The Endoplasmic Reticulum Chaperone Calnexin Is a NADPH Oxidase NOX4 Interacting Protein

Within the family of NADPH oxidases, NOX4 is unique as it is predominantly localized in the endoplasmic reticulum, has constitutive activity, and generates hydrogen peroxide (H₂O₂). We hypothesize that these features are consequences of a so far unidentified NOX4-interacting protein. Two-dimensional blue native (BN) electrophorese combined with SDS-PAGE yielded NOX4 to reside in macromolecular complexes. Interacting proteins were screened by quantitative SILAC (stable isotope labeling of amino acids in cell culture) co-immunoprecipitation (Co-IP) in HEK293 cells stably overexpressing NOX4. By this technique, several interacting proteins were identified with calnexin showing the most robust interaction. Calnexin also resided in NOX4-containing complexes as demonstrated by complexome profiling from BN-PAGE. The calnexin NOX4 interaction could be confirmed by reverse Co-IP and proximity ligation assay, whereas NOX1, NOX2, or NOX5 did not interact with calnexin. Calnexin deficiency as studied in mouse embryonic fibroblasts from calnexin^−/− mice or in response to calnexin shRNA reduced cellular NOX4 protein expression and reactive oxygen species formation. Our results suggest that endogenous NOX4 forms macromolecular complexes with calnexin, which are needed for the proper maturation, processing, and function of NOX4 in the endoplasmic reticulum.     

Self-Folding of Naked Segment 8 Genomic RNA of Influenza A Virus

Influenza A is a negative sense RNA virus that kills hundreds of thousands of humans each year. Base pairing in RNA is very favorable, but possibilities for RNA secondary structure of the influenza genomic RNA have not been investigated. This work presents the first experimentally-derived exploration of potential secondary structure in an influenza A naked (protein-free) genomic segment. Favorable folding regions are revealed by in vitro chemical structure mapping, thermodynamics, bioinformatics, and binding to isoenergetic microarrays of an entire natural sequence of the 875 nt segment 8 vRNA and of a smaller fragment. Segment 8 has thermodynamically stable and evolutionarily conserved RNA structure and encodes essential viral proteins NEP and NS1. This suggests that vRNA self-folding may generate helixes and loops that are important at one or more stages of the influenza life cycle.     

Effect of spectrin on structure properties of lipid bilayers formed from mixtures of phospholipids. Fluorescence and microcalorimetric studies.

Effect of spectrin from human erythrocytes on structure properties of lipid bilayers formed from a mixture of phosphatidylethanolamine/phosphatidylserine (PE/PS) and/or phosphatidylethanolamine/phosphatidylcholine (PE/PC) was studied with the use of fluorescence and microcalorimetric methods. Spectrin did not affect the order parameter of lipids in PE/PS vesicles. However, spectrin binding to liposomes did influence temperature, half-width and enthalpy of phase transitions in mixtures of dimyristoylphosphatidylethanolamine (DMPE) and dimyristoylphosphatidylcholine (DMPC), and this effect was dependent on DMPE to DMPC weight ratio. A change in miscibility of the components in the presence of spectrin was observed and it might be due to spectrin-PE interactions.     

The role of ion antiporters in the maintenance of intracellular pH in rat vascular smooth muscle cells

Vascular smooth muscle intracellular pH is maintained by the Na⁺/H⁺ and Cl^–/HCO ₃ ^– antiporters. The Na⁺/H⁺ exchanger is a major route of H⁺ extrusion in most eukaryotic cells and is present in vascular smooth muscle cells in a similar capacity. It extrudes H^– into the extracellular space in exchange for Na⁺. The Cl^–/HCO ₃ ^– exchanger plays an analogous role to lower the pH of vascular smooth muscle cells when increases in intracellular pH occur. Its activity has also been demonstrated in A7r5 and A10 vascular smooth muscle cells. The Na⁺/H⁺ exchanger is regulated by a number of agents which act through inositol trisphosphate/diacylglycerol, to stimulate the antiporter. Calcium-calmodulin dependent protein kinase may also activate the antiporter in vivo. Phosphorylation of the Cl^–/HCO ₃ ^– exchanger has also been observed but its physiological role is not known. Both these antiporters exist in the plasma membrane as integral proteins with free acidic cytoplasmic termini. These regions may be important in sensing changes in intracellular pH, to which these antiporters respond.Abbreviations CaM Calmodulin - DCCD Dicylohexyl-Carbodiimide - DG Diacylglycerol - DIDS-4 4-Diisthiocyanostilbene-2,2-Disulfonic Acid - IP₃ Inositol Trisphosphate - PKC protein Kinase C - SITS-4 4-Acetamido-4-Isothiocyanstilbene-2,2-Disulfonate - VSMC Vascular Smooth Muscle Cell     

Molecular cloning of the high affinity calcium-binding protein (calreticulin) of skeletal muscle sarcoplasmic reticulum

A cDNA clone encoding the high affinity Ca2+-binding protein (HACBP) of rabbit skeletal muscle sarcoplasmic reticulum was isolated and sequenced. The cDNA encoded a protein of 418 amino acids, but a comparison of the deduced amino acid sequence with the NH2-terminal amino acid sequence of the purified protein indicates that a 17-residue NH2-terminal signal sequence was removed during synthesis. This was confirmed by studies of in vitro translation of mRNA encoding the protein. Structural predictions did not reveal any potential transmembrane segments in the protein. The COOH-terminal sequence of the high affinity Ca2+-binding protein, Lys-Asp-Glu-Leu, is the same as that proposed to be an endoplasmic reticulum retention signal (Munro, S., and Pelham, H. R. B. (1987) Cell 48, 899-907). All of these characteristics suggest that the protein is localized in the lumen of the sarcoplasmic reticulum. The mature protein of Mr 46,567 contains 109 acidic and 52 basic amino acids. Structural predictions suggest that the first half of the molecule forms a globular domain of 8 anti-parallel beta-strands with a helix-turn-helix motif at the extreme NH2 terminus. The next one-third of the sequence is proline-rich. This segment can be subdivided into a charged region which contains a 17-amino acid repeat, followed by a proline, serine, and threonine-rich segment extending from Pro-246 to Thr-316. Thirty-seven acidic residues are clustered within 56 amino acids at the COOH terminus of the protein. Although the protein binds 1 mol of Ca2+/mol with high affinity, no "EF-hand" consensus sequence was observed in the protein. The acidic COOH terminus, however, could account for the low affinity, high capacity Ca2+ binding observed in the protein. In agreement with other involved laboratories, we have chosen the name calreticulin for the protein.     

Calreticulin in T-lymphocytes. Identification of calreticulin in T-lymphocytes and demonstration that activation of T cells correlates with increased levels of calreticulin mRNA and protein.

Ca2+ is an essential second messenger for T cell activation, but the exact mechanisms of its action are poorly understood. The cytosolic Ca2+ concentration is significantly increased upon the stimulation of T cells with either mitogen, cross-linking antibodies, or their cognate ligands. In this study, expression of calreticulin, a major Ca(2+)-binding (storage), KDEL protein of the endoplasmic reticulum was examined in resting and concanavalin A (ConA)-stimulated mouse and human T-lymphocytes. Both resting, mouse and human lymphocytes contain very low levels of calreticulin mRNA and protein. Mouse splenocytes stimulated with ConA exhibited an induction in calreticulin mRNA which peaked by Day 4. A 5-fold increase in the immunoreactive calreticulin protein band was also observed in the cells during this period of stimulation. Similarly when human lymphocytes were cultured with ConA a significant increase in the levels of the calreticulin mRNA and protein was observed. The peak of calreticulin mRNA was observed at Day 1 rather than Day 4 as seen for the mouse. These results clearly demonstrate the presence of calreticulin, a Ca(2+)-binding protein originally characterized in muscle tissue, in activated T-lymphocytes. Furthermore, we show that expression of calreticulin correlates with T-lymphocyte activation. Our results suggest that calreticulin may be involved in the signaling pathway for the induction of Ca(2+)-dependent processes and may represent one regulatory mechanism operating in activation of T-lymphocytes.     

Algae as production systems of bioactive compounds

Algal extracts are gaining increasing interest due to their unique composition and possibilities of wide industrial applications. Various extraction techniques are used for conversion of algal biomass into extracts. Recently, attention of scientists has been paid to novel methods, such as enzyme‐assisted extraction, microwave‐assisted extraction, pressurized liquid extraction, supercritical fluid extraction, and ultrasound‐assisted extraction, which enable the extraction of biologically active compounds without their degradation. In this review, the properties of biologically active compounds extracted from the biomass of algae reported in the literature are presented in a structured way. Algal extracts contain compounds such as carbohydrates, proteins, minerals, oil, fats, polyunsaturated fatty acids as well as bioactive compounds such as antioxidants (polyphenols, tocopherols [vitamin E], vitamin C, mycosporine‐like amino acids), and pigments, such as carotenoids (carotene xanthophyll), chlorophylls, and phycobilins (phycocyanin, phycoerythrin), which possess antibacterial, antiviral, antifungal, antioxidative, anti‐inflammatory, and antitumor properties. Finally, we assemble a list of applications of algal extracts in different developing branches of agriculture (biostimulants, bioregulators, feed additives) and in pharmaceutical industry.     

Genomic signatures of experimental adaptive radiation in Drosophila

Abiotic environmental factors play a fundamental role in determining the distribution, abundance and adaptive diversification of species. Empowered by new technologies enabling rapid and increasingly accurate examination of genomic variation in populations, researchers may gain new insights into the genomic background of adaptive radiation and stress resistance. We investigated genomic variation across generations of large‐scale experimental selection regimes originating from a single founder population of Drosophila melanogaster, diverging in response to ecologically relevant environmental stressors: heat shock, heat knock down, cold shock, desiccation and starvation. When compared to the founder population, and to parallel unselected controls, there were more than 100,000 single nucleotide polymorphisms (SNPs) displaying consistent allelic changes in response to selective pressures across generations. These SNPs were found in both coding and noncoding sequences, with the highest density in promoter regions, and involved a broad range of functionalities, including molecular chaperoning by heat‐shock proteins. The SNP patterns were highly stressor‐specific despite considerable variation among line replicates within each selection regime, as reflected by a principal component analysis, and co‐occurred with selective sweep regions. Only ~15% of SNPs with putatively adaptive changes were shared by at least two selective regimes, while less than 1% of SNPs diverged in opposite directions. Divergent stressors driving evolution in the experimental system of adaptive radiation left distinct genomic signatures, most pronounced in starvation and heat‐shock selection regimes.