Importance of lysosomal cysteine proteases in lung disease

The human lysosomal cysteine proteases are a family of 11 proteases whose members include cathepsins B, C, H, L, and S. The biology of these proteases was largely ignored for decades because of their lysosomal location and the belief that their function was limited to the terminal degradation of proteins. In the past 10 years, this view has changed as these proteases have been found to have specific functions within cells. This review highlights some of these functions, specifically their roles in matrix remodeling and in regulating the immune response, and their relationship to lung diseases

SmCL3, a Gastrodermal Cysteine Protease of the Human Blood Fluke Schistosoma mansoni

Parasitic infection caused by blood flukes of the genus Schistosoma is a major global health problem. More than 200 million people are infected. Identifying and characterizing the constituent enzymes of the parasite's biochemical pathways should reveal opportunities for developing new therapies (i.e., vaccines, drugs). Schistosomes feed on host blood, and a number of proteolytic enzymes (proteases) contribute to this process. We have identified and characterized a new protease, SmCL3 (for Schistosoma mansoni cathepsin L3), that is found within the gut tissue of the parasite. We have employed various biochemical and molecular biological methods and sequence similarity analyses to characterize SmCL3 and obtain insights into its possible functions in the parasite, as well as its evolutionary position among cathepsin L proteases in general. SmCL3 hydrolyzes major host blood proteins (serum albumin and hemoglobin) and is expressed in parasite life stages infecting the mammalian host. Enzyme substrate specificity detected by positional scanning-synthetic combinatorial library was confirmed by molecular modeling. A sequence analysis placed SmCL3 to the cluster of other cathepsins L in accordance with previous phylogenetic analyses

The Many Faces of Platelet Glycoprotein Ibα - Thrombin Interaction

The platelet glycoprotein receptor regulates the adhesion of blood platelets to damaged blood vessel walls and the subsequent platelet aggregation. One of the subunits, platelet glycoprotein Ibα (GpIbα), binds thrombin, a serine protease with both proc

The many faces of platelet glycoprotein Ib alpha - thrombin interaction

The platelet glycoprotein receptor regulates the adhesion of blood platelets to damaged blood vessel walls and the subsequent platelet aggregation. One of the subunits, platelet glycoprotein Ib alpha (GpIb alpha), binds thrombin, a serine protease with both procoagulant and anticoagulant activities. Two groups reported the crystal structures of the complex between thrombin and the N-terminal extracellular domain (leucine-rich repeat [LRR] domain) of GpIb alpha. In both these structures, GpIb alpha was reported to bind two thrombin molecules, but both the primary and secondary thrombin binding sites differed between them. We performed a detailed comparison of the two structures to look for insights that may explain the differences. Our results show that the 1: 1 GpIb alpha-thrombin complex detected in solution between the crystallized proteins is likely the only strong interaction. The anionic sequence ( residues 268-284) of GpIb alpha is likely responsible for the initial interaction with thrombin and the interaction with the rest of LRR domain of GpIb alpha occurs subsequently and may alternate between two or more different binding modes. Our modelling suggests the interaction between GpIb alpha and thrombin is highly pH-dependent and a small change in pH is likely to contribute to the formation of alternate binding modes. The differences in the crystal structures reported for the GpIb alpha-thrombin complex suggest a fascinating plasticity in this protein-protein interaction that may be biologically significant

Molecular basis of Colorado potato beetle adaptation to potato plant defence at the level of digestive cysteine proteinases

Potato synthesises high levels of proteinase inhibitors in response to insect attack. This can adversely affect protein digestion in the insects, leading to reduced growth, delayed development and lowered fecundity. Colorado potato beetle overcomes this defence mechanism by changing the composition of its digestive proteinases. The induced cysteine proteinases in the adapted gut sustain a normal rate of protein hydrolysis either by inactivating the inhibitors by cleavage or by insensitivity to the inhibitors as a result of high K(i)s. In this Study cDNA clones of cysteine proteinases in adapted guts were isolated by nested PCR on the basis of N-terminal sequences previously determined for purified enzymes (Gruden et al., 2003). The cysteine proteinase cDNAs call be classified into three groups: intestains A, B and C. The amino acid identity is more than 91% within and 35-62%, between the groups. They share 43-50% identity to mammalian cathepsins S, L, K, H, J and cathepsin-like enzymes from different arthropods. Homology modelling predicts that intestains A, B and C follow the general fold of papain-like proteinases. Intestains from each group, however, differ in some specific structural characteristics in the S1 and S2 binding sites that could influence enzyme-inhibitor interaction and thus, provide different mechanisms of resistance to inhibitors for the different enzymes. Gene expression analysis revealed that the intestains A and C, but not B, are induced twofold by potato plants with high levels of proteinase inhibitors. (C) 2004 Elsevier Ltd. All rights reserved

A medium or high throughput protein refolding assay

Expression of insoluble protein in E. Coli is a major bottleneck of high throughput structural biology projects. Refolding proteins into native conformations from inclusion bodies could significantly increase the number of protein targets that can be taken on to structural studies

A General Target Selection Method for Crystallographic Proteomics

Increasing the success in obtaining structures and maximizing the value of the structures determined are the two major goals of target selection in structural proteomics. This chapter presents an efficient and flexible target selection procedure supplemented with a Web-based resource that is suitable for small- to large-scale structural genomics projects that use crystallography as the major means of structure determination. Based on three criteria, biological significance, structural novelty, and "crystallizability," the approach first removes (filters) targets that do not meet minimal criteria and then ranks the remaining targets based on their "crystallizability" estimates. This novel procedure was designed to maximize selection efficiency, and its prevailing criteria categories make it suitable for a broad range of structural proteomics projects