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 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•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 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 μgml⁻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.

中文翻译：

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引入由酪氨酸解氨酶和 TAT 穿透肽结合介导的黑色素瘤 B16F10 细胞中黑色素生成抑制的递送系统

色素沉着过度症会对个人的生活质量产生负面影响，并可能导致情绪困扰。多年来，各种黑色素生成抑制剂（主要是酪氨酸酶抑制剂）被开发出来，其中大多数是低效或高毒性的。尽管通过底物流量偏差进行的代谢工程引起了相当大的兴趣，但缺乏开发基于 L-酪氨酸 (L-Tyr) 限制的黑素生成抑制剂的试验。我们提出了一种新的蛋白质黑色素生成抑制剂，称为酪氨酸解氨酶 (TAL)，这是一种催化 L-Tyr 转化为对香豆酸和氨的酶。由于细胞膜可以作为细胞内蛋白质传递的屏障，我们共价结合了来自球形红杆菌的重组 TAL酶(RsTAL) 到转录反式激活因子 (TAT) 细胞穿透肽 (CPP) 以提供细胞内递送。直接荧光显微镜证实，异源表达的 TAT-RsTAL 融合蛋白成功地递送到 B16F10 黑素细胞中，强度从 30 分钟增加到 180 分钟。TAT-RsTAL 显示出足够的细胞内活性，约为 0.83 ± 0.04 和 0.34 ± 0.03 nmol•mg ^-1 •s ^-1分别用于天然和包涵体提取的结合物。该偶联物以时间依赖性方式抑制 B16F10 细胞中的黑色素生物合成。在孵育 24、48 和 72 小时后，与未处理的对照组相比，黑色素积累分别受到 12.7 ± 6.2%、28.2 ± 5.7% 和 33.9 ± 2.9% 的抑制。即使在 72 小时后，L-Tyr 限制对高达 100 μgml ^{-1浓度的细胞活力也没有显着影响。}根据在本研究中观察到的偶联物的色素减退作用，TAT-RsTAL 可以作为黑色素生成抑制剂进行进一步研究。