Structure and dynamics of small soluble Aβ(1-40) oligomers studied by top-down hydrogen exchange mass spectrometry

Aβ peptides can assemble into amyloid fibrils, which represent one of the hallmarks of Alzheimer's disease. Recent studies, however, have focused on the behavior of small soluble Aβ oligomers that possess a much greater neurotoxicity than mature fibrils. The structural characterization of these oligomers remains difficult because of their highly dynamic and polymorphic nature. This work explores the behavior of Aβ(1-40) in a slightly basic solution (pH 9.3) at a low salt concentration (10 mM ammonium acetate). These conditions lead to the formation of small oligomers, without any signs of fibrillation for several hours. The structure and dynamics of these oligomers were characterized by circular dichroism spectroscopy, size exclusion chromatography, and millisecond time-resolved hydrogen exchange mass spectrometry (MS). Our results reveal rapid interconversion between Aβ(1-40) oligomers and monomers. The mole fraction of monomeric molecules is on the order of 40%. Oligomers consist of ~4 Aβ(1-40) molecules on average, and the resulting assemblies have a predominantly β-sheet secondary structure. Hydrogen exchange proceeds in the EX1 regime. This feature allows the application of conformer-specific top-down MS. Electron capture dissociation is used for interrogating the deuteration behavior of the Aβ(1-40) oligomers. This approach provides a spatial resolution of ~2 residues. The backbone amide deuteration pattern uncovered in this way is consistent with a β-turn-β motif for L17-M35. The N-terminus is involved in hydrogen bonding, as well, whereas protection gradually tapers off for C-terminal residues 35-40. Our data are consistent with earlier proposals, according to which Aβ(1-40) oligomers adopt a β-barrel structure. In general terms, this study demonstrates how top-down MS with precursor ion selection can be employed for structural studies of specific protein conformers within a heterogeneous mix.     

Binding of the three-repeat domain of tau to phospholipid membranes induces an aggregated-like state of the protein

In patients with Alzheimer's disease, the microtubule-associated protein tau is found aggregated into paired helical filaments (PHFs) in neurofibrillary deposits. In solution, tau is intrinsically unstructured. However, the tubulin binding domain consisting of three or four 31-32 amino acid repeat regions exhibits both helical and β-structure propensity and makes up the proteolysis resistant core of PHFs. Here, we studied the structure and dynamics of the three-repeat domain of tau (i.e. K19) when bound to membranes consisting of a phosphatidylcholine and phosphatidylserine mixture or phosphatidylserine alone. Tau K19 binds to phospholipid vesicles with submicromolar affinity as measured by fluorescence spectroscopy. The interaction is driven by electrostatic forces between the positively charged protein and the phospholipid head groups. The structure of the membrane-bound state of K19 was studied using CD spectroscopy and solid-state magic-angle spinning NMR spectroscopy. To this end, the protein was selectively (13)C-labeled at all valine and leucine residues. Isotropic chemical shift values of tau K19 were consistent with a β-structure. In addition, motionally averaged (1)H-(13)C dipolar couplings indicated a high rigidity of the protein backbone. The structure formation of K19 was also shown to depend on the charge density of the membrane. Phosphatidylserine membranes induced a gain in the α-helix structure along with an immersion of K19 into the phospholipid bilayer as indicated by a reduction of the lipid chain (2)H NMR order parameter. Our results provide structural insights into the membrane-bound state of tau K19 and support a potential role of phospholipid membranes in mediating the physiological and pathological functions of tau.     

Effects of systemic versus local administration of corticosteroids on mucosal tolerance

Respiratory exposure to allergen induces T cell tolerance and protection against the development of airway hyperactivity in animal models of asthma. Whereas systemic administration of dexamethasone during the delivery of respiratory Ag has been suggested to prevent the development of mucosal tolerance, the effects of local administration of corticosteroids, first-line treatment for patients with bronchial asthma, on mucosal tolerance remain unknown. To analyze the effects of systemic versus local administration of different types of corticosteroids on the development of mucosal tolerance, mice were exposed to respiratory allergen to induce mucosal tolerance with or without systemic or intranasal application of different doses of dexamethasone or prednisolone. After the induction of mucosal tolerance, proliferation of T cells was inhibited in tolerized mice, whereas systemic applications of corticosteroids restored T cell proliferation and secretion of Th2 cytokines. In contrast, inhaled corticosteroids showed no effect on both T cell proliferation and cytokine secretion. In addition, mice systemically treated with corticosteroids showed an increased airway hyperactivity with a significant lung inflammation, but also an increased T effector cells/regulatory T cells ratio in the second lymphoid organs when compared with mice that receive corticosteroids by inhalation. These results demonstrate that local administration of corticosteroids has no effect on the development of immune tolerance in contrast to systemically applied corticosteroids. Furthermore, although different concentrations of corticosteroids are administered to patients, our results demonstrated that the route of administration rather than the doses affects the effect of corticosteroids on respiratory tolerance induction. Considering the broad application of corticosteroids in patients with allergic disease and asthma, the route of administration of steroid substances seems crucial in terms of treatment and potential side effects. These findings may help elucidate the apparently contradicting results of corticosteroid treatment in allergic diseases.     

Caspase-4 is required for activation of inflammasomes

IL-1β and IL-18 are crucial regulators of inflammation and immunity. Both cytokines are initially expressed as inactive precursors, which require processing by the protease caspase-1 for biological activity. Caspase-1 itself is activated in different innate immune complexes called inflammasomes. In addition, caspase-1 activity regulates unconventional protein secretion of many other proteins involved in inflammation and repair. Human caspase-4 is a poorly characterized member of the caspase family, which is supposed to be involved in endoplasmic reticulum stress-induced apoptosis. However, its gene is located on the same locus as the caspase-1 gene, which raises the possibility that caspase-4 plays a role in inflammation. In this study, we show that caspase-4 expression is required for UVB-induced activation of proIL-1β and for unconventional protein secretion by skin-derived keratinocytes. These processes require expression of the nucleotide-binding domain leucine-rich repeat containing, Pyrin domain containing-3 inflammasome, and caspase-4 physically interacts with its central molecule caspase-1. As the active site of caspase-4 is required for activation of caspase-1, the latter most likely represents a substrate of caspase-4. Caspase-4 expression is also essential for efficient nucleotide-binding domain leucine-rich repeat containing, Pyrin domain containing-3 and for absent in melanoma 2 inflammasome-dependent proIL-1β activation in macrophages. These results demonstrate an important role of caspase-4 in inflammation and innate immunity through activation of caspase-1. Therefore, caspase-4 represents a novel target for the treatment of (auto)inflammatory diseases.     

The BUME method: a novel automated chloroform-free 96-well total lipid extraction method for blood plasma

Lipid extraction from biological samples is a critical and often tedious preanalytical step in lipid research. Primarily on the basis of automation criteria, we have developed the BUME method, a novel chloroform-free total lipid extraction method for blood plasma compatible with standard 96-well robots. In only 60 min, 96 samples can be automatically extracted with lipid profiles of commonly analyzed lipid classes almost identically and with absolute recoveries similar or better to what is obtained using the chloroform-based reference method. Lipid recoveries were linear from 10-100 μl plasma for all investigated lipids using the developed extraction protocol. The BUME protocol includes an initial one-phase extraction of plasma into 300 μl butanol:methanol (BUME) mixture (3:1) followed by two-phase extraction into 300 μl heptane:ethyl acetate (3:1) using 300 μl 1% acetic acid as buffer. The lipids investigated included the most abundant plasma lipid classes (e.g., cholesterol ester, free cholesterol, triacylglycerol, phosphatidylcholine, and sphingomyelin) as well as less abundant but biologically important lipid classes, including ceramide, diacylglycerol, and lyso-phospholipids. This novel method has been successfully implemented in our laboratory and is now used daily. We conclude that the fully automated, high-throughput BUME method can replace chloroform-based methods, saving both human and environmental resources.     

The plasma concentration of HDL-associated apoM is influenced by LDL receptor-mediated clearance of apoB-containing particles

ApoM is mainly associated with HDL. Nevertheless, we have consistently observed positive correlations of apoM with plasma LDL cholesterol in humans. Moreover, LDL receptor deficiency is associated with increased plasma apoM in mice. Here, we tested the idea that plasma apoM concentrations are affected by the rate of LDL receptor-mediated clearance of apoB-containing particles. We measured apoM in humans each carrying one of three different LDL receptor mutations (n = 9) or the apoB3500 mutation (n = 12). These carriers had increased plasma apoM (1.34 ± 0.13 μM, P = 0.003, and 1.23 ± 0.10 μM, P = 0.02, respectively) as compared with noncarriers (0.93 ± 0.04 μM). When we injected human apoM-containing HDL into Wt (n = 6) or LDL receptor-deficient mice (n = 6), the removal of HDL-associated human apoM was delayed in the LDL receptor-deficient mice. After 2 h, 54 ± 5% versus 90 ± 8% (P < 0.005) of the initial amounts of human apoM remained in the plasma of Wt and LDL receptor-deficient mice, respectively. Finally, we compared the turnover of radio-iodinated LDL and plasma apoM concentrations in 45 normocholesterolemic humans. There was a negative correlation between plasma apoM and the fractional catabolic rate of LDL (r = -0.38, P = 0.009). These data suggest that the plasma clearance of apoM, despite apoM primarily being associated with HDL, is influenced by LDL receptor-mediated clearance of apoB-containing particles.     

Small RNA deep sequencing identifies microRNAs and other small noncoding RNAs from human herpesvirus 6B

Roseolovirus, or human herpesvirus 6 (HHV-6), is a ubiquitous human pathogen infecting over 95% of the population by the age of 2 years. As with other herpesviruses, reactivation of HHV-6 can present with severe complications in immunocompromised individuals. Recent studies have highlighted the importance of herpesvirus-derived microRNAs (miRNAs) in modulating both cellular and viral gene expression. An initial report which computed the likelihood of various viruses to encode miRNAs did not predict HHV-6 miRNAs. To experimentally screen for small HHV-6-encoded RNAs, we conducted large-scale sequencing of Sup-T-1 cells lytically infected with a laboratory strain of HHV-6B. This revealed an abundant, 60- to 65-nucleotide RNA of unknown function derived from the lytic origin of replication (OriLyt) that gave rise to smaller RNA species of 18 or 19 nucleotides. In addition, we identified four pre-miRNAs whose mature forms accumulated in Argonaute 2. In contrast to the case for other betaherpesviruses, HHV-6B miRNAs are expressed from direct repeat regions (DR(L) and DR(R)) located at either side of the genome. All miRNAs are conserved in the closely related HHV-6A variant, and one of them is a seed ortholog of the human miRNA miR-582-5p. Similar to alphaherpesvirus miRNAs, they are expressed in antisense orientation relative to immediate-early open reading frames (ORFs) and thus have the potential to regulate key viral genes.     

Community-specific pH response of denitrification: experiments with cells extracted from organic soils

Denitrifying prokaryotes are phylogenetically and functionally diverse. Little is known about the relationship between soil denitrifier community composition and functional traits. We extracted bacterial cells from three cultivated peat soils with contrasting native pH by density gradient centrifugation and investigated their kinetics of oxygen depletion and NO2 -, NO, N(2) O and N(2) accumulation during initially hypoxic batch incubations (0.5-1 μM O(2)) in minimal medium buffered at either pH 5.4 or 7.1 (2 mM glutamate, 2 mM NO3 -). The three communities differed strikingly in NO2 - accumulation and transient N(2) O accumulation at the two pH levels, whereas NO peak concentrations (24-53 nM) were similar across all communities and pH treatments. The results confirm that the communities represent different denitrification regulatory phenotypes, as indicated by previous denitrification bioassays with nonbuffered slurries of the same three soils. The composition of the extracted cells resembled that of the parent soils (PCR-TRFLP analyses of 16S rRNA genes, nirK, nirS and nosZ), which were found to differ profoundly in their genetic composition (Braker et al., ). Together, this suggests that direct pH response of denitrification depends on denitrifier community composition, with implications for the propensity of soils to emit N(2) O to the atmosphere.     

Engineering yield and rate of reductive biotransformation in <Emphasis Type="Italic">Escherichia coli</Emphasis> by partial cyclization of the pentose phosphate pathway and PTS-independent glucose transport

Optimization of yields and productivities in reductive whole-cell biotransformations is an important issue for the industrial application of such processes. In a recent study with Escherichia coli, we analyzed the reduction of the prochiral β-ketoester methyl acetoacetate by an R-specific alcohol dehydrogenase (ADH) to the chiral hydroxy ester (R)-methyl 3-hydroxybutyrate (MHB) using glucose as substrate for the generation of NADPH. Deletion of the phosphofructokinase gene pfkA almost doubled the yield to 4.8 mol MHB per mole of glucose, and it was assumed that this effect was due to a partial cyclization of the pentose phosphate pathway (PPP). Here, this partial cyclization was confirmed by ¹³C metabolic flux analysis, which revealed a negative net flux from glucose 6-phosphate to fructose 6-phosphate catalyzed by phosphoglucose isomerase. For further process optimization, the genes encoding the glucose facilitator (glf) and glucokinase (glk) of Zymomonas mobilis were overexpressed in recombinant E. coli strains carrying ADH and deletions of either pgi (phosphoglucose isomerase), or pfkA, or pfkA plus pfkB. In all cases, the glucose uptake rate was increased (30–47%), and for strains Δpgi and ΔpfkA also, the specific MHB production rate was increased by 15% and 20%, respectively. The yield of the latter two strains slightly dropped by 11% and 6%, but was still 73% and 132% higher compared to the reference strain with intact pgi and pfkA genes and expressing glf and glk. Thus, metabolic engineering strategies are presented for improving yield and rate of reductive redox biocatalysis by partial cyclization of the PPP and by increasing glucose uptake, respectively.