Singlet-Oxygen Generation by Peroxidases and Peroxygenases for Chemoenzymatic Synthesis.

Singlet oxygen is a reactive oxygen species undesired in living cells but a rare and valuable reagent in chemical synthesis. We present a fluorescence spectroscopic analysis of the singlet-oxygen formation activity of commercial peroxidases and novel peroxygenases. Singlet-oxygen sensor green (SOSG) is used as fluorogenic singlet oxygen trap. Establishing a kinetic model for the reaction cascade to the fluorescent SOSG endoperoxide permits a kinetic analysis of enzymatic singlet-oxygen formation. All peroxidases and peroxygenases show singlet-oxygen formation. No singlet oxygen activity could be found for any catalase under investigation. Substrate inhibition is observed for all reactive enzymes. The commercial dye-decolorizing peroxidase industrially used for dairy bleaching shows the highest singlet-oxygen activity and the lowest inhibition. This enzyme was immobilized on a textile carrier and successfully applied for a chemical synthesis. Here, ascaridole was synthesized via enzymatically produced singlet oxygen.

Systemic administration of a proteasome inhibitor does not cause nigrostriatal dopamine degeneration.

Proteasomal dysfunction has been suggested to contribute to the degeneration of nigrostriatal dopamine neurons in Parkinson's disease. A recent study reported that systemic treatment of rats with the proteasome inhibitor Z-lle-Glu(OtBu)-Ala-Leu-al (PSI) causes a slowly progressive degeneration of nigrostriatal dopamine neurons, the presence of inclusion bodies in dopamine neurons, and motor impairment. We examined in vitro and in vivo the effects of PSI on nigrostriatal dopamine neurons. Mass spectrometric analysis was employed to verify the authenticity of the PSI compound. PSI was non-specifically toxic to neurons in ventral mesencephalic organotypic slice cultures, indicating that impairment of proteasome function in vitro is toxic. Moreover, systemic administration of PSI transiently decreased brain proteasome activity. Systemic treatment of rats with PSI did not, however, result in any biochemical or anatomical evidence of lesions of nigrostriatal dopamine neurons, nor were any changes in locomotor activity observed. These data suggest that systemic administration of proteasome inhibitors to normal adult rats does not reliably cause an animal model of parkinsonism.

Reciprocal effects of alpha-synuclein overexpression and proteasome inhibition in neuronal cells and tissue.

Defects in the 20S/26S proteasome and conformational changes in alpha-synuclein (alpha-syn) are implicated in the development of sporadic and familial cases of PD. The objective of this study was to evaluate whether alpha-syn affects proteolysis by the proteasome and, reciprocally, whether proteasome inhibition affects alpha-syn solubility and localization. Although alpha-syn directly inhibited purified 20S proteasomes reversibly in vitro, its overexpression in neuroblastoma (SH-SY5Y and SK-N-BE), embryonic kidney (HEK293) cells, or mouse brain did not affect proteasome activity. Proteasome inhibition with MG132 and epoxomicin in SH-SY5Y cells failed to induce alpha-syn aggregation, although it increased membrane bound forms of endogenous and overexpressed wild-type, but not mutant, alpha-syn. Concomitantly this treatment generated cytoplasmic alpha-syn inclusions devoid of polyubiquitin in a small percentage of cells. The combination of proteasome inhibition with serum deprivation, which induced oxidative stress and autophagy, caused the appearance of high molecular weight alpha-syn species, such as those found in Lewy bodies. Our data suggest that high concentrations of alpha-syn do not affect proteasome function in vivo, whereas proteasome inhibition can modify synuclein solubility, most prominently under conditions of cell stress which occur during aging. These results have implications for the convergence of age-related oxidative stress and impaired protein degradation in neurodegeneration.