Structural basis for the recognition and cleavage of histone H3 by cathepsin L.

Proteolysis of eukaryotic histone tails has emerged as an important factor in the modulation of cell-cycle progression and cellular differentiation. The recruitment of lysosomal cathepsin L to the nucleus where it mediates proteolysis of the mouse histone H3 tail has been described recently. Here, we report the three-dimensional crystal structures of a mature, inactive mutant of human cathepsin L alone and in complex with a peptide derived from histone H3. Canonical substrate-cathepsin L interactions are observed in the complex between the protease and the histone H3 peptide. Systematic analysis of the impact of posttranslational modifications at histone H3 on substrate selectivity suggests cathepsin L to be highly accommodating of all modified peptides. This is the first report of cathepsin L-histone H3 interaction and the first structural description of cathepsin L in complex with a substrate.

Displacement of the occluding loop by the parasite protein, chagasin, results in efficient inhibition of human cathepsin B.

Cathepsin B is a papain-like cysteine protease showing both endo- and exopeptidase activity, the latter due to a unique occluding loop that restricts access to the active site cleft. To clarify the mode by which natural protein inhibitors manage to overcome this obstacle, we have analyzed the structure and function of cathepsin B in complexes with the Trypanosoma cruzi inhibitor, chagasin. Kinetic analysis revealed that substitution of His-110e, which anchors the loop in occluding position, results in 3-fold increased chagasin affinity (Ki for H110A cathepsin B, 0.35 nm) due to an improved association rate (kon, 5 x 10(5) m(-1)s(-1)). The structure of chagasin in complex with cathepsin B was solved in two crystal forms (1.8 and 2.67 angstroms resolution), demonstrating that the occluding loop is displaced to allow chagasin binding with its three loops, L4, L2, and L6, spanning the entire active site cleft. The occluding loop is differently displaced in the two structures, indicating a large range of movement and adoption of conformations forced by the inhibitor. The area of contact is slightly larger than in chagasin complexes with the endopeptidase, cathepsin L. However, residues important for high affinity to both enzymes are mainly found in the outer loops L4 and L6 of chagasin. The chagasin-cathepsin B complex provides a structural framework for modeling and design of inhibitors for cruzipain, the parasite cysteine protease and a virulence factor in Chagas disease.

Crystal structure of the parasite protease inhibitor chagasin in complex with a host target cysteine protease.

Chagasin is a protein produced by Trypanosoma cruzi, the parasite that causes Chagas' disease. This small protein belongs to a recently defined family of cysteine protease inhibitors. Although resembling well-known inhibitors like the cystatins in size (110 amino acid residues) and function (they all inhibit papain-like (C1 family) proteases), it has a unique amino acid sequence and structure. We have crystallized and solved the structure of chagasin in complex with the host cysteine protease, cathepsin L, at 1.75 A resolution. An inhibitory wedge composed of three loops (L2, L4, and L6) forms a number of contacts responsible for high-affinity binding (K(i), 39 pM) to the enzyme. All three loops interact with the catalytic groove, with the central loop L2 inserted directly into the catalytic center. Loops L4 and L6 embrace the enzyme molecule from both sides and exhibit distinctly different patterns of protein-protein recognition. Comparison with a 1.7 A structure of uncomplexed chagasin, also determined in this study, demonstrates that a conformational change of the first binding loop (L4) allows extended binding to the non-primed substrate pockets of the enzyme active site cleft, thereby providing a substantial part of the inhibitory surface. The mode of chagasin binding is generally similar, albeit distinctly different in detail, when compared to those displayed by cystatins and the cysteine protease inhibitory p41 fragment of the invariant chain. The chagasin-cathepsin L complex structure provides details of how the parasite protein inhibits a host enzyme of possible importance in host defense. The high level of structural and functional similarity between cathepsin L and the T. cruzi enzyme cruzipain gives clues to how the cysteine protease activity of the parasite can be targeted. This information will aid in the development of synthetic inhibitors for use as potential drugs for the treatment of Chagas disease.

Quantitative continuous assay for hyaluronan synthase.

A rapid, continuous, and convenient three-enzyme coupled UV absorption assay was developed to quantitate the glucuronic acid and N-acetylglucosamine transferase activities of hyaluronan synthase from Pasteurella multocida (PmHAS). Activity was measured by coupling the UDP produced from the PmHAS-catalyzed transfer of UDP-GlcNAc and UDP-GlcUA to a hyaluronic acid tetrasaccharide primer with the oxidation of NADH. Using a fluorescently labeled primer, the products were characterized by gel electrophoresis. Our results show that a truncated soluble form of recombinant PmHAS (residues 1-703) can catalyze the glycosyl transfers in a time- and concentration-dependent manner. The assay can be used to determine kinetic parameters, inhibition constants, and mechanistic aspects of this enzyme. In addition, it can be used to quantify PmHAS during purification of the enzyme from culture media.

Use of anti-neoepitope antibodies for the analysis of degradative events in cartilage and the molecular basis for neoepitope specificity.

Degradation of the cartilage proteoglycan, aggrecan, is an essential aspect of normal growth and development, and of joint pathology. The roles of different proteolytic enzymes in this process can be determined from the sites of cleavage in the aggrecan core protein, which generates novel termini (neoepitopes). Antibodies specific for the different neoepitopes generated by such cleavage events provide powerful tools with which to analyse these processes. The same approach can be used to differentiate the processed, active forms of proteases from their inactive pro-forms. Since the proteolytic processing of these enzymes requires the removal of the inhibitory pro-region, it also results in the generation of N-terminal neoepitopes. Using the newborn rat long bone as a model system, it was shown that the active form of ADAMTS-4 [ADAM (a disintegrin and metalloproteinase) with thrombospondin motifs-4], but not ADAMTS-5, co-localizes with the aggrecan cleavage neoepitopes known to be produced by this metalloproteinase. Thus, in long bone growth, aggrecan turnover seems to be dependent on ADAMTS-4 activity. To demonstrate the molecular basis of the specificity of anti-neoepitope antibodies, the Fv region of a monoclonal antibody specific for a neoepitope generated by the ADAMTS-4-mediated cleavage of aggrecan has been modelled and the binding of the peptide epitope simulated. In the docked structure, the N-terminus of the peptide antigen is clearly buried in the binding-site cavity. The absence of an open cleft makes it impossible for the intact substrate to pass through the binding site, providing a rationale for the specificity of this class of antibodies.

Quantitative bead assay for hyaluronidase and heparinase I.

A novel, simple, and sensitive assay was developed to monitor, quantitatively, the hyaluronidase and heparinase I-catalyzed cleavage of fluoresceinamine-labeled hyaluronic acid and heparin, respectively. The fluoresceinamine-labeled substrates were hydrophobically absorbed onto 4-microm polystyrene beads. In the presence of enzyme, the change in fluorescence output of the substrate-absorbed beads was monitored in a noncontinuous manner using a flow cytometer. Our results show that hyaluronidase and heparinase I can cleave their respective substrates on the beads in a concentration- and time-dependent manner. The assay is suitable for detecting the presence of these glycosaminoglycan-degrading enzymes in cell lysates, extracts, or purified fractions, for quantifying their amounts, and for investigating the activity of potential inhibitors.

Structure and ligand-induced conformational change of the 39-kDa glycoprotein from human articular chondrocytes.

The 39-kDa human cartilage glycoprotein (HCGP39), a member of a novel family of chitinase-like lectins (Chilectins), is overexpressed in articular chondrocytes and certain cancers. Proposed functions of this protein include a role in connective tissue remodeling and defense against pathogens. Similar to other Chi-lectins, HCGP39 promotes the growth of connective tissue cells. The ability of HCGP39 to activate cytoplasmic signaling pathways suggests the presence of a ligand for this protein at the cell surface. There is currently no information regarding the identity of any physiological or pathological ligands of the Chi-lectins or the nature of the protein-ligand interaction. Here, we show that HCGP39 is able to bind chitooligosaccharides with micromolar affinity. Crystal structures of the native protein and a complex with GlcNAc8 show that the ligand is bound in identical fashion to family 18 chitinases. However, unlike the chitinases, binding of the oligosaccharide ligand to HCGP39 induces a large conformational change. Thus, HCGP39 could be a lectin that binds chitin-like oligosaccharide ligands and possibly plays a role in innate responses to chitinous pathogens, such as fungi and nematodes.

S2' substrate specificity and the role of His110 and His111 in the exopeptidase activity of human cathepsin B.

The ability of the lysosomal cysteine protease cathepsin B to function as a peptidyldipeptidase (removing C-terminal dipeptides) has been attributed to the presence of two histidine residues (His(110) and His(111)) present in the occluding loop, an extra peptide segment located in the primed side of the active-site cleft. Whereas His(111) is unpaired, His(110) is present as an ion pair with Asp(22) on the main body of the protease. This ion pair appears to act as a latch to hold the loop in a closed position. The exopeptidase activity of cathepsin B, examined using quenched fluorescence substrates, was shown to have a 20-fold preference for aromatic side chains in the P2' position relative to glutamic acid as the least favourable residue. Site-directed mutagenesis demonstrated that His(111) makes a positive 10-fold contribution to the exopeptidase activity, whereas His(110) is critical for this action with the Asp(22)-His(110) ion pair stabilizing the electrostatic interaction by a maximum of 13.9 kJ/mol (3.3 kcal/mol). These studies showed that cathepsin B is optimized to act as an exopeptidase, cleaving dipeptides from protein substrates in a successive manner, because of its relaxed specificity in P2' and its other subsites.