Global identification of peptidase specificity by multiplex substrate profiling.

We developed a simple and rapid multiplex substrate-profiling method to reveal the substrate specificity of any endo- or exopeptidase using liquid chromatography-tandem mass spectrometry sequencing. We generated a physicochemically diverse library of peptides by incorporating all combinations of neighbor and near-neighbor amino acid pairs into decapeptide sequences that are flanked by unique dipeptides at each terminus. Addition of a panel of evolutionarily diverse peptidases to a mixture of these tetradecapeptides generated information on prime and nonprime sites as well as on substrate specificity that matched or expanded upon known substrate motifs. This method biochemically confirmed the activity of the klassevirus 3C protein responsible for polypeptide processing and allowed granzyme B substrates to be ranked by enzymatic turnover efficiency using label-free quantitation of precursor-ion abundance. Additionally, the proteolytic secretions from schistosome parasitic flatworm larvae and a pancreatic cancer cell line were deconvoluted in a subtractive strategy using class-specific peptidase inhibitors.

Investigation of the proteolytic functions of an expanded cercarial elastase gene family in Schistosoma mansoni.

BACKGROUND: Cercarial elastase is the major invasive larval protease in Schistosoma mansoni, a parasitic blood fluke, and is essential for host skin invasion. Genome sequence analysis reveals a greatly expanded family of cercarial elastase gene isoforms in Schistosoma mansoni. This expansion appears to be unique to S. mansoni, and it is unknown whether gene duplication has led to divergent protease function. METHODS: Profiling of transcript and protein expression patterns reveals that cercarial elastase isoforms are similarly expressed throughout the S. mansoni life cycle. Computational modeling predicts key differences in the substrate-binding pockets of various cercarial elastase isoforms, suggesting a diversification of substrate preferences compared with the ancestral gene of the family. In addition, active site labeling of SmCE reveals that it is activated prior to exit of the parasite from its intermediate snail host. CONCLUSIONS: The expansion of the cercarial gene family in S. mansoni is likely to be an example of gene dosage. In addition to its critical role in human skin penetration, data presented here suggests a novel role for the protease in egress from the intermediate snail host. This study demonstrates how enzyme activity-based analysis complements genomic and proteomic studies, and is key in elucidating proteolytic function.

[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.