4 research outputs found
The catalytic reaction mechanism of drosophilid alcohol dehydrogenases
The present review describes the current knowledge about the reaction mechanism of drosophilid alcohol dehydrogenases (DADH), a member of the short chain dehydrogenase/reductase (SDR) superfamily. Included is the binding order of the substrates to the enzyme, rate limiting steps, stereochemistry of the reaction, active site topology, role of important amino acids and water molecules in the reaction and pH dependence of kinetic coefficients. We focus on the contribution from steady state kinetics where alternative substrates, dead end and product inhibitors, isotopes and mutated DADHs have been used as well as on the contributions from X-ray crystallography, NMR and theoretical calculations. Furthermore, we also raise some open questions in order to fully understand the reaction mechanism of this enzyme
Molecular Interactions Stabilizing the Promatrix Metalloprotease-9·Serglycin Heteromer
Previous studies have shown that THP-1 cells produced an SDS-stable and reduction-sensitive complex between proMMP-9 and a chondroitin sulfate proteoglycan (CSPG) core protein. The complex could be reconstituted in vitro using purified serglycin (SG) and proMMP-9 and contained no inter-disulfide bridges. It was suggested that the complex involved both the FnII module and HPX domain of proMMP-9. The aims of the present study were to resolve the interacting regions of the molecules that form the complex and the types of interactions involved. In order to study this, we expressed and purified full-length and deletion variants of proMMP-9, purified CSPG and SG, and performed in vitro reconstitution assays, peptide arrays, protein modelling, docking, and molecular dynamics (MD) simulations. ProMMP-9 variants lacking both the FnII module and the HPX domain did not form the proMMP-9∙CSPG/SG complex. Deletion variants containing at least the FnII module or the HPX domain formed the proMMP-9∙CSPG/SG complex, as did the SG core protein without CS chains. The interacting parts covered large surface areas of both molecules and implicated dynamic and complementary ionic, hydrophobic, and hydrogen bond interactions. Hence, no short single interacting linear motifs in the two macromolecules could explain the strong SDS-stable and reduction-sensitive binding
Zinc-Chelating Compounds as Inhibitors of Human and Bacterial Zinc Metalloproteases
Inhibition of bacterial virulence is believed to be a new treatment option for bacterial
infections. In the present study, we tested dipicolylamine (DPA), tripicolylamine (TPA), tris pyridine
ethylene diamine (TPED), pyridine and thiophene derivatives as putative inhibitors of the bacterial
virulence factors thermolysin (TLN), pseudolysin (PLN) and aureolysin (ALN) and the human zinc
metalloproteases, matrix metalloprotease-9 (MMP-9) and matrix metalloprotease-14 (MMP-14). These
compounds have nitrogen or sulfur as putative donor atoms for zinc chelation. In general, the
compounds showed stronger inhibition of MMP-14 and PLN than of the other enzymes, with Ki
values in the lower µM range. Except for DPA, none of the compounds showed significantly stronger
inhibition of the virulence factors than of the human zinc metalloproteases. TPA and Zn230 were
the only compounds that inhibited all five zinc metalloproteinases with a Ki value in the lower µM
range. The thiophene compounds gave weak or no inhibition. Docking indicated that some of the
compounds coordinated zinc by one oxygen atom from a hydroxyl or carbonyl group, or by oxygen
atoms both from a hydroxyl group and a carbonyl group, and not by pyridine nitrogen as in DPA
and TPA
Iterative Design and in Vivo Evaluation of an Oncolytic Antilymphoma Peptide
Oncolytic
peptides represent a promising new strategy within the
field of cancer immunotherapy. Here we describe the systematic design
and evaluation of short antilymphoma peptides within this paradigm.
The peptides were tested in vitro and in vivo to identify a lead compound
for further evaluation as novel oncolytic immunotherapeutic. In vitro
tests revealed peptides with high activity against several lymphoma
types and low cytotoxicity toward normal cells. Treated lymphoma cells
exhibited a reduced mitochondrial membrane potential that resulted
in an irreversible disintegration of their plasma membranes. No caspase
activation or ultrastructural features of apoptotic cell death were
observed. One of these peptides, <b>11</b>, was shown to induce
complete tumor regression and protective immunity following intralesional
treatment of murine A20 B-lymphomas. Due to its selectivity for lymphoma
cells and its ability to induce tumor-specific immune responses, <b>11</b> has the potential to be used in intralesional treatment
of accessible lymphoma tumors