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The investigation of luminal factors influencing zinc availability and accessibility in the intestine is of great interest when analyzing parameters regulating intestinal zinc resorption. Of note, intestinal mucins were suggested to play a beneficial role in the luminal availability of zinc. Their exact zinc binding properties, however, remain unknown and the impact of these glycoproteins on human intestinal zinc resorption has not been investigated in detail. Thus, the aim of this study is to elucidate the impact of intestinal mucins on luminal uptake of zinc into enterocytes and its transfer into the blood. In the present study, in vitro zinc binding properties of mucins were analyzed using commercially available porcine mucins and secreted mucins of the goblet cell line HT-29-MTX. The molecular zinc binding capacity and average zinc binding affinity of these glycoproteins demonstrates that mucins contain multiple zinc-binding sites with biologically relevant affinity within one mucin molecule. Zinc uptake into the enterocyte cell line Caco-2 was impaired by zinc-depleted mucins. Yet this does not represent their form in the intestinal lumen in vivo under zinc adequate conditions. In fact, zinc-uptake studies into enterocytes in the presence of mucins with differing degree of zinc saturation revealed zinc buffering by these glycoproteins, indicating that mucin-bound zinc is still available for the cells. Finally, the impact of mucins on zinc resorption using three-dimensional cultures was studied comparing the zinc transfer of a Caco-2/HT-29-MTX co-culture and conventional Caco-2 monoculture. Here, the mucin secreting co-cultures yielded higher fractional zinc resorption and elevated zinc transport rates, suggesting that intestinal mucins facilitate the zinc uptake into enterocytes and act as a zinc delivery system for the intestinal epithelium.
The primary function of leaves is to provide an interface between plants and their environment for gas exchange, light exposure and thermoregulation. Leaves have, therefore a central contribution to plant fitness by allowing an efficient absorption of sunlight energy through photosynthesis to ensure an optimal growth. Their final geometry will result from a balance between the need to maximize energy uptake while minimizing the damage caused by environmental stresses. This intimate relationship between leaf and its surroundings has led to an enormous diversification in leaf forms. Leaf shape varies between species, populations, individuals or even within identical genotypes when those are subjected to different environmental conditions. For instance, the extent of leaf margin dissection has, for long, been found to inversely correlate with the mean annual temperature, such that Paleobotanists have used models based on leaf shape to predict the paleoclimate from fossil flora. Leaf growth is not only dependent on temperature but is also regulated by many other environmental factors such as light quality and intensity or ambient humidity. This raises the question of how the different signals can be integrated at the molecular level and converted into clear developmental decisions. Several recent studies have started to shed the light on the molecular mechanisms that connect the environmental sensing with organ-growth and patterning. In this review, we discuss the current knowledge on the influence of different environmental signals on leaf size and shape, their integration as well as their importance for plant adaptation.
The plant pathogen Pseudomonas syringae is a gram-negative bacterium which infects a wide range of plant species including important crops plants. To suppress plant immunity and cause disease P.syringae injects type-III effector proteins (T3Es) into the plant cell cytosol. In this study, we identified a novel target of the well characterized bacterial T3E HopZ1a. HopZ1a is an acetyltransferase that was shown to disrupt vesicle transport during innate immunity by acetylating tubulin. Using a yeast-two-hybrid screen approach, we identified a REMORIN (REM) protein from tobacco as a novel HopZ1a target. HopZ1a interacts with REM at the plasma membrane (PM) as shown by split-YFP experiments. Interestingly, we found that PBS1, a well-known kinase involved in plant immunity also interacts with REM in pull-down assays, and at the PM as shown by BiFC. Furthermore, we confirmed that REM is phosphorylated by PBS1 in vitro. Overexpression of REM provokes the upregulation of defense genes and leads to disease-like phenotypes pointing to a role of REM in plant immune signaling. Further protein-protein interaction studies reveal novel REM binding partners with a possible role in plant immune signaling. Thus, REM might act as an assembly hub for an immune signaling complex targeted by HopZ1a. Taken together, this is the first report describing that a REM protein is targeted by a bacterial effector. How HopZ1a might mechanistically manipulate the plant immune system through interfering with REM function will be discussed.
Franco-Obregon, Alfredo ; Cambria, Elena ; Greutert, Helen ; Wernas, Timon ; Hitzl, Wolfgang ; Egli, Marcel ; Sekiguchi, Miho ; Boos, Norbert ; Hausmann, Oliver ; Ferguson, Stephen J. ; Kobayashi, Hiroshi ; Würtz-Kozak, Karin
Purpose Prolonged bed rest and microgravity in space cause intervertebral disc (IVD) degeneration. However, the underlying molecular mechanisms are not completely understood. Transient receptor potential canonical (TRPC) channels are implicated in mechanosensing of several tissues, but are poorly explored in IVDs. Methods Primary human IVD cells from surgical biopsies composed of both annulus fibrosus and nucleus pulposus (passage 1-2) were exposed to simulated microgravity and to the TRPC channel inhibitor SKF-96365 (SKF) for up to 5days. Proliferative capacity, cell cycle distribution, senescence and TRPC channel expression were analyzed. Results Both simulated microgravity and TRPC channel antagonism reduced the proliferative capacity of IVD cells and induced senescence. While significant changes in cell cycle distributions (reduction in G1 and accumulation in G2/M) were observed upon SKF treatment, the effect was small upon 3days of simulated microgravity. Finally, downregulation of TRPC6 was shown under simulated microgravity. Conclusions Simulated microgravity and TRPC channel inhibition both led to reduced proliferation and increased senescence. Furthermore, simulated microgravity reduced TRPC6 expression. IVD cell senescence and mechanotransduction may hence potentially be regulated by TRPC6 expression. This study thus reveals promising targets for future studies.
Trees control the flowering processes in response to both environmental and endogenous (mechanisms at cellular/tissue level) conditions. Dormancy of flower buds is characterized by the reduction of growth and the enhancement of frost and desiccation resistance. The release of endodormancy and the beginning of ontogenetic development, as two important dates for developing reliable phenological models, escape from any visible signs. Thus, we identified - to our knowledge as first - relevant proteins in sweet cherry buds occurring during these phenological phases at high time resolution in three seasons (2012/13–2014/15) under natural conditions in Northeast Germany. The protein content of buds from the first week of October to leaf fall, from leaf fall to the end of endodormancy (t1), from t1 to the beginning of ontogenetic development (t1*), and from t1* until swollen bud, was comparable in each of the seasons. The increase of the protein content began after swollen bud and markedly differences occurred at side green, green tip, tight and open cluster. SDS gel electrophoresis followed by peptide mass fingerprinting accomplished by MALDI-TOF MS was applied for protein identification. ‘Volume intensity’ has been used to demonstrate the pattern and changes of proteins. None of the analysed proteins like for cell proliferation/differentiation (Phytosulfokines 3), carbon fixation (Rubisco), and defense against pathogenes (Major allergen Pru sv 1) indicates the date of endodormancy release or the beginning of the (invisible) ontogenetic development. The stages around green tip, tight, and open cluster resulted in markedly increase of the volume intensity of the protein for cell proliferation/differentiation and the carbon fixation, whereas the volume intensity of a protein for defense against pathogens markedly decreased. The pattern and changes of the volume intensity of neoxanthin synthase (NXS) in sweet cherry buds followed the increasing demand during endo- and ecodormancy to produce neoxanthin, which is a prominent member of the group of reactive oxygen species (ROS) scavengers.
Folding and Lipid Composition Determine Membrane Interaction of the Disordered Protein COR15A
(2018)
Navarro-Retamal, Carlos ; Bremer, Anne ; Ingolfsson, Helgi I. ; Alzate-Morales, Jans ; Caballero, Julio ; Thalhammer, Anja ; Gonzalez, Wendy ; Hincha, Dirk K.
Plants from temperate climates, such as the model plant Arabidopsis thaliana, are challenged with seasonal low temperatures that lead to increased freezing tolerance in fall in a process termed cold acclimation. Among other adaptations, this involves the accumulation of cold-regulated (COR) proteins, such as the intrinsically disordered chloroplast-localized protein COR15A. Together with its close homolog COR15B, it stabilizes chloroplast membranes during freezing. COR15A folds into amphipathic alpha-helices in the presence of high concentrations of low-molecular-mass crowders or upon dehydration. Under these conditions, the (partially) folded protein binds peripherally to membranes. In our study, we have used coarse-grained molecular dynamics simulations to elucidate the details of COR15A-membrane binding and its effects on membrane structure and dynamics. Simulation results indicate that at least partial folding of COR15A and the presence of highly unsaturated galactolipids in the membranes are necessary for efficient membrane binding. The bound protein is stabilized on the membrane by interactions of charged and polar amino acids with galactolipid headgroups and by interactions of hydrophobic amino acids with the upper part of the fatty acyl chains. Experimentally, the presence of liposomes made from a mixture of lipids mimicking chloroplast membranes induces additional folding in COR15A under conditions of partial dehydration, in agreement with the simulation results.
Brügger, Sandra Olivia ; Gobet, Erika ; Sigl, Michael ; Osmont, Dimitri ; Papina, Tatyana ; Rudaya, Natalia ; Schwikowski-Gigar, Margit ; Tinner, Willy
Forest and steppe communities in the Altai region of Central Asia are threatened by changing climate and anthropogenic pressure. Specifically, increasing drought and grazing pressure may cause collapses of moisture-demanding plant communities, particularly forests. Knowledge about past vegetation and fire responses to climate and land use changes may help anticipating future ecosystem risks, given that it has the potential to disclose mechanisms and processes that govern ecosystem vulnerability. We present a unique paleoecological record from the high-alpine Tsambagarav glacier in the Mongolian Altai that provides novel large-scale information on vegetation, fire and pollution with an exceptional temporal resolution and precision. Our palynological record identifies several late-Holocene boreal forest expansions, contractions and subsequent recoveries. Maximum forest expansions occurred at 3000-2800 BC, 2400-2100 BC, and 1900-1800 BC. After 1800 BC mixed boreal forest communities irrecoverably declined. Fires reached a maximum at 1600 BC, 200 years after the final forest collapse. Our multiproxy data suggest that burning peaked in response to dead biomass accumulation resulting from forest diebacks. Vegetation and fire regimes partly decoupled from climate after 1700 AD, when atmospheric industrial pollution began, and land use intensified. We conclude that moisture availability was more important than temperature for past vegetation dynamics, in particular for forest loss and steppe expansion. The past Mongolian Altai evidence implies that in the future forests of the Russian Altai may collapse in response to reduced moisture availability.
DNA nanostructures enable the attachment of functional molecules to nearly any unique location on their underlying structure. Due to their single-base-pair structural resolution, several ligands can be spatially arranged and closely controlled according to the geometry of their desired target, resulting in optimized binding and/or signaling interactions. Here, the efficacy of SWL, an ephrin-mimicking peptide that binds specifically to EphrinA2 (EphA2) receptors, increased by presenting up to three of these peptides on small DNA nanostructures in an oligovalent manner. Ephrin signaling pathways play crucial roles in tumor development and progression. Moreover, Eph receptors are potential targets in cancer diagnosis and treatment. Here, the quantitative impact of SWL valency on binding, phosphorylation (key player for activation) and phenotype regulation in EphA2-expressing prostate cancer cells was demonstrated. EphA2 phosphorylation was significantly increased by DNA trimers carrying three SWL peptides compared to monovalent SWL. In comparison to one of EphA2’s natural ligands ephrin-A1, which is known to bind promiscuously to multiple receptors, pinpointed targeting of EphA2 by oligovalent DNA-SWL constructs showed enhanced cell retraction. Overall, we show that DNA scaffolds can increase the potency of weak signaling peptides through oligovalent presentation and serve as potential tools for examination of complex signaling pathways.
Same but different
(2018)
The maturation of bacterial molybdoenzymes is a complex process leading to the insertion of the bulky bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor into the apoenzyme. Most molybdoenzymes were shown to contain a specific chaperone for the insertion of the bis-MGD cofactor. Formate dehydrogenases (FDH) together with their molecular chaperone partner seem to display an exception to this specificity rule, since the chaperone FdhD has been proven to be involved in the maturation of all three FDH enzymes present in Escherichia colt. Multiple roles have been suggested for FdhD-like chaperones in the past, including the involvement in a sulfur transfer reaction from the L-cysteine desulfurase IscS to bis-MGD by the action of two cysteine residues present in a conserved CXXC motif of the chaperones. However, in this study we show by phylogenetic analyses that the CXXC motif is not conserved among FdhD-like chaperones. We compared in detail the FdhD-like homologues from Rhodobacter capsulatus and E. colt and show that their roles in the maturation of FDH enzymes from different subgroups can be exchanged. We reveal that bis-MGDbinding is a common characteristic of FdhD-like proteins and that the cofactor is bound with a sulfido-ligand at the molybdenum atom to the chaperone. Generally, we reveal that the cysteine residues in the motif CXXC of the chaperone are not essential for the production of active FDH enzymes.