Expression,purification, and crystallization of <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> eIF2B

Tight control of protein synthesis is necessary for cells to respond and adapt to environmental changes rapidly. Eukaryotic translation initiation factor (eIF) 2B, the guanine nucleotide exchange factor for eIF2, is a key target of translation control at the initiation step. The nucleotide exchange activity of eIF2B is inhibited by the stress-induced phosphorylation of eIF2. As a result, the level of active GTP-bound eIF2 is lowered, and protein synthesis is attenuated. eIF2B is a large multi-subunit complex composed of five different subunits, and all five of the subunits are the gene products responsible for the neurodegenerative disease, leukoencephalopathy with vanishing white matter. However, the overall structure of eIF2B has remained unresolved, due to the difficulty in preparing a sufficient amount of the eIF2B complex. To overcome this problem, we established the recombinant expression and purification method for eIF2B from the fission yeast Schizosaccharomyces pombe. All five of the eIF2B subunits were co-expressed and reconstructed into the complex in Escherichia coli cells. The complex was successfully purified with a high yield. This recombinant eIF2B complex contains each subunit in an equimolar ratio, and the size exclusion chromatography analysis suggests it forms a heterodecamer, consistent with recent reports. This eIF2B increased protein synthesis in the reconstituted in vitro human translation system. In addition, disease-linked mutations led to subunit dissociation. Furthermore, we crystallized this functional recombinant eIF2B, and the crystals diffracted to 3.0 Å resolution.     

Energy and electron transfer in the photosynthetic reaction center complex of Acidiphilium rubrum containing Zn-bacteriochlorophyll a studied by femtosecond up-conversion spectroscopy

A photosynthetic reaction center (RC) complex was isolated from a purple bacterium, Acidiphilium rubrum. The RC contains bacteriochlorophyll a containing Zn as a central metal (Zn-BChl a) and bacteriopheophytin a (BPhe a) but no Mg-BChl a. The absorption peaks of the Zn-BChl a dimer (P(Zn)), the accessory Zn-BChl a (B(Zn)), and BPhe a (H) at 4 K in the RC showed peaks at 875, 792, and 753 nm, respectively. These peaks were shorter than the corresponding peaks in Rhodobacter sphaeroides RC that has Mg-BChl a. The kinetics of fluorescence from P(Zn)(*), measured by fluorescence up-conversion, showed the rise and the major decay with time constants of 0.16 and 3.3 ps, respectively. The former represents the energy transfer from B(Zn)(*) to P(Zn), and the latter, the electron transfer from P(Zn) to H. The angle between the transition dipoles of B(Zn) and P(Zn) was estimated to be 36 degrees based on the fluorescence anisotropy. The time constants and the angle are almost equal to those in the Rb. sphaeroides RC. The high efficiency of A. rubrum RC seems to be enabled by the chemical property of Zn-BChl a and by the L168HE modification of the RC protein that modifies P(Zn).     

Aquaporin-11 containing a divergent NPA motif has normal water channel activity

Recently, two novel mammalian aquaporins (AQPs), AQPs 11 and 12, have been identified and classified as members of a new AQP subfamily, the "subcellular AQPs". In members of this subfamily one of the two asparagine-proline-alanine (NPA) motifs, which play a crucial role in selective water conduction, are not completely conserved. Mouse AQP11 (mAQP11) was expressed in Sf9 cells and purified using the detergent Fos-choline 10. The protein was reconstituted into liposomes, which were used for water conduction studies with a stopped-flow device. Single water permeability (pf) of AQP11 was measured to be 1.72+/-0.03x10(-13) cm(3)/s, suggesting that other members of the subfamily with incompletely conserved NPA motifs may also function as water channels.     

Conformational behavior of oxygenated mycobacterial mycolic acids from Mycobacterium bovis BCG

Phase diagrams of Langmuir monolayers of oxygenated mycolic acids, i.e. methoxy mycolic acid (MeO-MA), ketomycolic acid (Keto-MA), and artificially obtained deoxo-mycolic acid (deoxo-MA) from Mycobacterium bovis BCG were obtained by thermodynamic analysis of the surface pressure (pi) vs. average molecular area (A) isotherms. At lower temperatures and lower surface pressures, both Keto- and MeO-MAs formed rigid condensed monolayers where each MA molecule was considered to be in a 4-chain form, in which the three carbon chain segments due to bending of the 3-hydroxy aliphatic carboxylate chain and the 2-side chain were in compact parallel arrangement. At higher temperatures and surface pressures, MeO-MA and deoxo-MA tended to take stretched-out conformations in which the 3-hydroxy aliphatic carboxylate chain was more or less in an extended form, but Keto-MA retained the original 4-chain structure. The thickness measurement of the monolayers in situ by ellipsometry at different pi values and temperatures supported the above conclusions derived from the phase diagrams. The enthalpy changes associated with the phase transitions of MeO-MA and deoxo-MA implied that the MeO-MA needed larger energy to change from a compact conformation to an extended one, possibly and partly due to the dehydration of the methoxy group from water surface involved. Molecular dynamics studies of MA models derived from Monte Carlo calculations were also performed, which confirmed the conformational behavior of MAs suggested by the thermodynamic studies on the Langmuir monolayers.     

A mechanistic link between oxidative stress and membrane mediated amyloidogenesis revealed by infrared spectroscopy

The fully developed lesion of Alzheimer's disease is a dense plaque composed of fibrillar amyloid beta-proteins (Abeta) with a characteristic and well-ordered beta-sheet secondary structure. Because the incipient lesion most likely develops when these proteins are first induced to form beta-sheet structure, it is important to understand factors that induced Abeta to adopt this conformation. In this review, we describe the application of polarized attenuated total internal reflection infrared FT-IR spectroscopy for characterizing the conformation, orientation, and rate of accumulation of Abeta on lipid membranes. We also describe the application and yield of linked analysis, whereby multiple spectra are fit simultaneously with component bands that are constrained to share common fitting parameters. Results have shown that membranes promote beta-sheet formation under a variety of circumstances that may be significant to the pathogenesis of Alzheimer's disease.     

Vitamin B6 suppresses apoptosis of NM-1 bovine endothelial cells induced by homocysteine and copper

Hyperhomocysteinemia is an important risk factor for atherosclerosis. We previously reported that formation of early atherosclerosis in the rat aorta was associated with hyperhomocysteinemia and reduction of antioxidant activity caused by low concentration of vitamin B(6)in vivo. In the present study, we examined effects of vitamin B(6) on apoptosis of bovine endothelial cells (NM-1 cells) treated with homocysteine and copper. Homocysteine and copper induced extracellular hydrogen peroxide, intracellular ROS and cellular lipid peroxide levels. Cell viability was reduced to 30% compared to that of control cells. On the other hand, pyridoxal treatment as well as EDTA treatment increased viability of NM-1 cells treated with homocysteine and copper to about 60%, and significantly decreased extracellular hydrogen peroxide, intracellular ROS and cellular lipid peroxide levels. The treatment of catalase recovered cell viability and reduced the level of extracellular hydrogen peroxide and intracellular ROS. Cell death by homocysteine and copper was confirmed to be due to apoptosis by evaluation of DNA fragmentation and by TUNEL assay. However, apoptosis of NM-1 cells induced by homocysteine and copper was due to a caspase-independent pathway as it was not inhibited by the caspase inhibitor, Z-VAD-fmk. Apoptosis of NM-1 cells induced by homocysteine and copper accompanied with mitochondrial permeability but not cytochrome c release. These results suggest that pyridoxal treatment suppresses apoptosis of NM-1 cells induced by homocysteine and copper, most likely through antioxidant effects.     

Neuroligins 3 and 4X interact with syntrophin-gamma2, and the interactions are affected by autism-related mutations

Recently, neuroligins (NLs)3 and 4X have received much attention as autism-related genes. Here, we identified syntrophin-gamma2 (SNTG2) as a de novo binding partner of NL3. SNTG2 also bound to NL4X and NL4Y. Interestingly, the binding was influenced by autism-related mutations, implying that the impaired interaction between NLs and SNTG2 contributes to the etiology of autism.