Protein engineering of the colony-stimulating factor-1 receptor kinase domain for structural studies

A parallel approach to designing crystallization constructs for the c-FMS kinase domain was implemented, resulting in proteins suitable for structural studies. Sequence alignment and limited proteolysis were used to identify and eliminate unstructured and surface-exposed domains. A small library of chimeras was prepared in which the kinase insert domain of FMS was replaced with the kinase insert domain of previously crystallized receptor-tyrosine kinases. Characterization of the newly generated FMS constructs by enzymology and thermoshift assays demonstrated similar activities and compound binding to the FMS full-length cytoplasmic domain. Two chimeras were evaluated for crystallization in the presence and absence of a variety of ligands resulting in crystal structures, and leading to a successful structure-based drug design project for this important inflammation target.     

Multiple restrictions of human immunodeficiency virus type 1 in feline cells

The productive replication of human immunodeficiency virus type 1 (HIV-1) occurs exclusively in defined cells of human or chimpanzee origin, explaining why heterologous animal models for HIV replication, pathogenesis, vaccination, and therapy are not available. This lack of an animal model for HIV-1 studies prompted us to examine the susceptibility of feline cells in order to evaluate the cat (Felis catus) as an animal model for studying HIV-1. Here, we report that feline cell lines harbor multiple restrictions with respect to HIV-1 replication. The feline CD4 receptor does not permit virus infection. Feline T-cell lines MYA-1 and FeT-1C showed postentry restrictions resulting in low HIV-1 luciferase reporter activity and low expression of viral Gag-Pol proteins when pseudotyped vectors were used. Feline fibroblastic CrFK and KE-R cells, expressing human CD4 and CCR5, were very permissive for viral entry and HIV-long terminal repeat-driven expression but failed to support spreading infection. KE-R cells displayed a profound block with respect to release of HIV-1 particles. In contrast, CrFK cells allowed very efficient particle production; however, the CrFK cell-derived HIV-1 particles had low specific infectivity. We subsequently identified feline apolipoprotein B-editing catalytic polypeptide 3 (feAPOBEC3) proteins as active inhibitors of HIV-1 particle infectivity. CrFK cells express at least three different APOBEC3s: APOBEC3C, APOBEC3H, and APOBEC3CH. While the feAPOBEC3C did not significantly inhibit HIV-1, the feAPOBEC3H and feAPOBEC3CH induced G to A hypermutations of the viral cDNA and reduced the infectivity approximately 10- to approximately 40-fold.     

The developmental expression of fluorescent proteins in organotypic hippocampal slice cultures from transgenic mice and its use in the determination of excitotoxic neurodegeneration

Transgenic mice, expressing fluorescent proteins in neurons and glia, provide new opportunities for real-time microscopic monitoring of degenerative and regenerative structural changes. We have previously validated and compared a number of quantifiable markers for neuronal damage and cell death in organotypic brain slice cultures, such as cellular uptake of propidium iodide (PI), loss of microtubule-associated protein 2 (MAP2), Fluoro-Jade (FJ) cell staining, and the release of cytosolic lactate dehydrogenase (LDH). An important supplement to these markers would be data on corresponding morphological changes, as well as the opportunity to monitor reversible changes or long-term effects in the event of minor damage. As a first step, we present: a) the developmental expression in organotypic hippocampal brain slice cultures of transgenic fluorescent proteins, useful for the visualisation of neuronal subpopulations and astroglial cells; and b) examples of excitotoxic, glutamate receptor-induced degeneration of hippocampal CA1 pyramidal cells, with corresponding astroglial reactivity in such cultures. The slice cultures were set up according to standard techniques, by using one-week old pups from four transgenic mouse strains which express fluorescent proteins in their neurons and/or astroglial cells. From the time of explantation, and subsequently for up to nine weeks in culture, the transgenic neuronal fluorescence displayed the expected characteristics of a developmental, in vivo-like increase, including both the number and localisation of cells, as well as the intensity of fluorescence. At that stage and later, the transgenic fluorescence clearly permitted the visualisation of cell bodies, larger and smaller dendritic branches, spines and axons. In separate experiments, with a 24-hour exposure of matured sliced cultures to 100 microM of the glutamate agonist, N-methyl-D-aspartate (NMDA), we observed, by time-lapse recording, a gradual, but rapid loss of fluorescent CA1 pyramidal cells, accompanied by astrogliosis of transgene fluorescent astroglial cells. Based on these results, we consider that organotypic brain slice cultures from transgenic mice, with fluorescent neurons and glia, combined with detailed visualisation by time-lapse fluorescence microscopy, have great potential for investigating both major irreversible and minor reversible structural changes in neurons and glia, induced by neurotoxins and other neurodegenerative compounds and conditions.     

Formation of giant spicules in the deep-sea hexactinellid <Emphasis Type="Italic">Monorhaphis chuni</Emphasis> (Schulze 1904): electron-microscopic and biochemical studies

The siliceous sponge Monorhaphis chuni (Hexactinellida) synthesizes the largest biosilica structures on earth (3 m). Scanning electron microscopy has shown that these spicules are regularly composed of concentrically arranged lamellae (width: 3–10 μm). Between 400 and 600 lamellae have been counted in one giant basal spicule. An axial canal (diameter: ~2 μm) is located in the center of the spicules; it harbors the axial filament and is surrounded by an axial cylinder (100–150 μm) of electron-dense homogeneous silica. During dissolution of the spicules with hydrofluoric acid, the axial filament is first released followed by the release of a proteinaceous tubule. Two major proteins (150 kDa and 35 kDa) have been visualized, together with a 24-kDa protein that cross-reacts with antibodies against silicatein. The spicules are surrounded by a collagen net, and the existence of a hexactinellidan collagen gene has been demonstrated by cloning it from Aphrocallistes vastus. During the axial growth of the spicules, silicatein or the silicatein-related protein is proposed to become associated with the surface of the spicules and to be finally internalized through the apical opening to associate with the axial filament. Based on the data gathered here, we suggest that, in the Hexactinellida, the growth of the spicules is mediated by silicatein or by a silicatein-related protein, with the orientation of biosilica deposition being controlled by lectin and collagen. Carsten Eckert was previously with the Museum für Naturkunde, Invalidenstrasse 43, 10115 Berlin, Germany. The collagen sequence from Aphrocallistes vastus reported here, viz., [COL_APHRO] APHVACOL (accession number AM411124), has been deposited in the EMBL/GenBank data base. This work was supported by grants from the European Commission, the Deutsche Forschungsgemeinschaft, the Bundesministerium für Bildung und Forschung Germany (project: Center of Excellence BIOTECmarin), the National Natural Science Foundation of China (grant no. 50402023), and the International Human Frontier Science Program.     

Recharacterization of ancient DNA miscoding lesions: insights in the era of sequencing-by-synthesis

Although ancient DNA (aDNA) miscoding lesions have been studied since the earliest days of the field, their nature remains a source of debate. A variety of conflicting hypotheses exist about which miscoding lesions constitute true aDNA damage as opposed to PCR polymerase amplification error. Furthermore, considerable disagreement and speculation exists on which specific damage events underlie observed miscoding lesions. The root of the problem is that it has previously been difficult to assemble sufficient data to test the hypotheses, and near-impossible to accurately determine the specific strand of origin of observed damage events. With the advent of emulsion-based clonal amplification (emPCR) and the sequencing-by-synthesis technology this has changed. In this paper we demonstrate how data produced on the Roche GS20 genome sequencer can determine miscoding lesion strands of origin, and subsequently be interpreted to enable characterization of the aDNA damage behind the observed phenotypes. Through comparative analyses on 390 965 bp of modern chloroplast and 131 474 bp of ancient woolly mammoth GS20 sequence data we conclusively demonstrate that in this sample at least, a permafrost preserved specimen, Type 2 (cytosine→thymine/guanine→adenine) miscoding lesions represent the overwhelming majority of damage-derived miscoding lesions. Additionally, we show that an as yet unidentified guanine→adenine analogue modification, not the conventionally argued cytosine→uracil deamination, underpins a significant proportion of Type 2 damage. How widespread these implications are for aDNA will become apparent as future studies analyse data recovered from a wider range of substrates.     

Small molecule ago-allosteric modulators of the human glucagon-like peptide-1 (hGLP-1) receptor

Following our previous publication describing the biological profiles, we herein describe the structure-activity relationships of a core set of quinoxalines as the hGLP-1 receptor agonists. The most potent and efficacious compounds are 6,7-dichloroquinoxalines bearing an alkyl sulfonyl group at the C-2 position and a secondary alkyl amino group at the C-3 position. These findings serve as a valuable starting point for the discovery of more drug-like small molecule agonists for the hGLP-1 receptor.     

Novel tetrahydroisoquinolines are histamine H3 antagonists and serotonin reuptake inhibitors

A series of novel 4-aryl-1,2,3,4-tetrahydroisoquinoline-based histamine H(3) ligands that also have serotonin reuptake transporter inhibitor activity is described. The synthesis, in vitro biological data, and select pharmacokinetic data for these novel compounds are discussed.     

Quantitative chemical proteomics reveals mechanisms of action of clinical ABL kinase inhibitors

We describe a chemical proteomics approach to profile the interaction of small molecules with hundreds of endogenously expressed protein kinases and purine-binding proteins. This subproteome is captured by immobilized nonselective kinase inhibitors (kinobeads), and the bound proteins are quantified in parallel by mass spectrometry using isobaric tags for relative and absolute quantification (iTRAQ). By measuring the competition with the affinity matrix, we assess the binding of drugs to their targets in cell lysates and in cells. By mapping drug-induced changes in the phosphorylation state of the captured proteome, we also analyze signaling pathways downstream of target kinases. Quantitative profiling of the drugs imatinib (Gleevec), dasatinib (Sprycel) and bosutinib in K562 cells confirms known targets including ABL and SRC family kinases and identifies the receptor tyrosine kinase DDR1 and the oxidoreductase NQO2 as novel targets of imatinib. The data suggest that our approach is a valuable tool for drug discovery.     

Cleavage of CXCR1 on neutrophils disables bacterial killing in cystic fibrosis lung disease

Interleukin-8 (IL-8) activates neutrophils via the chemokine receptors CXCR1 and CXCR2. However, the airways of individuals with cystic fibrosis are frequently colonized by bacterial pathogens, despite the presence of large numbers of neutrophils and IL-8. Here we show that IL-8 promotes bacterial killing by neutrophils through CXCR1 but not CXCR2. Unopposed proteolytic activity in the airways of individuals with cystic fibrosis cleaved CXCR1 on neutrophils and disabled their bacterial-killing capacity. These effects were protease concentration-dependent and also occurred to a lesser extent in individuals with chronic obstructive pulmonary disease. Receptor cleavage induced the release of glycosylated CXCR1 fragments that were capable of stimulating IL-8 production in bronchial epithelial cells via Toll-like receptor 2. In vivo inhibition of proteases by inhalation of alpha1-antitrypsin restored CXCR1 expression and improved bacterial killing in individuals with cystic fibrosis. The cleavage of CXCR1, the functional consequences of its cleavage, and the identification of soluble CXCR1 fragments that behave as bioactive components represent a new pathophysiologic mechanism in cystic fibrosis and other chronic lung diseases.