The chloroplast lumen and stromal proteomes of Arabidopsis thaliana show differential sensitivity to short- and long-term exposure to low temperature

Cold acclimation and over-wintering by herbaceous plants are energetically expensive and are dependent on functional plastid metabolism. To understand how the stroma and the lumen proteomes adapt to low temperatures, we have taken a proteomic approach (difference gel electrophoresis) to identify proteins that changed in abundance in Arabidopsis chloroplasts during cold shock (1 day), and short- (10 days) and long-term (40 days) acclimation to 5 degrees C. We show that cold shock (1 day) results in minimal change in the plastid proteomes, while short-term (10 days) acclimation results in major changes in the stromal but few changes in the lumen proteome. Long-term acclimation (40 days) results in modulation of the proteomes of both compartments, with new proteins appearing in the lumen and further modulations in protein abundance occurring in the stroma. We identify 43 differentially displayed proteins that participate in photosynthesis, other plastid metabolic functions, hormone biosynthesis and stress sensing and signal transduction. These findings not only provide new insights into the cold response and acclimation of Arabidopsis, but also demonstrate the importance of studying changes in protein abundance within the relevant cellular compartment.     

Hedgehog signalling: how to get from Smo to Ci and Gli

The secreted morphogens of the Hedgehog family have important roles in normal development as well as in associated pathologies, including cancer. The Hedgehog signalling pathway has been studied in Drosophila and is thought to be conserved in vertebrates. Hedgehog elicits a signalling response that activates Smoothened (Smo). There is evidence of differences between Drosophila and vertebrates concerning signalling downstream of Smo, as well as in Smo itself. Here, we discuss this evidence and its importance for investigations of the pathway and related biology, as well as for the development of drugs targeting components of the pathway for treatment of associated pathologies.     

Structural basis for target protein recognition by the protein disulfide reductase thioredoxin

Thioredoxin is ubiquitous and regulates various target proteins through disulfide bond reduction. We report the structure of thioredoxin (HvTrxh2 from barley) in a reaction intermediate complex with a protein substrate, barley alpha-amylase/subtilisin inhibitor (BASI). The crystal structure of this mixed disulfide shows a conserved hydrophobic motif in thioredoxin interacting with a sequence of residues from BASI through van der Waals contacts and backbone-backbone hydrogen bonds. The observed structural complementarity suggests that the recognition of features around protein disulfides plays a major role in the specificity and protein disulfide reductase activity of thioredoxin. This novel insight into the function of thioredoxin constitutes a basis for comprehensive understanding of its biological role. Moreover, comparison with structurally related proteins shows that thioredoxin shares a mechanism with glutaredoxin and glutathione transferase for correctly positioning substrate cysteine residues at the catalytic groups but possesses a unique structural element that allows recognition of protein disulfides.     

Stevioside does not cause increased basal insulin secretion or beta-cell desensitization as does the sulphonylurea, glibenclamide: studies in vitro

We have shown that stevioside (SVS) enhances insulin secretion and thus may have a potential role as antihyperglycemic agent in the treatment of type 2 diabetes mellitus. However, whether SVS stimulates basal insulin secretion (BIS) and/or cause desensitization of beta cells like sulphonylureas (SU), e.g. glibenclamide (GB), is not known. To explore and compare the effects of SVS pretreatment with those of GB and glucagon-like peptide-1 (GLP-1), we exposed isolated mouse islets to low or high glucose for 1 h after short-term (2 h) or long-term (24 h) pretreatment with SVS, GB or GLP-1, respectively. BIS at 3.3 or 5.5 mM glucose were not changed after short-term pretreatment with SVS (10(-7) M), while it increased about three folds after pretreatment with GB (10(-7) M). Glucose stimulated insulin secretion (GSIS) (16.7 mM) increased dose-dependently after long-term pretreatment with SVS at concentrations from 10(-7) to 10(-5) M. Pretreatment for 24 h with GB (10(-7) M) increased the subsequent BIS (3.3 mM glucose) (p < 0.001), but decreased GSIS (16.7 mM glucose) (p < 0.001). In contrast SVS (10(-7) M) and GLP-1 (10(-7) M) did not stimulate BIS but both enhanced the subsequent GSIS (16.7 mM glucose) (p < 0.05 and p < 0.05, respectively). While SVS pretreatment increased the intracellular insulin content, GB pretreatment decreased the insulin content. Our study suggests that SVS pretreatment does not cause a stimulation of BIS and does not desensitize beta-cells, i.e. SVS seems to have advantageous characteristics to GB as a potential treatment of type 2 diabetes.     

Modeling the impact of store-operated Ca2+ entry on intracellular Ca2+ oscillations

Calcium (Ca2+) oscillations play fundamental roles in various cell signaling processes and have been the subject of numerous modeling studies. Here we have implemented a general mathematical model to simulate the impact of store-operated Ca2+ entry on intracellular Ca2+ oscillations. In addition, we have compared two different models of the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and their influences on intracellular Ca2+ oscillations. Store-operated Ca2+ entry following Ca2+ depletion of endoplasmic reticulum (ER) is an important component of Ca2+ signaling. We have developed a phenomenological model of store-operated Ca2+ entry via store-operated Ca2+ (SOC) channels, which are activated upon ER Ca2+ depletion. The depletion evokes a bi-phasic Ca2+ signal, which is also produced in our mathematical model. The IP3R is an important regulator of intracellular Ca2+ signals. This IP3 sensitive Ca2+ channel is also regulated by Ca2+. We apply two IP3R models, the Mak-McBride-Foskett model and the De Young and Keizer model, with significantly different channel characteristics. Our results show that the two separate IP3R models evoke intracellular Ca2+ oscillations with different frequencies and amplitudes. Store-operated Ca2+ entry affects the oscillatory behavior of these intracellular Ca2+ oscillations. The IP3 threshold is altered when store-operated Ca2+ entry is excluded from the model. Frequencies and amplitudes of intracellular Ca2+ oscillations are also altered without store-operated Ca2+ entry. Under certain conditions, when intracellular Ca2+ oscillations are absent, excluding store-operated Ca2+ entry induces an oscillatory response. These findings increase knowledge concerning store-operated Ca2+ entry and its impact on intracellular Ca2+ oscillations.     

Proteomic profiling of the Baltic Sea cyanobacterium Nodularia spumigena strain AV1 during ammonium supplementation

The cyanobacterium Nodularia spumigena dominates the annual, toxic summer blooms in the Baltic Sea. Although Nodularia has been receiving attention due to its production of the hepatotoxin nodularin, molecular data regarding the regulation of nitrogen fixation is lacking. We have previously reported that N. spumigena strain AV1, unlike model filamentous cyanobacteria, differentiates heterocysts in the absence of detectable nitrogen fixation activity. To further analyze the uncoupling between these two linked processes, we assessed the impact of ammonium ions on the N. spumigena metabolism using a proteomic approach. Proteomic profiling was performed at three different times during ammonium supplementation using quantitative 2-dimensional gel electrophoresis followed by MS/MS analysis. Using this approach, we identified 34 proteins, 28 of which were unique proteins that changed successively in abundance during growth on ammonium. Our results indicate that N. spumigena generally exhibits lower energy production and carbon fixation in the presence of ammonium and seems to be inefficient in utilizing ammonium as an external nitrogen source. The possibility of ammonium toxicity due to PSII damage was investigated and the results are discussed. Our findings have implications in regard to the strategies considered to manage the cyanobacterial blooms in the Baltic Sea.     

Neurotransmitter synthesis in poststroke cortical neurogenesis in adult rats

Neurogenesis occurs in the cerebral cortex of adult rats after focal cerebral ischemia. Whether or not the newborn neurons could synthesize neurotransmitters is unknown. To elucidate such a possibility, a photothrombotic ring stroke model with spontaneous reperfusion was induced in adult male Wistar rats. The DNA duplication marker BrdU was repeatedly injected, and the rats were sacrificed at various times after stroke. To detect BrdU nuclear incorporation and various neurotransmitters, brain sections were processed for single/double immunocytochemistry and single/double/triple immunofluorescence. Stereological cell counting was performed to assess the final cell populations. At 48 h, 5 days, 7 days, 30 days, 60 days and 90 days after stroke, numerous cells were BrdU-immunolabeled in the penumbral cortex. Some of these were doubly immunopositive to the cholinergic neuron-specific marker ChAT or GABAergic neuron-specific marker GAD. As analyzed by 3-D confocal microscopy, the neurotransmitters acetylcholine and GABA were colocalized with BrdU in the same cortical cells. In addition, GABA was colocalized with the neuron-specific marker Neu N in the BrdU triple-immunolabeled cortical cells. This study suggests that the newborn neurons are capable of synthesizing the neurotransmitters acetylcholine and GABA in the penumbral cortex, which is one of the fundamental requisites for these neurons to function in the poststroke recovery.     

Reactive oxygen species inhibit catalytic activity of peptidylarginine deiminase

Protein citrullination catalysed by peptidylarginine deiminase (PAD) may play an important pathogenic role in several chronic inflammatory diseases and malignancies. PAD2, PAD4, and citrullinated proteins are found in the synovium of rheumatoid arthritis patients. PAD activity is dependent on calcium and reducing conditions. However, reactive oxygen species (ROS) have been shown to induce citrullination of histones in granulocytes. Here we examine the ability of H₂O₂ and leukocyte-derived ROS to regulate PAD activity using citrullination of fibrinogen as read-out. H₂O₂ at concentrations above 40?µM inhibited the catalytic activity of PAD2 and PAD4 in a dose-dependent manner. PMA-stimulated leukocytes citrullinated fibrinogen and this citrullination was markedly enhanced when ROS formation was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium (DPI). In contrast, PAD released from stimulated leukocytes was unaffected by exogenously added H₂O₂ at concentrations up to 1000?µM. The role of ROS in regulating PAD activity may play an important part in preventing hypercitrullination of proteins.     

Fibrillation of the major curli subunit CsgA under a wide range of conditions implies a robust design of aggregation

The amyloid fold is usually considered a result of protein misfolding. However, a number of studies have recently shown that the amyloid structure is also used in nature for functional purposes. CsgA is the major subunit of Escherichia coli curli, one of the most well-characterized functional amyloids. Here we show, using a highly efficient approach to prepare monomeric CsgA, that in vitro fibrillation of CsgA occurs under a wide variety of environmental conditions and that the resulting fibrils exhibit similar structural features. This highlights how fibrillation is "hardwired" into amyloid that has evolved for structural purposes in a fluctuating extracellular environment and represents a clear contrast to disease-related amyloid formation. Furthermore, we show that CsgA polymerization in vitro is preceded by the formation of thin needlelike protofibrils followed by aggregation of the amyloid fibrils.     

Real-time detection of central carbon metabolism in living Escherichia coli and its response to perturbations

The direct tracking of cellular reactions in vivo has been facilitated with recent technologies that strongly enhance NMR signals in substrates of interest. This methodology can be used to assay intracellular reactions that occur within seconds to few minutes, as the NMR signal enhancement typically fades on this time scale. Here, we show that the enhancement of (13)C nuclear spin polarization in deuterated glucose allows to directly follow the flux of glucose signal through rather extended reaction networks of central carbon metabolism in living Escherichia coli. Alterations in central carbon metabolism depending on the growth phase or upon chemical perturbations are visualized with minimal data processing by instantaneous observation of cellular reactions.