominant to eliminate damaged proteins.

Comparative studies of differential expression of chitinolytic enzymes encoded by chiA, chiB, chiC and nagA genes in Aspergillus nidulans

N-Acetyl-d-glucosamine, chito-oligomers and carbon starvation regulatedchiA, chiB, andnagA gene expressions inAspergillus nidulans cultures. The gene expression patterns of the main extracellular endochitinase ChiB and theN-acetyl-β-d-glucosaminidase NagA were similar, and the ChiB-NagA enzyme system may play a morphological and/or nutritional role during autolysis. Alterations in the levels of reactive oxygen species or in the glutathione-glutathione disulfide redox balance, characteristic physiological changes developing in ageing and autolyzing fungal cultures, did not affect the regulation of either the growth-relatedchiA or the autolysis-coupledchiB genes although both of them were down-regulated under diamide stress. The transcription of thechiC gene with unknown physiological function was repressed by increased intracellular superoxide concentration.

Plant Rho-type (Rop) GTPase-dependent activation of receptor-like cytoplasmic kinases in vitro.

Plants have evolved distinct mechanisms to link Rho‐type (Rop) GTPases to downstream signaling pathways as compared to other eukaryotes. Here, experimental data are provided that members of the Medicago, as well as Arabidopsis, receptor‐like cytoplasmic kinase family (RLCK Class VI) were strongly and specifically activated by GTP‐bound Rop GTPases in vitro. Deletion analysis indicated that the residues implicated in the interaction might be distributed on various parts of the kinases. Using a chimaeric Rop GTPase protein, the importance of the Rho‐insert region in kinase activation could also be verified. These data strengthen the possibility that RLCKs may serve as Rop GTPase effectors in planta.