Synthesis and evaluation of the platelet antiaggregant properties of phenolic antioxidants structurally related to rosmarinic acid

Polyphenols, such as rosmarinic acid, are widely distributed natural products with relevant antioxidant activity. Oxidative stress plays an important role in the pathogenesis of a number of disorders. Here, we report on the synthesis and biological effects of the polyphenolic esters hydroxytyrosyl gallate (1), hydroxytyrosyl protocatechuate (2) and hydroxytyrosyl caffeate (3), structurally related to rosmarinic acid. The three compounds showed a greater free radical scavenging activity than their precursors and also than rosmarinic acid. Esters 1 and 3 significantly reduced thrombin-evoked platelet aggregation, which is likely mediated to the attenuation of thrombin-stimulated Ca²⁺ release and entry. The three compounds reduced the ability of platelets to accumulate Ca²⁺ in the intracellular stores, probably by enhancing the Ca²⁺ leakage rate and reduced store-operated Ca²⁺ entry in these cells. These observations suggest that the structurally-simplified analogs to rosmarinic acid, compounds 1 and 3, might be the base of therapeutic strategies to prevent thrombotic complications associated to platelet hyperaggregability due to oxidative stress.     

A plant peptide: N-glycanase orthologue facilitates glycoprotein ER-associated degradation in yeast

Background

The cytoplasmic peptide:N-glycanase (PNGase) is a deglycosylating enzyme involved in the ER-associated degradation (ERAD) process, while ERAD-independent activities are also reported. Previous biochemical analyses indicated that the cytoplasmic PNGase orthologue in Arabidopsis thaliana (AtPNG1) can function as not only PNGase but also transglutaminase, while its in vivo function remained unclarified.

Methods

AtPNG1 was expressed in Saccharomyces cerevisiae and its in vivo role on PNGase-dependent ERAD pathway was examined.

Results

AtPNG1 could facilitate the ERAD through its deglycosylation activity. Moreover, a catalytic mutant of AtPNG1 (AtPNG1(C251A)) was found to significantly impair the ERAD process. This result was found to be N-glycan-dependent, as the AtPNG(C251A) did not affect the stability of the non-glycosylated RTA? (ricin A chain non-toxic mutant). Tight interaction between AtPNG1(C251A) and the RTA? was confirmed by co-immunoprecipitation analysis.

Conclusion

The plant PNGase facilitates ERAD through its deglycosylation activity, while the catalytic mutant of AtPNG1 impair glycoprotein ERAD by binding to N-glycans on the ERAD substrates.

General significance

Our studies underscore the functional importance of a plant PNGase orthologue as a deglycosylating enzyme involved in the ERAD.     

The endoplasmic reticulum Ca2+-pump SERCA2b interacts with G protein-coupled receptors and enhances their expression at the cell surface

Calcium (Ca(2+)) plays a pivotal role in both cellular signaling and protein synthesis. However, it is not well understood how calcium metabolism and synthesis of secreted and membrane-bound proteins are related. Here we demonstrate that the sarco(endo)plasmic reticulum Ca(2+) ATPase 2b (SERCA2b), which maintains high Ca(2+) concentration in the lumen of the endoplasmic reticulum, interacts specifically with the human delta opioid receptor during early steps of receptor biogenesis in human embryonic kidney 293 cells. The interaction involves newly synthesized incompletely folded receptor precursors, because the association between the delta opioid receptor and SERCA2b (i) was short-lived and took place soon after receptor translation, (ii) was not affected by misfolding of the receptor, and (iii) decreased if receptor folding was enhanced by opioid receptor pharmacological chaperone. The physical association with SERCA2b was found to be a universal feature among G protein-coupled receptors within family A and was shown to occur also between the endogenously expressed luteinizing hormone receptor and SERCA2b in rat ovaries. Importantly, active SERCA2b rather than undisturbed Ca(2+) homeostasis was found to be essential for delta opioid receptor biogenesis, as inhibition of its Ca(2+) pumping activity by thapsigargin reduced the interaction and impaired the efficiency of receptor maturation, two phenomena that were not affected by a Ca(2+) ionophore A23187. Nevertheless, inhibition of SERCA2b did not compromise the functionality of receptors that were able to mature. Thus, we propose that the association with SERCA2b is required for efficient folding and/or membrane integration of G protein-coupled receptors.     

Characterization of the novel cardiolipin binding regions identified on the protease and lipid activated PKC‐related kinase 1

Protein kinase C‐related kinase 1 (PRK1) or PKN is a protease and lipid activated protein kinase that acted downstream of the RhoA or Rac1 pathway. PRK1 comprises a unique regulatory domain and a PKC homologous kinase domain. The regulatory domain of PRK1 consists of homologous region ?1 (HR1) and ?2 (HR2). PRK1‐(HR1) features a pseudosubstrate motif that overlapped with the putative cardiolipin and known RhoA binding sites. In fact, cardiolipin is the most potent lipid activator for PRK1 in respect of its either auto‐ or substrate phosphorylation activity. This study was thus aimed to characterize the binding region(s) of cardiolipin that was previously suggested for the regulatory domain of PRK1. The principal findings of this work established (i) PRK1‐(HR1) folded into an active conformation where high affinity binding sites (mainly located in HR1a subdomain) were accessible for cardiolipin binding to protect against limited Lys‐C digestion, (ii) the binding nature between acidic phospholipids and PRK1 (HR1) involved both polar and nonpolar components consistent with the amphipathic nature of the known cardiolipin‐binding motifs, (iii) identification of the molecule masses of the Lys‐C fragments of PRK1‐(HR1) complexed with cardiolipin molecule, and (iv) appreciable reductions in the secondary structural contents at 222 nm measured by circular dichroism analyses demonstrated the binding of cardiolipin elicited the disruptive effect that was most evident among all phospholipids tested, suggestive of a functional correlation between the extents of helical disruption and PRK1 activation.     

Significant enhancement in the binding of p-nitrophenyl-β- -xylobioside by the E128H mutant F/10 xylanase from Streptomyces olivaceoviridis E-86

Mutagenesis studies were carried out to examine the effects of replacement of either the nucleophile Glu-236 or the acid/base Glu-128 residue of the F/10 xylanase by a His residue. To our surprise, the affinity for the p-nitrophenyl-β- -xylobioside substrate was increased by 10³-fold in the case of the mutant E128H enzyme compared with that of the wild-type F/10 xylanase. The catalytic activity of the mutant enzymes was low, despite the fact that the distance between the nucleophilic atom (an oxygen in the native xylanase and a nitrogen in the mutant) and the α-carbon was barely changed. Thus, the alteration of the acid/base functionality (Glu-128 to His mutation) provided a significantly favorable interaction within the E128H enzyme/substrate complex in the ground state, accompanying a reduction in the stabilization effect in the transition state.     

Functional analysis of the anaphase promoting complex activator CCS52A highlights the crucial role of endo‐reduplication for fruit growth in tomato

Tomato fruit growth is characterized by the occurrence of numerous rounds of DNA endo‐reduplication in connection with cell expansion and final fruit size determination. Endo‐reduplication is an impairment of mitosis that originates from the selective degradation of M phase‐specific cyclins via the ubiquitin‐mediated proteolytic pathway, requiring the E3 ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). Two types of APC/C activators, namely CCS52 and CDC20 proteins, exist in plants. We report here the molecular characterization of such APC/C activators during fruit development, and provide an in planta functional analysis of SlCCS52A, a gene that is specifically associated with endo‐reduplication in tomato. Altering SlCCS52A expression in either a negative or positive manner had an impact on the extent of endo‐reduplication in fruit, and fruit size was reduced in both cases. In SlCCS52A over‐expressing fruits, endo‐reduplication was initially delayed, accounting for the altered final fruit size, but resumed and was even enhanced at 15 days post anthesis (dpa), leading to fruit growth recovery. This induction of growth mediated by endo‐reduplication had a considerable impact on nitrogen metabolism in developing fruits. Our data contribute to unravelling of the physiological role of endo‐reduplication in growth induction during tomato fruit development.     

Chemical Diversity in the Genus Alpinia (Zingiberaceae): Comparative Composition of Four Alpinia Species Grown in Northern India

The essential‐oil compositions of leaves, flowers, and rhizomes of Alpinia galanga (L.) Willd ., Alpinia calcarata Rosc ., Alpinia speciosa K. Schum. , and Alpinia allughas Rosc . were examined and compared by capillary GC and GC/MS. Monoterpenoids were the major oil constituents identified. 1,8‐Cineole, α‐terpineol, (E)‐methyl cinnamate, camphor, terpinen‐4‐ol, and α‐ and β‐pinenes were the major constituents commonly distributed in leaf and flower essential oils. The presence of endo‐fenchyl acetate, exo‐fenchyl acetate, and endo‐fenchol was the unique feature of rhizome essential oils of A. galanga, A. calcarata, and A. speciosa. On contrary, the rhizome oil of A. allughas was dominated by β‐pinene. Significant qualitative and quantitative variations were observed in essential‐oil compositions of different parts of Alpinia species growing in subtemperate and subtropical regions of Northern India. Cluster analysis was performed to find similarities and differences in essential‐oil compositions based on representative molecular skeletons. Monoterpenoids, viz., 1,8‐cineole, terpinen‐4‐ol, camphor, pinenes, (E)‐methyl cinnamate, and fenchyl derivatives, were used as chemotaxonomic markers.     

Structural characterization of the Acetobacter xylinum endo-beta-1,4-glucanase CMCax required for cellulose biosynthesis

Previous studies have demonstrated that endoglucanase is required for cellulose biosynthesis both in bacteria and plants. However, it has yet to be elucidated how the endoglucanases function in the mechanism of cellulose biosynthesis. Here we describe the crystal structure of the cellulose biosynthesis-related endo-beta-1,47-glucanase (CMCax; EC 3.2.1.4) from the cellulose-producing Gramnegative bacterium, Acetobacter xylinum (= Gluconacetobacter xylinus), determined at 1.65-A resolution. CMCax falls into the glycoside hydrolase family 8 (GH-8), and the structure showed that the overall fold of the CMCax is similar to those of other glycoside hydrolases belonging to GH-8. Structure comparison with Clostridium thermocellum CelA, the best characterized GH-8 endoglucanase, revealed that sugar recognition subsite +3 is completely missing in CMCax. The absence of the subsite +3 leads to significant broadness of the cleft at the cellooligosaccharide reducing-end side. CMCax is known to be a secreted enzyme and is present in the culture medium. However, electron microscopic analysis using immunostaining clearly demonstrated that a portion of CMCax is localized to the cell surface, suggesting a link with other known membrane-anchored endoglucanases that are required for cellulose biosynthesis.     

Cellular repressor of E1A-stimulated genes is a bona fide lysosomal protein which undergoes proteolytic maturation during its biosynthesis

Cellular repressor of E1A-stimulated genes (CREG) has been reported to be a secretory glycoprotein implicated in cellular growth and differentiation. We now show that CREG is predominantly localized within intracellular compartments. Intracellular CREG was found to lack an N-terminal peptide present in the secreted form of the protein. In contrast to normal cells, CREG is largely secreted by fibroblasts missing both mannose 6-phosphate receptors. This is not observed in cells lacking only one of them. Mass spectrometric analysis of recombinant CREG revealed that the protein contains phosphorylated oligosaccharides at either of its two N-glycosylation sites. Cellular CREG was found to cosediment with lysosomal markers upon subcellular fractionation by density-gradient centrifugation. In fibroblasts expressing a CREG-GFP fusion construct, the heterologous protein was detected in compartments containing lysosomal proteins. Immunolocalization of endogenous CREG confirmed that intracellular CREG is localized in lysosomes. Proteolytic processing of intracellular CREG involves the action of lysosomal cysteine proteinases. These results establish that CREG is a lysosomal protein that undergoes proteolytic maturation in the course of its biosynthesis, carries the mannose 6-phosphate recognition marker and depends on the interaction with mannose 6-phosphate receptors for efficient delivery to lysosomes.