Changes of enzyme activity in lipid signaling pathways related to substrate reordering

The static fluid mosaic model of biological membranes has been progressively complemented by a dynamic membrane model that includes phospholipid reordering in domains that are proposed to extend from nanometers to microns. Kinetic models for lipolytic enzymes have only been developed for homogeneous lipid phases. In this work, we develop a generalization of the well-known surface dilution kinetic theory to cases where, in a same lipid phase, both domain and nondomain phases coexist. Our model also allows understanding the changes in enzymatic activity due to a decrease of free substrate concentration when domains are induced by peptides. This lipid reordering and domain dynamics can affect the activity of lipolytic enzymes, and can provide a simple explanation for how basic peptides, with a strong direct interaction with acidic phospholipids (such as beta-amyloid peptide), may cause a complex modulation of the activities of many important enzymes in lipid signaling pathways.     

Identification and molecular characterization of Mnk1b, a splice variant of human MAP kinase-interacting kinase Mnk1

In this paper, we report the identification and molecular characterization of a splice variant of human Mnk1 which has been named as Mnk1b. Human Mnk1b mRNA is homologous to human Mnk1 mRNA but lacking a region corresponding to exon 19, which causes a change in the reading frame generating a stop codon. The resulting protein lacks the last 89 amino acids at the C-terminal region that are replaced by 12 amino acids with an entirely new sequence. The C-terminal end in Mnk1 corresponds to the extracellular signal-regulated kinase (ERK1/2) binding site. Although Mnk1b lacks this domain and, consequently, is not phosphorylated by ERK1/2, it is able, however, to phosphorylate eIF4E in vitro and in vivo in a mitogen-activated protein kinases-independent manner. This result suggests that Mnk1b may play a key role in regulating protein translation in the absence of stimuli. Interestingly, a significant population of cells shows Mnk1b within the nucleus whereas Mnk1 is always detected in the cytoplasm. This fact may be explained because Mnk1b maintains the nuclear localization signal (NLS) but lacks the nuclear export sequence (NES).     

Ischaemic preconditioning in the rat brain: effect on the activity of several initiation factors, Akt and extracellular signal-regulated protein kinase phosphorylation, and GRP78 and GADD34 expression

Translational repression induced during reperfusion of the ischaemic brain is significantly attenuated by ischaemic preconditioning. The present work was undertaken to identify the components of the translational machinery involved and to determine whether translational attenuation selectively modifies protein expression patterns during reperfusion. Wistar rats were preconditioned by 5-min sublethal ischaemia and 2 days later, 30-min lethal ischaemia was induced. Several parameters were studied after lethal ischaemia and reperfusion in rats with and without acquired ischaemic tolerance (IT). The phosphorylation pattern of the alpha subunit of eukaryotic initiation factor 2 (eIF2) in rats with IT was exactly the same as in rats without IT, reaching a peak after 30 min reperfusion and returning to control values within 4 h in both the cortex and hippocampus. The levels of phosphorylated eIF4E-binding protein after lethal ischaemia and eIF4E at 30 min reperfusion were higher in rats with IT, notably in the hippocampus. eIF4G levels diminished slightly after ischaemia and reperfusion, paralleling calpain-mediated alpha-spectrin proteolysis in rats with and without IT, but they did not show any further decrease after 30 min reperfusion in rats with IT. The phosphorylated levels of eIF4G, phosphatidylinositol 3-kinase-protein B (Akt) and extracellular signal-regulated kinases (ERKs) were very low after lethal ischaemia and increased following reperfusion. Ischaemic preconditioning did not modify the observed changes in eIF4G phosphorylation. All these results support that translation attenuation may occur through multiple targets. The levels of the glucose-regulated protein (78 kDa) remained unchanged in rats with and without IT. Conversely, our data establish a novel finding that ischaemia induces strong translation of growth arrest and DNA damage protein 34 (GADD34) after 4 h of reperfusion. GADD34 protein was slightly up-regulated after preconditioning, besides, as in rats without IT, GADD34 levels underwent a further clear-cut increase during reperfusion, this time as earlier as 30 min and coincident with translation attenuation.     

Depot- and gender-related differences in the lipolytic pathway of adipose tissue from severely obese patients.

The present study was performed to analyze in detail gender- and site-related alterations in the adrenergic signal transduction pathway of lipolysis in fat cells isolated from subcutaneous abdominal and visceral fat depots from severely obese patients. The study group consisted of 30 morbidly obese subjects (9 men and 21 women) aged 41.1+/-1.9 years, with a body mass index (BMI) of 54.7+/-1.7 kg/m2, who had undergone abdominal surgery. Protein levels of hormone-sensitive lipase (HSL) and adrenergic receptors (AR), as well as HSL activity and the lipolytic response to adrenergic agents were analyzed. Both fat depots had similar basal lipolysis, but the capacity of catecholamines to activate lipolysis was greater in visceral fat, both at AR and postreceptor levels. Basal lipolysis and lipolytic activity induced by dibutyryl cyclic AMP were higher in men than in women. However, the visceral depot of women showed a higher maximal stimulation by noradrenaline than that of men, in accordance with higher beta1- and beta3-AR protein levels. In conclusion, the main gender-related differences were located in the visceral depot, with women exhibiting a higher sensitivity to catecholamines associated with an increased provision of beta-AR, while men showed an enhanced lipolytic capacity at the postreceptor level.     

New Hierarchical Phosphorylation Pathway of the Translational Repressor eIF4E-binding Protein 1 (4E-BP1) in Ischemia-Reperfusion Stress

Eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1) is a translational repressor that is characterized by its capacity to bind specifically to eIF4E and inhibit its interaction with eIF4G. Phosphorylation of 4E-BP1 regulates eIF4E availability, and therefore, cap-dependent translation, in cell stress. This study reports a physiological study of 4E-BP1 regulation by phosphorylation using control conditions and a stress-induced translational repression condition, ischemia-reperfusion (IR) stress, in brain tissue. In control conditions, 4E-BP1 was found in four phosphorylation states that were detected by two-dimensional gel electrophoresis and Western blotting, which corresponded to Thr⁶⁹-phosphorylated alone, Thr⁶⁹- and Thr³⁶/Thr⁴⁵-phosphorylated, all these plus Ser⁶⁴ phosphorylation, and dephosphorylation of the sites analyzed. In control or IR conditions, no Thr³⁶/Thr⁴⁵ phosphorylation alone was detected without Thr⁶⁹ phosphorylation, and neither was Ser⁶⁴ phosphorylation without Thr³⁶/Thr⁴⁵/Thr⁶⁹ phosphorylation detected. Ischemic stress induced 4E-BP1 dephosphorylation at Thr⁶⁹, Thr³⁶/Thr⁴⁵, and Ser⁶⁴ residues, with 4E-BP1 remaining phosphorylated at Thr⁶⁹ alone or dephosphorylated. In the subsequent reperfusion, 4E-BP1 phosphorylation was induced at Thr³⁶/Thr⁴⁵ and Ser⁶⁴, in addition to Thr⁶⁹. Changes in 4E-BP1 phosphorylation after IR were according to those found for Akt and mammalian target of rapamycin (mTOR) kinases. These results demonstrate a new hierarchical phosphorylation for 4E-BP1 regulation in which Thr⁶⁹ is phosphorylated first followed by Thr³⁶/Thr⁴⁵ phosphorylation, and Ser⁶⁴ is phosphorylated last. Thr⁶⁹ phosphorylation alone allows binding to eIF4E, and subsequent Thr³⁶/Thr⁴⁵ phosphorylation was sufficient to dissociate 4E-BP1 from eIF4E, which led to eIF4E-4G interaction. These data help to elucidate the physiological role of 4E-BP1 phosphorylation in controlling protein synthesis.     

Hyperacidity of secreted fluid from submucosal glands in early cystic fibrosis

Prior studies have shown that fluid secretions from airway submucosal glands in cystic fibrosis (CF) are reduced and hyperviscous, possibly contributing to the pathogenesis of CF airway disease. Because the CF transmembrane conductance regulator (CFTR) protein can transport both chloride and bicarbonate, we investigated whether gland fluid pH is abnormal in early CF, using nasal biopsies from pediatric subjects having minimal CF lung disease. Gland fluid pH, measured in freshly secreted droplets under oil stained with BCECF-dextran, was 6.57 ± 0.09 (mean ± SE) in biopsies from six CF subjects, significantly lower than 7.18 ± 0.06 in eight non-CF biopsies (P < 0.01). To rule out the possibility that the apparent gland fluid hyperacidity in CF results from modification of fluid pH by the airway surface, a microcannulation method was used to measure pH in fluid exiting gland orifices. In pig trachea and human bronchi, gland fluid pH was reduced by up to 0.45 units by CFTR inhibitors, but was not affected by amiloride. Acid base transport in the surface epithelium of pig trachea was studied from pH changes in 300-nl fluid droplets deposited onto the oil-covered airway surface. The droplets had specified ionic composition/pH and/or contained transporter activators/inhibitors. We found evidence for CFTR-dependent bicarbonate transport by the tracheal surface epithelium as well as ATP/histamine-stimulated proton secretion, but not for sodium/proton or chloride/bicarbonate exchange. These results provide evidence for intrinsic hyperacidity in CF gland fluid secretions, which may contribute to CF airway pathology. cystic fibrosis transmembrane conductance regulator; airway; fluorescence microscopy; pH regulation     

Calpain-induced Proteolysis After Transient Global Cerebral Ischemia and Ischemic Tolerance in a Rat Model

The activation of the [Ca²⁺]-dependent cysteine protease calpain plays an important role in ischemic injury. Here, the levels of two calpain-specific substrates, p35 protein and eukaryotic initiation factor 4G (eIF4G), as well as its physiological regulator calpastatin, were investigated in a rat model of transient global cerebral ischemia with or without ischemic tolerance (IT). Extracts of the cerebral cortex, whole hippocampus and hippocampal subregions after 30 min of ischemia and different reperfusion times (30 min and 4 h) were used. In rats without IT, the p35 levels slightly decreased after ischemia or reperfusion, whereas the levels of p25 (the truncated form of p35) were much higher than those in sham control rats after ischemia and remained elevated during reperfusion. The eIF4G levels deeply diminished after reperfusion and the decrease was significantly greater in CA1 and the rest of the hippocampus than in the cortex. By contrast, the calpastatin levels did not significantly decrease during ischemia or early reperfusion, but were upregulated after 4 h of reperfusion in the cortex. Although IT did not promote significant changes in p35 and p25 levels, it induced a slight increase in calpastatin and eIF4G levels in the hippocampal subregions after 4 h of reperfusion.     

Protein subcellular localization prediction using a hybrid of similarity search and error-correcting output code techniques that produces interpretable results

In silico prediction of protein subcellular localization based on amino acid sequence can reveal valuable information about the protein's innate roles in the cell. Unfortunately, such prediction is made difficult because of complex protein sorting signals. Some prediction methods are based on searching for similar proteins with known localization, assuming that known homologs exist. However, it may not perform well on proteins with no known homolog. In contrast, machine learning-based approaches attempt to infer a predictive model that describes the protein sorting signals. Alas, in doing so, it does not take advantage of known homologs (if they exist) by doing a simple "table lookup". Here, we capture the best of both worlds by combining both approaches. On a dataset with 12 locations, similarity-based and machine learning independently achieve an accuracy of 83.8% and 72.6%, respectively. Our hybrid approach yields an improved accuracy of 85.9%. We compared our method with three other methods' published results. For two of the methods, we used their published datasets for comparison. For the third we used the 12 location dataset. The Error Correcting Output Code algorithm was used to construct our predictive model. This algorithm gives attention to all the classes regardless of number of instances and led to high accuracy among each of the classes and a high prediction rate overall. We also illustrated how the machine learning classifier we use, built over a meaningful set of features can produce interpretable rules that may provide valuable insights into complex protein sorting mechanisms.     

Regulation of sensory neuron-specific acid-sensing ion channel 3 by the adaptor protein Na+/H+ exchanger regulatory factor-1

Acid-sensing ion channels (ASICs) are cationic channels activated by extracellular protons. The ASIC3 subunit is largely expressed in the peripheral nervous system, where it contributes to pain perception and to some aspects of mechanosensation. We report here a PDZ-dependent and protein kinase C-modulated association between ASIC3 and the Na+/H+ exchanger regulatory factor-1 (NHERF-1) adaptor protein. We show that NHERF-1 and ASIC3 are co-expressed in dorsal root ganglion neurons. NHERF-1 enhances the ASIC3 peak current in heterologous cells, including F-11 dorsal root ganglion cells, by increasing the amount of channel at the plasma membrane. Perhaps more importantly, we show that the plateau current of ASIC3 can be dramatically increased (10-30-fold) by association with NHERF-1, leading to a significant sustained current at pH 6.6. In the presence of NHERF-1, the ASIC3 subcellular localization is modified, and the channel co-localizes with ezrin, a member of the ezrin-radixin-moesin family of actin-binding proteins, providing the first direct link between ASIC3 and the cortical cytoskeleton. Given the importance of the ASIC3 sustained current in nociceptor excitability, it is likely that NHERF-1 participates in channel functions associated with nociception and mechanosensation.