Salicylic Acid and Phenylalanine Ammonia-Lyase in Potato Plants Infected with the Causal Agent of Late Blight

In tuber tissues of potato (Solanum tuberosum L.) infected with an incompatible race of Phytophthora infestans (Mont.) de Bary, the activity of phenylalanine ammonia-lyase and the contents of free and bound salicylic acid (SA) considerably exceeded the corresponding indices in the tissues infected with a compatible race of the oomycete. The accumulation of the free form of SA apparently resulted from both enhanced SA biosynthesis and the liberation from the bound SA forms. SA accumulation in the incompatible host-pathogen combination presumes that SA participated in the local potato resistance to late blight.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 573–577.Original Russian Text Copyright © 2005 by Panina, Gerasimova, Chalenko, Vasyukova, Ozeretskovskaya.     

Multiple Roles of VARP in Endosomal Trafficking: Rabs,Retromer Components and R‐SNARE VAMP7 Meet on VARP

VARP (VPS9‐ankyrin‐repeat protein, also known as ANKRD27) was originally identified as an N‐terminal VPS9 (vacuolar protein sorting 9)‐domain‐containing protein that possesses guanine nucleotide exchange factor (GEF) activity toward small GTPase Rab21 and contains two ankyrin repeat (ANKR) domains in its central region. A number of VARP‐interacting molecules have been identified during the past five years, and considerable attention is now being directed to the multiple roles of VARP in endosomal trafficking. More specifically, VARP is now known to interact with three different types of key membrane trafficking regulators, i.e. small GTPase Rabs (Rab32, Rab38 and Rab40C), the retromer complex (a sorting nexin dimer, VPS26, VPS29 and VPS35) and R‐SNARE VAMP7. By binding to several of these molecules, VARP regulates endosomal trafficking, which underlies a variety of cellular events, including melanogenic enzyme trafficking to melanosomes, dendrite outgrowth of melanocytes, neurite outgrowth and retromer‐mediated endosome‐to‐plasma membrane sorting of transmembrane proteins.      

Comparative genomics enabled the isolation of the R3a late blight resistance gene in potato

Comparative genomics provides a tool to utilize the exponentially increasing sequence information from model plants to clone agronomically important genes from less studied crop species. Plant disease resistance (R) loci frequently lack synteny between related species of cereals and crucifers but appear to be positionally well conserved in the Solanaceae. In this report, we adopted a local RGA approach using genomic information from the model Solanaceous plant tomato to isolate R3a, a potato gene that confers race-specific resistance to the late blight pathogen Phytophthora infestans. R3a is a member of the R3 complex locus on chromosome 11. Comparative analyses of the R3 complex locus with the corresponding I2 complex locus in tomato suggest that this is an ancient locus involved in plant innate immunity against oomycete and fungal pathogens. However, the R3 complex locus has evolved after divergence from tomato and the locus has experienced a significant expansion in potato without disruption of the flanking colinearity. This expansion has resulted in an increase in the number of R genes and in functional diversification, which has probably been driven by the co-evolutionary history between P. infestans and its host potato. Constitutive expression was observed for the R3a gene, as well as some of its paralogues whose functions remain unknown.     

RabX1 Organizes a Late Endosomal Compartment that Forms Tubular Connections to Lysosomes Consistent with a “Kiss and Run” Mechanism

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Eng2 Is a Component of a Dynamic Protein Complex Required for Endocytic Uptake in Fission Yeast

Eng2 is a glucanase required for spore release, although it is also expressed during vegetative growth, suggesting that it might play other cellular functions. Its homology to the Saccharomyces cerevisiae Acf2 protein, previously shown to promote actin polymerization at endocytic sites in vitro, prompted us to investigate its role in endocytosis. Interestingly, depletion of Eng2 caused profound defects in endocytic uptake, which were not due to the absence of its glucanase activity. Analysis of the dynamics of endocytic proteins by fluorescence microscopy in the eng2Δ strain unveiled a previously undescribed phenotype, in which assembly of the Arp2/3 complex appeared uncoupled from the internalization of the endocytic coat and resulted in a fission defect. Strikingly also, we found that Eng2‐GFP dynamics did not match the pattern of other endocytic proteins. Eng2‐GFP localized to bright cytosolic spots that moved around the cellular poles and occasionally contacted assembling endocytic patches just before recruitment of Wsp1, the Schizosaccharomyces pombe WASP. Interestingly, Csh3‐YFP, a WASP‐interacting protein, interacted with Eng2 by co‐immunoprecipitation and was recruited to Eng2 in bright cytosolic spots. Altogether, our work defines a novel endocytic functional module, which probably couples the endocytic coat to the actin module.      

Control of Late Blight (Phytophthora capsici ) in Pepper Plant with a Compost Containing Multitude of Chitinase-producing Bacteria

Compost sustaining a multitude of chitinase-producing bacteria was evaluated in a greenhouse study as a soil amendment for the control of late blight (Phytophthora capsici L.) in pepper (Capsicum annuum L.). Microbial population and exogenous enzyme activity were measured in the rhizosphere and correlated to the growth and health of pepper plant. Rice straw was composted with and without a chitin source, after having been inoculated with an aliquot of coastal area soil containing a known titer of chitinase-producing bacteria. P. capsici inoculated plants cultivated in chitin compost-amended soil exhibited significantly higher root and shoot weights and lower root mortality than plants grown in pathogen-inoculated control compost. Chitinase and β-1,3-glucanase activities in rhizosphere of plants grown in chitin compost-amended soil were twice that seen in soil amended with control compost. Colony forming units of chitinase-producing bacteria isolated from rhizosphere of plants grown in chitin compost-amended soil were 10³ times as prevalent as bacteria in control compost. These results indicate that increasing the population of chitinase-producing bacteria and soil enzyme activities in rhizosphere by compost amendment could alleviate pathogenic effects of P. capsici.     

Routes and mechanisms of post‐endosomal cholesterol trafficking: A story that never ends

Mammalian cells acquire most exogenous cholesterol through receptor‐mediated endocytosis of low‐density lipoproteins (LDLs). After internalization, LDL cholesteryl esters are hydrolyzed to release free cholesterol, which then translocates to late endosomes (LEs)/lysosomes (LYs) and incorporates into the membranes by co‐ordinated actions of Niemann‐Pick type C (NPC) 1 and NPC2 proteins. However, how cholesterol exits LEs/LYs and moves to other organelles remain largely unclear. Growing evidence has suggested that nonvesicular transport is critically involved in the post‐endosomal cholesterol trafficking. Numerous sterol‐transfer proteins (STPs) have been identified to mediate directional cholesterol transfer at membrane contact sites (MCSs) formed between 2 closely apposed organelles. In addition, a recent study reveals that lysosome‐peroxisome membrane contact (LPMC) established by a non‐STP synaptotagmin VII and a specific phospholipid phosphatidylinositol 4,5‐bisphosphate also serves as a novel and important path for LDL‐cholesterol trafficking. These findings highlight an essential role of MCSs in intracellular cholesterol transport, and further work is needed to unveil how various routes are regulated and integrated to maintain proper cholesterol distribution and homeostasis in eukaryotic cells.      

Modulation of lipid-induced ER stress by fatty acid shape

Exposure of pancreatic β cells to long-chain saturated fatty acids (SFA) induces a so-called endoplasmic reticulum (ER) stress that can ultimately lead to cell death. This process is believed to participate in insulin deficiency associated with type 2 diabetes, via a decrease in β-cell mass. By contrast, some unsaturated fatty acid species appear less toxic to the cells and can even alleviate SFA-induced ER stress. In the present study, we took advantage of a simple yeast-based model, which brings together most of the trademarks of lipotoxicity in human cells, to screen fatty acids of various structures for their capacity to counter ER stress. Here we demonstrate that the tendency of a free fatty acid (FFA) to reduce SFA toxicity depends on a complex conjunction of parameters, including chain length, level of unsaturation, position of the double bonds and nature of the isomers (cis or trans). Interestingly, potent FFA act as building blocks for phospholipid synthesis and help to restore an optimal membrane organization, compatible with ER function and normal protein trafficking.     

The geometry and efficacy of cation-pi interactions in a diagonal position of a designed beta-hairpin

Cation-pi interactions are common in proteins, but their contribution to the stability and specificity of protein structure has not been well established. In this study, we examined the impact of cation-pi interactions in a diagonal position of a beta-hairpin peptide through comparison of the interaction of Phe or Trp with Lys or Arg. The diagonal interactions ranged from -0.20 to -0.48 kcal/mole. Our experimental values for the diagonal cation-pi interactions are similar to those found in alpha-helical studies. Upfield shifting of the Lys and Arg side chains indicates that the geometries of cation-pi interactions adopted in the 12-residue beta-hairpin are comparable to those found in protein structures. The Lys was found to interact through the polarized Cepsilon, and the Arg is stacked against the aromatic ring of Phe or Trp. Folding of these peptides was found to be enthalpically favorable (DeltaH degrees equals approximately -3 kcal/mole) and entropically unfavorable (DeltaS degrees equals approximately -8 cal mole(-1) K(-1)).     

Crystal structure of yeast YHR049W/FSH1, a member of the serine hydrolase family

Yhr049w/FSH1 was recently identified in a combined computational and experimental proteomics analysis for the detection of active serine hydrolases in yeast. This analysis suggested that FSH1 might be a serine-type hydrolase belonging to the broad functional alphabeta-hydrolase superfamily. In order to get insight into the molecular function of this gene, it was targeted in our yeast structural genomics project. The crystal structure of the protein confirms that it contains a Ser/His/Asp catalytic triad that is part of a minimal alpha/beta-hydrolase fold. The architecture of the putative active site and analogies with other protein structures suggest that FSH1 may be an esterase. This finding was further strengthened by the unexpected presence of a compound covalently bound to the catalytic serine in the active site. Apparently, the enzyme was trapped with a reactive compound during the purification process.     

Increased connexin 43 expression improves the migratory and proliferative ability of H9c2 cells by Wnt-3a overexpression

The change of connexin 43 (Cx43) expression and the biological behaviors of Cx43 in rat heart cell line H9c2, expressing Wnt-3a (wingless-type MMTV integration site family, member 3A) were evaluated in the present study. Plasmid pcDNA3.1/Wnt-3a was constructed and transferred into H9c2 cells. The cell model Wnt-3a~ -H9c2 steadily expressing Wnt-3a was obtained. Compared with H9c2 and pcDNA3.1-H9c2 cells, the expression of Cx43 in Wnt-3a~ -H9c2 cells was clearly increased, the proliferation of Wnt-3a~ -H9c2 cells was significantly changed, and cell migration abilities were also improved (P<0.05). In comparison with H9c2 and pcDNA3.1-H9c2 cells, the G_2 phase of the cell cycle increased by 11% in Wnt-3a~ -H9c2 cells. Thus, Wnt-3a overexpression is associated with an increase in Cx43 expression and altered migratory and proliferative activity in H9c2 cells. Cx43 might be one of the downstream target genes regulated by Wnt-3a.     

A Lysosomal Proteinase, the Late Infantile Neuronal Ceroid Lipofuscinosis Gene (CLN2) Product, Is Essential for Degradation of a Hydrophobic Protein, the Subunit c of ATP Synthase

The specific accumulation of the hydrophobic protein, subunit c of ATP synthase, in lysosomes from the cells of patients with the late infantile form of neuronal ceroid lipofuscinosis (LINCL) is caused by lysosomal proteolytic dysfunction. The defective gene in LINCL (CLN2 gene) has been identified recently. To elucidate the mechanism of lysosomal storage of subunit c, antibodies against the human CLN2 gene product (Cln2p) were prepared. Immunoblot analysis indicated that Cln2p is a 46-kDa protein in normal control skin fibroblasts and carrier heterozygote cells, whereas it was absent in cells from four patients with LINCL. RT-PCR analysis indicated the presence of mRNA for CLN2 in cells from the four different patients tested, suggesting a low efficiency of translation of mRNA or the production of the unstable translation products in these patient cells. Pulse-chase analysis showed that Cln2p was synthesized as a 67-kDa precursor and processed to a 46-kDa mature protein (t(1/2) = 1 h). Subcellular fractionation analysis indicated that Cln2p is localized with cathepsin B in the high-density lysosomal fractions. Confocal immunomicroscopic analysis also revealed that Cln2p is colocalized with a lysosomal soluble marker, cathepsin D. The immunodepletion of Cln2p from normal fibroblast extracts caused a loss in the degradative capacity of subunit c, but not the beta subunit of ATP synthase, suggesting that the absence of Cln2p provokes the lysosomal accumulation of subunit c.     

Toward a general method to observe the phosphate groups of phosphoenzymes with infrared spectroscopy

A general method to study the phosphate group of phosphoenzymes with infrared difference spectroscopy by helper enzyme-induced isotope exchange was developed. This allows the selective monitoring of the phosphate P-O vibrations in large proteins, which provides detailed information on several band parameters. Here, isotopic exchange was achieved at the oxygen atoms of the catalytically important phosphate group that transiently binds to the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a). [gamma-(18)O(3)]ATP phosphorylated the ATPase, which produced phosphoenzyme that was initially isotopically labeled. The helper enzyme adenylate kinase regenerated the substrate ATP from ADP (added or generated upon ATP hydrolysis) with different isotopic composition than used initially. With time this produced the unlabeled phosphoenzyme. The method was tested on the ADP-insensitive phosphoenzyme state of the Ca(2+)-ATPase for which the vibrational frequencies of the phosphate group are known, and it was established that the helper enzyme is effective in mediating the isotope exchange process.     

Use of Rifampicin in T7 RNA Polymerase-Driven Expression of a Plant Enzyme: Rifampicin Improves Yield and Assembly

Expression systems based on high selectivity and activity of T7 RNA polymerase and presence of a strong T7 promoter have been commonly used for cloning and expression of various recombinant proteins in Escherichia coli. When the expression system is designed in such a way that the produced protein is not being transferred into periplasm, bacterial cells must be lysed in order to isolate and purify the protein. The final yield and quality of the synthesized protein then depend on various factors, protein size, amino acid sequence, solubility in cytoplasm, and folding requirements among them. The yield in the T7 RNA polymerase/promoter system can be positively influenced by use of rifampicin. In this report we demonstrate usefulness of the antibiotic in detail. We describe rifampicin-enhanced expression of a plant cytokinin-specific beta-glucosidase. Two bacterial cultures are compared, one expressing the enzyme without and one in the presence of rifampicin. The antibiotic not only increased the yield of the recombinant protein, which seems to be a general phenomenon, but also favored the final assembly of the protein's subunits into a catalytically active dimer form.     

Molecular dynamics (MD) investigations of preformed structures of the transmembrane domain of the oncogenic Neu receptor dimer in a DMPC bilayer

The critical Val/Glu mutation in the membrane spanning domain of the rat Neu receptor confers the ability for ligand-independent signaling and leads to increased dimerization and transforming ability. There is evidence that the two transmembrane interacting helices play a role in receptor activation by imposing orientation constraints to the intracellular tyrosine kinase domains. By using MD simulations we have attempted to discriminate between correct and improper helix-helix packing by examining the structural and energetic properties of preformed left-handed and right-handed structures in a fully hydrated DMPC bilayer. The best energetic balance between the residues at the helix-helix interface and the residues exposed to the lipids is obtained for helices in symmetrical left-handed interactions packed together via Glu side chain/Ala backbone interhelical hydrogen bonds. Analyses demonstrate the importance of the ATVEG motif in helix-helix packing and point to additional contacting residues necessary for association. Our findings, all consistent with experimental data, suggest that a symmetrical left-handed structure of the helices could be the transmembrane domain configuration that promotes receptor activation and transformation. The present study may provide further insight into signal transduction mechanisms of the ErbB/Neu receptors.