Proteases of Tannerella forsythia, a pathogen associated with periodontal disease, are implicated as virulence factors. Here, we characterized a matrix metalloprotease (MMP)-like enzyme of T. forsythia referred to as karilysin. Full length (without a signal peptide) recombinant karilysin (49.9 kDa) processed itself into the mature 18 kDa enzyme through a sequential autoproteolytic cleavages both at N-and C-terminal profragments. The first cleavage at the Asn14-Tyr15 peptide bond generated the fully active enzyme (47.9 kDa) and subsequent truncations at the C-termini did not affect proteolytic activity. Mutation of Tyr15 to Ala generated a prokarilysin variant that processed itself into the final 18 kDa form with greatly reduced kinetics. Inactive prokarilysin with the mutated catalytic Glu residue (E136A) was processed by active karilysin at the same sites as the active enzymes. The karilysin proteolytic activity and autoprocessing were inhibited by 1,10-phenanthroline and EDTA. Calcium ions were found to be important for both activity and thermal stability of karilysin. Using the CLiPS technology, specificity of karilysin was found to be similar to that of MMPs with preference for Leu/Tyr/Met at P1′ and Pro/Ala at P3. This specificity and the ability to degrade elastin, fibrinogen and fibronectin may contribute to the pathogenicity of periodontitis.
A quantitative methodology was developed to identify protein interactions in a broad range of cell types by using FRET between fluorescent proteins. Genetic fusions of a target receptor to a FRET acceptor and a large library of candidate peptide ligands to a FRET donor enabled high-throughput optical screening for optimal interaction partners in the cytoplasm of Escherichia coli. Flow cytometric screening identified a panel of peptide ligands capable of recognizing the target receptors in the intracellular environment. For both SH3 and PDZ domain-type target receptors, physiologically meaningful consensus sequences were apparent among the isolated ligands. The relative dissociation constants of interacting partners could be measured directly by using a dilution series of cell lysates containing FRET hybrids, providing a previously undescribed high-throughput approach to rank the affinity of many interaction partners. FRET hybrid interaction screening provides a powerful tool to discover protein ligands in the cellular context with potential applications to a wide variety of eukaryotic cell types.cell sorting ͉ protein-ligand interactions T he ability to identify and quantitatively characterize proteinprotein interactions in living cells is essential for developing a detailed, system-level understanding of cellular function. The yeast two-hybrid (Y2H) system has served as the primary genetic tool to discover potential biological interaction partners for an enormous number of proteins (1, 2). Application of Y2H assays to each of the nearly 6,000 yeast ORFs enabled construction of the first large-scale protein interaction network in yeast (3, 4), and similar interaction studies were performed subsequently in Drosophila melanogaster and Caenorhabditis elegans (5, 6). Human-protein interaction networks are less well characterized and present difficult challenges to interaction mapping approaches. In fact, only a small fraction of an estimated 10 5 to 10 6 human-proteome interactions (7) have been unambiguously identified, primarily by using Y2H and mass spectrometry approaches (7,8). Unfortunately, high-throughput Y2H assays are typically prone to a high frequency of false positives that complicate the interpretation of interaction data. In some studies, it has been estimated that Ͼ50% of putative interactions identified are false positives (9).Several important challenges remain in the elucidation of protein-protein interaction networks and their influence on cell function (10). Large-scale interaction maps can portray basic network structure but lack quantitative features needed to understand network function. A predictive understanding of network function likely will require the elucidation of equilibrium and kinetic properties within a network, including individual equilibrium-binding constants. Furthermore, existing highthroughput approaches (e.g., Y2H) are not well suited to investigate real-time dynamics in protein networks essential for understanding cell function (11) because detection, in these cases, is ba...
As a general strategy to selectively target antibody activity in vivo, a molecular architecture was designed to render binding activity dependent upon proteases in disease tissues. A protease-activated antibody (pro-antibody) targeting vascular cell adhesion molecule 1 (VCAM-1), a marker of atherosclerotic plaques, was constructed by tethering a binding site-masking peptide to the antibody via a matrix metalloprotease (MMP) susceptible linker. Pro-antibody activation in vitro by MMP-1 yielded a 200-fold increase in binding affinity and restored anti-VCAM-1 binding in tissue sections from ApoE(−/−) mice ex vivo. The pro-antibody was efficiently activated by native proteases in aorta tissue extracts from ApoE(−/−), but not from normal mice, and accumulated in aortic plaques in vivo with enhanced selectivity when compared to the unmodified antibody. Pro-antibody accumulation in aortic plaques was MMP-dependant, and significantly inhibited by a broad-spectrum MMP inhibitor. These results demonstrate that the activity of disease-associated proteases can be exploited to site-specifically target antibody activity in vivo.
The ability to engineer bioactive sites within the biopolymer collagen has significant potential to dictate cellular microenvironments and processes. We have developed a novel recombinant DNA platform that enables such molecular-level control over this important material. In this investigation, we demonstrated the production of synthetic human collagen using yeast strains that were engineered with human prolyl hydroxylase α and β genes integrated into the genome and a codon-optimized collagen gene carried on a plasmid. To understand the extent to which this synthetic collagen can mimic native human collagen, we examined the relationships between the structural topology and physical stability with the ability to support adhesion of HT-1080 cells. Characterization of these biopolymers included evaluation using circular dichroism spectroscopy, atomic force microscopy, and MTT metabolic activity assays. Although the apparent melting temperatures of the recombinant collagens were ∼3-5 less than native sources, the recombinant and native collagens exhibited comparable triple helical structure, polymeric dimensions, adsorption on polystyrene, and cellular adhesion properties below their respective melting temperature values. These results support the feasibility of producing molecularly-engineered collagens that can mimic native substrates for therapeutic and tissue engineering applications.
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