Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.22.60 (caspase-7)
 920 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Caspases are a family of cysteine proteases that have critical roles in the apoptotic pathway. Caspase-7 is a well-known apoptotic effector that cleaves a variety of cellular substrates, and is known to be an important target in the treatment of many diseases. For efficient research, large amounts of the protein are required. However, it has been difficult to obtain sufficient quantities of either the precursor or active caspase-7 from Escherichia coli strain. In the present study, we constructed thrombin-activatable caspase-7 precursors by changing the auto-activation sites of the caspase-7 precursor into sequences susceptible to thrombin cleavage. These engineered precursors were highly expressed as soluble proteins in E. coli, and were easily purified by affinity chromatography (to levels of 10-15 mg per liter of E. coli culture), and were then readily activated by treatment with thrombin. In vitro cleavage assays and kinetic analyses revealed that the engineered active caspase-7 proteins had characteristics similar to those of wild-type caspase-7. This novel method is valuable for obtaining both precursor and active caspase-7, thereby contributing to the development of caspase-7-specific drugs to treat various diseases, including cancer and neurodegenerative conditions.
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PMID:Large-scale expression in Escherichia coli and efficient purification of precursor and active caspase-7 by introduction of thrombin cleavage sites. 1978 54

Many peptidases are thought to require non-active site interaction surfaces, or exosites, to recognize and cleave physiological substrates with high specificity and catalytic efficiency. However, the existence and function of protease exosites remain obscure owing to a lack of effective methods to identify and characterize exosite-interacting substrates. To address this need, we modified the cellular libraries of peptide substrates (CLiPS) methodology to enable the discovery of exosite-interacting peptide ligands. Invariant cleavage motifs recognized by the active sites of thrombin and caspase-7 were displayed on the outer surface of bacteria adjacent to a candidate exosite-interacting peptide. Exosite peptide libraries were then screened for ligands that accelerate cleavage of the active site recognition motif using two-color flow cytometry. Exosite CLiPS (eCLiPS) identified exosite-binding peptides for thrombin that were highly similar to a critical exosite interaction motif in the thrombin substrate, protease-activated receptor 1. Protease activity probes incorporating exosite-binding peptides were cleaved ten-fold faster than substrates without exosite ligands, increasing their sensitivity to thrombin activity in vitro. For comparison, screening with caspase-7 yielded peptides that modestly enhanced (two-fold) substrate cleavage rates. The eCLiPS method provides a new tool to profile the ligand specificity of protease exosites and to develop improved substrates.
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PMID:Identification of protease exosite-interacting peptides that enhance substrate cleavage kinetics. 2294 93

We have created models to predict cleavage sites for several human proteases including caspase-1, caspase-3, caspase-6, caspase-7, cathepsin B, cathepsin D, cathepsin G, cathepsin K, cathepsin L, elastase-2, granzyme A, granzyme B, matrix metallopeptidase-2 (MMP2), MMP7, MMP9, thrombin, and trypsin-1. Rather than representing the sequence pattern around the potential cleavage site through a series of flags with each flag representing one of the 20 standard amino acids, we first represent each amino acid by its calculated properties. For these calculated properties, we use validated cheminformatic descriptors, such as molecular weight, logP, and polar surface area, of the individual amino acids. Finally, the cleavage site-specific descriptors are calculated through various combinations of the individual amino acid descriptors for the residues surrounding the cleavage site. Some of these combinations do not take into account the location of the residue, as long as it is in a prescribed neighborhood of the potential cleavage site, whereas others are sensitive to the precise order of the residues in the sequence. The key advantage of this approach is that it allows one to perform meaningful calculations with nonstandard amino acids for which little or no data exists. Finally, using both docking and molecular dynamics simulations, we examine the potential for and limitations of protease crystal structures to impact the design of proteolytically stable peptides.
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PMID:A combined cheminformatic and bioinformatic approach to address the proteolytic stability challenge in peptide-based drug discovery. 2627 Mar 98