Peroxidasin protein expression and enzymatic activity in metastatic melanoma cell lines are associated with invasive potential

Peroxidasin, a heme peroxidase, has been shown to play a role in cancer progression. mRNA expression has been reported to be upregulated in metastatic melanoma cell lines and connected to the invasive phenotype, but little is known about how peroxidasin acts in cancer cells. We have analyzed peroxidasin protein expression and activity in eight metastatic melanoma cell lines using an ELISA developed with an in-house peroxidasin binding protein. RNAseq data analysis confirmed high peroxidasin mRNA expression in the five cell lines classified as invasive and low expression in the three non-invasive cell lines. Protein levels of peroxidasin were higher in the cell lines with an invasive phenotype. Active peroxidasin was secreted to the cell culture medium, where it accumulated over time, and peroxidasin protein levels in the medium were also much higher in invasive than non-invasive cell lines. The only well-established physiological role of peroxidasin is in the formation of a sulfilimine bond, which cross-links collagen IV in basement membranes via catalyzed oxidation of bromide to hypobromous acid. We found that peroxidasin secreted from melanoma cells formed sulfilimine bonds in uncross-linked collagen IV, confirming peroxidasin activity and hypobromous acid formation. Moreover, 3-bromotyrosine, a stable product of hypobromous acid reacting with tyrosine residues, was detected in invasive melanoma cells, substantiating that their expression of peroxidasin generates hypobromous acid, and showing that it does not exclusively react with collagen IV, but also with other biomolecules

Antiseizure Activity of Novel γ-Aminobutyric Acid (A) Receptor Subtype-Selective Benzodiazepine Analogues in Mice and Rat Models

The antiseizure activity of benzodiazepines (BDZs) 1-5 in mice and rats as animal models is described. These BDZs have selective efficacy for α2β3γ2 and α3β3γ2 GABAA-receptors. Significant anticonvulsant activity with little or no motor impairment and therapeutic indexes (TI) of 2.8-44 (mice, ip) were observed for compounds 2-4 in the subcutaneous metrazole seizure (scMET) test. In rats orally (po) the TI was >5 to 105. These compounds represent novel leads in the search for anticonvulsants devoid of sedative, ataxic and amnestic side effects

J. Mol. Biol.

Cyanobacteria are shown to be unique in containing membrane- bound manganese superoxide dismutases (MnSOD). They are homodimeric type 2 membrane proteins that protect this Phototrophic organism against oxidative stress. We have determined, for the first time, the 2.0 Angstrom resolution structure of the catalytic portion of the MnSOD from the filamentous cyanobacterium Anabaena PCC 7120. Within each subunit, both the N-terminal helical hairpin (His94 and His145) and the C-terminal alpha/beta domain (His232 and Asp228) contribute ligands to the catalytic manganese site. Together with a water or hydroxide ion (OHx) a five-coordinated trigonal bipyramidal geometry is formed, with OHx and His90 forming the axial ligands and manganese shifted out of the equatorial plane in the direction of OHx. The ligands including OHx are tightly constrained by hydrogen bonding with surrounding residues either from the same monomer (Tyr98, Asn144, Trp194, Gln213, Val229, Trp230) or from the neighbouring subunit (Glu231, Tyr235). This underlines the important role of the symmetric dimeric structure of MnSODs in contributing elements to both the active site and the substrate funnel. The Mn...Mn distance (18.4Angstrom) is bridged by the hydrogen-bonded His232 of one monomer with Glu231 of the other monomer. A detailed discussion of the structure, a comparison with known structures of soluble MnSODs as well as a model of the cyanobacterial membrane-bound MnSOD is presented. (C) 2002 Elsevier Science Ltd. All rights reserved

The 2.0 angstrom resolution structure of the catalytic portion of a cyanobacterial membrane-bound manganese superoxide dismutase

Cyanobacteria are shown to be unique in containing membrane- bound manganese superoxide dismutases (MnSOD). They are homodimeric type 2 membrane proteins that protect this Phototrophic organism against oxidative stress. We have determined, for the first time, the 2.0 Angstrom resolution structure of the catalytic portion of the MnSOD from the filamentous cyanobacterium Anabaena PCC 7120. Within each subunit, both the N-terminal helical hairpin (His94 and His145) and the C-terminal alpha/beta domain (His232 and Asp228) contribute ligands to the catalytic manganese site. Together with a water or hydroxide ion (OHx) a five-coordinated trigonal bipyramidal geometry is formed, with OHx and His90 forming the axial ligands and manganese shifted out of the equatorial plane in the direction of OHx. The ligands including OHx are tightly constrained by hydrogen bonding with surrounding residues either from the same monomer (Tyr98, Asn144, Trp194, Gln213, Val229, Trp230) or from the neighbouring subunit (Glu231, Tyr235). This underlines the important role of the symmetric dimeric structure of MnSODs in contributing elements to both the active site and the substrate funnel. The Mn...Mn distance (18.4Angstrom) is bridged by the hydrogen-bonded His232 of one monomer with Glu231 of the other monomer. A detailed discussion of the structure, a comparison with known structures of soluble MnSODs as well as a model of the cyanobacterial membrane-bound MnSOD is presented. (C) 2002 Elsevier Science Ltd. All rights reserved

Common Reactivity and Properties of Heme Peroxidases: A DFT Study of Their Origin

Electronic structure calculations using the density-functional theory (DFT) have been performed to analyse the effect of water molecules and protonation on the heme group of peroxidases in different redox (ferric, ferrous, compounds I and II) and spin states. Shared geometries, spectroscopic properties at the Soret region, and the thermodynamics of peroxidases are discussed. B3LYP and M06-2X density functionals with different basis sets were employed on a common molecular model of the active site (Fe-centred porphine and proximal imidazole). Computed Gibbs free energies indicate that the corresponding aquo complexes are not thermodynamically stable, supporting the five-coordinate Fe(III) centre in native ferric peroxidases, with a water molecule located at a non-bonding distance. Protonation of the ferryl oxygen of compound II is discussed in terms of thermodynamics, Fe–O bond distances, and redox properties. It is demonstrated that this protonation is necessary to account for the experimental data, and computed Gibbs free energies reveal pKa values of compound II about 8.5–9.0. Computation indicates that the general oxidative properties of peroxidase intermediates, as well as their reactivity towards water and protons and Soret bands, are mainly controlled by the iron porphyrin and its proximal histidine ligand

Introduction of germline residues improves the stability of anti-HIV mAb 2G12-IgM.

Immunoglobulins M (IgMs) are gaining increasing attention as biopharmaceuticals since their multivalent mode of binding can give rise to high avidity. Furthermore, IgMs are potent activators of the complement system. However, they are frequently difficult to express recombinantly and can suffer from low conformational stability. Here, the broadly neutralizing anti-HIV-1 antibody 2G12 was class-switched to IgM and then further engineered by introduction of 17 germline residues. The impact of these changes on the structure and conformational stability of the antibody was then assessed using a range of biophysical techniques. We also investigated the effects of the class switch and germline substitutions on the ligand-binding properties of 2G12 and its capacity for HIV-1 neutralization. Our results demonstrate that the introduced germline residues improve the conformational and thermal stability of 2G12-IgM without altering its overall shape and ligand-binding properties. Interestingly, the engineered protein was found to exhibit much lower neutralization potency than its wild-type counterpart, indicating that potent antigen recognition is not solely responsible for IgM-mediated HIV-1 inactivation