Preparation of Purified Tyrosinase Devoid of Dopachrome Tautomerase From Mammalian Malignant Melanocytes

Although tyrosinase has been considered for a long time the only enzyme involved in mammalian melanosynthesis, it has been shown that mouse melanoma melanosomes contain high levels of dopachrome tautomerase (DCT²), an enzyme catalyzing DC tautomerization to DHICA. At least in B16 mouse melanoma, DCT is present in higher catalytic amounts than tyrosinase. Moreover, it can be anticipated that tyrosinase and DCT should be very difficult to resolve by most conventional biochemical techniques because of the structural similarity between these enzymes, as predicted from the sequence of their corresponding cDNAs. It is shown that the presence of DCT can cause serious artifacts when tyrosinase activity is determined by most of the currently available methods, such as the Dopa oxidase and melanin formation assays. We describe a simple and convenient method for the preparation of tyrosinase devoid of DCT. The method takes advantage of the different thermal stability of both enzymes. Heating of crude melanosomal extracts at 60°C for 1 hr results in a complete denaturation of DCT, while tyrosinase activity is recovered almost quantitatively. The resulting tyrosinase preparation is considerably purified and the electrophoretic, immunologic and kinetic characteristics of the enzyme appear unaltered. Because if its high yield and simplicity, the method can be used for the microscale partial purification of DCT-free tyrosinase from mammalian malignant melanocytes grown in culture.     

Complex Formation Between Mushroom Tyrosinase and Manduca Dopachrome Isomerase

Melanin biosynthesis in animals is initiated by the ubiquitously present tyrosinase and is aided by dopachrome isomerase. We have characterized a novel dopachrome isomerase (decarboxylating) from the hemolymph of Manduca sexta that generates a new quinone methide intermediate during melanogenesis (Sugumaran, M. and Semensi, V. (1991) J. Biol. Chem. 266, 6073–6078). This enzyme has the ability to form a complex with mushroom tyrosinase as judged by a number of physicochemical studies. The isomerase exhibited a marked inhibitory effect on tyrosinase and tyrosinase reciprocated by inhibiting the isomerase. While the isomerase showed no activity toward preformed dopaminechrome, it readily influenced the stability of dopaminechrome generated in situ by tyrosinase. Moreover, mushroom tyrosinase, which lacked specific binding to Concanavalin A Sepharose column, after complexing with the isomerase exhibited binding to this column. The complex formation also affected the pi value as well as mobility on a size exclusion column of these enzymes. Enzymes executing sequential metabolic transformation are known to form complexes called metabolons. Based on these above studies, it is concluded that both the enzymes involved in insect melanogenic pathway—phenoloxidase and dopachrome isomerase—are able to form a metabolon complex.     

Immunofluorescent Identification of Melanocytes in Murine Hair Follicles

Summary Immunocytochemical identification of skin cells are difficult due to numerous endogenous autofluorescent components within the cell and the environment. This is particularly evident in hair follicles. This paper reports on a serendipitous modification to an existing method which results in a drastically reduced background fluorescence. Immediately after antigen retrieval, sections exposed to 0.3% hydrogen peroxide in methanol for 30 min at room temperature exhibited low background fluorescence, increased antigenicity and revealed quantifiable numbers of melanocytes. This method is applicable to both human and mouse melanocytes particularly in the hair follicle.     

小鼠正常黑素细胞和小鼠B16黑素瘤细胞的环状RNAs表达谱

黑素瘤是一种多发于皮肤的恶性肿瘤,因其侵袭性强,预后差等特点一直是科研人员关注的热点。环状RNAs(circRNAs)是一种新型内源性非编码RNA,广泛参与动物生长发育、细胞分化和信号转导等生理过程,但circRNAs在黑素瘤细胞内的分子机制尚未被充分解析。本研究以小鼠(C57BL/6J)正常黑素细胞及B16黑素瘤细胞为研究对象,采用二代测序技术分析两种细胞间circRNAs表达特性。测序结果显示,小鼠正常黑素细胞和黑素瘤细胞中共有851个circRNAs,其中195个差异表达circRNAs(DECs)。GO及KEGG数据库注释发现,DECs的来源基因主要参与细胞周期(cell cycle)、紧密结合(tight junction)、Rap1信号通路(Rap1 signaling pathway)、TGF-beta信号通路(TGF-beta signaling pathway)等与细胞增殖、迁移相关的信号通路;探究发现,黑素瘤细胞中显著性高表达的circE2F5(circ-3:14578602|14606309)通过上调E2F5的表达促进黑素瘤细胞增殖。circRNA靶基因预测发现,...     

Comparison of TRPs From Murine and Human Malignant Melanocytes

Most of our knowledge of the mammalian tyrosinase related protein (TRP) activities is derived from studies using murine melanoma models, such as B16 or Cloudman S-91 melanocytes. Owing to the high degree of homology between the murine and human enzymes, it has been assumed that their kinetic behavior could be similar. However, the protein sequences at the metal binding sites of the murine and human enzymes show some differences of possible functional relevance. These differences are more significant in the metal-A site than in the metal-B site. By using three human melanoma cell lines (HBL, SCL, and BEU), we have studied the catalytic abilities of the human melanogenic enzymes in comparison to those obtained for the counterpart murine enzymes isolated from B16 melanoma. We have found that TRP2 extracted from all cell lines show dopachrome tautomerase activity, although the activity levels in human malignant melanocytes are much lower than in mouse cells. Reconstitution experiments of the human enzyme indicate that TRP2 has Zn at its metal binding-sites. Although mouse tyrosinase does not show DHICA oxidase activity, and this step of the melanogenesis pathway is specifically catalyzed by mouse TRP1, the human enzyme seems to recognize carboxylated indoles. Thus, human tyrosinase could display some residual DHICA oxidase activity, and the function of human TRP1 could differ from that of the murine protein. Attempts to clarify the nature of the metal cofactor in TRP1 were unsuccessful. The enzyme contains mostly Fe and Cu, but the reconstitution of the enzymatic activity from the apoprotein with these ions was not possible.     

Melanosomal Tyrosine Transport in Normal and Pink-eyed Dilution Murine Melanocytes

Tyrosine is the endogenous substrate for melanin production within melanosomes, but the method of tyrosine transport into the melanosome has not been investigated. In the mouse, melanogenesis is disrupted by mutations in the p gene resulting in the pink-eyed dilution phenotype; it has been suggested that the p gene codes for a tyrosine transport protein. We determined that normal (melan-a) melanosome-rich granular fractions take up 10 μm [³H]tyrosine at 21.1 ± 6.1 (SEM, standard error of the mean) pmol/min/mg protein (N=7) compared with 21.3 ± 5.8 SEM pmol/min/mg protein (N=5) for pink-eyed dilution, whose plasma membrane tyrosine transport was also normal (K_m 89 μM; V_max 302 pmol/min/mg cell protein). We also demonstrated that pink-eyed dilution melanosomes are immature by virtue of their low density, high hexosaminidase activity, and lack of pigment. These data indicate that a tyrosine transport system exists in the melanosomal membrane and that the p gene does not encode a tyrosine transporter of critical importance.     

Effects of 4-Tertiary Butylphenol on the Tyrosinase Activity in Human Melanocytes

Vitiligo is a common dermatological disorder characterized by the development of complete pigment loss from focal lesions that tends to increase in size over time. The etiology of vitiligo, resulting in the disappearance of functional melanocytes from involved skin, is not clearly understood. As a consequence, no satisfactory therapy has been developed. A subtype of vitiligo, termed‘occupational’or‘contact’vitiligo, is increased in individuals who are exposed to materials containing phenolic derivatives, such as 4-tertiary butylphenol (4-TBP). Phenolic derivatives are structurally similar to tyrosine, the initial substrate of tyrosinase in the biochemical synthesis of melanin. Therefore, it has been proposed that phenolic derivatives compete with tyrosine for hydroxyla-tion by tyrosinase and interfere with the completion of melanin synthesis and/or generate cytotoxic intermediates. Our results demonstrated that 4-TBP competitively inhibited both tyrosine hydroxylase and dihydrox-yphenylalanine (DOPA) oxidase activities of tyrosinase, i.e., the first two catalytic steps in the biochemical conversion of tyrosine to melanin in cultured human melanocytes. This inhibition occurred at concentrations that did not influence the viability of melanocytes. The tyrosinase activity inhibited by 4-TBP was recovered after removing the treatment. 4-TBP did not affect the function of other enzymes, such as succinate-tetra-zolium reductase, acid phosphatase and sulfatase. Since depigmentation occurred without a cytotoxic response after exposure of melanocytes to low concentration of 4-TBP, it is unclear whether the interaction between 4-TBP and tyrosinase leads to the destruction of the melanocytes in‘contact/occupational’vitiligo.     

AP-3 Mediates Tyrosinase but Not TRP-1 Trafficking in Human Melanocytes

Patients with Hermansky-Pudlak syndrome type 2 (HPS-2) have mutations in the beta 3A subunit of adaptor complex-3 (AP-3) and functional deficiency of this complex. AP-3 serves as a coat protein in the formation of new vesicles, including, apparently, the platelet's dense body and the melanocyte's melanosome. We used HPS-2 melanocytes in culture to determine the role of AP-3 in the trafficking of the melanogenic proteins tyrosinase and tyrosinase-related protein-1 (TRP-1). TRP-1 displayed a typical melanosomal pattern in both normal and HPS-2 melanocytes. In contrast, tyrosinase exhibited a melanosomal (i.e., perinuclear and dendritic) pattern in normal cells but only a perinuclear pattern in the HPS-2 melanocytes. In addition, tyrosinase exhibited a normal pattern of expression in HPS-2 melanocytes transfected with a cDNA encoding the beta 3A subunit of the AP-3 complex. This suggests a role for AP-3 in the normal trafficking of tyrosinase to premelanosomes, consistent with the presence of a dileucine recognition signal in the C-terminal portion of the tyrosinase molecule. In the AP-3-deficient cells, tyrosinase was also present in structures resembling late endosomes or multivesicular bodies; these vesicles contained exvaginations devoid of tyrosinase. This suggests that, under normal circumstances, AP-3 may act on multivesicular bodies to form tyrosinase-containing vesicles destined to fuse with premelanosomes. Finally, our studies demonstrate that tyrosinase and TRP-1 use different mechanisms to reach their premelanosomal destination.     

Up‐regulation of Tyrosinase Gene by Nitric Oxide in Human Melanocytes

Ultraviolet light (UV) radiation causes skin‐tanning, which is thought to be mediated by stimulating the release of melanogenic factors from keratinocytes as well as other cells. Nitric oxide (NO) has been reported to be generated after UV radiation and to stimulate melanocytes as one of the melanogens. In a previous experiment by another group on melanogenesis induced by NO, increases in both tyrosinase activity and tyrosinase protein levels were observed after daily stimulation of NO for 4 days. In the present study, we investigated tyrosinase gene expression within the first 24 hr of NO‐induced melanogenesis. Tyrosinase mRNA expression was found to be induced 2 hr after a single treatment with S‐nitroso‐N‐acetyl‐ l ‐arginine. An increase of tyrosinase activity was also detected time‐dependently within the 24‐hr period, accompanied by an increase of tyrosinase protein levels. The induction of mRNA expression was suppressed by a cyclic guanosine 3′,5′‐monophosphate (cGMP)‐dependent protein kinase (cGMP/PKG) inhibitor. These results suggest that the enhancement of tyrosinase gene expression via the cGMP pathway may be a primary mechanism for NO‐induced melanogenesis.     

miR-146a抑制酪氨酸酶基因家族在小鼠黑色素细胞中表达

miRNA是在许多生物过程中都起着至关重要作用的一类内源性非编码的小RNA,与癌症、肿瘤的发生有关。现发现很多miRNA在黑色素生成中都有重要的调控作用,但miR-146a是否对黑色素的生成具有影响未见报道。本研究发现miR-146a通过靶向抑制酪氨酸酶相关蛋白1(tyrosinase related protein 1,TYRP1)的表达而使黑色素生成降低。在小鼠黑色素细胞中分别转染miR-146a mimic和miR-146a 抑制剂,通过qRT-PCR与Western印迹分析比较各实验组中TYRP1基因与酪氨酸家族相关基因酪氨酸酶(tyrosinase, TYR)、酪氨酸酶相关蛋白2(tyrosinase related protein 2, TYRP2)的表达差异。双荧光报告实验验证TYRP1与miR-146a的靶向关系,双荧光酶活性结果显示,实验组相比对照组,荧光素酶活性明显降低,说明TYRP1是miR-146a的靶基因之一;qRT-PCR和Western印迹结果显示实验组TYR、TYRP1及TYRP2 在mRNA水平和蛋白质水平表达均显著降低;紫外分光光度法检测黑色素含量,结果显示miR-146a mimic转染组黑色素含量明显下降,而抑制组的黑色素含量呈上升趋势。综上所述,miR-146a通过靶向抑制TYRP1基因的表达,而影响TYR家族成员的表达,调控黑色素的生物合成。     

小鼠正常黑色素细胞与黑色素瘤细胞(B16)mRNA表达差异分析

黑色素瘤是一种高侵袭性的恶性皮肤肿瘤,转移率高、预后差。研究黑素瘤细胞生物学特性对黑素瘤的治疗和控制具有重要的意义。本研究以C57BL/6J小鼠的正常黑色素细胞及B16黑色素瘤细胞为研究对象,采用二代测序技术分析两种细胞间的转录组表达差异,筛选差异基因,为后续黑色素瘤的形成机制研究提供理论依据。采用差异倍数及错误率分析测序数据,鉴定出1 436个新的mRNA和4 086个差异表达的已知mRNA。GO数据库和KEGG数据库分析显示,差异表达的mRNAs参与了149个调控途径,主要集中在疾病调控、细胞周期调节和环境信息调控方面。qRT-PCR及Western印迹检测发现,调节细胞增殖、迁移的Pdgf-B、Integrinβ1和Integrinβ5以及调节黑色素颗粒增加的Mitf、Tyr、Tyrp1和Tyrp2在B16细胞中的表达量显著高于在正常黑色素细胞中的表达。本研究获得的差异基因为后续黑色素瘤的研究提供了新的候选基因。     

小鼠正常黑素细胞与黑色素瘤细胞（B16）mRNA表达差异分析

黑色素瘤是一种高侵袭性的恶性皮肤肿瘤,转移率高、预后差。研究黑素瘤细胞生物学特性对黑素瘤的治疗和控制具有重要的意义。本研究以C57BL/6J小鼠的正常黑色素细胞及B16黑色素瘤细胞为研究对象,采用二代测序技术分析两种细胞间的转录组表达差异,筛选差异基因,为后续黑色素瘤的形成机制研究提供理论依据。采用差异倍数及错误率分析测序数据,鉴定出1 436个新的mRNA和4 086个差异表达的已知mRNA。GO数据库和KEGG数据库分析显示,差异表达的mRNAs参与了149个调控途径, 主要集中在疾病调控、细胞周期调节和环境信息调控方面。qRT-PCR及Western印迹检测发现,调节细胞增殖、迁移的Pdgf-B、Integrin-β1和Integrin-β5以及调节黑色素颗粒增加的Mitf、Tyr、Tyrp1和Tyrp2在B16细胞中的表达量显著高于在正常黑色素细胞中的表达。本研究获得的差异基因为后续黑色素瘤的研究提供了新的候选基因。     

Tyrosinase Protein Is Expressed Also in Some Neural Crest Derived Cells Which Are Not Melanocytes

Some neural crest cells give rise to pigment cells in early ontogenesis. We tested here whether tyrosinase-a key enzyme in melanogenesis—was present in some nonpigment neural crest derivatives in adult hamsters. Interestingly enough, inactive tyrosinase protein was detected, using indirect immunofluorescence, in the satellite cells of spinal ganglia and Schwann cells of sciatic and facial nerves in normal adult animals. The results of cell blotting from spinal ganglia were similar to the fluorescence findings. Thus, our results seem to support the hypothesis that Schwann cells, satellite cells of spinal ganglia, and melanocytes may be more intimately related developmentally than other neural-crest-derived cells. Moreover, since we detected tyrosinase protein in cells which normally do not produce melanosomes, it could be deduced that, during the melanocyte's differentiation from its cell precursor, the expression of tyrosinase protein might precede the point when melanosomes begin to differentiate from known cytoplasmic structures.     

Induction of Growth Factor Receptors on Cultured Human Melanocytes

Using a sensitive and selective culture system for human epidermal melanocytes, we have demonstrated that the morphologic changes induced by addition of phorbol 12-tetradecanoate 13-acetate (TPA) to proliferating newborn melanocytes are associated with induction of nerve growth factor (NGF) receptors, as measured by messenger RNA (mRNA) levels and protein accumulation and by cell surface immunofluorescent staining. Growth factor deprivation or addition of NGF similarly results in NGF receptor induction. NGF is believed to function in vivo and in vitro as a survival factor for many neural crest-derived cells and has been demonstrated to promote specific neural cell functions ranging from neurite outgrowth to enzyme induction, but to date no role for NGF has been identified with regard to normal human melanocytes. Our data demonstrate that, given appropriate stimulation, cultured human melanocytes may express the NGF receptor gene and therefore suggest that NGF may modulate human melanocyte behavior in vivo. This first demonstration of a growth factor receptor on human melanocytes provides an important opportunity to explore signal transduction relevant to their growth, differentiation, and malignant transformation.     

不同剂量L-酪氨酸（L—TYR）对羊驼黑素细胞增殖及酪氨酸酶mRNA表达的影响

目的：探索不同剂量的L-酪氨酸对羊驼黑素细胞增殖以及酪氨酸酶mRNA表达的影响。方法：体外培养正常羊驼皮肤黑素细胞,显微镜下观察不同剂量的L-酪氨酸（100μmol／L、200μmol／L、300μmol／L、400μmol／L）对羊驼皮肤黑素细胞增殖的影响,利用实时荧光定量PCR技术分析不同剂量L-酪氨酸对酪氨酸酶mRNA的表达量的影响。结果：研究结果显示：在培养的黑素细胞中添加了不同剂量L-酪氨酸3d后,镜检观察,黑素细胞数量相比较空白对照组有所减少,酪氨酸酶mRNA表达量增加且显著高于对照组（P〈0．05）,在200μmol／L时,酪氨酸酶mRNA表达量达到最高峰值。结论：L-酪氨酸能诱导羊驼皮肤黑素细胞酪氨酸酶mRNA表达量增高。     

Correction of Defective Early Tyrosinase Processing by Bafilomycin A1 and Monensin in Pink‐Eyed Dilution Melanocytes

Mutations in the human P gene result in oculocutaneous albinism type 2, the most common form of albinism. Mouse melan‐p1 melanocytes, cultured from mice null at the homologous pink‐eyed dilution (p) locus, exhibit defective melanin production. A variety of compounds including tyrosine, NH₄Cl, bafilomycin A1, concanamycin, monensin, and nigericin are capable of restoring melanin synthesis in these cells. In the current study, we investigated the subcellular effects of bafilomycin A1 and monensin treatment of melan‐p1 cells. Both agents play two roles in the processing of tyrosinase (Tyr) in melan‐p1 cells. First, combined glycosidase digestion and immunoblotting analysis showed that these agents reduce levels of Tyr retained in the endoplasmic reticulum (ER) and facilitate the release of Tyr from the ER to the Golgi. Secondly, treatment with these compounds resulted in the stabilization of Tyr. Surprisingly, induction of melanin synthesis corresponds more closely with diminution of ER‐retained Tyr, rather than the absolute amount of Tyr. Our results suggest that bafilomycin A1 and monensin induce melanin synthesis in melan‐p1 cells mainly by facilitating Tyr processing from the ER to the Golgi by increasing the pH in either the ER or the ER–Golgi intermediate compartment.