The effects of curcumin, mangiferin, resveratrol and other natural plant products on aminopeptidase B activity.

Aminopeptidase B (Ap-B) is a Zn2+-aminopeptidase of the M1 family which is implicated, in conjunction with the nardilysin endoprotease, in the generation of miniglucagon, a peptide involved in the maintenance of glucose homeostasis. Other in vivo physiological roles have been established for this vertebrate enzyme, such as the processing of Arg-extended forms of human insulin and cholecystokinin 9 and the degradation of viral epitopes in the cytoplasm. Among M1 family members, Ap-B is phylogenetically close to leukotriene A4 hydrolase (LTA4H), a bi-functional aminopeptidase also able to transform LTA4 in LTB4 (a lipid mediator of inflammation). As the activities of LTA4H are reported to be inhibited by resveratrol, a polyphenolic molecule from red wine, the effect of this molecule was investigated on the Ap-B activity. Several other active phenolic compounds produced in plants were also tested. Among them, curcumin and mangiferin are the most effective inhibitors. Dixon analysis indicates that curcumin is a non-competitive inhibitor with a Ki value of 46 µmol.L-1. Dixon and Lineweaver-Burk representations with mangiferin show a mixed non-competitive inhibition with Ki' and Ki values of 194 µmol.L-1 and 105 µmol.L-1, respectively. At 200 µmol.L-1, no significant effect was observed with caffeic, chlorogenic, ferulic, salicylic and sinapic acids as well as with resveratrol. Analyses on the 3D-structure of LTA4H with resveratrol (pdb: 3FTS) and the Ap-B 3D-model allow hypothesis to explain theses results.

Proteomic and functional analysis of proline dehydrogenase 1 link proline catabolism to mitochondrial electron transport in Arabidopsis thaliana.

Proline accumulates in many plant species in response to environmental stresses. Upon relief from stress, proline is rapidly oxidized in mitochondria by proline dehydrogenase (ProDH) and then by pyrroline-5-carboxylate dehydrogenase (P5CDH). Two ProDH genes have been identified in the genome of the model plant Arabidopsis thaliana To gain a better understanding of ProDH1 functions in mitochondria, proteomic analysis was performed. ProDH1 polypeptides were identified in Arabidopsis mitochondria by immunoblotting gels after 2D blue native (BN)-SDS/PAGE, probing them with an anti-ProDH antibody and analysing protein spots by MS. The 2D gels showed that ProDH1 forms part of a low-molecular-mass (70-140 kDa) complex in the mitochondrial membrane. To evaluate the contribution of each isoform to proline oxidation, mitochondria were isolated from wild-type (WT) and prodh1, prodh2, prodh1prodh2 and p5cdh mutants. ProDH activity was high for genotypes in which ProDH, most likely ProDH1, was strongly induced by proline. Respiratory measurements indicate that ProDH1 has a role in oxidizing excess proline and transferring electrons to the respiratory chain.

The aminopeptidase B (Ap-B) is phosphorylated in HEK293 cells.

Proteolysis is a post-translational modification (PTM) that affects the whole proteome. First regarded as only destructive, it is more precise than expected. It is finely regulated by other PTMs like phosphorylation. Aminopeptidase B (Ap-B), a M1 metallopeptidase, hydrolyses the peptide bond on the carbonyl side of basic residues at the NH2-terminus of peptides. 2D electrophoresis (2DE) was used to show that Ap-B is modified by phosphorylation. Detection of Ap-B by western blot after 2DE reveals several isoforms with different isoelectric points. Using alkaline phosphatase, Pro-Q Diamond phosphorylation-specific dye and kinase-specific inhibitors, we confirmed that Ap-B is phosphorylated. Phosphorylation can alter the structure of proteins leading to changes in their activity, localization, stability and association with other interacting molecules. We showed that Ap-B phosphorylation might delay its turnover. Our study illustrates the central role of the crosstalk between kinases and proteases in the regulation of many biological processes.

Proline dehydrogenase: a key enzyme in controlling cellular homeostasis.

Proline dehydrogenase (ProDH), also called proline oxidase (POX), is a universal enzyme in living organisms. It catalyzes the oxidation of L-proline to delta1-pyrroline-5-carboxylate leading to the release of electrons, which can be transferred to either electron transfer systems or to molecular oxygen. ProDH is not only essential for proline catabolism but also plays key roles in providing energy, shuttling redox potential between cellular compartments and reactive oxygen species production. Structural analysis of prokaryotic ProDHs already gives some insights into the biochemical activity and biological functions of this enzyme, which can be extended to eukaryotic ProDHs based on sequence similarities. Here we report the most recent investigations on the biochemical and regulation of ProDH at transcriptional, post-transcriptional and translational levels. The biological roles of ProDH in cell homeostasis and adaptation through energetic, developmental, adaptive, physiological and pathological processes in eukaryotes are presented and discussed to create a framework for future research direction.

Signal processing by protein tyrosine phosphorylation in plants.

Protein phosphorylation is a reversible post-translational modification controlling many biological processes. Most phosphorylation occurs on serine and threonine, and to a less extend on tyrosine (Tyr). In animals, Tyr phosphorylation is crucial for the regulation of many responses such as growth or differentiation. Only recently with the development of mass spectrometry, it has been reported that Tyr phosphorylation is as important in plants as in animals. The genes encoding protein Tyr kinases and protein Tyr phosphatases have been identified in the Arabidopsis thaliana genome. Putative substrates of these enzymes, and thus Tyr-phosphorylated proteins have been reported by proteomic studies based on accurate mass spectrometry analysis of the phosphopeptides and phosphoproteins. Biochemical approaches, pharmacology and genetic manipulations have indicated that responses to stress and developmental processes involve changes in protein Tyr phosphorylation. The aim of this review is to present an update on Tyr phosphorylation in plants in order to better assess the role of this post-translational modification in plant physiology.

Protein tyrosine kinases and protein tyrosine phosphatases are involved in abscisic acid-dependent processes in Arabidopsis seeds and suspension cells.

Protein tyrosine (Tyr) phosphorylation plays a central role in many signaling pathways leading to cell growth and differentiation in animals. Tyr phosphorylated proteins have been detected in higher plants, and the roles of protein Tyr phosphatases and protein Tyr kinases in some physiological responses have been shown. We investigated the involvement of Tyr phosphorylation events in abscisic acid (ABA) signaling using a pharmacological approach. Phenylarsine oxide, a specific inhibitor of protein Tyr phosphatase activity, abolished the ABA-dependent accumulation of RAB18 (responsive to ABA 18) transcripts. Protein Tyr kinase inhibitors like genistein, tyrphostin A23, and erbstatin blocked the RAB18 expression induced by ABA in Arabidopsis (Arabidopsis thaliana). Stomatal closure induced by ABA was also inhibited by phenylarsine oxide and genistein. We studied the changes in the Tyr phosphorylation levels of proteins in Arabidopsis seeds after ABA treatment. Proteins were separated by two-dimensional gel electrophoresis, and those phosphorylated on Tyr residues were detected using an anti-phosphotyrosine antibody by western blot. Changes were detected in the Tyr phosphorylation levels of 19 proteins after ABA treatment. Genistein inhibited the ABA-dependent Tyr phosphorylation of proteins. The 19 proteins were analyzed by matrix-assisted laser-desorption ionization time-of-flight/time-of-flight mass spectrometry. Among the proteins identified were storage proteins like cruciferins, enzymes involved in the mobilization of lipid reserves like aconitase, enolase, aldolase, and a lipoprotein, and enzymes necessary for seedling development like the large subunit of Rubisco. Additionally, the identification of three putative signaling proteins, a peptidyl-prolyl isomerase, an RNA-binding protein, and a small ubiquitin-like modifier-conjugating enzyme, enlightens how Tyr phosphorylation might regulate ABA transduction pathways in plants.

Plasmalemma abscisic acid perception leads to RAB18 expression via phospholipase D activation in Arabidopsis suspension cells.

Abscisic acid (ABA) plays a key role in the control of stomatal aperture by regulating ion channel activities and water exchanges across the plasma membrane of guard cells. Changes in cytoplasmic calcium content and activation of anion and outward-rectifying K(+) channels are among the earliest cellular responses to ABA in guard cells. In Arabidopsis suspension cells, we have demonstrated that outer plasmalemma perception of ABA triggered similar early events. Furthermore, a Ca(2+) influx and the activation of anion channels are part of the ABA-signaling pathway leading to the specific expression of RAB18. Here, we determine whether phospholipases are involved in ABA-induced RAB18 expression. Phospholipase C is not implicated in this ABA pathway. Using a transphosphatidylation reaction, we show that ABA plasmalemma perception results in a transient stimulation of phospholipase D (PLD) activity, which is necessary for RAB18 expression. Further experiments showed that PLD activation was unlikely to be regulated by heterotrimeric G proteins. We also observed that ABA-dependent stimulation of PLD was necessary for the activation of plasma anion current. However, when ABA activation of plasma anion channels was inhibited, the ABA-dependent activation of PLD was unchanged. Thus, we conclude that in Arabidopsis suspension cells, ABA stimulation of PLD acts upstream from anion channels in the transduction pathway leading to RAB18 expression.