Karin U. Schallreuter

The role of thioredoxin reductase in the reduction of free radicals at the surface of the epidermis

A study of guinea pig and human skin in vivo has revealed that keratinocytes contain a thioenzyme which reduces radicals. This enzyme has been purified by affinity column chromatography and identified as thioredoxin reductase. In vivo and in vitro bioassays were performed by using a spin-labelled surfactant as the radical substrate, because it can diffuse through the stratum corneum and react by surface complexation with the epidermis and also on the outer plasma membrane of keratinocytes from cell cultures. Thioredoxin, the native substrate for thioredoxin reductase effectively competes for electrons with radical substrates. Nicotinamide adenine dinucleotide phosphate (NADPH) is the electron donating coenzyme in both the reduction of radicals and thioredoxin. Reduced thioredoxin has been shown to be an inhibitor of tyrosinase, whereas oxidized thioredoxin has no effect on this enzyme activity. Taken together these results indicate that the thioredoxin/thioredoxin reductase system plays an important role in preventing cell damage from UV-generated free radicals on the skin.

The role of calcium in the regulation of free radical reduction by thioredoxin reductase at the surface of the skin

Membrane associated thioredoxin reductase has been previously shown to reduce free radicals on the outer plasma membranes of human keratinocytes and melanocytes to provide a possible first line of defense against free radical damage at the surface of the skin. Preliminary experiments with cell cultures of human keratinocytes and melanocytes grown in serum-free medium showed that the enzyme activity depends on extracellular calcium concentration in the medium. Thioredoxin reductase activity at the surface of the skin, at the surface of human keratinocytes and melanocytes, and purified thioredoxin reductase from E. coli and adult human keratinocytes all exhibited calcium-dependent allosteric control. Since thioredoxin reductase contains two extremely reactive thiolate groups at the active site with pK values close to neutrality, both of these anions can form covalent complexes with N-ethylmaleimide by nucleophilic attack on the double bond. In our experiments we used spin-labeled maleimide [4-maleimido-tempo] to examine the local environment in the active site of thioredoxin reductase in the presence and absence of calcium. Both spin-labeled thioethers are distinguishable by EPR spectroscopy, with one site being significantly more immobilized than the other. Hence, it has been possible to observe direct evidence for active site closure by calcium. These results suggest that extracellular calcium may play an important role in regulation of thioredoxin reductase activity for the defense mechanism against UV-mediated free radical damage at the surface of human skin.

Thioredoxin reductase in control of the pigmentary system

Abstract Free radical reactions involving molecular oxygen have been implicated in a variety of diseases 1 as well as in the biologic process of aging. 2 The surface of the skin is especially susceptible to free radical damage as a consequence of photochemical reduction reactions between ultraviolet (UV) light and molecular oxygen producing active oxygen intermediates, such as Superoxide anion radical (O 2 ÷), peroxide (O 2 = ) and hydroxyl radical (OH•). 3 It is believed that the prevention of excessive oxygen radical synthesis in the dermis occurs by the natural UV-absorbing polymers eumelanin and pheomelanin, and that the biosynthesis of these pigments in the melanosomes depends on the activity of the copper-containing enzyme tyrosinase. 4 The expression of tyrosinase activity in human skin seems to vary according to the individuals skin type. 5 The connection between free radical defense on the external plasma membranes of cells in the epidermis and the biosynthesis of the melanins had escaped definition, although it had been obvious that such a connection had to exist between free radical damage to the skin (eg, sunburn) and pigmentation. 5,6 Only recently the function of membrane-associated thioredoxin reductase as a free radical scavenger at the surface of the skin and its possible role in the regulation of melanin biosynthesis have been suggested. 6 The following results have provided some direct evidence to support this postulation.

EF-Hands Calcium Binding Regulates the Thioredoxin Reductase/Thioredoxin Electron Transfer in Human Keratinocytes and Melanoma

Thioredoxin reductases purified from Escherichia coli from human metastatic melanoma tissue and from human keratinocytes are subject to allosteric inhibition by calcium. 45Calcium has been used to show that this enzyme contains a single binding site. Bound calcium does not exchange from thioredoxin reductase upon dialysis for 48 hours or upon exposure to 10(-3) M EGTA. An intelligenetics computer analysis yielded a single EF-hands calcium binding site on E. coli thioredoxin reductase with homology to the first EF-hands site on calmodulin. Calcium exchange from the enzyme requires the addition of the natural electron acceptor oxidized thioredoxin which causes a concentration dependent slow exchange. Due to the large conformational change caused by calcium binding to thioredoxin reductase it has been possible to separate Calcium-free and Calcium-bound enzyme by FPLC chromatography. Human keratinocytes contain 5% thioredoxin reductase in their acidic protein cytosol fraction. The influence of extracellular calcium concentration on the intracellular equilibrium between calcium bound versus calcium free thioredoxin reductase has been assessed. This equilibrium was shown to determine the redox status of keratinocytes via the reduction of thioredoxin. Our results provide the first evidence for calcium dependent regulation of redox conditions in the human epidermis.