Volatile constituents of Cupressus dupreziana and the sesquiterpenes of Cupressus sempervirens

Analysis of wood essential oil of Cupressus dupreziana revealed 26 components: 13 monoterpenes and 13 sesquiterpenes. The main components were carv     

APC Inhibits Ligand-Independent Wnt Signaling by the Clathrin Endocytic Pathway

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Targeting and post‐translational processing of human α1‐antichymotrypsin in BY‐2 tobacco cultured cells†

The post‐translational processing of human α₁‐antichymotrypsin (AACT) in Bright Yellow‐2 (BY‐2) tobacco cells was assessed in relation to the cellular compartment targeted for accumulation. As determined by pulse‐chase labelling experiments and immunofluorescence microscopy, AACT sent to the vacuole or the endoplasmic reticulum (ER) was found mainly in the culture medium, similar to a secreted form targeted to the apoplast. Unexpectedly, AACT expressed in the cytosol was found in the nucleus under a stable, non‐glycosylated form, in contrast with secreted variants undergoing multiple post‐translational modifications during their transit through the secretory pathway. All secreted forms of AACT were N‐glycosylated, with the presence of complex glycans as observed naturally on human AACT. Proteolytic trimming was also observed for all secreted variants, both during their intracellular transit and after their secretion in the culture medium. Overall, the targeting of human AACT to different compartments of BY‐2 tobacco cells led to the production of two protein products: (i) a stable, non‐glycosylated protein accumulated in the nucleus; and (ii) a heterogeneous mixture of secreted variants resulting from post‐translational N‐glycosylation and proteolytic processing. Overall, these data suggest that AACT is sensitive to resident proteases in the ER, the Golgi and/or the apoplast, and that the production of intact AACT in the plant secretory pathway will require innovative approaches to protect its structural integrity in vivo. Studies are now needed to assess the activity of the different AACT variants, and to identify the molecular determinants for the nuclear localization of AACT expressed in the cytosol.     

Modulating the proteinase inhibitory profile of a plant cystatin by single mutations at positively selected amino acid sites

Cysteine proteinase inhibitors of the cystatin superfamily have several important functions in plants, including the inhibition of exogenous cysteine proteinases during herbivory or infection. Here we used a maximum-likelihood approach to assess whether plant cystatins, like other proteins implicated in host-pest interactions, have been subject to positive selection during the course of their evolution. Several amino acid sites were identified as being positively selected in cystatins from either Poaceae (monocots) and Solanaceae (dicots). These hypervariable sites were located at strategic positions on the protein: on each side of the conserved glycine residues in the N-terminal trunk, within the first and second inhibitory loops entering the active site of target enzymes, and surrounding the larfav motif, a sequence of unknown function conserved among plant cystatins. Supporting the assumption that positively selected, hypervariable sites are indicative of amino acid sites implicated in functional diversity, mutants of the 8th cystatin unit of tomato multicystatin including alternative residues at positively selected sites in the N-terminal trunk exhibited highly variable affinities for the cysteine proteases papain, cathepsin B and cathepsin H. Overall, these observations support the hypothesis that plant cystatins have been under selective pressure to evolve in response to predatory challenges by herbivorous enemies. They also indicate the potential of site-directed mutagenesis at positively selected sites for the generation of cystatins with improved binding properties.     

Nucleocytoplasmic transit of human α1‐antichymotrypsin in tobacco leaf epidermal cells†

Recently, we have observed a nuclear localization for human α₁‐antichymotrypsin (AACT) expressed in the cytosol of transgenic Bright Yellow‐2 (BY‐2) tobacco cultured cells (see accompanying paper: Benchabane, M., Saint‐Jore‐Dupas, C., Bardor, M., Faye, L., Michaud, D. and Gomord, V. (2008a) Targeting and post‐translational processing of human α₁‐antichymotrypsin in BY‐2 tobacco cultured cells. Plant Biotechnol. J. doi: 10.1111/j.1467‐7652.2008.00382.x). In the present article, we assess whether the intrinsic DNA‐binding activity of AACT can explain its nuclear localization, and whether this same activity has an impact on its protease inhibitory potency and stability in planta. An engineered form of AACT with no DNA‐binding activity, rAACTΔK, was compared with the wild‐type polypeptide, rAACT, in terms of chymotrypsin inhibitory potency, stability in planta and distribution in tobacco cells. In accordance with available data reporting distinct sites for protease inhibition and DNA binding, rAACT and rAACTΔK showed similar antichymotrypsin activity, similar to the activity of native AACT purified from human plasma. As observed for AACT in BY‐2 tobacco cells, a green fluorescent protein (GFP)‐AACT fusion transiently expressed in the cytosol of tobacco leaf epidermal cells was detected mainly in the nucleus by confocal laser microscopy. By contrast, rAACTΔK expressed as a GFP fusion showed a balanced distribution between the cytosol and the nucleus, similar to the distribution pattern of free GFP exhibiting no DNA‐binding affinity. In line with immunodetection data showing higher accumulation levels for GFP‐AACT in tobacco leaf cells, rAACTΔK was more susceptible than rAACT to tryptic digestion in the presence of DNA. Overall, these observations suggest the following: (i) a retention effect of DNA on AACT in the nucleus; and (ii) a stabilizing effect of the AACT–DNA interaction on rAACT challenged with non‐target proteases, which, possibly, may be useful in protecting this protein in plant expression platforms.