Potent and selective HIV-1 ribonuclease H inhibitors based on a 1-hydroxy-1,8-naphthyridin-2(1H)-one scaffold

Optimization studies using an HIV RNase H active site inhibitor containing a 1-hydroxy-1,8-naphthyridin-2(1H)-one core identified 4-position substituents that provided several potent and selective inhibitors. The best compound was potent and selective in biochemical assays (IC₅₀ = 0.045 μM, HIV RT RNase H; 13 μM, HIV RT-polymerase; 24 μM, HIV integrase) and showed antiviral efficacy in a single-cycle viral replication assay in P4-2 cells (IC₅₀ = 0.19 μM) with a modest window with respect to cytotoxicity (CC₅₀ = 3.3 μM).     

Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Background  

The assembly and spatial organization of enzymes in naturally occurring multi-protein complexes is of paramount importance for the efficient degradation of complex polymers and biosynthesis of valuable products. The degradation of cellulose into fermentable sugars by Clostridium thermocellum is achieved by means of a multi-protein "cellulosome" complex. Assembled via dockerin-cohesin interactions, the cellulosome is associated with the cell surface during cellulose hydrolysis, forming ternary cellulose-enzyme-microbe complexes for enhanced activity and synergy. The assembly of recombinant cell surface displayed cellulosome-inspired complexes in surrogate microbes is highly desirable. The model organism Lactococcus lactis is of particular interest as it has been metabolically engineered to produce a variety of commodity chemicals including lactic acid and bioactive compounds, and can efficiently secrete an array of recombinant proteins and enzymes of varying sizes.     

Thyroid hormone production rates in rat liver and intestine in vivo: a novel graph theory and experimental solution

We develop a novel method for finding sufficient experimental conditions for discriminating and quantifying individual biomolecule production sources in distributed, inhomogeneous multisource systems in vivo, and we apply it experimentally to a complex, unsolved problem in endocrinology. The majority of hormonal triiodothyronine (T3) is produced from prohormone thyroxine (T4) in numerous nonthyroidal organs and, with one exception, the T3 production rate has not been fully resolved in any single extrathyroidal organ of any species. Using a readily generalized graphic method called cut-set analysis, we show here that measured steady-state responses in several organs to three independent tracer infusions, two into blood and one directly into the organ(s) of interest, are sufficient to resolve this problem for organs fully accessible to direct infusion in vivo. We evaluated local T3 production in rat liver and intestine, which also required T3 bile flux measurements, and we found that liver produces approximately 31% and whole intestine approximately 6% of whole body T3 from T4. With thyroidal production included, liver contributes approximately 15% and intestine approximately 3% of whole rat T3 production. This new methodology is broadly applicable, especially to biosystems that include molecular interconversions at multiple sites.     

Functional characterization of Narc 1, a novel proteinase related to proteinase K

The NARC 1 gene encodes a novel proteinase K family proteinase. The domain structure of rat Narc 1 resembles that of the subtilisin-like proprotein convertases (SPCs), except that rNarc 1 lacks the canonical P-domain of SPCs, retaining only the RGD motif as part of what might be a cryptically functioning P-domain. Narc 1 undergoes autocatalytic intramolecular processing at the site LVFAQ/, resulting in the cleavage of its prosegment and the generation of an active proteinase with a broad alkaline pH optimum and no apparent calcium requirement for activity. Both primary and secondary structural determinants influence Narc 1 substrate recognition. Our functional characterization of Narc 1 reinforces the inference drawn from the analysis of its predicted structure that this enzyme is most closely related to representatives of the proteinase K family, but that it is also sufficiently different to warrant its possible classification in a separate sub-family.     

Functional screening of five PYPAF family members identifies PYPAF5 as a novel regulator of NF-kappaB and caspase-1

PYRIN-containing Apaf-1-like proteins (PYPAFs) are a recently identified family of proteins thought to function in apoptotic and inflammatory signaling pathways. PYPAF1 and PYPAF7 proteins have been found to assemble with the PYRIN–CARD protein ASC and coordinate the activation of NF-κB and pro-caspase-1. To determine if other PYPAF family members function in pro-inflammatory signaling pathways, we screened five other PYPAF proteins (PYPAF2, PYPAF3, PYPAF4, PYPAF5 and PYPAF6) for their ability to activate NF-κB and pro-caspase-1. Co-expression of PYPAF5 with ASC results in a synergistic activation of NF-κB and the recruitment of PYPAF5 to punctate structures in the cytoplasm. The expression of PYPAF5 is highly restricted to granulocytes and T-cells, indicating a role for this protein in inflammatory signaling. In contrast, PYPAF2, PYPAF3, PYPAF4 and PYPAF6 failed to colocalize with ASC and activate NF-κB. PYPAF5 also synergistically activated caspase-1-dependent cytokine processing when co-expressed with ASC. These findings suggest that PYPAF5 functions in immune cells to coordinate the transduction of pro-inflammatory signals to the activation of NF-κB and pro-caspase-1.     

The abundance of additional N-acetyllactosamine units in N-linked tetraantennary oligosaccharides of human Tamm-Horsfall glycoprotein is a donor-specific feature

Previously, treatment of Tamm-Horsfall glycoprotein (THp) from different donors with endo-beta-galactosidase has been shown to liberate a tetra- and a Sd(a)-active pentasaccharide, concluding the presence of N-linked carbohydrate chains containing additional N - acetyllactosamine units. These type of oligosaccharides were not found in a detailed structure elucidation of the carbohydrate moiety of THp of one male donor, suggesting a donor-specific feature for these type of structures. Therefore, THp was isolated from four healthy male donors and each subjected to endo-beta-galactosidase treatment in order to release these tetra- and Sd(a)-active pentasaccharide. Differences were observed in the total amount of released tetra- and Sda-active pentasaccharide of the used donors (42, 470, 478, 718 microg/100 mg THp), indicating that the presence of repeating N-acetyllactosamine units incorporated into the N-glycan moiety of THp is donor specific. Furthermore, a higher expression of the Sd(a) determinant on antennae which display N-acetyllactosamine elongation was observed, suggesting a better accessibility for the beta-N-acetylgalactosaminyltransferase. In order to characterize the N-glycans containing repeating N- acetyllactosamine units, carbohydrate chains were enzymatically released from THp and isolated. The tetraantennary fraction, which accounts for more than 33% of the total carbohydrate moiety of THp, was used to isolate oligosaccharides containing additional N - acetyllactosamine units. Five N-linked tetraantennary oligosaccharides containing a repeating N-acetyllactosamine unit were identified, varying from structures bearing four Sd(a) determinants to structures containing no Sd(a) determinant (see below). One compound was used in order to specify the branch location of the additional N- acetyllactosamine unit, and it appeared that only the Gal-6' and Gal-8' residues were occupied by a repeating N -acetyllactosamine unit.