Disruption of two putative nuclear localization sequences is required for cytosolic localization of mitogen-activated protein kinase phosphatase-2

MAP kinase phosphatase-2 (MKP-2) is a member of a family of dual specificity phosphatases (DSPs) that function in both the cytosol and nucleus to inactivate the MAP kinases. The mechanism that controls the subcellular distribution of these proteins is currently unclear. In this study, we have used site-directed mutagenesis to remove two novel nuclear localization sequences, NLS-1 and -2, either alone or in combination (DNLS). Loss of NLS-1 or NLS-2 alone did not alter the nuclear targeting of MKP-2 but mutation of both resulted in MKP-2 being retained within the cytosol. Furthermore, whilst expression of WT-MKP-2, NLS-1 or NLS-2 reduced both sorbitol- or UV-stimulated nuclear c-Jun N-terminal kinase (JNK) activity in HEK293 cells, this effect was absent in cells expressing DNLS-MKP-2. Similarly, transient transfection of WT-MKP-2, NLS-1 or NLS-2, but not DNLS-MKP-2 was able to substantially reduce agonist-stimulated ANF reporter activity in rat cardiac myocytes. Taken together, these results indicate that whilst both novel NLS participate in the nuclear localization of MKP-2, the expression of either sequence is sufficient to retain nuclear targeting.     

Characterization of the oligomer structure of recombinant human mannan-binding lectin

Mannan-binding lectin (MBL) belongs to a family of proteins called the collectins, which show large differences in their ultrastructures. These differences are believed to be determined by different N-terminal disulfide-bonding patterns. So far only the bonding pattern of two of the simple forms (recombinant rat MBL-C and bovine CL-43) have been determined. Recombinant MBL expressed in human cells was purified, and the structure of the N-terminal region was determined. Preliminary results on human plasma-derived MBL revealed high similarity to the recombinant protein. Here we report the structure of the N-terminal part of recombinant human MBL and present a model to explain the oligomerization pattern. Using a strategy of consecutive enzymatic digestions and matrix-assisted laser desorption ionization mass spectrometry, we succeeded in identifying a number of disulfide-linked peptides from the N-terminal cysteine-rich region. Based on these building blocks, we propose a model that can explain the various oligomeric forms found in purified MBL preparations. Furthermore, the model was challenged by the production of cysteine to serine mutants of the three N-terminally situated cysteines. The oligomerization patterns of these mutants support the proposed model. The model indicates that the polypeptide dimer is the basic unit in the oligomerization.     

Binding of antitumor antibiotic daunomycin to histones in chromatin and in solution

Daunomycin is an anticancer drug that is well-known to interact with DNA in chromatin. Using a compositionally defined chicken erythrocyte chromatin fraction, we have obtained conclusive evidence that the drug is also able to interact with chromatin-bound linker histones without any noticeable binding to core histones. The drug can interact in an equal fashion with both histone H1 and H5 and to a greater extent with core histones H3/H4 and H2A/H2B as free proteins in solution. Thus, the binding of daunomycin to linker histones in the chromatin fiber is most likely due to the well-known higher accessibility of these histones to the surrounding environment of the fiber. Binding of daunomycin to linker histones appears to primarily involve the trypsin-resistant (winged-helix) domain of these proteins. The studies described here reveal the occurrence of a previously undisclosed mechanism for the antitumor activity of anthracycline drugs at the chromatin level.     

Geographic variation in patterns of nestedness among local stream fish assemblages in Virginia

Nestedness of faunal assemblages is a multi-scale phenomenon, potentially influenced by a variety of factors. Prior small-scale studies have found freshwater fish species assemblages to be nested along stream courses as a result of either selective colonization or extinction. However, within-stream gradients in temperature and other factors are correlated with the distributions of many fish species and may also contribute to nestedness. At a regional level, strongly nested patterns would require a consistent set of structuring mechanisms across streams, and correlation among species tolerances of the environmental factors that influence distribution. Thus, nestedness should be negatively associated with the spatial extent of the region analyzed and positively associated with elevational gradients (a correlate of temperature and other environmental factors). We examined these relationships for the freshwater fishes of Virginia. Regions were defined within a spatial hierarchy and included whole river drainages, portions of drainages within physiographic provinces, and smaller subdrainages. In most cases, nestedness was significantly stronger in regions of smaller spatial extent and in regions characterized by greater topographic relief. Analysis of hydrologic variability and patterns of faunal turnover provided no evidence that inter-annual colonization/extinction dynamics contributed to elevational differences in nestedness. These results suggest that, at regional scales, nestedness is influenced by interactions between biotic and abiotic factors, and that the strongest nestedness is likely to occur where a small number of organizational processes predominate, i.e., over small spatial extents and regions exhibiting strong environmental gradients.The Virginia Cooperate Fish and Wildlife Research Unit is jointly sponsored by United States Geological Survey, Virginia Polytechnic Institute and State University, and Virginia Department of Game and Inland Fisheries.     

High-throughput synthesis optimization of sulfonamide NPY Y5 antagonists

A series of sulfonamide neuropeptide Y Y5 antagonists was optimized by preparation of sets of analogues using high-throughput synthesis and purification techniques. Testing of these compounds for their ability to bind to the human NPY Y5 receptor revealed separate SAR trends for sulfonamide amides versus sulfonamide ureas versus sulfonamide amines. By understanding these SAR trends, potent compounds were identified in all three series.     

Synthesis and biological activity of N-Acylated ornithine analogues of daptomycin

N-Acylated ornithine analogues of daptomycin were synthesized and tested for their antibacterial efficacy.     

U-2012: An improved Lowry protein assay, insensitive to sample color, offering reagent stability and enhanced sensitivity

Traditional colorimetric protein assays such as Biuret, Lowry, and modified Lowry (U-1988) are unsuitable for colored biological samples. Here we describe an improved Lowry protein assay (U-2012), which utilizes stable reagents and offers enhanced sensitivity over the U-1988 assay. U-2012 circumvents interference from colored pigments and other substances (for example sugars) bound to perchloric acid (PCA) precipitated proteins by hydrogen peroxide (H2O2) induced oxidation at 50°C. Unused hydrogen peroxide is neutralized with sodium pyruvate before protein estimation for a stable end color. The U-2012 assay is carried out on the PCA precipitated protein pellet after neutralization (with Na2CO3 plus NaOH), solubilization (in Triton-NaCl), decolorization (by H2O2) and pyruvate treatment. Protein contents in red wine and homogenates of beetroot and blueberry are calculated from standard curves established for various proteins and generated using a rectangular hyperbola with parameters estimated with Microsoft Excel's Solver add-in. The U-2012 protein assay represents an improvement over U-1988 and gives a more accurate estimation of protein content.     

Insights into the regulation of eukaryotic elongation factor 2 kinase and the interplay between its domains

eEF2K (eukaryotic elongation factor 2 kinase) is a Ca2+/CaM (calmodulin)-dependent protein kinase which regulates the translation elongation machinery. eEF2K belongs to the small group of so-called 'α-kinases' which are distinct from the main eukaryotic protein kinase superfamily. In addition to the α-kinase catalytic domain, other domains have been identified in eEF2K: a CaM-binding region, N-terminal to the kinase domain; a C-terminal region containing several predicted α-helices (resembling SEL1 domains); and a probably rather unstructured 'linker' region connecting them. In the present paper, we demonstrate: (i) that several highly conserved residues, implicated in binding ATP or metal ions, are critical for eEF2K activity; (ii) that Ca2+/CaM enhance the ability of eEF2K to bind to ATP, providing the first insight into the allosteric control of eEF2K; (iii) that the CaM-binding/α-kinase domain of eEF2K itself possesses autokinase activity, but is unable to phosphorylate substrates in trans; (iv) that phosphorylation of these substrates requires the SEL1-like domains of eEF2K; and (v) that highly conserved residues in the C-terminal tip of eEF2K are essential for the phosphorylation of eEF2, but not a peptide substrate. On the basis of these findings, we propose a model for the functional organization and control of eEF2K.     

Optimization of a novel kinase inhibitor scaffold for the dual inhibition of JAK2 and FAK kinases

The elaboration of a novel scaffold for the inhibition of JAK2 and FAK kinases was targeted in order to provide a dual inhibitor that could target divergent pathways for tumor cell progression.