Mycosporine-like amino acids (MAAs) are UVR-absorbing metabolites typically produced by cyanobacteria and marine algae, but their properties are not limited to direct sun screening protection. Herein, we examine the antioxidant activities of porphyra-334 and shinorine and demonstrate that these MAAs are prospective activators of the cytoprotective Keap1-Nrf2 pathway. The ability of porphyra-334 and shinorine to bind with Keap1 was determined using fluorescence polarization (FP) and thermal shift assays to detect Keap1 receptor antagonism. Concomitantly, the ability of porphyra-334 and shinorine to dissociate Nrf2 from Keap1 was confirmed also by measurement of increased mRNA expression of Nrf2 targeted genes encoding oxidative stress defense proteins in primary skin fibroblasts prior and post UVR exposure. Surprisingly, enhanced transcriptional regulation was only promoted by MAAs in cells after exposure to UVR-induced oxidative stress. Furthermore, the in-vitro antioxidant activities of porphyra-334 and shinorine determined by the DPPH free-radical quenching assay were low in comparison to ascorbic acid. However, their antioxidant capacity determined by the ORAC assay to quench free radicals via hydrogen atom transfer is substantial. Hence, the dual nature of MAAs to provide antioxidant protection may offer a prospective chemotherapeutic strategy to prevent or retard the progression of multiple degenerative disorders of ageing.
Hydrolysis of histones by proteinases from rat liver, skin and other sources was studied by using a rat thymus histone preparation as the substrate and polyacrylamide-gel electrophoresis and densitometric analysis as the methods to detect histone subtypes and their hydrolysis. The rat mast-cell proteinase I effectively hydrolysed histones except type H4. Thrombin hydrolysed effectively histones H1 and H2A, whereas plasmin hydrolysed all types of histones. Cathepsin D hydrolysed especially histone H2A. Cathepsins B and L hydrolysed all histones more slowly, and cathepsin H hydrolysed them extremely slowly. Epidermal aminoendopeptidase did not hydrolyse histones. Trypsin and chymotrypsin were used as reference enzymes, which hydrolysed all types of histones in very low concentrations. This study suggests that a variety of proteinases could play a role in histone hydrolysis. Hydrolysis of a specific subtype of histones, such as histone H2A at pH 6 by cathepsin D, may be directly involved in regulation of epidermal-cell differentiation.
An aminoendopeptidase isolated from 2-day-old rat epidermis was purified to apparent homogeneity by the procedures of ammonium sulfate fractionation, DE-52 column chromatography, Sephadex G-200 gel filtration, and CM-52 and DEAE-Sepharose 6B column chromatography. Enzymatic activity was exhibited only in the presence of sulfhydryl compounds and further enhanced by addition of 5 mM EDTA. It was inhibited by p-chloromercuribenzoate, other sulfhydryl blocking reagents, and o-phenanthroline. The monomer form of the enzyme is Mr = 52,000 +/- 2,300 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, but a native form was considered to be Mr = 400,000 +/- 26,000 having an isoelectric point of pH 5.25. Among synthetic substrates the enzyme hydrolyzed amino acid 2-naphthylamide derivatives and L-leucine amine (L-LeuNH2) most effectively. N-alpha-benzoyl-DL-arginine-2-naphthylamide (BANA) was the only endopeptidase substrate for the enzyme and a competitive inhibitor for its aminopeptidase activity. Protein substrates have not yet been found. The pH optimum is 7.5 and in a range of pH 6.5-7.5 it is stable at 37 degrees C for 30 min but loses about 50% of its activity at 50 degrees C.
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