ApoE4 upregulates the activity of mitochondria‐associated ER membranes

In addition to the appearance of senile plaques and neurofibrillary tangles, Alzheimer''s disease (AD) is characterized by aberrant lipid metabolism and early mitochondrial dysfunction. We recently showed that there was increased functionality of mitochondria‐associated endoplasmic reticulum (ER) membranes (MAM), a subdomain of the ER involved in lipid and cholesterol homeostasis, in presenilin‐deficient cells and in fibroblasts from familial and sporadic AD patients. Individuals carrying the ε4 allele of apolipoprotein E (ApoE4) are at increased risk for developing AD compared to those carrying ApoE3. While the reason for this increased risk is unknown, we hypothesized that it might be associated with elevated MAM function. Using an astrocyte‐conditioned media (ACM) model, we now show that ER–mitochondrial communication and MAM function—as measured by the synthesis of phospholipids and of cholesteryl esters, respectively—are increased significantly in cells treated with ApoE4‐containing ACM as compared to those treated with ApoE3‐containing ACM. Notably, this effect was seen with lipoprotein‐enriched preparations, but not with lipid‐free ApoE protein. These data are consistent with a role of upregulated MAM function in the pathogenesis of AD and may help explain, in part, the contribution of ApoE4 as a risk factor in the disease.     

Does the energy state of mitochondria influence the surface potential of the inner mitochondrial membrane? A critical appraisal

Binding of 8-anilino-1-naphthalene sulphonate (ANS) to rat liver mitochondria and submitochondrial inside-out particles was measured under energized and de-energized conditions. In mitochondria, energization/de-energization changed the binding capacity for ANS extrapolated for its infinitely high concentration, whereas the apparent Kd value remained unchanged. In submitochondrial particles apparent Kd was changed but the extrapolated maximum binding was not altered. These results are compatible with theoretical considerations assuming a free permeability of mitochondrial membranes to ANS and its distribution according to the transmembrane potential. The spin-labelled cationic amphiphile, 4-(dodecyl dimethyl ammonium)-1-oxyl-2,2,6,6-tetramethyl piperidine bromide (CAT12), was trapped by de-energized mitochondria in such a way that about half of the bound probe became inaccessible to reduction by externally added ascorbate. This inaccessible fraction was increased by energization. This indicates that this cationic probe can penetrate through the inner mitochondrial membrane. De-energization produced a parallel shift of the Lineweaver-Burk plots for the oxidation of external ferrocytochrome c by mitoplasts and of succinate by submitochondrial particles. A similar shift was obtained by a partial inhibition of succinate oxidation by antimycin A. Thus, the observed changes of the kinetics of the two membrane-bound enzyme systems on de-energization can be interpreted as reflecting changes of the control points of mitochondrial respiration rather than changes of the surface potential. It is concluded that neither the fluorescent probe ANS, the spin-labelled amphiphilic cation CAT12, nor the kinetics of some respiratory enzyme systems provide a sufficient proof for changes of the surface potential of the inner mitochondrial membrane upon energization.     

Does the function of adenine nucleotide translocase in fatty acid uncoupling depend on the type of mitochondria?

The stimulation of respiration by long-chain fatty acids and FCCP was studied with oligomycin-inhibited mitochondria from rat liver, heart and kidney tissue. By addition of equal amounts of palmitate and oleate, mitochondrial respiration was increased in the order RLM less than RKM less than RHM. Using the classical protonophore FCCP, this difference could not be observed. Inhibition of oleate-stimulated respiration by carboxyatractyloside decreased in the order RHM greater than RKM greater than RLM. As CAT sensitivity of oleate-stimulated respiration and the mitochondrial ANT content were found to be correlated, it is suggested that the weak CAT sensitivity of oleate-stimulated respiration of RLM [(1989) Biochim. Biophys. Acta 977, 266-272] is due to the low content of ANT.     

BamE structure: the assembly of β-barrel proteins in the outer membranes of bacteria and mitochondria

A study recently published in EMBO reports solves the solution structure of E. coli BamE, thus providing the basis for a better understanding of the mechanism of β-barrel assembly in bacterial and mitochondrial outer membranes.EMBO Rep (2011) advance online publication. doi: 10.1038/embor.2010.202β-barrel membrane proteins are found exclusively in the outer membrane of Gram-negative bacteria and the outer membranes of eukaryotic organelles of prokaryotic origin, mitochondria and chloroplasts. In contrast to the inner membrane, the bacterial outer membrane is an asymmetrical bilayer that consists mainly of lipopolysaccharides in the outer leaflet and phospholipids in the inner leaflet. Bacterial β-barrel outer membrane proteins (OMPs) mediate many cellular functions, for example, passive or selective diffusion of small molecules through the β-barrel pores across the outer membrane. By contrast, only a few mitochondrial β-barrel outer membrane proteins (MBOMPs) have been identified so far. The central machineries that mediate insertion and assembly of OMPs/MBOMPs are the β-barrel assembly machine (BAM) complex in the bacterial outer membrane and the topogenesis of outer-membrane β-barrel proteins (TOB)/sorting and assembly machinery (SAM) complex in the mitochondrial outer membrane (Knowles et al, 2009; Endo & Yamano, 2010; Stroud et al, 2010; Fig 1). However, the molecular mechanisms of β-barrel protein topogenesis in bacterial and mitochondrial outer membranes remain poorly understood.Open in a separate windowFigure 1β-barrel protein assembly in bacterial and mitochondrial outer membranes. (A) Bacteria. Ribbon models of the structures of the Sec complex, SurA, BamA (Clantin et al, 2007; Kim et al, 2007), BamE and OMP. The upper and lower inserts show the surface of BamE (residues 20–108; viewed after approximately 90° rotation of the ribbon model around the horizontal axis toward the reader). Residues important for BamD binding are shown in red and residues with NMR signals that were perturbed by BamD binding are shown in yellow. The residue (Phe 74) important for PG binding is shown in red and the residues with NMR signals that were perturbed by PG binding are shown in yellow. (B) Mitochondria. Ribbon models were drawn for the structures of small Tim and MBOMP. IM, inner membrane; IMS, intermembrane space; MBOMP, mitochondrial β-barrel outer membrane protein; OM, outer membrane; OMP, outer membrane protein; PG, phosphatidylglycerol; POTRA, polypeptide transport-associated domain.Bacterial OMPs are synthesized in the cytosol as precursor proteins with an amino-terminal signal sequence that guides the proteins to the Sec machinery for crossing the inner membrane and is cleaved off in the periplasm. Periplasmic chaperones then escort OMPs through the aqueous periplasmic space in a partly unfolded state. On reaching the outer membrane, OMPs assemble into a β-barrel structure and insert into the outer membrane with the help of the BAM complex. The bacterial OMP insertion pathway can be compared to the assembly pathway of MBOMPs from the mitochondrial intermembrane space into the outer membrane. MBOMPs are synthesized in the cytosol and imported into the intermembrane space by the outer membrane translocator TOM40. The subsequent chaperone-mediated escort across the intermembrane space and insertion into the outer membrane by the TOB complex is similar to the OMP assembly process. Notably, the BAM and TOB complexes share the homologous β-barrel proteins BamA and Tob55/Sam50, respectively, as the central components of their insertion machineries. The BAM complex in Escherichia coli consists of BamA (YaeT/Omp85) and four accessory lipoproteins: BamB (YfgL), BamC (NlpB), BamD (YfiO) and BamE (SmpA). BamA and BamD are essential for cell growth, yet deletion of dispensable BamB, BamC or BamE leads to outer membrane defects manifested in hypersensitivity to antibiotics. Although BamAB and BamCDE can form distinct subcomplexes, they become functional only after formation of the entire BAM complex with all five subunits (Hagan et al, 2010).In this issue of EMBO reports, Knowles et al (2011) solve the nuclear magnetic resonance (NMR) solution structure of E. coli BamE, which sheds light on the roles of one of the Bam subunits in β-barrel protein assembly. The structure of BamE consists of a three-stranded antiparallel β-sheet packed against a pair of α-helices (Fig 1).As the ΔbamE mutant cannot grow in the presence of vancomycin, the authors identify functionally important residues of BamE by testing the effects of amino-acid substitutions in BamE on its inability to complement the growth defects of ΔbamE, without destabilizing BamE itself. Many of the identified residues are conserved among BamE proteins from different organisms and map to a single surface area on BamE. Interestingly, NMR signals of the residues around this region are sensitive to the addition of micelles containing the lipid phosphatidylglycerol, but not phosphatidylethanolamine or cardiolipin. In parallel, the authors analyse perturbation of the NMR spectra of BamE after the addition of purified BamB, C and D proteins. Only BamD affects the NMR spectra of BamE, and the BamD interacting region of BamE is found to overlap partly with the residues involved in phosphatidylglycerol binding. As the addition of BamD and phosphatidylglycerol have different effects on the NMR spectra of BamE, the binding of BamD and phosphatidylglycerol to BamE seem to take place simultaneously. What is the biological relevance of the observed interactions of BamE with both BamD and phosphatidylglycerol? As phosphatidylglycerol was found to help the insertion of OMPs into lipid liposomes (Patel et al, 2009), BamE might recruit the BAM complex through BamD to phosphatidylglycerol-rich regions in the outer membrane, or might directly recruit phosphatidylglycerol to form assembly points for OMP insertion and folding.What are the roles of other subunits of the BAM complex in β-barrel protein assembly? The essential subunit of the E. coli BAM complex BamA consists of two domains: the N-terminal polypeptide transport-associated (POTRA) domain repeat in the periplasm and the carboxy-terminal β-barrel domain, embedded in the outer membrane. The number of POTRA domains ranges from one to five in BamA homologues from different organisms. Of these POTRA domains, the one nearest to the C-terminal that is most connected to the β-barrel domain is essential for cell viability and its deletion leads to disassembly of the BAM complex (Kim et al, 2007). Structural studies of the E. coli BamA POTRA domains suggest that each POTRA domain has a common fold, whereas conformational rigidity might differ between inter-domain linkers (Gatzeva-Topalova et al, 2010; Fig 1). As individual POTRA domains have some affinity for unfolded substrate proteins, the periplasmic tandem POTRA repeat probably provides several substrate binding sites that slide the substrate progressively towards the BamA β-barrel domain. The β-barrel domain of BamA probably functions as a scaffold to facilitate the formation of β-strands, possibly through β-augmentation and subsequent spontaneous membrane insertion of the β-barrel. Yet, it is not clear whether this cradle for β-strand formation is provided by the pore formed within the monomer or oligomeric forms of the BamA β-barrel domain. Alternatively, membrane insertion and folding of OMPs might take place at the interface between BamA and the outer membrane lipid bilayer.How much of the β-barrel assembly process is conserved during the evolution of mitochondria from Gram-negative bacteria? Although the central subunits BamA and Tob55 of the BAM and TOB complexes are conserved, other subunits of these complexes are unrelated to each other. The POTRA domains of BamA are essential for recognition and assembly of bacterial OMPs, whereas that of Tob55 is dispensable for MBOMP assembly in the mitochondrial outer membrane. Nevertheless, the mitochondrial TOB complex facilitates assembly of bacterial OMPs at low efficiency (Walther et al, 2009) and, in turn, the bacterial BAM complex can mediate assembly of mitochondrial porin. Therefore, the basic mechanism of β-barrel assembly in the outer membranes of bacteria and mitochondria seems to be conserved. High-resolution structures of each component of the BAM and TOB complexes—including that of BamE in this study—will thus provide the basis for a better understanding of the mechanism of β-barrel assembly in evolutionarily related bacterial and mitochondrial outer membranes.     

What is the real crystallographic structure of the L photointermediate of bacteriorhodopsin?

In the last few years, three laboratories have reported three entirely different crystallographic models for the L photointermediate of bacteriorhodopsin. All are from X-ray diffraction of illuminated crystals that contain L in photostationary states created at similar cryogenic temperatures. This article compares the models and their implications, the crystallographic statistics and the methods used to derive them, as well as their agreement with non-crystallographic information.     

Does green light influence the fluorescence properties and structure of phototrophic biofilms?

Artificial illumination can harm works of art by inducing the development of photosynthetic biofilms. With the aim of preventing biodeterioration or esthetic damage to such surfaces, we evaluated and compared the effects of illuminating biofilms formed by Gloeothece membranacea (cyanobacteria) and Chlorella sorokiniana (Chlorophyta) using exclusively white or green light.     

Does cholesterol suppress the antimicrobial peptide induced disruption of lipid raft containing membranes?

The activity of antimicrobial peptides has been shown to depend on the composition of the target cell membrane. The bacterial selectivity of most antimicrobial peptides has been attributed to the presence of abundant acidic phospholipids and the absence of cholesterol in bacterial membranes. The high amount of cholesterol present in eukaryotic cell membranes is thought to prevent peptide-induced membrane disruption by increasing the cohesion and stiffness of the lipid bilayer membrane. While the role of cholesterol on an antimicrobial peptide-induced membrane disrupting activity has been reported for simple, homogeneous lipid bilayer systems, it is not well understood for complex, heterogeneous lipid bilayers exhibiting phase separation (or "lipid rafts"). In this study, we show that cholesterol does not inhibit the disruption of raft-containing 1,2-dioleoyl-sn-glycero-3-phosphocholine:1,2-dipalmitoyol-sn-glycero-3-phosphocholine model membranes by four different cationic antimicrobial peptides, MSI-78, MSI-594, MSI-367 and MSI-843 which permeabilize membranes. Conversely, the presence of cholesterol effectively inhibits the disruption of non-raft containing 1,2-dioleoyl-sn-glycero-3-phosphocholine or 1,2-dipalmitoyol-sn-glycero-3-phosphocholine lipid bilayers, even for antimicrobial peptides that do not show a clear preference between the ordered gel and disordered liquid-crystalline phases. Our results show that the peptide selectivity is not only dependent on the lipid phase but also on the presence of phase separation in heterogeneous lipid systems.     

Does the morphology of animal foraging pits influence secondary seed dispersal by ants?

Secondary seed dispersal by ants (myrmecochory) is an important process in semi‐arid environments where seeds are transported from the soil surface to an ant nest. Microsites from which ants often remove seeds are the small pits and depressions made by native and exotic animals that forage in the soil. Previous studies have demonstrated greater seed retention in the pits of native than exotic animals, but little is known about how biotic factors such as secondary seed dispersal by ants affect seed removal and therefore retention in these foraging pits. We used an experimental approach to examine how the morphology of burrowing bettong (Bettongia lesueur), greater bilby (Macrotis lagotis), short‐beaked echidna (Tachyglossus aculeatus) and European rabbit (Oryctolagus cuniculus) foraging pits and ant body size influenced ant locomotion and seed removal from pits along an aridity gradient. Ants took 3.7‐times longer to emerge from echidna pits (19.6 s) and six‐times longer to emerge from bettong pits (30.5 s) than from rabbit pits (5.2 s), resulting in lower seed removal from bettong pits than other pit types. Fewer seeds were removed from pits when cages were used to exclude large body‐sized (>2 mm) ants. Few seeds were removed from the pits or surface up to aridity values of 0.5 (humid and dry sub‐humid), but removal increased rapidly in semi‐arid and arid zones. Our study demonstrates that mammal foraging pit morphology significantly affects ant locomotion, the ability of ants to retrieve seeds, and therefore the likelihood that seeds will be retained within foraging pits.     

Is the rate of photosynthesis under blue light altered in the 7B-1 tomato mutant?

Changes of photosynthesis under blue light were examined in the ABA-overproducing 7B-1 mutant in tomato. Net photosynthetic rate (P _N), stomatal conductance (g _s), intrinsic water-use efficiency (WUE_i) and chlorophyll (a+b) [Chl (a+b)] content in leaves of different insertion (1^st, 4^th and 9^th ones) were measured in 5-, 7- and 9-week-old plants. P _N, g _s, and Chl (a+b) content were mostly similar in young leaves of 7B-1 and wild type (WT) plants. With the aging of leaves, a blue-light-induced increase in P _N and g _s to steady-state was delayed and steady-state values of P _N and g _s were lower in 7B-1 plants compared with WT. Steady-state values of WUE_i were increased in 4^th and 9^th leaves of 7B-1 plants compared with WT. The results can be explained by the higher endogenous level of ABA in 7B-1 plants and their lower sensitivity to ABA in earlier growth stage.     

Is the purple color of bacteriorhodopsin maintained by lipid-protein interactions?

When bacteriorhodopsin is delipidated and purified in detergents, its purple chromophore can be reversibly titrated to a red one. The pK_a of this equilibrium depends on the nature of the detergent in which bacteriorhodopsin is dispersed. In the absence of solvating amphiphiles, lipid-free detergent-free bacteriorhodopsin is red (λ_max = 480 nm) at pH higher than 3.5.     

Can the low-resolution structures of photointermediates of bacteriorhodopsin explain their crystal structures?

To understand the molecular mechanism of light-driven proton pumps, the structures of the photointermediates of bacteriorhodopsin have been intensively investigated. Low-resolution diffraction techniques have demonstrated substantial conformational changes at the helix level in the M and N intermediates, between which there are noticeable differences. The intermediate structures at atomic resolution have also been solved by x-ray crystallography. Although the crystal structures have demonstrated local structural changes, such as hydrogen bond network rearrangements including water molecules, the large conformational changes at the helix level are not necessarily observed. Furthermore, the two reported crystal structures of an intermediate accumulated using a common method were distinct. To reconcile these apparent discrepancies, low-resolution projection maps were calculated from the crystal structures and compared to the low-resolution intermediate structures obtained using native membranes. The crystal structures can be categorized into three groups, which qualitatively correspond to the low-resolution structures of the M1-type, M2-type, and N-type determined in the native membrane. Based on these results, we conclude that at least three types of intermediate structures play a role during the photocycle.     

Does pupil constriction under blue and green monochromatic light exposure change with age?

Many nonvisual functions are regulated by light through a photoreceptive system involving melanopsin-expressing retinal ganglion cells that are maximally sensitive to blue light. Several studies have suggested that the ability of light to modulate circadian entrainment and to induce acute effects on melatonin secretion, subjective alertness, and gene expression decreases during aging, particularly for blue light. This could contribute to the documented changes in sleep and circadian regulatory processes with aging. However, age-related modification in the impact of light on steady-state pupil constriction, which regulates the amount of light reaching the retina, is not demonstrated. We measured pupil size in 16 young (22.8±4 years) and 14 older (61±4.4 years) healthy subjects during 45-second exposures to blue (480 nm) and green (550 nm) monochromatic lights at low (7×10(12) photons/cm2/s), medium (3×10(13) photons/cm2/s), and high (10(14) photons/cm2/s) irradiance levels. Results showed that young subjects had consistently larger pupils than older subjects for dark adaptation and during all light exposures. Steady-state pupil constriction was greater under blue than green light exposure in both age groups and increased with increasing irradiance. Surprisingly, when expressed in relation to baseline pupil size, no significant age-related differences were observed in pupil constriction. The observed reduction in pupil size in older individuals, both in darkness and during light exposure, may reduce retinal illumination and consequently affect nonvisual responses to light. The absence of a significant difference between age groups for relative steady-state pupil constriction suggests that other factors such as tonic, sympathetic control of pupil dilation, rather than light sensitivity per se, account for the observed age difference in pupil size regulation. Compared to other nonvisual functions, the light sensitivity of steady-state pupil constriction appears to remain relatively intact and is not profoundly altered by age.     

Does the natriuretic peptide system exist throughout the animal and plant kingdom?

Natriuretic peptides (NPs) and their receptors have been identified in vertebrate species ranging from elasmobranchs to mammals. Atrial, brain and ventricular NP (ANP, BNP and VNP) are endocrine hormones secreted from the heart, while C-type NP (CNP) is principally a paracrine factor in the brain and periphery. In elasmobranchs, only CNP is present in the heart and brain and it functions as a circulating hormone as well as a paracrine factor. Four types of NP receptors are cloned in vertebrates. NPR-A and NPR-B are guanylyl cyclase-coupled receptors, whereas NPR-C and NPR-D have only a short cytoplasmic domain. NPs are hormones important for volume regulation in mammals, while they act more specifically for Na(+) regulation in fishes. The presence of NP and its receptor has also been suggested in the most primitive vertebrate group, cyclostomes, and its molecular identification is in progress. The presence of ANP or its mRNA has been reported in the hearts and ganglia of various invertebrate species such as mollusks and arthropods using either antisera raised against mammalian ANP or rat ANP cDNA as probes. Immunoreactive ANP has also been detected in the unicellular Paramecium and in various species of plants including Metasequoia. Furthermore, the N-terminal prosegments of ANP, whose sequences are scarcely conserved even in vertebrates, have also been detected by the radioimmunoassay for human ANP prosegments in all invertebrate and plant species examined including Paramecium. Although these data are highly attractive, the current evidence is too circumstantial to be convincing that the immunoreactivity truly originates from ANP and its prosegments in such diverse organisms. The caution that has to be exercised in identification of vertebrate hormones from phylogenetically distant organisms is discussed.     

Does probabilistic modelling of linkage disequilibrium evolution improve the accuracy of QTL location in animal pedigree?

Background

Since 2001, the use of more and more dense maps has made researchers aware that combining linkage and linkage disequilibrium enhances the feasibility of fine-mapping genes of interest. So, various method types have been derived to include concepts of population genetics in the analyses. One major drawback of many of these methods is their computational cost, which is very significant when many markers are considered. Recent advances in technology, such as SNP genotyping, have made it possible to deal with huge amount of data. Thus the challenge that remains is to find accurate and efficient methods that are not too time consuming. The study reported here specifically focuses on the half-sib family animal design. Our objective was to determine whether modelling of linkage disequilibrium evolution improved the mapping accuracy of a quantitative trait locus of agricultural interest in these populations. We compared two methods of fine-mapping. The first one was an association analysis. In this method, we did not model linkage disequilibrium evolution. Therefore, the modelling of the evolution of linkage disequilibrium was a deterministic process; it was complete at time 0 and remained complete during the following generations. In the second method, the modelling of the evolution of population allele frequencies was derived from a Wright-Fisher model. We simulated a wide range of scenarios adapted to animal populations and compared these two methods for each scenario.

Results

Our results indicated that the improvement produced by probabilistic modelling of linkage disequilibrium evolution was not significant. Both methods led to similar results concerning the location accuracy of quantitative trait loci which appeared to be mainly improved by using four flanking markers instead of two.

Conclusions

Therefore, in animal half-sib designs, modelling linkage disequilibrium evolution using a Wright-Fisher model does not significantly improve the accuracy of the QTL location when compared to a simpler method assuming complete and constant linkage between the QTL and the marker alleles. Finally, given the high marker density available nowadays, the simpler method should be preferred as it gives accurate results in a reasonable computing time.