Niki Yoko, Adachi Naoko, Fukata Masaki, Fukata Yuko, Oku Shinichiro, Makino-Okamura Chieko, Takeuchi Seiji, Wakamatsu Kazumasa, Ito Shosuke, Declercq Lieve, Yarosh Daniel B, Mammone Tomas, Nishigori Chikako, Saito Naoaki, Ueyama Takehiko
Kobe Skin Research Department, Biosignal Research Center, Kobe University, Kobe, Japan; School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women's University, Nishinomiya, Japan.
Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe, Japan.
J Invest Dermatol. 2023 Feb;143(2):317-327.e6. doi: 10.1016/j.jid.2022.08.040. Epub 2022 Sep 5.
Palmitoylation is a lipid modification involving the attachment of palmitic acid to a cysteine residue, thereby affecting protein function. We investigated the effect of palmitoylation of tyrosinase, the rate-limiting enzyme in melanin synthesis, using a human three-dimensional skin model system and melanocyte culture. The palmitoylation inhibitor, 2-bromopalmitate, increased melanin content and tyrosinase protein levels in melanogenic cells by suppressing tyrosinase degradation. The palmitoylation site was Cysteine in the C-terminal cytoplasmic tail of tyrosinase. The nonpalmitoylatable mutant, tyrosinase (C500A), was slowly degraded and less ubiquitinated than wild-type tyrosinase. Screening for the Asp-His-His-Cys (DHHC) family of proteins for tyrosinase palmitoylation suggested that DHHC2, 3, 7, and 15 are involved in tyrosinase palmitoylation. Knockdown of DHHC2, 3, or 15 increased tyrosinase protein levels and melanin content. Determination of their subcellular localization in primary melanocytes revealed that DHHC2, 3, and 15 were localized in the endoplasmic reticulum, Golgi apparatus, and/or melanosomes, whereas only DHHC2 was localized in the melanosomes. Immunoprecipitation showed that DHHC2 and DHHC3 predominantly bind to mature and immature tyrosinase, respectively. Taken together, tyrosinase palmitoylation at Cysteine by DHHC2, 3, and/or 15, especially DHHC2 in trans-Golgi apparatus and melanosomes and DHHC3 in the endoplasmic reticulum and cis-Golgi apparatus, regulate melanogenesis by modulating tyrosinase protein levels.
棕榈酰化是一种脂质修饰，涉及将棕榈酸附着到半胱氨酸残基上，从而影响蛋白质功能。我们使用人三维皮肤模型系统和黑素细胞培养物，研究了黑色素合成中的限速酶酪氨酸酶的棕榈酰化作用。棕榈酰化抑制剂2-溴棕榈酸酯通过抑制酪氨酸酶降解，增加了黑素生成细胞中的黑色素含量和酪氨酸酶蛋白水平。棕榈酰化位点是酪氨酸酶C末端细胞质尾巴中的半胱氨酸。与野生型酪氨酸酶相比，不可棕榈酰化的突变体酪氨酸酶（C500A）降解缓慢且泛素化程度较低。对参与酪氨酸酶棕榈酰化的天冬氨酸-组氨酸-组氨酸-半胱氨酸（DHHC）蛋白家族进行筛选表明，DHHC2、3、7和15参与了酪氨酸酶的棕榈酰化。敲低DHHC2、3或15可增加酪氨酸酶蛋白水平和黑色素含量。对它们在原代黑素细胞中的亚细胞定位进行测定，结果显示DHHC2、3和15定位于内质网、高尔基体和/或黑素小体，而只有DHHC2定位于黑素小体。免疫沉淀表明，DHHC2和DHHC3分别主要与成熟和未成熟的酪氨酸酶结合。综上所述，DHHC2、3和/或15在半胱氨酸处对酪氨酸酶进行的棕榈酰化，尤其是反式高尔基体和黑素小体中的DHHC2以及内质网和顺式高尔基体中的DHHC3，通过调节酪氨酸酶蛋白水平来调控黑色素生成。
