Original Contribution UVA IRRADIATION INDUCES L-ISOASPARTYL FORMATION IN MELANOMA CELL PROTEINS STEFANIA D’ANGELO,* DIEGO INGROSSO,* BRUNELLA PERFETTO, ² ADONE BARONI, ‡ MARCELLO ZAPPIA,* LUCIA LUBRANO LOBIANCO,* MARIA ANTONIETTA TUFANO, ² and PATRIZIA GALLETTI* *Institute of Biochemistry of Macromolecules, ² Institute of Microbiology, and ‡ Department of Dermatology, Medical School, Second University of Naples, Naples, Italy (Received 2 January 2001; Accepted 23 February 2001) Abstract—It has been reported that UVA effects are partly mediated by production of reactive oxygen species. Moreover, oxidative stress increases protein damage, involving the occurrence of isoaspartyl residues, a product of protein deamidation/isomerization reactions. This work was undertaken in order to study the effects of UVA irradiation, mediated by oxidation, on sensitive protein targets. Melanoma cells exposed to UVA rays have been chosen as a model for monitoring the occurrence of L-isoaspartyl sites. A dramatic increase of these abnormal residues, specifically recognized and methylated by the enzyme L-isoaspartate(D-aspartate) O-methyltransferase (PCMT; EC 2.1.1.77), can be detected after exposure of M14 cells to raising doses of UVA. The effect of UVA on NO and TBARS accumulation, as well as on DNA fragmentation, has also been investigated. NO formation parallels the increase in isoaspartyl formation, while lipid peroxidation occurs only at the highest UVA doses. No DNA fragmentation has been detected under the employed experimental conditions. These results, as a whole, indicate that protein damages are one of the early events on UVA-induced cell injury. The endogenous activity of PCMT remains remarkably stable under UVA treatment, suggesting that this enzyme might play a crucial role in the repair and/or disposal of damaged proteins in UVA-irradiated cells. © 2001 Elsevier Science Inc. Keywords—Isoaspartyl methylation, Melanoma cells, Oxidative damage, Protein deamidation, Protein repair, UVA radiation, Free radicals INTRODUCTION Ultraviolet radiation represents 5% of solar electromag- netic emission. Only UVA (320 – 400 nm) and UVB (290 –320 nm) can penetrate stratosphere, with average irradiation values, at sea level, of 5.6 mW/cm 2 and 0.16 mW/cm 2 , respectively [1]. A number of epidemiological studies show a relationship between solar exposure and all types of skin cancer [2]. As a matter of fact, the recent lessening of the ozone layer, a natural screen against UVR, has been related with an increased incidence of skin damages [3– 4]. The interest in the study of UVA irradiation risks has recently grown due to applications in the phototherapy of psoriasis and vitiligo, in the treatment of a variety of dermatoses (UVA affects both epidermal and dermal cell population), as well as for cosmetic tanning [5–7]. Ultraviolet radiation and particularly UVA have a capacity to generate in cells reactive oxygen species (ROS), which can act as an initiator in the pathogenesis of UV-induced skin cancer and photoaging [8,9]. DNA has been identified as a molecular target of UVA-induced damage, consisting of single strand breaks, cyclobutane-type pyrimidine dimmers, and py- rimidine 6-4 adducts [1]. Other cellular components, as lipids [10,11], cell membranes [12], RNA [13], DNA repair enzymes, carrier proteins, and enzymes [14] can also be affected by UVA radiation. Nucleated mamma- lian cells exposed to UVA radiation show structural damage at the level of microtubules [15], nuclear mem- brane, and rough endoplasmic reticulum [16]. Moreover, sulphydryl groups of bovine serum albumin and human gamma-globulin are oxidized upon UVA irradiation in vitro [11]. Address correspondence to: Dr. Stefania D’Angelo, Dipartimento di Biochimica delle Macromolecole, Facolta ` di Medicina e Chirurgia, Via Costantinopoli 16, 80138 Napoli, Italy; Tel: +39 (081) 566-5816; Fax: +39 (081) 441-688; E-Mail: stefania.dangelo@unina2.it. Free Radical Biology & Medicine, Vol. 31, No. 1, pp. 1–9, 2001 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/01/$–see front matter PII S0891-5849(01)00518-4 1