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ABSTRACT: Tyrosinase and tyrosinase-related protein-2 (Tyrp2) play a pivotal role in melanin production within melanocytes. Both share a high degree of structural and sequence similarity, with a Root-Mean-Square Deviation (RMSD) of 0.75 Å and 62% sequence similarity, including 43% sequence identity. Despite these similarities, differences in the allosteric sites, particularly the residues that constitute these sites, contribute to distinct regulatory mechanisms. This study elucidates the contrasting allosteric modulation of tyrosinase and Tyrp2 by two flavonoids: (a) luteolin and (b) apigenin, respectively. Luteolin demonstrated its allosteric inhibitory property against tyrosinase by disturbing the spatial arrangement of histidine residues critical for copper coordination, essential for enzymatic activity. Conversely, apigenin serves as an allosteric activator for Tyrp2, enhancing structural stability and maintaining the active site conformation. Despite the structural resemblance between luteolin and apigenin, differentiated by a single hydroxyl group, their impact on melanin synthesis diverges — luteolin inhibits, whereas apigenin stimulates the process. Both showed high binding affinity at the allosteric site of tyrosinase and Tyrp2. Luteolin significantly alters the spatial arrangement of key histidine residues (HIS180, HIS202 and HIS211), forming coordination bonds in tyrosinase, affecting copper coordination and coordination with the peroxide (O₂2⁻) ion. However, apigenin did not remain bound to tyrosinase for an extended period, ruling out its role as an allosteric modulator. In contrast, apigenin stayed at the allosteric site of the Tyrp2 protein and showed a positive allosteric regulator role. The findings offer a mechanistic view of melanin synthesis regulation, demonstrating the potential of subtle structural variations in modulators to selectively influence enzyme activity in related biosynthetic pathways. This underscores the intricate control within melanogenic systems and the possibility of targeting these pathways for therapeutic and cosmetic applications.

Ligusticum sinensis Oliv. (LSO) is an herbal drug commonly used as a topical treatment of epidermal hyperdepigmentation in Chinese medicine. However, the mechanism underlying the depigmentation by LSO is still unclear. The purpose of this study was to investigate the effects of LSO on the process of melanogenesis and its possible underlying mechanism. Suppressed DOPA oxidase activity of mushroom tyrosinase was first noted when incubated with aqueous extracts of LSO, demonstrating the direct inhibitory effect of LSO on mushroom tyrosinase. Further experiments were carried out in murine B16/F10 melanoma cells and the effects of LSO extract on melanin formation, tyrosinase activity and tyrosinase gene expression were tested. Under conditions without affecting the viability of murine B16/F10 melanoma cells, LSO extract significantly reduced the cellular melanin content in a dose-dependent manner. The DOPA oxidase activity of tyrosinase in B16/F10 cells was dose-dependently inhibited by LSO treatment, possibly mediated by the suppressed tyrosinase mRNA expression in LSO-treated B16/F10 cells. In conclusion, the inhibitory effect of LSO on melanogenesis is likely associated with decreased DOPA oxidase activity of tyrosinase that is most likely the result of the down-regulation of tyrosinase mRNA expression.

Down-regulation of melanin synthesis and\or melanin transfer are\is required for recovery of pigmentary disorders. It is known that direct inhibitors of tyrosinase, the key enzyme in melanin synthesis, such as hydroquinone with a phenol structure, suppress melanin synthesis. We screened some herbal monomers using human melanocytes and found that paeonol, a major phenolic component of Moutan Cortex, down-regulated melanin synthesis. The melanin synthesis and tyrosinase activity were inhibited by paeonol in a dose-dependent manner. The expression levels of tyrosinase mRNA and protein were also reduced by paeonol. We further studied the inhibitory effects of paeonol on melanin transfer in co-culture of melanocytes and keratinocytes. More than 50% of inhibition of melanin transfer was observed at concentration of 200 μM of paeonol and the increased melanin transfer induced by SLIGRL, the PAR-2 activating peptide, was also reduced by paeonol. However, paeonol did not influence the expression level of PAR-2 mRNA in co-culture cells. These results indicate that the depigmenting effect of paeonol might be due to its down-regulation of melanogenesis and melanin transfer.

Tyrosinase and its transcriptional regulator microphthalmia-associated transcription factor (MITF) play critical roles in regulation of melanogenesis, and are required for environmental cues or agents in modulation of melanin synthesis. Identifying the signals regulating tyrosinase and MITF is crucial to understanding how pigmentation responds to extracellular stimuli. In this report, we discovered that paeonol down-regulated melanin production via decreasing MITF expression and consequent mRNA and protein levels of tyrosinase. We also found that paeonol reduced phosphorylation of a cAMP responsive element binding protein (phospho-CREB), which binds and activates MITF. A selective inhibitor of c-jun N-terminal or stress-activated protein kinases (JNK/SAPK)-SP600125 significantly reversed paeonol-induced down-regulation of melanogenesis. Inhibition of cAMP/PKA pathway intensified the hypopigmentation response to paeonol. These results identify a mechanism in which paeonol induces the down-regulation of melanogenesis through inhibition of CREB phosphorylation, leading to the expression reduction of MITF and subsequently tyrosinase. The key kinase mediating the effects of paeonol on melanogenesis in B16F10 cells is JNK/SAPK. Additionally, the cAMP/PKA pathway may take part in this process.

For cosmetic reasons, the demand for effective and safe skin-whitening agents is high. Since the key enzyme in the melanin synthetic pathway is tyrosinase, many depigmenting agents in the treatment of hyperpigmentation act as tyrosinase inhibitors. In this study, we have investigated the hypo-pigmentary mechanism of royal jelly in a mouse melanocyte cell line, B16F1. Treatment of B16F1 cells with royal jelly markedly inhibited melanin biosynthesis in a dose-dependent manner. Decreased melanin content occurred through the decrease of tyrosinase activity. The mRNA levels of tyrosinase were also reduced by royal jelly. These results suggest that royal jelly reduces melanin synthesis by down-regulation of tyrosinase mRNA transcription and serves as a new candidate in the design of new skin-whitening or therapeutic agents.

A phenol biosensor based on the skillful immobilization of tyrosinase on zinc oxide (ZnO) nanorods was proposed. The ZnO nanorods fabricated by a simple vapor-phase transport method possess a high aspect ratio, good electron communication, chemical purity, smooth and positive charged surface and are ready for immobilization of biochemicals with low isoelectric point (IEP). Electrochemical measurement and Scanning Electron Microscopic (SEM) analysis showed that the enzyme of tyrosinase with IEP 4.5 can be adsorbed on the surface of ZnO nanorods and kept its bioactivity of the oxidation of phenol to a large extent. This led us to develop phenol biosensor with good stability and reproducibility. The proposed method creates a new way to develop biosensors using nanostructured materials with high IEP.