Introducing a delivery system for melanogenesis inhibition in melanomaB16F10cells mediated by the conjugation of tyrosine ammonia-lyase and aTAT-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 top-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 fromRhodobacter sphaeroides(RsTAL) to a trans-activator of transcription (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 to 180 min. TAT-RsTAL showed sufficient intracellular activity of about 0.83 +/- 0.04 and 0.34 +/- 0.03 nmol center dot mg(-1)center dot 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 nontreated control groups after 24, 48, and 72 hr of incubation, respectively. L-Tyr restriction had no significant effect on the cell viability up to a concentration of 100 mu gml(-1)even after 72 hr. According to the observed hypopigmentary effect of the conjugate in this study, TAT-RsTAL can be suggested as a melanogenesis inhibitor for further investigations.

Automated summary

This study introduces a novel proteinaceous melanogenesis inhibitor called TAL, successfully delivered into melanocytes and demonstrated effective inhibition of melanin biosynthesis.