Search

Rapid evolution and high intrahost sequence diversity are hallmarks of human and simian immunodeficiency virus (HIV/SIV) infection. Minor viral variants have important implications for drug resistance, receptor tropism, and immune evasion. Here, we used ultradeep pyrosequencing to sequence complete HIV/SIV genomes, detecting variants present at a frequency as low as 1%. This approach provides a more complete characterization of the viral population than is possible with conventional methods, revealing low-level drug resistance and detecting previously hidden changes in the viral population. While this work applies pyrosequencing to immunodeficiency viruses, this approach could be applied to virtually any viral pathogen.

AMP activated protein kinase (AMPK) is a key regulator of cellular metabolism. AMPK activity is modulated in part by binding of AMP to the gamma-subunit of the kinase, which increases the activity of the catalytic alpha-subunit. Because increased AMPK activity in the liver and in skeletal muscle leads to increased fatty acid oxidation and decreased cholesterol and fatty acid biosynthesis, activators of AMPK are being sought for treatment of type-2 diabetes and other metabolic disorders. The unique mechanism of AMPK activation offers an opportunity to develop small molecules that directly upregulate AMPK activity, and there exists a need for simplified methods to identify and characterize small-molecules that show isoform-specific effects on AMPK. We have developed a suite of fluorescence-based assays to identify and characterize such compounds, and have used these to characterize and compare activity of recombinant AMPK alpha(1)beta(1)gamma(1) and alpha(2)beta(1)gamma(1) isoforms in response to small molecule activators and inhibitors.

Tryptases betaI and betaII were heterologously expressed and purified in yeast to functionally characterize the substrate specificity of each enzyme. Three positional scanning combinatorial tetrapeptide substrate libraries were used to determine the primary and extended substrate specificity of the proteases. Both enzymes have a strict primary preference for cleavage after the basic amino acids, lysine and arginine, with only a slight preference for lysine over arginine. betaI and betaII tryptase share similar extended substrate specificity, with preference for proline at P4, preference for arginine or lysine at P3, and P2 showing a slight preference for asparagine. Measurement of kinetic constants with multiple substrates designed for beta-tryptases reveal that selectivity is highly dependent on ground state substrate binding. Coupled with the functional determinants, structural determinants of tryptase substrate specificity were identified. Molecular docking of the preferred substrate sequence to the three-dimensional tetrameric tryptase structure reveals a novel extended substrate binding mode that involves interactions from two adjacent protomers, including P4 Thr-96', P3 Asp-60B' and Glu-217, and P1 Asp-189. Based on the determined substrate information, a mechanism-based tetrapeptide-chloromethylketone inhibitor was designed and shown to be a potent tryptase inhibitor. Finally, the cleavage sites of several physiologically relevant substrates of beta-tryptases show consistency with the specificity data presented here.

Human mast cell tryptase beta (EC 3.4.21.59) is a trypsin-like serine protease that is stored in and released from mast cell granules. This enzyme has been expressed in Pichia pastoris via homologous recombination of the cDNA coding for the mature active tryptase with the addition of a KEX 2 processing site into the Pichia genome. Cells producing recombinant human tryptase (rHT) were selected by screening with antibodies. Induction with methanol resulted in the secretion of rHT into the Pichia growth medium; tryptase activity was stabilized by the addition of heparin to the culture medium. Increasing levels of enzyme were detected in the medium for up to 3 days. Fully active enzyme was purified from the culture medium with a 100% yield of activity via a simple two-step procedure, with hydrophobic interaction chromatography followed by affinity chromatography on immobilized heparin. Bands of 33 (faint), 34.2, 35.9 and 50 kDa (diffuse) were observed on SDS/PAGE. These multiple forms were due to differences in post-translational glycosylation of asparagine residues, because enzymic deglycosylation resulted in only one band at 33 kDa. A single symmetrical peak with an estimated size of 197 kDa was obtained on gel filtration. Kinetic analyses in comparison with native human lung mast cell tryptase (HLT) yielded similar Km values, but the kcat of rHT was more than twice that of HLT.