Biological activity of CXCL8 forms generated by alternative cleavage of the signal peptide or by aminopeptidase-mediated truncation

BackgroundPosttranslational modification of chemokines is one of the mechanisms that regulate leukocyte migration during inflammation. Multiple natural NH₂-terminally truncated forms of the major human neutrophil attractant interleukin-8 or CXCL8 have been identified. Although differential activity was reported for some CXCL8 forms, no biological data are available for others.ConclusionsIn terms of their ability to induce neutrophil recruitment in vivo, the multiple CXCL8 forms may be divided in three groups. The first group includes CXCL8 proteins consisting of 75 to 79 amino acids, cleaved by aminopeptidases, with intermediate activity on neutrophils. The second group, generated through proteolytic cleavage (e.g. by Ser proteases), contains 69 to 72 amino acid forms which are highly potent neutrophil attractants in vivo. A third category is generated through the modification of the arginine in the NH₂-terminal region into citrulline by peptidylarginine deiminases and has weak potency to induce neutrophil extravasation.     

A yeast-based model of alpha-synucleinopathy identifies compounds with therapeutic potential

We have developed a yeast-based model recapitulating neurotoxicity of alpha-synuclein fibrilization. This model recognized metal ions, known risk factors of alpha-synucleinopathy, as stimulators of alpha-synuclein aggregation and cytotoxicity. Elimination of Yca1 caspase activity augmented both cytotoxicity and inclusion body formation, suggesting the involvement of apoptotic pathway components in toxic alpha-synuclein amyloidogenesis. Deletion of hydrophobic amino acids at positions 66-74 in alpha-synuclein reduced its cytotoxicity but, remarkably, did not lower the levels of insoluble alpha-synuclein, indicating that noxious alpha-synuclein species are different from insoluble aggregates. A compound screen aimed at finding molecules with therapeutic potential identified flavonoids with strong activity to restrain alpha-synuclein toxicity. Subsequent structure-activity analysis elucidated that these acted by virtue of anti-oxidant and metal-chelating activities. In conclusion, this yeast-cell model as presented allows not only fundamental studies related to mechanisms of alpha-synuclein-instigated cellular degeneration, but is also a valid high-throughput identification tool for novel neuroprotective agents.     

Understanding the development of roots exposed to contaminants and the potential of plant-associated bacteria for optimization of growth

Background and Scope

Plant responses to the toxic effects of soil contaminants, such as excess metals or organic substances, have been studied mainly at physiological, biochemical and molecular levels, but the influence on root system architecture has received little attention. Nevertheless, the precise position, morphology and extent of roots can influence contaminant uptake. Here, data are discussed that aim to increase the molecular and ecological understanding of the influence of contaminants on root system architecture. Furthermore, the potential of plant-associated bacteria to influence root growth by their growth-promoting and stress-relieving capacities is explored.

Methods

Root growth parameters of Arabidopsis thaliana seedlings grown in vertical agar plates are quantified. Mutants are used in a reverse genetics approach to identify molecular components underlying quantitative changes in root architecture after exposure to excess cadmium, copper or zinc. Plant-associated bacteria are isolated from contaminated environments, genotypically and phenotypically characterized, and used to test plant root growth improvement in the presence of contaminants.

Key Results

The molecular determinants of primary root growth inhibition and effects on lateral root density by cadmium were identified. A vertical split-root system revealed local effects of cadmium and copper on root development. However, systemic effects of zinc exposure on root growth reduced both the avoidance of contaminated areas and colonization of non-contaminated areas. The potential for growth promotion and contaminant degradation of plant-associated bacteria was demonstrated by improved root growth of inoculated plants exposed to 2,4-di-nitro-toluene (DNT) or cadmium.

Conclusions

Knowledge concerning the specific influence of different contaminants on root system architecture and the molecular mechanisms by which this is achieved can be combined with the exploitation of plant-associated bacteria to influence root development and increase plant stress tolerance, which should lead to more optimal root systems for application in phytoremediation or safer biomass production.     

Comparative anatomy of intervessel pits in two mangrove species growing along a natural salinity gradient in Gazi bay, Kenya

BACKGROUND AND AIMS: According to the air-seeding hypothesis, embolism vulnerability in xylem elements is linked directly to bordered pit structure and functioning. To elucidate the adaptive potential of intervessel pits towards fluctuating environmental conditions, two mangrove species with a distinct ecological distribution growing along a natural salinity gradient were investigated. METHODS: Scanning and transmission electron microscopic observations were conducted to obtain qualitative and quantitative characteristics of alternate intervessel pits in A. marina and scalariform intervessel pits in Rhizophora mucronata. Wood samples from three to six trees were collected at seven and five sites for A. marina and R. mucronata, respectively, with considerable differences between sites in soil water salinity. KEY RESULTS: Vestured pits without visible pores in the pit membrane were observed in A. marina, the mangrove species with the widest geographical distribution on global as well as local scale. Their thick pit membranes (on average 370 nm) and minute pit apertures may contribute to reduced vulnerability to cavitation of this highly salt-tolerant species. The smaller ecological distribution of R. mucronata was in accordance with wide pit apertures and a slightly higher pitfield fraction (67 % vs. 60 % in A. marina). Nonetheless, its outer pit apertures were observed to be funnel-shaped shielding non-porous pit membranes. No trends in intervessel pit size were observed with increasing soil water salinity of the site. CONCLUSIONS: The contrasting ecological distribution of two mangrove species was reflected in the geometry and pit membrane characteristics of their intervessel pits. Within species, intervessel pit size seemed to be independent of spatial variations in environmental conditions and was only weakly correlated with vessel diameter. Further research on pit formation and function has to clarify the large variations in intervessel pit size within trees and even within single vessels.     

Kinetic and equilibrium intermediate states are different in LYLA1, a chimera of lysozyme and alpha-lactalbumin.

For several proteins, a striking resemblance has been observed between the equilibrium partially folded state and the kinetic burst-phase intermediate, observed just after the dead-time in refolding experiments. This has led to the general statement that the conformation of both types of intermediates is similar. We show, at least for one of the proteins investigated here, that, although both states have some common characteristics, they are not identical. LYLA1 is a chimeric protein resulting from the transplantation of the Ca(2+)-binding loop and the adjacent helix C of bovine alpha-lactalbumin into the homologous position (residues 76-102) in human lysozyme. The apo-form of LYLA1 unfolds through a partially folded state, in analogy with the folding behaviour of the structurally homologous alpha-lactalbumin. The folding kinetics of LYLA1 and of its wild-type homologue, human lysozyme, are investigated by means of stopped-flow fluorescence and CD spectroscopy. In the case of human lysozyme, refolding involves parallel pathways as indicated by experiments in the presence of a fluorescent inhibitor. For apo-LYLA1, the burst-phase intermediate is compared with the equilibrium intermediate. At neutral pH, both states correspond, in that an important amount of secondary structure has been established, but the burst-phase intermediate is shown to be significantly less stable than the equilibrium intermediate. At pH 1.85, in the presence of 1.5 M guanidinium hydrochloride (GdnHCl) and at 25 degrees C, the equilibrium partially folded state of LYLA1 is 100% populated. When LYLA1 is rapidly diluted from 6 M GdnHCl to 1.5 M under these conditions, a time-dependent evolution of the fluorescence signal is observed, reflecting the transition from a burst-phase to a different equilibrium intermediate. These results provide strong evidence for the non-identity of both states in this protein.     

Dehydroascorbate influences the plant cell cycle through a glutathione-independent reduction mechanism

Glutathione is generally accepted as the principal electron donor for dehydroascorbate (DHA) reduction. Moreover, both glutathione and DHA affect cell cycle progression in plant cells. But other mechanisms for DHA reduction have been proposed. To investigate the connection between DHA and glutathione, we have evaluated cellular ascorbate and glutathione concentrations and their redox status after addition of dehydroascorbate to medium of tobacco (Nicotiana tabacum) L. cv Bright Yellow-2 (BY-2) cells. Addition of 1 mm DHA did not change the endogenous glutathione concentration. Total glutathione depletion of BY-2 cells was achieved after 24-h incubation with 1 mm of the glutathione biosynthesis inhibitor l-buthionine sulfoximine. Even in these cells devoid of glutathione, complete uptake and internal reduction of 1 mm DHA was observed within 6 h, although the initial reduction rate was slower. Addition of DHA to a synchronized BY-2 culture, or depleting its glutathione content, had a synergistic effect on cell cycle progression. Moreover, increased intracellular glutathione concentrations did not prevent exogenous DHA from inducing a cell cycle shift. It is therefore concluded that, together with a glutathione-driven DHA reduction, a glutathione-independent pathway for DHA reduction exists in vivo, and that both compounds act independently in growth control.     

Kinase Substrate Sensor (KISS), a Mammalian In Situ Protein Interaction Sensor

Probably every cellular process is governed by protein-protein interaction (PPIs), which are often highly dynamic in nature being modulated by in- or external stimuli. Here we present KISS, for KInase Substrate Sensor, a mammalian two-hybrid approach designed to map intracellular PPIs and some of the dynamic features they exhibit. Benchmarking experiments indicate that in terms of sensitivity and specificity KISS is on par with other binary protein interaction technologies while being complementary with regard to the subset of PPIs it is able to detect. We used KISS to evaluate interactions between different types of proteins, including transmembrane proteins, expressed at their native subcellular location. In situ analysis of endoplasmic reticulum stress-induced clustering of the endoplasmic reticulum stress sensor ERN1 and ligand-dependent β-arrestin recruitment to GPCRs illustrated the method''s potential to study functional PPI modulation in complex cellular processes. Exploring its use as a tool for in cell evaluation of pharmacological interference with PPIs, we showed that reported effects of known GPCR antagonists and PPI inhibitors are properly recapitulated. In a three-hybrid setup, KISS was able to map interactions between small molecules and proteins. Taken together, we established KISS as a sensitive approach for in situ analysis of protein interactions and their modulation in a changing cellular context or in response to pharmacological challenges.A protein''s function is largely mediated through its interactions with other proteins, hence the critical importance of protein-protein interaction (PPI)¹ maps for understanding cellular mechanisms of action in health and disease. Whereas many proteins are organized in stable multi-protein complexes, the majority of cellular processes are governed by transient protein encounters, the dynamics of which are directed by a diversity of both intra- and extracellular signals. Our view of protein networks is still, however, mainly a static one (1). Current interactomes consist mainly of data generated by yeast 2-hybrid (Y2H) (2) and (tandem) affinity purification combined with mass spectrometry (3) and should be interpreted as scaffolds of potential PPIs that might occur at a certain time and place in the cell or as snapshots of PPIs taking place under a specific cellular condition. Although very robust and highly efficient, these approaches do not allow studying PPI modulation because they do not offer the proper context for mammalian PPI analysis, e.g. they operate in yeast cells (Y2H) or make use of cell lysates (affinity purification-based methods). Moreover, because these interactome mapping tools are biased against interactions that involve transmembrane proteins, the latter are underrepresented in current interactome network versions (4). Yet, membrane-associated proteins constitute around one third of the entire proteome and their significance is underscored by the fact that over half of currently marketed drugs target membrane proteins (5). These observations support the need for approaches that allow PPIs, including those involving transmembrane proteins, to be assayed in their native cellular environment.Apart from the high-throughput methods mentioned above, a diverse arsenal of other PPI technologies has been developed, a number of which actually operate in mammalian cells. FRET and BRET, which rely on fluorescence or bioluminescence energy transfer between interacting fusion proteins, make assays with high spatiotemporal resolution (6, 7). A variety of PCAs have been reported, including split fluorescent protein or reporter enzyme technologies, that are able to capture aspects of PPI dynamics in a mammalian background (8, 9). A recent addition is an infrared fluorescent PCA that, unlike previous fluorescent PCAs, exhibits reversible complementation, thus enabling spatiotemporal analysis of dynamic PPIs (10). Another binary interaction assay, luminescence-based mammalian interactome mapping (LUMIER), has been applied to map TGFβ induced modulation of PPIs with components of the TGFβ signaling pathway (11). MaMTH, a mammalian version of the split ubiquitin approach, was designed particularly for the analysis of PPIs involving integral membrane proteins, also allowing the detection of functional PPI modulation (12). Efforts to apply purification-based methods for detecting context-dependent PPI modulation recently resulted in the development of AP-SRM (13) and AP-SWATH (14).Our group previously conceived mammalian protein-protein interaction trap (MAPPIT) (supplemental Fig. S1A) (15, 16), a mammalian two-hybrid approach based on complementation of a cytokine receptor that was developed into a broad platform for PPI analysis (17, 18), screening for small molecule PPI disruptors (19, 20) and drug target profiling (21, 22). Although MAPPIT operates in intact human cells, thus providing the natural environment for human protein analysis, the interaction sensor is anchored to the plasma membrane, precluding the analysis of PPIs at their native subcellular localization. In addition, MAPPIT is incompatible with full size transmembrane proteins. Here we describe KInase Substrate Sensor (KISS), a novel binary PPI mapping approach that enables in situ analysis in living mammalian cells of protein interactions and their responses to physiological or pharmacological challenges.