Introducing a delivery system for melanogenesis inhibition in melanoma B16F10 cells mediated by the conjugation of tyrosine ammonia-lyase and a TAT penetrating peptide

Hyperpigmentation disorders negatively influence an individual's quality of life and may cause emotional distress. Over the years, various melanogenesis inhibitors (mainly tyrosinase inhibitors) have been developed, most of which with low efficacy or high toxicity. Although metabolic engineering by deviation in the flux of substrate is of considerable interest, trials to develop a melanogenesis inhibitor based on L-tyrosine (L-Tyr) restriction are missing. We propose a novel proteinaceous melanogenesis inhibitor called tyrosine ammonia-lyase (TAL), an enzyme that catalyzes the conversion of L-Tyr to p-coumaric acid and ammonia. Since the cell membrane can act as a barrier for intracellular protein delivery, we have covalently conjugated a recombinant TAL enzyme from Rhodobacter sphaeroides (RsTAL) to a TAT cell-penetrating peptide (CPP) to afford the intracellular delivery. The heterologously expressed TAT-RsTAL fusion protein was delivered successfully into B16F10 melanocytes as confirmed by the direct fluorescence microscopy with increased intensity from 30-180 min. TAT-RsTAL showed sufficient intracellular activity of about 0.83±0.04 and 0.34±0.03 nmol•mg^-1 •s^-1 for the native and inclusion body-extracted conjugates, respectively. The conjugate inhibited melanin biosynthesis in B16F10 cells in a time-dependent manner. Melanin accumulation was inhibited by 12.7±6.2%, 28.2±5.7%, and 33.9±2.9% compared to the non-treated control groups after 24, 48, and 72 h of incubation, respectively. L-Tyr restriction had no significant effect on the cell viability up to a concentration of 100 μgml^-1 even after 72 h. According to the observed hypopigmentary effect of the conjugate in this study, TAT-RsTAL can be suggested as a melanogenesis inhibitor for further investigations. This article is protected by copyright. All rights reserved.

Keywords: cell-penetrating peptide; melanin; metabolic engineering; protein delivery; tyrosine ammonia-lyase.