Leishmanicidal Triterpenes from Lantana camara

Two new natural triterpenes, lantaninilic acid and lantoic acid, along with the known triterpenes lantadene A, and oleanolic, ursolic, betulinic, lantanolic, and camaric acid, were obtained from the aerial parts of Lantana camara through bioassay‐guided isolation, monitoring the in vitro antileishmanial activity against promastigotes of Leishmania major. Oleanolic acid ( 3 ), ursolic acid ( 4 ), lantadene A ( 5 ), and lantanilic acid ( 7 ) showed significant leishmanicidal activities with IC₅₀ values of 53.0, 12.4, 20.4, and 21.3 μM , respectively. The IC₅₀ value of ursolic acid ( 4 ; 12.4 μM ) was found to be comparable with that of the standard drugs, pentamidine (IC₅₀ 15.0 μM ) and amphotericin B (IC₅₀ 0.31 μM ). The in vitro activities of L. camara and its constituents against promastigotes of Leishmania major are reported here for the first time.     

Targeted tandem affinity purification of PSD‐95 recovers core postsynaptic complexes and schizophrenia susceptibility proteins

The molecular complexity of mammalian proteomes demands new methods for mapping the organization of multiprotein complexes. Here, we combine mouse genetics and proteomics to characterize synapse protein complexes and interaction networks. New tandem affinity purification (TAP) tags were fused to the carboxyl terminus of PSD‐95 using gene targeting in mice. Homozygous mice showed no detectable abnormalities in PSD‐95 expression, subcellular localization or synaptic electrophysiological function. Analysis of multiprotein complexes purified under native conditions by mass spectrometry defined known and new interactors: 118 proteins comprising crucial functional components of synapses, including glutamate receptors, K⁺ channels, scaffolding and signaling proteins, were recovered. Network clustering of protein interactions generated five connected clusters, with two clusters containing all the major ionotropic glutamate receptors and one cluster with voltage‐dependent K⁺ channels. Annotation of clusters with human disease associations revealed that multiple disorders map to the network, with a significant correlation of schizophrenia within the glutamate receptor clusters. This targeted TAP tagging strategy is generally applicable to mammalian proteomics and systems biology approaches to disease.     

Proteome-wide Analysis of Lysine Acetylation Suggests its Broad Regulatory Scope in Saccharomyces cerevisiae

Post-translational modification of proteins by lysine acetylation plays important regulatory roles in living cells. The budding yeast Saccharomyces cerevisiae is a widely used unicellular eukaryotic model organism in biomedical research. S. cerevisiae contains several evolutionary conserved lysine acetyltransferases and deacetylases. However, only a few dozen acetylation sites in S. cerevisiae are known, presenting a major obstacle for further understanding the regulatory roles of acetylation in this organism. Here we use high resolution mass spectrometry to identify about 4000 lysine acetylation sites in S. cerevisiae. Acetylated proteins are implicated in the regulation of diverse cytoplasmic and nuclear processes including chromatin organization, mitochondrial metabolism, and protein synthesis. Bioinformatic analysis of yeast acetylation sites shows that acetylated lysines are significantly more conserved compared with nonacetylated lysines. A large fraction of the conserved acetylation sites are present on proteins involved in cellular metabolism, protein synthesis, and protein folding. Furthermore, quantification of the Rpd3-regulated acetylation sites identified several previously known, as well as new putative substrates of this deacetylase. Rpd3 deficiency increased acetylation of the SAGA (Spt-Ada-Gcn5-Acetyltransferase) complex subunit Sgf73 on K33. This acetylation site is located within a critical regulatory domain in Sgf73 that interacts with Ubp8 and is involved in the activation of the Ubp8-containing histone H2B deubiquitylase complex. Our data provides the first global survey of acetylation in budding yeast, and suggests a wide-ranging regulatory scope of this modification. The provided dataset may serve as an important resource for the functional analysis of lysine acetylation in eukaryotes.Lysine acetylation is a dynamic and reversible post-translational modification. Acetylation of lysines on their ε-amino group is catalyzed by lysine acetyltransferases (KATs¹, also known as histone acetyltrasferases (HATs)), and reversed by lysine deacetylases (KDACs, also known as histone deacetylases (HDACs)) (1). The enzymatic machinery involved in lysine acetylation is evolutionary conserved in all forms of life (2–4). The role of acetylation has been extensively studied in the regulation of gene expression via modification of histones (5). Acetylation also plays important roles in controlling cellular metabolism (6–10), protein folding (11), and sister chromatid cohesion (12). Furthermore, acetylation has been implicated in regulating the beneficial effects of calorie restriction (13), a low nutrient diet without starvation, and aging. Based on these findings, it is proposed that the functional roles of acetylation in these processes are evolutionary conserved from yeast to mammals.Advancements in mass spectrometry (MS)-based proteomics have greatly facilitated identification of thousands of post-translational modification (PTM) sites in eukaryotic cells (14–18). Proteome-wide mapping of PTM sites can provide important leads for analyzing the functional relevance of individual sites and a systems-wide view of the regulatory scope of post-translational modifications. Also, large-scale PTM datasets are an important resource for the in silico analysis of PTMs, which can broaden the understanding of modification site properties and their evolutionary trajectories.The budding yeast Saccharomyces cerevisiae is a commonly used unicellular eukaryotic model organism. Yeast has been used in many pioneering “-omics” studies, including sequencing of the first eukaryotic genome (19), systems-wide genetic interactions analysis (20, 21), MS-based comprehensive mapping of a eukaryotic proteome (22), and proteome-wide analysis of protein-protein interactions (23, 24). In addition, S. cerevisiae has been extensively used to study the molecular mechanisms of acetylation. Many lysine acetyltransferases and deacetylases were discovered in this organism (2, 25), and their orthologs were subsequently identified in higher eukaryotes. Furthermore, the functional roles of many well-studied acetylation sites on histones are conserved from yeast to mammals. Recent data from human and Drosophila cells show that acetylation is present on many highly conserved metabolic enzymes (26–28). However, only a few dozen yeast acetylation sites are annotated in the Uniprot database. Given the presence of a well-conserved and elaborate acetylation machinery in yeast, we reasoned that many more acetylation sites exist in this organism that remained to be identified.Here we used high resolution mass spectrometry-based proteomics to investigate the scope of acetylation in S. cerevisiae. We identified about 4000 unique acetylation sites in this important model organism. Bioinformatic analysis of yeast acetylation sites and comparison with previously identified human and Drosophila acetylation sites indicates that many acetylation sites are evolutionary conserved. Furthermore, quantitative analysis of the Rpd3-regulated acetylation sites identified several nuclear proteins that showed increased acetylation in rpd3 knockout cells. Our results provide a systems-wide view of acetylation in budding yeast, and a rich dataset for functional analysis of acetylation sites in this organism.     

The caveolin-binding motif of the pathogen-related yeast protein Pry1, a member of the CAP protein superfamily,is required for in vivo export of cholesteryl acetate

Proteins belonging to the CAP superfamily are present in all kingdoms of life and have been implicated in different physiological processes. Their molecular mode of action, however, is poorly understood. Saccharomyces cerevisiae expresses three members of this superfamily, pathogen-related yeast (Pry)1, -2, and -3. We have recently shown that Pry function is required for the secretion of cholesteryl acetate and that Pry proteins bind cholesterol and cholesteryl acetate, suggesting that CAP superfamily members may generally act to bind sterols or related small hydrophobic compounds. Here, we analyzed the mode of sterol binding by Pry1. Computational modeling indicates that ligand binding could occur through displacement of a relatively poorly conserved flexible loop, which in some CAP family members displays homology to the caveolin-binding motif. Point mutations within this motif abrogated export of cholesteryl acetate but did not affect binding of cholesterol. Mutations of residues located outside the caveolin-binding motif, or mutations in highly conserved putative catalytic residues had no effect on export of cholesteryl acetate or on lipid binding. These results indicate that the caveolin-binding motif of Pry1, and possibly of other CAP family members, is crucial for selective lipid binding and that lipid binding may occur through displacement of the loop containing this motif.     

Improving Heat Stress Tolerance in Camelina sativa and Brassica napus Through Thiourea Seed Priming

Journal of Plant Growth Regulation - Heat stress is a major detriment to crop growth and yield under the current patterns of climate change. Two independent experiments of camelina and canola were...     

Distribution of Cu2+-diamine oxidase during ontogeny of seedlings of Vigna radiata cultivars

Activity of Cu2+-diamine oxidase (DAO; E.C.1.4.3.6.) was measured in Vigna radiata (L.) Wilczek cultivars K851, MH8320 and Pusa Baisakhi in light and in dark during ontogeny of seedlings. DAO activity was always the highest in cv. K851. In both light and dark grown seedlings maximum DAO activity was detected on day 2 after germination. Thereafter, in light grown seedlings it declined consistently upto non-detectable levels. In dark, DAO activity was higher than in light and it had the second maximum on day 7 following a similar declining pattern as observed for the light grown seedlings. The DAO activity was higher in a shoot apex alongwith leaves than in roots and shoot axis. This revised version was published online in August 2006 with corrections to the Cover Date.