Phosphopeptides with improved cellular uptake properties as ligands for the polo-box domain of polo-like kinase 1

Human polo-like kinase 1 (Plk1) is involved in cell proliferation and overexpressed in a broad variety of different cancer types. Due to its crucial role in cancerogenesis Plk1 is a potential target for diagnostic and therapeutic applications. Peptidic ligands can specifically interact with the polo-box domain (PBD) of Plk1, a C-terminal located phosphoepitope binding motif. Recently, phosphopeptide MQSpTPL has been identified as ligand with high binding affinity. However, a radiolabeled version of this peptide showed only insufficient cellular uptake. The present study investigated peptide dimers consisting of PBD-targeting phosphopeptide MQSpTPL and a cell-penetrating peptide (CPP) moiety. The new constructs demonstrate superior uptake in different cancer cell-lines compared to the phosphopeptide alone. Furthermore, we could demonstrate binding of phosphopeptide-CPP dimers to PBD of Plk1 making the compounds interesting leads for the development of molecular probes for imaging Plk1 in cancer.     

Substrate profiling of IGF-1R and InsR: identification of a potent pentamer substrate

Protein kinases are widely recognized as important therapeutic targets due to their involvement in signal transduction pathways. These pathways are tightly controlled and regulated, notably by the ability of kinases to selectively phosphorylate a defined set of substrates. As part of a study on the substrate requirements of Insulin-like Growth Factor 1 Receptor (IGF-1R) and Insulin Receptor (InsR), we evaluated and applied a universal assay system able to monitor the phosphorylation of unlabelled peptides of any length in real time. In contrast to already reported profiling methodologies, we were able to assess the k(cat)/K(M) ratio of peptides as short as tetramers. Notably, we were able to identify an efficient pentamer substrate that exhibited kinetic properties close to those of a 250-amino acid protein derived from IRS-1, a natural substrate of IGF-1R and InsR.     

An automated growth enclosure for metabolic labeling of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> with <Superscript>13</Superscript>C-carbon dioxide - an <Emphasis Type="Italic">in vivo</Emphasis> labeling system for proteomics and metabolomics research

Background  

Labeling whole Arabidopsis (Arabidopsis thaliana) plants to high enrichment with ¹³C for proteomics and metabolomics applications would facilitate experimental approaches not possible by conventional methods. Such a system would use the plant's native capacity for carbon fixation to ubiquitously incorporate ¹³C from ¹³CO₂ gas. Because of the high cost of ¹³CO₂ it is critical that the design conserve the labeled gas.     

Solid-solid interface adsorption of proteins and enzymes in nanophase-separated amphiphilic conetworks

Amphiphilic polymer conetworks (APCNs) are materials with a very large interface between their hydrophilic and hydrophobic phases due to their nanophase-separated morphologies. Proteins were found to enrich in APCNs by up to 2 orders of magnitude when incubated in aqueous protein solutions, raising the question of the driving force of protein uptake into APCNs. The loading of poly(2-hydroxyethyl acrylate)-linked by-poly(dimethylsiloxane) (PHEA-l-PDMS) with heme proteins (myoglobin, horseradish peroxidase, hemoglobin) and lipases was studied under variation of parameters such as incubation time, pH, concentration of the protein solution, and conetwork composition. Adsorption of enzymes to the uncharged interface is the main reason for protein uptake, resulting in protein loading of up to 23 wt %. Experimental results were supported by computation of electrostatic potential maps of a lipase, indicating that hydrophobic patches are responsible for the adsorption to the interface. The findings underscore the potential of enzyme-loaded APCNs in biocatalysis and as sensors.     

Starch-related carbon fluxes in roots and leaves of Arabidopsis thaliana

Both photoautotrophic and heterotrophic tissues from plants are capable of synthesizing and degrading starch. To analyze starch metabolism in the two types of tissue from the same plant, several starch-related mutants from Arabidopsis thaliana were grown hydroponically together with the respective wild-type control. Starch contents, patterns of starch-related enzymes and the monomer patterns of the cytosolic starch-related heteroglycans were determined. Based on the phenotypical data obtained, three comparisons were made: First, data from leaves and roots of the mutants were compared with the respective wild-type controls. Secondly, data from leaves and roots from the same plant were compared. Third, we included data obtained from soil-grown plants and compared them with those from hydroponically grown plants. Thus, phenotypical features reflecting altered gene expression can be distinguished from those that are due to the specific growth conditions. Implications on the carbon fluxes in photoautotrophic and heterotrophic cells are discussed.Key words: starch metabolism, cytosolic heteroglycans, cytosolic glucosyl transferases, carbon fluxes     

Atg1 allows second-signaled autophagic cell death in Dictyostelium

We investigated the role of Atg1 in autophagic cell death (ACD) in a Dictyostelium monolayer model. The model is especially propitious, not only because of genetic tractability and absence of apoptosis machinery, but also because induction of ACD requires two successive exogenous signals, first the combination of starvation and cAMP, second the differentiation factor DIF-1. This enables one to analyze separately first-signal-induced autophagy and subsequent second-signal-induced ACD. We used mutants of atg1, a gene that plays an essential role in the initiation of autophagy. Upon starvation/cAMP, in contrast to parental cells, atg1 mutant cells showed irreversible lesions, clearly establishing a protective role for Atg1. Upon subsequent exposure to DIF-1 or to more ACD-specific second signals, starved parental cells progressed to ACD, but starved atg1 mutant cells did not, showing that Atg1 was required for ACD. Thus, in the same cells Atg1 was required in two apparently opposite ways, upon first-signaling for cell survival and upon second-signaling for ACD. Our findings strongly suggest that Atg1, thus presumably autophagy, protects the cells from starvation-induced cell death, allowing subsequent induction of ACD by the second signal. ACD is therefore not only "with" autophagy (since it showed signs of autophagy throughout), but is also "allowed by" autophagy. This does not exclude a role for autophagy also after second signaling. These results may account for discrepancies reported in the literature, encourage searches for second signals in different developmental models of ACD, and incite caution in autophagy-related therapeutic attempts.     

Brown adipose tissue activity controls triglyceride clearance

Brown adipose tissue (BAT) burns fatty acids for heat production to defend the body against cold and has recently been shown to be present in humans. Triglyceride-rich lipoproteins (TRLs) transport lipids in the bloodstream, where the fatty acid moieties are liberated by the action of lipoprotein lipase (LPL). Peripheral organs such as muscle and adipose tissue take up the fatty acids, whereas the remaining cholesterol-rich remnant particles are cleared by the liver. Elevated plasma triglyceride concentrations and prolonged circulation of cholesterol-rich remnants, especially in diabetic dyslipidemia, are risk factors for cardiovascular disease. However, the precise biological role of BAT for TRL clearance remains unclear. Here we show that increased BAT activity induced by short-term cold exposure controls TRL metabolism in mice. Cold exposure drastically accelerated plasma clearance of triglycerides as a result of increased uptake into BAT, a process crucially dependent on local LPL activity and transmembrane receptor CD36. In pathophysiological settings, cold exposure corrected hyperlipidemia and improved deleterious effects of insulin resistance. In conclusion, BAT activity controls vascular lipoprotein homeostasis by inducing a metabolic program that boosts TRL turnover and channels lipids into BAT. Activation of BAT might be a therapeutic approach to reduce elevated triglyceride concentrations and combat obesity in humans.     

Habitat specificity and diversity of tree species in an African wet tropical forest

Niche differentiation with respect to habitat has been hypothesized to shape patterns of diversity and species distributions in plant communities. African forests have been reported to be relatively less diverse compared to highly diversed regions of the Amazonian or Southeast Asian forests, and might be expected to have less niche differentiation. We examined patterns of structural and floristic differences among five topographically defined habitats for 494 species with stems ≥1 cm dbh in a 50-ha plot in Korup National Park, Cameroon. In addition, we tested for species–habitat associations for 272 species (with more than 50 individuals in the plot) using Torus translation randomization tests. Tree density and basal area were lowest in areas with negative convexity, which contained streams or were inundated during rainy periods and highest in moist well-drained habitats. Species composition and diversity varied along the topographical gradient from low flat to ridge top habitats. The low depression and low flat habitats were characterized by high diversity and similar species composition, relative to slopes, high gullies and ridge tops. Sixty-three percent of the species evaluated showed significant positive associations with at least one of the five habitat types. The majority of associations were with low depressions (75 species) and the fewest with ridge tops (8 species). The large number of species–habitat associations and the pronounced contrast between low (valley) and elevated (ridgetop) habitats in the Korup plot shows that niche differentiation with respect to edaphic variables (e.g., soil moisture, nutrients) contributes to local scale tree species distributions and to the maintenance of diversity in African forests.