49 research outputs found
Dissecting the Active Site of the Collagenolytic Cathepsin L3 Protease of the Invasive Stage of Fasciola hepatica
Background: A family of secreted cathepsin L proteases with differential activities is essential for host colonization and survival in the parasitic flatworm Fasciola hepatica. While the blood feeding adult secretes predominantly FheCL1, an enzyme with a strong preference for Leu at the S2 pocket of the active site, the infective stage produces FheCL3, a unique enzyme with collagenolytic activity that favours Pro at P2. Methodology/Principal Findings: Using a novel unbiased multiplex substrate profiling and mass spectrometry methodology (MSP-MS), we compared the preferences of FheCL1 and FheCL3 along the complete active site cleft and confirm that while the S2 imposes the greatest influence on substrate selectivity, preferences can be indicated on other active site subsites. Notably, we discovered that the activity of FheCL1 and FheCL3 enzymes is very different, sharing only 50% of the cleavage sites, supporting the idea of functional specialization. We generated variants of FheCL1 and FheCL3 with S2 and S3 residues by mutagenesis and evaluated their substrate specificity using positional scanning synthetic combinatorial libraries (PS-SCL). Besides the rare P2 Pro preference, FheCL3 showed a distinctive specificity at the S3 pocket, accommodating preferentially the small Gly residue. Both P2 Pro and P3 Gly preferences were strongly reduced when Trp67 of FheCL3 was replaced by Leu, rendering the enzyme incapable of digesting collagen. In contrast, the inverse Leu67Trp substitution in FheCL1 only slightly reduced its Leu preference and improved Pro acceptance in P2, but greatly increased accommodation of Gly at S3. Conclusions/Significance: These data reveal the significance of S2 and S3 interactions in substrate binding emphasizing the role for residue 67 in modulating both sites, providing a plausible explanation for the FheCL3 collagenolytic activity essential to host invasion. The unique specificity of FheCL3 could be exploited in the design of specific inhibitors selectively directed to specific infective stage parasite proteinases. © 2013 Corvo et al
Investigation of the Proteolytic Functions of an Expanded Cercarial Elastase Gene Family in Schistosoma mansoni
Schistosome parasites are a major cause of disease in the developing world. The larval stage of the parasite transitions between an intermediate snail host and a definitive human host in a dramatic fashion, burrowing out of the snail and subsequently penetrating human skin. This process is facilitated by secreted proteases. In Schistosoma mansoni, cercarial elastase is the predominant secreted protease and essential for host skin invasion. Genomic analysis reveals a greatly expanded cercarial elastase gene family in S. mansoni. Despite sequence divergence, SmCE isoforms show similar expression profiles throughout the S. mansoni life cycle and have largely similar substrate specificities, suggesting that the majority of protease isoforms are functionally redundant and therefore their expansion is an example of gene dosage. However, activity-based profiling also indicates that a subset of SmCE isoforms are activated prior to the parasite's exit from its intermediate snail host, suggesting that the protease may also have a role in this process
[Mn(bpb)(DMAP)(NO)], an {Mn–NO} 6
The structure of the title compound octaÂkisÂ{[4-(dimethylÂamino)Âpyridine](nitrosÂyl)[N,N′-(o-phenylÂene)bisÂ(pyridine-2-carboxamidato)]manganese(II)} ethanol heptaÂsolvate 3.5-hydrate, [Mn(C(18)H(12)N(4)O(2))(C(7)H(10)N(2))(NO)](8)·7C(2)H(5)OH·3.5H(2)O, or 8[Mn(bpb)(DMAP)(NO)]·7EtOH·3.5H(2)O, is an unusual example of a structure with Z′ = 8. The tetraÂdentate bpb ligand, together with the nitrosyl and dimethylÂaminoÂpyridine ligands, gives rise to a distorted octaÂhedral coordination environment for the Mn(II) ion. The average Mn—N((N=O)) bond length is 1.631 (13) Å. The eight molÂecules in the asymmetric unit differ mainly in the rotation of the DMAP pyridine plane with respect to a reference plane of the Mn and three N atoms, one of which is the N atom of the NO group. The dihedral angles between the normals to these planes range from a minimum of 28.0 (2)° to a maximum of 64.2 (2)°. There are also some differences in O—H⋯O hydrogen bonding interÂactions. For example, of the sixteen C=O acceptors, there are seven different interÂactions with EtOH donors and two interÂactions with H(2)O donors. The crystal studied was found to be a two-component twin, with a 179.9° rotation about the real axis [−0.535, 0.004, 1.000]. Due to the presence of a superlattice and, consequently, the large number of weak reflections, the refinement utilized rigid solvate groups and isotropic displacement parameters for all except the Mn atoms. H atoms were not located for hydrate molecules
[Mn(bpb)(DMAP)(NO)], an {Mn-NO} nitrosyl with Z' = 8.
The structure of the title compound octa-kis-{[4-(dimethyl-amino)-pyridine](nitros-yl)[N,N'-(o-phenyl-ene)bis-(pyridine-2-carboxamidato)]manganese(II)} ethanol hepta-solvate 3.5-hydrate, [Mn(C(18)H(12)N(4)O(2))(C(7)H(10)N(2))(NO)](8)·7C(2)H(5)OH·3.5H(2)O, or 8[Mn(bpb)(DMAP)(NO)]·7EtOH·3.5H(2)O, is an unusual example of a structure with Z' = 8. The tetra-dentate bpb ligand, together with the nitrosyl and dimethyl-amino-pyridine ligands, gives rise to a distorted octa-hedral coordination environment for the Mn(II) ion. The average Mn-N((N=O)) bond length is 1.631 (13) Å. The eight mol-ecules in the asymmetric unit differ mainly in the rotation of the DMAP pyridine plane with respect to a reference plane of the Mn and three N atoms, one of which is the N atom of the NO group. The dihedral angles between the normals to these planes range from a minimum of 28.0 (2)° to a maximum of 64.2 (2)°. There are also some differences in O-H⋯O hydrogen bonding inter-actions. For example, of the sixteen C=O acceptors, there are seven different inter-actions with EtOH donors and two inter-actions with H(2)O donors. The crystal studied was found to be a two-component twin, with a 179.9° rotation about the real axis [-0.535, 0.004, 1.000]. Due to the presence of a superlattice and, consequently, the large number of weak reflections, the refinement utilized rigid solvate groups and isotropic displacement parameters for all except the Mn atoms. H atoms were not located for hydrate molecules
[Mn(bpb)(DMAP)(NO)], an {Mn–NO}6 nitrosyl with Z′ = 8
The structure of the title compound octakis{[4-(dimethylamino)pyridine](nitrosyl)[N,N′-(o-phenylene)bis(pyridine-2-carboxamidato)]manganese(II)} ethanol heptasolvate 3.5-hydrate, [Mn(C18H12N4O2)(C7H10N2)(NO)]8·7C2H5OH·3.5H2O, or 8[Mn(bpb)(DMAP)(NO)]·7EtOH·3.5H2O, is an unusual example of a structure with Z′ = 8. The tetradentate bpb ligand, together with the nitrosyl and dimethylaminopyridine ligands, gives rise to a distorted octahedral coordination environment for the Mn(II) ion. The average Mn—N(N=O) bond length is 1.631 (13) Å. The eight molecules in the asymmetric unit differ mainly in the rotation of the DMAP pyridine plane with respect to a reference plane of the Mn and three N atoms, one of which is the N atom of the NO group. The dihedral angles between the normals to these planes range from a minimum of 28.0 (2)° to a maximum of 64.2 (2)°. There are also some differences in O—H...O hydrogen bonding interactions. For example, of the sixteen C=O acceptors, there are seven different interactions with EtOH donors and two interactions with H2O donors. The crystal studied was found to be a two-component twin, with a 179.9° rotation about the real axis [−0.535, 0.004, 1.000]. Due to the presence of a superlattice and, consequently, the large number of weak reflections, the refinement utilized rigid solvate groups and isotropic displacement parameters for all except the Mn atoms. H atoms were not located for hydrate molecules
Nitric oxide-donating materials and their potential in pharmacological applications for site-specific nitric oxide delivery
Syntheses, Structures, and Photochemistry of Manganese Nitrosyls Derived from Designed Schiff Base Ligands: Potential NO Donors That Can Be Activated by Near-Infrared Light
Peptide Length and Leaving-Group Sterics Influence Potency of Peptide Phosphonate Protease Inhibitors
The ability to follow enzyme activity in a cellular context represents a challenging technological frontier that impacts fields ranging from disease pathogenesis to epigenetics. Activity-based probes (ABPs) label the active form of an enzyme via covalent modification of catalytic residues. Here we present an analysis of parameters influencing potency of peptide phosphonate ABPs for trypsin-fold S1A proteases, an abundant and important class of enzymes with similar substrate specificities. We find that peptide length and stability influence potency more than sequence composition and present structural evidence that steric interactions at the prime-side of the substrate-binding cleft affect potency in a protease-dependent manner. We introduce guidelines for the design of peptide phosphonate ABPs and demonstrate their utility in a live-cell labeling application that specifically targets active S1A proteases at the cell surface of cancer cells