Macrocyclic peptidomimetic β-secretase (BACE-1) inhibitors with activity in vivo

The macrocyclic peptidic BACE-1 inhibitors 2a–c show moderate enzymatic and cellular activity. By exchange of the hydroxyethylene- to ethanolamine-transition state mimetic the peptidic character was reduced, providing the highly potent and selective inhibitor 3. Variation of the P′ moiety resulted in the macrocyclic inhibitor 14. Both macrocycles show inhibition of BACE-1 in the brain of APP51/16 transgenic mice, 3 (NB-544) after intravenous and 14 (NB-533) after oral application.     

Morphine and HIV-Tat increase microglial-free radical production and oxidative stress: possible role in cytokine regulation

Opiate abuse alters the progression of human immunodeficiency virus and may increase the risk of neuroAIDS. As neuroAIDS is associated with altered microglial reactivity, the combined effects of human immunodeficiency virus-Tat and morphine were determined in cultured microglia. Specifically, experiments determined the effects of Tat and morphine on microglial-free radical production and oxidative stress, and on cytokine release. Data show that combined Tat and morphine cause early and synergistic increases in reactive oxygen species, with concomitant increases in protein oxidation. Furthermore, combined Tat and morphine, but not Tat or morphine alone, cause reversible decreases in proteasome activity. The effects of morphine on free radical production and oxidative stress are prevented by pre-treatment with naloxone, illustrating the important role of opioid receptor activation in these phenomena. While Tat is well known to induce cytokine release from cultured microglia, morphine decreases Tat-induced release of the cytokines tumor necrosis factor-α and interleukin-6, as well as the chemokine monocyte chemoattractant protein-1 (MCP-1). Finally, experiments using the reversible proteasome inhibitor MG115 show that temporary, non-cytotoxic decreases in proteasome activity increase protein oxidation and decrease tumor necrosis factor-α, interleukin-6, and MCP-1 release from microglia. Taken together, these data suggest that oxidative stress and proteasome inhibition may be involved in the immunomodulatory properties of opioid receptor activation in microglia.     

Prolectin, a Glycan-binding Receptor on Dividing B Cells in Germinal Centers

Prolectin, a previously undescribed glycan-binding receptor, has been identified by re-screening of the human genome for genes encoding proteins containing potential C-type carbohydrate-recognition domains. Glycan array analysis revealed that the carbohydrate-recognition domain in the extracellular domain of the receptor binds glycans with terminal α-linked mannose or fucose residues. Prolectin expressed in fibroblasts is found at the cell surface, but unlike many glycan-binding receptors it does not mediate endocytosis of a neoglycoprotein ligand. However, compared with other known glycan-binding receptors, the receptor contains an unusually large intracellular domain that consists of multiple sequence motifs, including phosphorylated tyrosine residues, that allow it to interact with signaling molecules such as Grb2. Immunohistochemistry has been used to demonstrate that prolectin is expressed on a specialized population of proliferating B cells in germinal centers. Thus, this novel receptor has the potential to function in carbohydrate-mediated communication between cells in the germinal center.Membrane-bound mammalian glycan-binding receptors, often referred to as lectins, are believed to play multiple distinct roles in the immune system, decoding information in complex oligosaccharide structures on cell surfaces and soluble glycoproteins (1, 2). A host of glycan-binding receptors on dendritic cells and macrophages function in pathogen recognition, often resulting in uptake of microbes through endocytic mechanisms. Examples include the mannose receptor, DC-SIGN,³ langerin, and the macrophage galactose receptor. Glycan-binding receptors can also recognize glycans found on the surfaces of mammalian cells. Some of these receptors, such as the selectins, mediate adhesion between leukocytes and endothelia (3, 4). A small number of receptors, notably members of the siglec family, bind mammalian-type glycans and have been shown to have potential signaling functions (5). While multiple glycan-binding receptors have been described on cells of the myeloid lineage, the complement of such receptors on lymphocytes is much more restricted. The best characterized examples are the T-cell adhesion molecule L-selectin (4) and the B-cell receptor CD22, also designated siglec-2 (5).Genomic screening for potential glycan-binding receptors has usually been undertaken by initially searching for the presence of one of the several types of structural domains that are known to support sugar-binding activity (6). Knowledge of the structures of multiple families of modular carbohydrate-recognition domains (CRDs) has facilitated identification of proteins with potential sugar-binding activity and can lead to predictions of what types of ligands might be bound. Although the human genome has been extensively screened with profile-recognition algorithms that identify common sequence motifs associated with CRDs, refinements to the genome sequence and improvements in gene-recognition algorithms occasionally result in detection of novel proteins that contain putative CRDs.We describe a previously undetected glycan-binding receptor identified by re-screening of the human genome and provide characterization of its molecular and cellular properties. Based on its expression in a specialized population of proliferating B cells in germinal centers, we propose that it be designated prolectin. Our results suggest that prolectin functions in carbohydrate-mediated communication between cells in the germinal center.     

Delipidation of insect lipoprotein,lipophorin, affects its binding to the lipophorin receptor,LpR: Implications for the role of LpR-mediated endocytosis

The insect lipophorin receptor (LpR), an LDL receptor (LDLR) homologue that is expressed during restricted periods of insect development, binds and endocytoses high-density lipophorin (HDLp). However, in contrast to LDL, HDLp is not lysosomally degraded, but recycled in a transferrin-like manner, leaving a function of receptor-mediated uptake of HDLp to be uncovered. Since a hallmark of circulatory HDLp is its ability to function as a reusable shuttle that selectively loads and unloads lipids at target tissues without being endocytosed or degraded, circulatory HDLp can exist in several forms with respect to lipid loading. To investigate whether lipid content of the lipoprotein affects binding and subsequent endocytosis by LpR, HDLp was partially delipidated in vitro by incubation with α-cyclodextrin, yielding a particle of buoyant density 1.17 g/mL (HDLp-1.17). Binding experiments demonstrated that LpR bound HDLp-1.17 with a substantially higher affinity than HDLp both in LpR-transfected Chinese hamster ovary (CHO) cells and isolated insect fat body tissue endogenously expressing LpR. Similar to HDLp, HDLp-1.17 was targeted to the endocytic recycling compartment after endocytosis in CHO(LpR) cells. The complex of HDLp-1.17 and LpR appeared to be resistant to endosomal pH, as was recently demonstrated for the LpR–HDLp complex, corroborating that HDLp-1.17 is recycled similar to HDLp. This conclusion was further supported by the observation of a significant decrease with time of HDLp-1.17-containing vesicles after endocytosis of HDLp-1.17 in LpR-expressing insect fat body tissue. Collectively, our results indicate that LpR favors the binding and subsequent endocytosis of HDLp-1.17 over HDLp, suggesting a physiological role for LpR in selective endocytosis of relatively lipid-unloaded HDLp particles, while lipid reloading during their intracellular itinerary might result in decreased affinity for LpR and thus allows recycling.     

A role for the 30S subunit E site in maintenance of the translational reading frame

The exit (E) site has been implicated in several ribosomal activities, including translocation, decoding, and maintenance of the translational reading frame. Here, we target the 30S subunit E site by introducing a deletion in rpsG that truncates the β-hairpin of ribosomal protein S7. This mutation (S7ΔR77–Y84) increases both −1 and +1 frameshifting but does not increase miscoding, providing evidence that the 30S E site plays a specific role in frame maintenance. Mutation S7ΔR77–Y84 also stimulates +1 programmed frameshifting during prfB′-lacZ translation in many synthetic contexts. However, no effect is seen when the E codon of the frameshift site corresponds to those found in nature, suggesting that E-tRNA release does not normally limit the rate of prfB frameshifting. Ribosomes containing S7ΔR77–Y84 exhibit an elevated rate of spontaneous reverse translocation and an increased K_1/2 for E-tRNA. These effects are of similar magnitude, suggesting that both result from destabilization of E-tRNA. Finally, this mutation of the 30S E site does not inhibit EF-G-dependent translocation, consistent with a primary role for the 50S E site in the mechanism.     

Prion Protein-Detergent Micelle Interactions Studied by NMR in Solution

Cellular prion proteins, PrP^C, carrying the amino acid substitutions P102L, P105L, or A117V, which confer increased susceptibility to human transmissible spongiform encephalopathies, are known to form structures that include transmembrane polypeptide segments. Herein, we investigated the interactions between dodecylphosphocholine micelles and the polypeptide fragments 90–231 of the recombinant mouse PrP variants carrying the amino acid replacements P102L, P105L, A117V, A113V/A115V/A118V, K110I/H111I, M129V, P105L/M129V, and A117V/M129V. Wild-type mPrP-(90–231) and mPrP[M129V]-(91–231) showed only weak interactions with dodecylphosphocholine micelles in aqueous solution at pH 7.0, whereas discrete interaction sites within the polypeptide segment 102–127 were identified for all other aforementioned mPrP variants by NMR chemical shift mapping. These model studies thus provide evidence that amino acid substitutions within the polypeptide segment 102–127 affect the interactions of PrP^C with membranous structures, which might in turn modulate the physiological function of the protein in health and disease.Transmissible spongiform encephalopathies (TSEs),² such as Creutzfeldt-Jakob disease and the Gerstmann-Sträussler-Scheinker syndrome in humans, are accompanied by the appearance in the brain of an aggregated “scrapie” isoform of the host-encoded prion protein, PrP^Sc (1–3). The cellular form, PrP^C, consists of an unstructured N-terminal “tail” of residues 23–125 and a globular domain of residues 126–231, and is attached by a C-terminal glycosylphosphatidylinositol (GPI) anchor to the outer plasma membrane. This structure ensures a role of membrane interactions in the physiological function of PrP^C and probably also in the disease-related events leading to TSEs. For example, transgenic mice expressing a prion protein variant lacking the GPI membrane anchor did not develop the typical clinical signs of TSE after inoculation with infectious brain homogenate, although significant amounts of PrP^Sc accumulated in the brain (4). This finding led to the conclusion that membrane-association of PrP^C is necessary for the development of a TSE. Independent evidence for the importance of membrane interactions for the onset of prion diseases was derived from cell-free conversion assays and cell culture experiments (5, 6).Data have also been presented that indicate that in addition to the normal form with the C terminus linked to a GPI anchor and the C-terminal domain located on the cell surface, PrP^C can adopt two different transmembrane topologies, ^CtmPrP and ^NtmPrP, which have the C-terminal polypeptide segment located in the lumen of the endoplasmic reticulum (^CtmPrP) or in the cytoplasm (^NtmPrP) (7–9). The population of the ^CtmPrP variant is <10% of the total wild-type prion protein present during cellular biosynthesis but is increased to 20–30% for the pathogenic mutations P102L, P105L, and A117V of human PrP and the designed variant mouse PrPs obtained with the amino acid exchanges A113V/A115V/A118V and K110I/H111I (10–13). The population of ^CtmPrP was further increased when an additional mutation, L9R, was present in the N-terminal signal sequence (14), so that ∼50% of the PrP was synthesized as the ^CtmPrP variant in granule neurons obtained from transgenic mice expressing a prion protein construct carrying the four amino acid replacements L9R, A113V, A115V, and A118V (15). Quite generally, an increase in the population of ^CtmPrP was also shown to be associated with severe neurodegeneration in transgenic mice, and it has been suggested that ^CtmPrP may be the proximate cause of neuronal death in certain prion disorders (10, 11, 15).In vitro studies on interactions of full-length and N-terminally truncated forms of recombinant PrP showed that acidic membranes caused the N-terminal part of the protein to become more structured, whereas the C-terminal domain was destabilized (16–19). Furthermore, zwitterionic gel-phase dipalmitoylphosphatidylcholine or raft-like membranes were shown to induce increased α-helical structure in recombinant Syrian hamster PrP-(90–231) at pH 7.0 (18, 19). Membrane interactions of polypeptides representing sequence motifs found in the prion protein have also been studied (20–23).In this report we describe investigations of PrP interactions with a membrane mimetic and focus on the mutations P102L, P105L, and A117V, which have been linked with familial Gerstmann-Sträussler-Scheinker syndrome in humans (2, 24, 25). Our interest in these variant proteins is related to open questions about the mechanisms by which pathogenic mutations predispose humans for prion diseases. We studied the interactions of a recombinant wild-type mouse prion protein fragment, mPrP-(90–231), and the variants mPrP[P102L]-(91–231), mPrP[P105L]-(91–231), mPrP[A117V]-(90–231), mPrP[A113V,A115V,A118V]-(90–231), and mPrP[K110I,H111I]-(90–231). For these studies, we used the N-terminally truncated protein composed of residues 90–231. This region contains the transmembrane segment, all known disease-associated point mutations, the entire polypeptide fragment with proteinase K-resistance in PrP^Sc, which is also sufficient to transmit disease (1, 25, 26). The amino acid substitutions in these variant PrPs are located either within a hydrophobic stretch of residues 112–127, which is highly conserved in mammalian PrPs (27, 28), or in the positively charged segment of residues 95–111 (Fig. 1, B and C). We also included the M129V polymorphism into this study, which was reported to have a significant influence on the susceptibility of humans to prion diseases and on the disease phenotype. For example, the mutations P105L and A117V are only pathogenic in the presence of valine at position 129 (2, 24). The zwitterionic detergent dodecylphosphocholine (DPC, Fig. 1A) was used as a biomembrane mimetic model system, and NMR spectroscopy was employed to screen for protein-detergent micelle interactions, and for the structural characterization of the various prion protein constructs interacting with the detergent micelles.Open in a separate windowFIGURE 1.Detergent and proteins used in this study. A, zwitterionic form of DPC. B, schematic diagram of the mPrP-(90–231) polypeptide indicating the locations of the regular secondary structures, i.e. three α-helices and two strands of an antiparallel β-sheet, a “positively charged cluster” (CC) of amino acid residues in positions 95–111, and a “hydrophobic polypeptide segment” (HPS) comprising residues 112–127. C, amino acid sequence alignment of residues 90–135 for wild-type mPrP-(90–231) and the protein variants studied in this paper, where for each variant mPrP the amino acid replacements are given and identical residues are indicated by dots; the numbering is according to Schätzl et al. (27).     

Species-specific responses to atmospheric carbon dioxide and tropospheric ozone mediate changes in soil carbon

We repeatedly sampled the surface mineral soil (0–20 cm depth) in three northern temperate forest communities over an 11-year experimental fumigation to understand the effects of elevated carbon dioxide (CO₂) and/or elevated phyto-toxic ozone (O₃) on soil carbon (C). After 11 years, there was no significant main effect of CO₂ or O₃ on soil C. However, within the community containing only aspen ( Populus tremuloides Michx.), elevated CO₂ caused a significant decrease in soil C content. Together with the observations of increased litter inputs, this result strongly suggests accelerated decomposition under elevated CO_2. In addition, an initial reduction in the formation of new (fumigation-derived) soil C by O₃ under elevated CO₂ proved to be only a temporary effect, mirroring trends in fine root biomass. Our results contradict predictions of increased soil C under elevated CO₂ and decreased soil C under elevated O₃ and should be considered in models simulating the effects of Earth's altered atmosphere.     

miR‐29a suppresses tristetraprolin,which is a regulator of epithelial polarity and metastasis

Several microRNAs (miRNAs) have recently been described as crucial regulators of epithelial‐to‐mesenchymal transition (EMT) and metastasis. By comparing the expression profiles of miRNAs, we found upregulation of miR‐29a in mesenchymal, metastatic RasXT cells relative to epithelial EpRas cells. Overexpression of miR‐29a suppressed the expression of tristetraprolin (TTP), a protein involved in the degradation of messenger RNAs with AU‐rich 3′‐untranslated regions, and led to EMT and metastasis in cooperation with oncogenic Ras signalling. We also observed enhanced miR‐29a and reduced TTP levels in breast cancer patient samples, indicating relevance for human disease. Previously, miR‐29 family members were shown to have tumour‐suppressive effects in haematopoietic, cholangiocytic and lung tumours. Therefore, miRNAs can act as either oncogenes or tumour suppressors, depending on the context.     

Progress in spondylarthritis. Mechanisms of new bone formation in spondyloarthritis

Targeted therapies that neutralize tumour necrosis factor are often able to control the signs and symptoms of spondyloarthritis. However, recent animal model data and clinical observations indicate that control of inflammation may not be sufficient to impede disease progression toward ankylosis in these patients. Bone morphogenetic proteins and WNTs (wingless-type like) are likely to play an important role in ankylosis and could be therapeutic targets. The relationship between inflammation and new bone formation is still unclear. This review summarizes progress made in our understanding of ankylosis and offers an alternative view of the relationship between inflammation and ankylosis.