The co-crystal structure of ubiquitin carboxy-terminal hydrolase L1 (UCHL1) with a tripeptide fluoromethyl ketone (Z-VAE(OMe)-FMK)

UCHL1 is a 223 amino acid member of the UCH family of deubiquitinating enzymes (DUBs), found abundantly and exclusively expressed in neurons and the testis in normal tissues. Two naturally occurring variants of UCHL1 are directly involved in Parkinson’s disease (PD). Not only has UCHL1 been linked to PD, but it has oncogenic properties, having been found abnormally expressed in lung, pancreatic, and colorectal cancers. Although inhibitors of UCHL1 have been described previously the co-crystal structure of the enzyme bound to any inhibitor has not been reported. Herein, we report the X-ray structure of UCHL1 co-crystallized with a peptide-based fluoromethylketone inhibitor, Z-VAE(OMe)-FMK (VAEFMK) at 2.35 Å resolution. The co-crystal structure reveals that the inhibitor binds in the active-site cleft, irreversibly modifying the active-site cysteine; however, the catalytic histidine is still misaligned as seen in the native structure, suggesting that the inhibitor binds to an inactive form of the enzyme. Our structure also reveals that the inhibitor approaches the active-site cleft from the opposite side of the crossover loop as compared to the direction of approach of ubiquitin’s C-terminal tail, thereby occupying the P1′ (leaving group) site, a binding site perhaps used by the unknown C-terminal extension of ubiquitin in the actual in vivo substrate(s) of UCHL1. This structure provides a view of molecular contacts at the active-site cleft between the inhibitor and the enzyme as well as furnishing structural information needed to facilitate further design of inhibitors targeted to UCHL1 with high selectivity and potency.     

Biotechnological methods of <Emphasis Type="Italic">in vitro</Emphasis> propagation in willows (<Emphasis Type="Italic">Salix</Emphasis> spp.)

Many populations of high-mountainous relic dioecious willows in Central Europe only consist of female individuals and are thus limited in their reproductive potential. We completed micropropagation experiments with shoot apexes and nodal segments of common and endangered willow (Salix) species, which can help to reintroduce autochthonous genotypes to their natural sites. Until recently, cultivation of green young shoot apexes of S. alba and S. lapponum showed the highest percentage of regeneration. We successfully applied the two-times-sterilisation due to high contamination of natural explants. The OK medium was the most efficient culture medium. In vitro propagation of willows with unisexual catkins, anther and ovule cultures were tested and optimised. Isolated anthers were cultivated on selected media and then microcallus and calluses of S. caprea and calluses of S. viminalis were formed on the A medium. Among various tested and optimised media for the ovule culture, the CP medium was the most efficient one. In this case, only the microcalluses of S. viminalis were observed. We developed biotechnological procedures that can be useful in conserving fragmented populations of high-mountainous willows.     

Regulation of Toll-like receptor 4-mediated immune responses through Pasteurella multocida toxin-induced G protein signalling

Background

Lipopolysaccharide (LPS)-triggered Toll-like receptor (TLR) 4-signalling belongs to the key innate defence mechanisms upon infection with Gram-negative bacteria and triggers the subsequent activation of adaptive immunity. There is an active crosstalk between TLR4-mediated and other signalling cascades to secure an effective immune response, but also to prevent excessive inflammation. Many pathogens induce signalling cascades via secreted factors that interfere with TLR signalling to modify and presumably escape the host response. In this context heterotrimeric G proteins and their coupled receptors have been recognized as major cellular targets. Toxigenic strains of Gram-negative Pasteurella multocida produce a toxin (PMT) that constitutively activates the heterotrimeric G proteins G??_q, G??₁₃ and G??_i independently of G protein-coupled receptors through deamidation. PMT is known to induce signalling events involved in cell proliferation, cell survival and cytoskeleton rearrangement.

Results

Here we show that the activation of heterotrimeric G proteins through PMT suppresses LPS-stimulated IL-12p40 production and eventually impairs the T cell-activating ability of LPS-treated monocytes. This inhibition of TLR4-induced IL-12p40 expression is mediated by G??_i-triggered signalling as well as by G?|?-dependent activation of PI3kinase and JNK. Taken together we propose the following model: LPS stimulates TLR4-mediated activation of the NF?B-pathway and thereby the production of TNF-??, IL-6 and IL-12p40. PMT inhibits the production of IL-12p40 by G??_i-mediated inhibition of adenylate cyclase and cAMP accumulation and by G?|?-mediated activation of PI3kinase and JNK activation.

Conclusions

On the basis of the experiments with PMT this study gives an example of a pathogen-induced interaction between G protein-mediated and TLR4-triggered signalling and illustrates how a bacterial toxin is able to interfere with the host??s immune response.     

Persistence versus escape: Aspergillus terreus and Aspergillus fumigatus employ different strategies during interactions with macrophages

Invasive bronchopulmonary aspergillosis (IBPA) is a life-threatening disease in immunocompromised patients. Although Aspergillus terreus is frequently found in the environment, A. fumigatus is by far the main cause of IBPA. However, once A. terreus establishes infection in the host, disease is as fatal as A. fumigatus infections. Thus, we hypothesized that the initial steps of disease establishment might be fundamentally different between these two species. Since alveolar macrophages represent one of the first phagocytes facing inhaled conidia, we compared the interaction of A. terreus and A. fumigatus conidia with alveolar macrophages. A. terreus conidia were phagocytosed more rapidly than A. fumigatus conidia, possibly due to higher exposure of β-1,3-glucan and galactomannan on the surface. In agreement, blocking of dectin-1 and mannose receptors significantly reduced phagocytosis of A. terreus, but had only a moderate effect on phagocytosis of A. fumigatus. Once phagocytosed, and in contrast to A. fumigatus, A. terreus did not inhibit acidification of phagolysosomes, but remained viable without signs of germination both in vitro and in immunocompetent mice. The inability of A. terreus to germinate and pierce macrophages resulted in significantly lower cytotoxicity compared to A. fumigatus. Blocking phagolysosome acidification by the v-ATPase inhibitor bafilomycin increased A. terreus germination rates and cytotoxicity. Recombinant expression of the A. nidulans wA naphthopyrone synthase, a homologue of A. fumigatus PksP, inhibited phagolysosome acidification and resulted in increased germination, macrophage damage and virulence in corticosteroid-treated mice. In summary, we show that A. terreus and A. fumigatus have evolved significantly different strategies to survive the attack of host immune cells. While A. fumigatus prevents phagocytosis and phagolysosome acidification and escapes from macrophages by germination, A. terreus is rapidly phagocytosed, but conidia show long-term persistence in macrophages even in immunocompetent hosts.     

Adipose triglyceride lipase in immune response, inflammation, and atherosclerosis

Abstract Consistent with its central importance in lipid and energy homeostasis, lipolysis occurs in essentially all tissues and cell types, including macrophages. The hydrolytic cleavage of triacylglycerol by adipose triglyceride lipase (ATGL) generates non-esterified fatty acids, which are subsequently used as essential precursors for lipid and membrane synthesis, mediators in cell signaling processes or as energy substrate in mitochondria. This review summarizes the current knowledge concerning the consequences of ATGL deficiency in macrophages with particular emphasis on macrophage (dys)-function, apoptosis, and atherosclerosis.     

Identification of novel benzimidazole derivatives as inhibitors of leukotriene biosynthesis by virtual screening targeting 5-lipoxygenase-activating protein (FLAP)

Pharmacological suppression of leukotriene biosynthesis by 5-lipoxygenase (5-LO)-activating protein (FLAP) inhibitors is a promising strategy to intervene with inflammatory, allergic and cardiovascular diseases. Virtual screening targeting FLAP based on a combined ligand- and structure-based pharmacophore model led to the identification of 1-(2-chlorobenzyl)-2-(1-(4-isobutylphenyl)ethyl)-1H-benzimidazole (7) as developable candidate. Compound 7 potently suppressed leukotriene formation in intact neutrophils (IC(50)=0.31 μM) but essentially failed to directly inhibit 5-LO suggesting that interaction with FLAP causes inhibition of leukotriene synthesis. For structural optimization, a series of 46 benzimidazole-based derivatives of 7 were synthesized leading to more potent analogues (70-72, 82) with IC(50)=0.12-0.19 μM in intact neutrophils. Together, our results disclose the benzimidazole scaffold bearing an ibuprofen fingerprint as a new chemotype for further development of anti-leukotriene agents.     

Long-term immunity against actual poxviral HLA ligands as identified by differential stable isotope labeling

Viral peptides are presented by HLA class I on infected cells to activate CD8(+) T cells. Several immunogenic peptides have been identified indirectly by epitope prediction and screening of T cell responses to poxviral vectors, including modified vaccinia virus Ankara (MVA) currently being tested as recombinant or smallpox vaccines. However, for the development of optimal vaccination and immunomonitoring strategies, it is essential to characterize the actual viral HLA ligand repertoire of infected cells. We used an innovative approach to identify naturally processed MVA HLA ligands by differential HPLC-coupled mass spectrometry. We describe 12 viral peptides presented by HLA-A*0201 and 3 by HLA-B*0702. All HLA-A*0201 ligands participated in the memory response of MVA-immune donors, and several were immunogenic in Dryvax vaccinees. Eight epitopes were novel. Viral HLA ligand presentation and viral protein abundance did not correlate. All ligands were expressed early during the viral life cycle, and a pool of three of these mediated stronger protection against a lethal challenge in mice as compared with late epitopes. This highlights the reliability of the comparative mass spectrometry-based technique to identify relevant viral CD8(+) T cell epitopes for optimizing the monitoring of protective immune responses and the development of effective peptide-based vaccines.     

Antioxidant protection during ageing and senescence in chloroplasts of tobacco with modulated life span

We studied changes in antioxidant protection during ageing and senescence in chloroplasts of tobacco (Nicotiana tabacum L., cv. Wisconsin) with introduced SAG(12) promoter fused with ipt gene for cytokinin synthesis (transgenic plants with increased levels of cytokinins, SAG) or without it (control). Old leaves of SAG plants as well as their chloroplasts maintained higher physiological parameters compared to controls; accordingly, we concluded that their ageing was diverted due to increased cytokinin content. The chloroplast antioxidant protection did not decrease as well. Although antioxidant protection usually decreased in whole leaves of senescing control plants, ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR) activity, which maintained the high redox state of ascorbate, increased in chloroplasts of old control leaves.     

Cryo-section immunolabelling of difficult to preserve specimens: advantages of cryofixation, freeze-substitution and rehydration

BACKGROUND INFORMATION: Electron microscopic immunolabelling of ultrathin thawed cryo-sections, according to the method of Tokuyasu, is widely used as a very sensitive high-resolution localization technique. Its main advantages are that antigens remain in a hydrated environment prior to immunolabelling, and that antigen accessibility is improved compared with resin section labelling. However, the quality of structural appearance and antigenicity depends highly on the limitations of the initial conventional chemical fixation step, such as slow diffusion and selective reaction/cross-linking of fixative molecules. RESULTS AND CONCLUSIONS: Cryofixation, instead of conventional chemical fixation, followed by freeze-substitution/chemical fixation, rehydration and further processing for Tokuyasu cryo-sectioning leads to an improved preservation of both ultrastructure and antigenicity. This is especially true for tissues which are difficult to preserve by conventional chemical fixation at ambient temperatures, such as plant material, Drosophila embryos or nematode tissue. In particular labile and highly dynamic structures (for example, microtubules and Golgi apparatus) are remarkably better preserved. These improvements are also valid for light microscopic applications.     

The putative RNase P motif in the DEAD box helicase Hera is dispensable for efficient interaction with RNA and helicase activity

DEAD box helicases use the energy of ATP hydrolysis to remodel RNA structures or RNA/protein complexes. They share a common helicase core with conserved signature motifs, and additional domains may confer substrate specificity. Identification of a specific substrate is crucial towards understanding the physiological role of a helicase. RNA binding and ATPase stimulation are necessary, but not sufficient criteria for a bona fide helicase substrate. Here, we report single molecule FRET experiments that identify fragments of the 23S rRNA comprising hairpin 92 and RNase P RNA as substrates for the Thermus thermophilus DEAD box helicase Hera. Both substrates induce a switch to the closed conformation of the helicase core and stimulate the intrinsic ATPase activity of Hera. Binding of these RNAs is mediated by the Hera C-terminal domain, but does not require a previously proposed putative RNase P motif within this domain. ATP-dependent unwinding of a short helix adjacent to hairpin 92 in the ribosomal RNA suggests a specific role for Hera in ribosome assembly, analogously to the Escherichia coli and Bacillus subtilis helicases DbpA and YxiN. In addition, the specificity of Hera for RNase P RNA may be required for RNase P RNA folding or RNase P assembly.