The trafficking protein SYP121 of Arabidopsis connects programmed stomatal closure and K⁺ channel activity with vegetative growth

The vesicle‐trafficking protein SYP121 (SYR1/PEN1) was originally identified in association with ion channel control at the plasma membrane of stomatal guard cells, although stomata of the Arabidopsis syp121 loss‐of‐function mutant close normally in ABA and high Ca²⁺. We have now uncovered a set of stomatal phenotypes in the syp121 mutant that reduce CO₂ assimilation, slow vegetative growth and increase water use efficiency in the whole plant, conditional upon high light intensities and low relative humidity. Stomatal opening and the rise in stomatal transpiration of the mutant was delayed in the light and following Ca²⁺‐evoked closure, consistent with a constitutive form of so‐called programmed stomatal closure. Delayed reopening was observed in the syp121, but not in the syp122 mutant lacking the homologous gene product; the delay was rescued by complementation with wild‐type SYP121 and was phenocopied in wild‐type plants in the presence of the vesicle‐trafficking inhibitor Brefeldin A. K⁺ channel current that normally mediates K⁺ uptake for stomatal opening was suppressed in the syp121 mutant and, following closure, its recovery was slowed compared to guard cells of wild‐type plants. Evoked stomatal closure was accompanied by internalisation of GFP‐tagged KAT1 K⁺ channels in both wild‐type and syp121 mutant guard cells, but their subsequently recycling was slowed in the mutant. Our findings indicate that SYP121 facilitates stomatal reopening and they suggest that K⁺ channel traffic and recycling to the plasma membrane underpins the stress memory phenomenon of programmed closure in stomata. Additionally, they underline the significance of vesicle traffic for whole‐plant water use and biomass production, tying SYP121 function to guard cell membrane transport and stomatal control.     

Synthesis and anti-parasitic activity of a novel quinolinone–chalcone series

A series of novel quinolinone–chalcone hybrids and analogues were designed, synthesized and their biological activity against the mammalian stages of Trypanosoma brucei and Leishmania infantum evaluated. Promising molecular scaffolds with significant microbicidal activity and low cytotoxicity were identified. Quinolinone–chalcone 10 exhibited anti-parasitic properties against both organisms, being the most potent anti-L. infantum agent of the entire series (IC₅₀ value of 1.3 ± 0.1 μM). Compounds 4 and 11 showed potency toward the intracellular, amastigote stage of L. infantum (IC₅₀ values of 2.1 ± 0.6 and 3.1 ± 1.05 μM, respectively). Promising trypanocidal compounds include 5 and 10 (IC₅₀ values of 2.6 ± 0.1 and 3.3 ± 0.1 μM, respectively) as well as 6 and 9 (both having IC₅₀ values of <5 μM). Chemical modifications on the quinolinone–chalcone scaffold were performed on selected compounds in order to investigate the influence of these structural features on antiparasitic activity.     

Design,synthesis, and structure–activity relationships of a series of novel N-aryl-2-phenylcyclopropanecarboxamide that are potent and orally active orexin receptor antagonists

Herein we describe the design, synthesis, and structure–activity relationships (SARs) of a novel phenylcyclopropane series represented by 7 and 33b as antagonists of orexin 1 and orexin 2 receptors. With 4 serving as the initial lead for the development of orexin antagonists, exploration of SAR resulted in improved binding affinity for orexin 1 and orexin 2 receptors. Among the synthesized compounds, 33b ((−)-N-(5-cyanopyridin-2-yl)-2-[(3,4-dimethoxyphenyl)oxymethyl]-2-phenylcyclopropanecarboxamide) exhibited potent in vitro activity and oral efficacy in animal sleep measurement experiments. The results of our study suggest that compound 33b may serve as a valuable template for the development of new orexin receptor antagonists.     

8-Br-cADPR, a TRPM2 ion channel antagonist,inhibits renal ischemia–reperfusion injury

The transient receptor potential melastatin-2 (TRPM2) channel belongs to the transient receptor potential channel superfamily and is a cation channel permeable to Na⁺ and Ca ²⁺. The TRPM2 ion channel is expressed in the kidney and can be activated by various molecules such as hydrogen peroxide, calcium, and cyclic adenosine diphosphate (ADP)-ribose (cADPR) that are produced during acute kidney injury. In this study, we investigated the role of 8-bromo-cyclic ADP-ribose (8-Br-cADPR; a cADPR antagonist) in renal ischemia–reperfusion injury using biochemical and histopathological parameters. CD38, cADPR, tumor necrosis factor-α, interleukin-1β, and myeloperoxidase (inflammatory markers), urea and creatinine, hydrogen peroxide (oxidant), and catalase (antioxidant enzyme) levels that increase with ischemia–reperfusion injury decreased in the groups treated with 8-Br-cADPR. In addition, renin levels were elevated in the groups treated with 8-Br-cADPR. Histopathological examination revealed that 8-Br-cADPR reduced renal damage and the expression of caspase-3 and TRPM2. Our results suggest that the inhibition of TRPM2 ion channel may be a new treatment modality for ischemic acute kidney injury.     

Isolation of a novel protein,P12—from adult Drosophila melanogaster that inhibits deoxyribonucleoside and protein kinase activities and activates 3′-5′- exonuclease activity

ABSTRACT

We have previously found that Drosophila melanogaster only has one deoxyribonucleoside kinase, Dm-dNK, however, capable to phosphorylate all four natural deoxyribonucleosides. Dm-dNK was originally isolated from an embryonic cell line. We wanted to study the expression of Dm-dNK during development from embryonic cells to adult flies and found declining Dm-dNK activity during development and no activity in adult flies. Surprisingly, the extract from adult flies exhibited a strong inhibitory effect on deoxyribonucloside kinase activity. The dNK-inhibitor was precipitable with ammonium sulfate, and was purified to a high degree by gel-filtration as indicated by LC-MS/MS analysis. Since the inhibitor eluted from G-200 gel-filtration with a size of 10–13 kDa, we named it P12. We tested the purified fraction for specificity towards various enzymes and found that both mammalian and bacterial dNKs were inhibited, whereas there was no effect on hexokinase and pyruvate kinases and acidic phosphatase. However, when tested against cyclin B-dependent kinase, we found a strong inhibitory effect. Both with human Cdk1/CycB and S. pombe Cdc2/B-type cyclin the purified fraction from Superdex 200 that inhibited Dm-dNK, also inhibited the two protein kinases to the same degree. Furthermore, testing P12 in a DNA polymerase based assay we found that the 3′-5′-exonuclease part of the DNA polymerase (Klenow polymerase) was activated.     

Discovery and structure–activity relationships of a novel class of quinazoline glucokinase activators

We describe design, syntheses and structure–activity relationships of a novel class of 4,6-disubstituted quinazoline glucokinase activators. Prototype quinazoline leads (4 and 5) were designed based on the X-ray analyses of the previous 2-aminobenzamide lead classes. Modifications of the quinazoline leads led to the identification of a potent GK activator (21d).     

Comparing protein structures and inferring functions with a novel three-dimensional Yau–Hausdorff method

Abstract

Structures and functions of proteins play various essential roles in biological processes. The functions of newly discovered proteins can be predicted by comparing their structures with that of known-functional proteins. Many approaches have been proposed for measuring the protein structure similarity, such as the template-modeling (TM)-score method, GRaphlet (GR)-Align method as well as the commonly used root-mean-square deviation (RMSD) measures. However, the alignment comparisons between the similarity of protein structure cost much time on large dataset, and the accuracy still have room to improve. In this study, we introduce a new three-dimensional (3D) Yau–Hausdorff distance between any two 3D objects. The (3D) Yau–Hausdorff distance can be used in particular to measure the similarity/dissimilarity of two proteins of any size and does not need aligning and superimposing two structures. We apply structural similarity to study function similarity and perform phylogenetic analysis on several datasets. The results show that (3D) Yau–Hausdorff distance could serve as a more precise and effective method to discover biological relationships between proteins than other methods on structure comparison.

Communicated by Ramaswamy H. Sarma     

Discovery and structure–activity relationships of small molecules that block the human immunoglobulin G–human neonatal Fc receptor (hIgG–hFcRn) protein–protein interaction

The neonatal Fc receptor, FcRn, prolongs the half-life of IgG in the serum and represents a potential therapeutic target for the treatment of autoimmune disease. Small molecules that block the protein–protein interactions of human IgG–human FcRn may lower pathogenic autoantibodies and provide effective treatment. A novel class of quinoxalines has been discovered as antagonists of the IgG:FcRn protein–protein interaction through optimization of a hit derived from a virtual ligand-based screen.     

TRAM,LAG1 and CLN8: members of a novel family of lipid-sensing domains?

A family of membrane-associated proteins related to yeast Lag1p and mammalian TRAM has been identified. The family includes the protein product of CLN8, a gene mutated in progressive epilepsy with mental retardation. Mouse CLN8 is also mutated in the mnd/mnd mouse, a model for neuronal ceroid lipofuscinoses. The identification of these homologues has potential implications for our understanding of ceramide synthesis, lipid regulation and protein translocation in the endoplasmic reticulum.     

Acyl-lipid thioesterase1–4 from Arabidopsis thaliana form a novel family of fatty acyl–acyl carrier protein thioesterases with divergent expression patterns and substrate specificities

Hydrolysis of fatty acyl thioester bonds by thioesterases to produce free fatty acids is important for dictating the diversity of lipid metabolites produced in plants. We have characterized a four-member family of fatty acyl thioesterases from Arabidopsis thaliana, which we have called acyl-lipid thioesterase1 (ALT1), ALT2, ALT3, and ALT4. The ALTs belong to the Hotdog fold superfamily of thioesterases. ALT-like genes are present in diverse plant taxa, including dicots, monocots, lycophytes, and microalgae. The four Arabidopsis ALT genes were found to have distinct gene expression profiles with respect to each other. ALT1 was expressed specifically in stem epidermal cells and flower petals. ALT2 was expressed specifically in root endodermal and peridermal cells as well as in stem lateral organ boundary cells. ALT3 was ubiquitously expressed in aerial and root tissues and at much higher levels than the other ALTs. ALT4 expression was restricted to anthers. All four proteins were localized in plastids via an N-terminal targeting sequence of about 48 amino acids. When expressed in Escherichia coli, the ALT proteins used endogenous fatty acyl–acyl carrier protein substrates to generate fatty acids that varied in chain length (C6–C18), degree of saturation (saturated and monounsaturated), and oxidation state (fully reduced and β-ketofatty acids). Despite their high amino acid sequence identities, each enzyme produced a different profile of lipids in E. coli. The biological roles of these proteins are unknown, but they potentially generate volatile lipid metabolites that have previously not been reported in Arabidopsis.     

Both the N-terminal fragment and the protein–protein interaction domain (PDZ domain) are required for the pro-apoptotic activity of presenilin-associated protein PSAP

Presenilin-associated protein (PSAP) was originally identified as a PS1-associated, PDZ domain protein. In a subsequent study, PSAP was found to be a mitochondrial apoptotic molecule. In this study, we cloned the PSAP gene and found that it is composed of 12 exons and localizes on chromosome 6. To better understand the structure and function of PSAP, we have generated a series of antibodies that recognize different regions of PSAP. Using these antibodies, we found that PSAP is expressed in four isoforms as a result of differential splicing of exon 8 in addition to the use of either the first or the second ATG codon as the start codon. We also found that all these isoforms are localized in the mitochondria and are pro-apoptotic. Furthermore, our data revealed that the PDZ domain and N-terminal fragment are required for the pro-apoptotic activity of PSAP.