Homologous Overexpression of Tyrosinase in Trichoderma reesei and Its Application in Glycinin Cross-Linking.

Tyrosinase is capable of oxidizing tyrosine residues in proteins, leading to intermolecular protein cross-linking, which could modify the protein network of food and improve the texture of food. To obtain the recombinant tyrosinase with microbial cell factory instead of isolation tyrosinase from the mushroom Agaricus bisporus, a TYR expression cassette was constructed in this study. The expression cassette was electroporated into Trichoderma reesei Rut-C30 and integrated into its genome, resulting in a recombinant strain C30-TYR. After induction with microcrystalline cellulose for 7 days, recombinant tyrosinase could be successfully expressed and secreted by C30-TYR, corresponding to approximately 2.16 g/L tyrosinase in shake-flask cultures. The recombinant TYR was purified by ammonium sulfate precipitation and gel filtration, and the biological activity of purified TYR was 45.6 U/mL. The purified TYR could catalyze the cross-linking of glycinin, and the emulsion stability index of TYR-treated glycinin emulsion was increased by 30.6% compared with the untreated one. The cross-linking of soy glycinin by TYR resulted in altered properties of oil-in-water emulsions compared to emulsions stabilized by native glycinin. Therefore, cross-linking with this recombinant tyrosinase is a feasible approach to improve the properties of protein-stabilized emulsions and gels.

Tyrosinase regulates the motility of human melanoma cell line A375 through its hydroxylase enzymatic activity.

Melanoma, originating from melanocytes, is a highly aggressive tumor. Tyrosinase is involved in melanin production in melanocytes, and its overexpression is noted in malignant melanomas. However, the role of tyrosinase in melanomas remains unclear. Therefore, this study aimed to evaluate the potential functions of tyrosinase in the human melanoma cell line A375. The expression level of tyrosinase in A375 cells was undetectable. However, markedly increased expression level was observed in the mouse melanoma cell line B16F10 and the human melanoma cell line WM266-4. Subsequently, we investigated the effect of ectopic tyrosinase expression on A375 cell motility using wound-healing assay. The overexpression of tyrosinase resulted in enhanced cell migration in both stable and transient tyrosinase expression cells. The levels of filamentous actin were decreased in tyrosinase-expressing A375 cells, suggesting that tyrosinase regulates cell motility by modulating actin polymerization. Histidine residues in tyrosinase are important for its enzymatic activity for synthesizing melanin. Substitution of these histidine residues to alanine residues mitigated the promotion of tyrosinase-induced A375 cell metastasis. Furthermore, melanin treatment enhanced A375 cell metastasis and phosphorylation of Cofilin. Thus, our findings suggest that tyrosinase increases the migration of A375 cells by regulating actin polymerization through its enzymatic activity.

(2-Methylbutyryl)shikonin Naturally Occurring Shikonin Derivative Ameliorates the α-MSH-Induced Melanogenesis via ERK1/2 and p38 MAP Kinase-Mediated Down-Regulation of the MITF Transcription Factor.

Cutaneous pigmentation is an important phenotypic trait whose regulation, despite recent advances, has yet to be completely elucidated. Melanogenesis, a physiological process of melanin production, is imperative for organism survival as it provides protection against the environmental insults that majorly involve sunlight-induced skin photodamage. However, immoderate melanin synthesis can cause pigmentation disorders associated with a psychosocial impact. In this study, the hypopigmentation effect of (2-methylbutyryl)shikonin, a natural product present in the root extract of Lithospermum erythrorhizon, and the underlying mechanisms responsible for the inhibition of melanin synthesis in α-MSH-stimulated B16F10 cells and C57BL/6J mice was studied. Non-cytotoxic concentrations of (2-methylbutyryl)shikonin significantly repressed cellular tyrosinase activity and melanin synthesis in both in vitro and in vivo models (C57BL/6J mice). (2-Methylbutyryl)shikonin remarkably abolished the protein expression of MITF, tyrosinase, tyrosinase-related protein 1, and tyrosinase-related protein 2, thereby blocking the production of pigment melanin via modulating the phosphorylation status of MAPK proteins, viz., ERK1/2 and p38. In addition, specific inhibition of ERK1/2 attenuated the inhibitory effects of (2-methylbutyryl)shikonin on melanin synthesis, whereas selective inhibition of p38 augmented the inhibitory effect of BSHK on melanin synthesis. Moreover, topical application of (2-methylbutyryl)shikonin on C57BL/6J mouse tails remarkably induced tail depigmentation. In conclusion, with these findings, we, for the first time, report the hypopigmentation effect of (2-methylbutyryl)shikonin via inhibition of cellular tyrosinase enzyme activity, subsequently ameliorating the melanin production, thereby indicating that (2-methylbutyryl)shikonin is a potential natural therapy for hyperpigmentation disorders.

Generation and characterization of retinal pigment epithelium from patient iPSC line to model oculocutaneous albinism (OCA)1A disease.

Oculocutaneous albinism (OCA) is characterized by reduced melanin biosynthesis affecting the retina, thus impairing visual function. The disease pathology of OCA is poorly understood at the cellular level due to unavailability of suitable biological model systems. This study aimed to develop a disease-specific in vitro model for OCA type 1A, the most severe form caused by TYR (tyrosinase) gene mutations, using retinal pigment epithelium (RPE) differentiated from patient-derived human-induced pluripotent stem cells (hiPSCs). A comparative study between healthy and OCA1A RPE cells revealed that while healthy RPE cells exhibited timely onest of pigmentation during differentiation, OCA1A RPE cells failed to pigment even after an extended culture period. This observation was validated by ultrastructural studies using electron microscopy, hinting at melanosome-specific defects. Immunocytochemistry demonstrated abnormal expression patterns of melanogenesis-specific protein markers in OCA1A RPE cells, indicating reduced or absence of melanin synthesis. Next, a quantitative assay was performed to confirm the absence of melanin production in OCA1A RPE cells. Tyrosinase assay showed no activity in OCA1A compared with healthy RPE, suggesting non-functionality of TYR, further corroborated by western blot analysis showing complete absence of the protein. Gene expression by RNA sequencing of healthy and OCA1A RPE cells uncovered differential gene expression associated with lens development, visual perception, transmembrane transporter activity, and key signaling pathways. This disease-in-a-dish model of OCA1A provides an excellent platform to understand disease mechanism, identify potential therapeutic targets, and facilitate gene therapy or gene correction.

Melanogrit potentiates melanogenesis by escalating cellular tyrosinase activity and MITF levels via pERK inhibition.

Vitiligo is characterized by the development of white patches on the skin either due to the loss of functional melanocytes or perturbations in the melanogenesis pathway. In the present study, we investigated the therapeutic potential of herbo-mineral formulation, Melanogrit in neutralizing the white patches in the skin. The study utilized UPLC/MS-QToF technique to determine the diversified phytochemical profile in Melanogrit. The murine B16F10 cells when treated with Melanogrit underwent morphological changes, including increased angularity, enlarged cell size, and greater dendritic protrusions. To establish an equivalent model to study melanogenesis, we carefully optimized the dosage of α-melanocyte stimulating hormone (αMSH) in B16F10 cells as an alternative to using melanocyte-keratinocyte cocultures. The study determined a sub-optimal dose of αMSH (0.2 nM) in B16F10 cells that does not manifest any measurable effects on melanogenesis. In contrast, Melanogrit when used in conjunction with 0.2 nM αMSH, induced a dose-dependent increase in extracellular and intracellular melanin levels. Melanogrit transcriptionally up-regulated the decisive genes of the melanogenesis pathway, MITF, TYR, and TRP1, which was evident from the increased cellular tyrosine activity. Our findings also demonstrated that Melanogrit ameliorated the MITF protein levels by inhibiting pERK; notably without involving GSK3ß in the process. Taken together, our findings strongly suggest that Melanogrit has the potential to stimulate melanogenesis, making it a promising candidate for clinical applications in the treatment of white skin patches that develop in vitiligo patients.

Copper ion (Cu2+) is involved in the transcription of the tyrosinase-encoding melB gene of Aspergillus oryzae in solid-state culture.

In Aspergillus oryzae, the tyrosinase-encoding gene melB causes undesirable browning of sake and sake lees. This gene is known to be expressed specifically in solid-state culture; however, its expression mechanisms remain unknown. Here, we evaluated the possible factors affecting the transcription of melB and found that the copper ion (Cu2+) significantly enhanced the transcription level of melB in solid-state culture.

Comparative transcriptome analysis reveals growth and molecular pathway of body color regulation in turbot (Scophthalmus maximus) exposed to different light spectrum.

Fish body color changes play vital roles in adapting to ecological light environment and influencing market value. However, the initial mechanisms governing the changes remain unknown. Here, we scrutinized the impact of light spectrum on turbot (Scophthalmus maximus) body coloration, exposing them to red, blue, and full light spectra from embryo to 90 days post hatch. Transcriptome and quantitative real-time PCR (qRT-PCR) analyses were employed to elucidate underlying biological processes. The results showed that red light induced dimorphism in turbot juvenile skin pigmentation: some exhibited black coloration (Red_Black_Surface, R_B_S), while others displayed lighter skin (Red_White_Bottom, R_W_B), with red light leading to reduced skin lightness (L*) and body weight, particularly in R_B_S group. Transcriptomic and qRT-PCR analyses showcased upregulated gene expressions related to melanin synthesis in R_B_S individuals, notably tyrosinase (tyr), tyrosinase-related protein 1 (tyrp1), and dopachrome tautomerase (dct), alongside solute carrier family 24 member 5 (slc24a5) and oculocutaneous albinism type II (oca2) as pivotal regulators. Nervous system emerged as a critical mediator in spectral environment-driven color regulation. N-methyl d-aspartate (NMDA) glutamate receptor, and calcium signaling pathway emerged as pivotal links intertwining spectral conditions, neural signal transduction, and color regulation. The individual differences in NMDA glutamate receptor expression and subsequent neural excitability seemed responsible for dichromatic body coloration in red light-expose juveniles. This study provides new insights into the comprehending of fish adaptation to environment and methods for fish body color regulation and could potentially help enhance the economic benefit of fish farming industry.

TYR mutation in a Chinese population with oculocutaneous albinism: Molecular characteristics and ophthalmic manifestations.

Oculocutaneous albinism (OCA) is a rare inherited disorder characterized by a partial or complete reduction of melanin biosynthesis that leads to hypopigmentation in the skin, hair and eyes. The OCA1 subtype is caused by mutations in TYR. The purpose of this study was to investigate the genetic and clinical ophthalmic characteristics of TYR mutations in patients with OCA. Herein, 51 probands with a clinical diagnosis of OCA were enrolled. Whole-exome sequencing and comprehensive ophthalmic examinations were performed. Overall, TYR mutations were detected in 37.3% (19/51) in the patients with OCA. Fifteen patients had compound heterozygous variants, and four cases had homozygous variants. Eleven different pathogenic variants in TYR were detected in these 19 patients, with missense, insertion, delins and nonsense in 71.1% (27/38), 15.8% (6/38), 2.6% (1/38), and 10.5% (4/38), respectively. Clinical examinations revealed that 84.2% (16/19) of patients were OCA1A, and 15.8% (3/19) were OCA1B. Most TYR probands (52.6%, 10/19) had moderate vision impairment, 15.8% (3/19) had severe visual impairment, 10.5% (2/19) exhibited blindness, only 5.3% (1/19) had mild visual impairment and 15.8% (3/19) were not available. Photophobia and nystagmus were found in 100% (19/19) of the patients. In addition, grade 4 foveal hypoplasia was detected in 100% (12/12) of the patients. In conclusion: The TYR patients exhibited severe ocular phenotypes: the majority (93.8%, 15/16) of them had a moderate vision impairment or worse, and 100% (12/12) had severe grade 4 foveal hypoplasia. These novel findings could provide insight into the understanding of OCA.

Mitf involved in shell pigmentation by activating tyrosinase-mediated melanin synthesis in Pacific oyster (Crassostrea gigas).

Pigmentation is frequently observed in the molluscan shells, whereas the molecular regulation about these shell pigments formation is not clear. The microphthalmia-associated transcription factor (Mitf) is an important transactivator in melanin synthesis in vertebrates. Here, the Mitf containing a highly conserved basic helix-loop-helixleucine zipper (bHLH-LZ) domain was identified in an economically important marine bivalve Pacific oyster Crassostrea gigas. The Mitf was found to widespread tissue distribution and the expression was higher in the marginal mantle than in the central mantle. Particularly, the expression level of Mitf was high in black shell color oysters compared with white shell oysters. After injecting siRNA, the expression of Mitf decreased significantly, and the efficiency of RNA interference reached 53%. Besides, knockdown Mitf obviously decreased expression of tyrosinase family genes and tyrosinase activity of mantles, indicating a potential regulatory relationship between Mitf and Tyr or Typs. Simultaneously, there was a sharply reduce in the number of the melanosomes in the outer fold of mantle by silencing of Mitf. Luciferase assays in cell culture further verified that Mitf was involved in transcriptional regulation of Typ-2 and Typ-3 genes through binding to their specific promoter regions. These data argue that Mitf is involved in shell pigmentation through activating tyrosinase-mediated melanin synthesis in C. gigas.

Silencing of Amylomyces rouxii aspartic II protease by siRNA to increase tyrosinase activity.

Amylomyces rouxii is a zygomycete that produces extracellular protease and tyrosinase. The tyrosinase activity is negatively regulated by the proteases and, which attempts to purify the tyrosinase (tyr) enzyme that has been hampered by the presence of a protease that co-purified with it. In this work we identified genes encoding aspartic protease II (aspII) and VI of A. rouxii. Using an RNAi strategy based on the generation of a siRNA by transcription from two opposite-orientated promoters, the expression of these two proteases was silenced, showing that this molecular tool is suitable for gene silencing in Amylomyces. The transformant strains showed a significant attenuation of the transcripts (determined by RT-qPCR), with respective inhibition of the protease activity. In the case of aspII, inhibition was in the range of 43-90 % in different transformants, which correlated well with up to a five-fold increase in tyr activity with respect to the wild type and control strains. In contrast, silencing of aspVI caused a 43-65 % decrease in protease activity but had no significant effect on the tyr activity. The results show that aspII has a negative effect on tyr activity, and that the silencing of this protease is important to obtain strains with high levels of tyr activity.

Peptide OA-VI12 restrains melanogenesis in B16 cells and C57B/6 mouse ear skin via the miR-122-5p/Mitf/Tyr axis.

Excessive melanogenesis leads to hyperpigmentation, which is one of the common skin conditions in humans. Existing whitening cosmetics cannot meet market needs due to their inherent limitations. Thus, the development of novel skin-whitening agents continues to be a challenge. The peptide OA-VI12 from the skin of amphibians at high altitude has attracted attention due to its remarkable anti light damage activity. However, whether OA-VI12 has the skin-whitening effect of inhibiting melanogenesis is still. Mouse melanoma cells (B16) were used to study the effect of OA-VI12 on cell viability and melanin content. The pigmentation model of C57B/6 mouse ear skin was induced by UVB and treated with OA-VI12. Melanin staining was used to observe the degree of pigmentation. MicroRNA sequencing, quantitative real-time PCR (qRT-PCR), immunofluorescence analysis and Western blot were used to detect the change of factor expression. Double luciferase gene report experiment was used to prove the regulatory relationship between miRNA and target genes. OA-VI12 has no effect on the viability of B16 cells in the concentration range of 1-100 µM and significantly inhibits the melanin content of B16 cells. Topical application of OA-VI12, which exerted transdermal potency, prevented UVB-induced pigmentation of ear skin. MicroRNA sequencing and double luciferase reporter analysis results showed that miR-122-5p, which directly regulated microphthalmia-associated transcription factor (Mitf), had significantly different expression before and after treatment with OA-VI12. Mitf is a simple helix loop and leucine zipper transcription factor that regulates tyrosinase (Tyr) expression by binding to the M-box promoter element of Tyr. qRT-PCR, immunofluorescence analysis and Western blot showed that OA-VI12 up-regulated the expression of miR-122-5p and inhibited the expression of Mitf and Tyr. The effects of OA-VI12 on melanogenesis inhibition in vitro and in vivo may involve the miR-122-5p/Mitf/tyr axis. OA-VI12 represents the first report on a natural amphibian-derived peptide with skin-whitening capacity and the first report of miR-122-5p as a target for regulating melanogenesis, thereby demonstrating its potential as a novel skin-whitening agent and highlighting amphibian-derived peptides as an underdeveloped resource.

Evaluating the Cysteine-Rich and Catalytic Subdomains of Human Tyrosinase and OCA1-Related Mutants Using 1 µs Molecular Dynamics Simulation.

The inherited disorder oculocutaneous albinism type 1 (OCA1) is caused by mutations in the TYR gene encoding tyrosinase (Tyr), an enzyme essential to producing pigments throughout the human body. The intramelanosomal domain of Tyr consists of the cysteine-rich and tyrosinase catalytic subdomains, which are essential for enzymatic activity. In protein unfolding, the roles of these subdomains are not well established. Here, we performed six molecular dynamics simulations at room temperature for Tyr and OCA1-related mutant variants P406L and R402Q intramelanosomal domains. The proteins were simulated for 1 µs in water and urea to induce unfolding. In urea, we observed increases in surface area, decreases in intramolecular hydrogen bonding, and decreases in hydrophobic interactions, suggesting a 'molten globule' state for each protein. Between all conditions, the cysteine-rich subdomain remains stable, whereas the catalytic subdomain shows increased flexibility. This flexibility is intensified by the P406L mutation, while R402Q increases the catalytic domain's rigidity. The cysteine-rich subdomain is rigid, preventing the protein from unfolding, whereas the flexibility of the catalytic subdomain accommodates mutational changes that could inhibit activity. These findings match the conclusions from our experimental work suggesting the function alteration by the P406L mutation, and the potential role of R402Q as a polymorphism.

Combine with RNA-seq Reveals the Effect of Melatonin in the Synthesis of Melanin in Primary Melanocytes of Silky Fowls Black-Bone Chicken.

(1) Background: It was found that the melanin of black-bone chicken has various effects such as scavenging DPPH free radicals and anti-oxidation, and the synthesis of melanin is affected by various factors including hormones. In addition, several studies have found that melatonin affects the melanoma cell synthesis of melanin, which has not been reported in chicken primary melanocytes; so, relevant studies were conducted. (2) Methods: In this study, chicken primary melanocytes were isolated and characterized, and then melanocytes were treated with different concentrations of melatonin to investigate the effects of melatonin on melanin synthesis in chicken melanocytes in terms of melanin synthesis-related genes, melanin content, and tyrosinase activity, and combined with RNA seq to detect the change in gene expression level of chicken melanocytes after melatonin treatment. (3) Results: We isolated and characterized primary melanocytes, and indirect immunofluorescence assay results showed positive melanocyte marker genes. RT-qPCR results showed that melatonin decreased the expression of melanin synthesis-related genes. In addition, melatonin reduced the melanin content and decreased the tyrosinase activity of melanocytes in the treated group. A total of 1703 differentially expressed genes were screened by RNA-seq, and in addition, in the KEGG results, the signaling pathway associated with melanin synthesis, and the mTOR signaling pathway were enriched. (4) Conclusions: Melatonin could decrease the synthesis of melanin in chicken primary melanocytes.

Genetic variants in melanogenesis proteins TYRP1 and TYR are associated with the golden rhesus macaque phenotype.

Nonhuman primates (NHPs) are vital translational research models due to their high genetic, physiological, and anatomical homology with humans. The "golden" rhesus macaque (Macaca mulatta) phenotype is a naturally occurring, inherited trait with a visually distinct pigmentation pattern resulting in light blonde colored fur. Retinal imaging also reveals consistent hypopigmentation and occasional foveal hypoplasia. Here, we describe the use of genome-wide association in 2 distinct NHP populations to identify candidate variants in genes linked to the golden phenotype. Two missense variants were identified in the Tyrosinase-related protein 1 gene (Asp343Gly and Leu415Pro) that segregate with the phenotype. An additional and distinct association was also found with a Tyrosinase variant (His256Gln), indicating the light-colored fur phenotype can result from multiple genetic mechanisms. The implicated genes are related through their contribution to the melanogenesis pathway. Variants in these 2 genes are known to cause pigmentation phenotypes in other species and to be associated with oculocutaneous albinism in humans. The novel associations presented in this study will permit further investigations into the role these proteins and variants play in the melanogenesis pathway and model the effects of genetic hypopigmentation and altered melanogenesis in a naturally occurring nonhuman primate model.

Identification and characterization of the compound heterozygous variants of TYR gene in a northern Chinese family with Oculocutaneous albinism type 1.

Oculocutaneous albinism (OCA) is a genetically heterogeneous disease and is most inherited in an autosomal recessive manner. The characteristic manifestation of OCA is due to disfunction of melanin synthesis. OCA1 is the most severe subtype of OCA and is caused by homozygous or compound heterozygous variants in tyrosinase (TYR) gene, which is the key gene for melanin synthesis. This study aimed to identify the genetic variants of a northern Chinese family with OCA1. Clinical information and peripheral blood samples were collected. PCR amplification and Sanger sequencing were used to detect the entire exons and adjacent flanking sequences of TYR gene. Functional prediction of variants was performed by various bioinformatic analyses, while the pathogenicity classification of variants was evaluated according to ACMG standards and guidelines. A missense variant NM_000372.5:c.107G > C;NP_000363.1:p.C36S was discovered in TYR gene which converted cysteine to serine. Another variant in intron, NM_000372.5:c.1037-7 T > A, also affected the function of TYR gene. We verified the pathogenicity of the intron variant with a pCAS2 mini-gene based splicing assay and found that c.1037-7 T > A led to an insertion of 5 bp upstream from the common acceptor site of exon 3, which caused a frameshift TYR:c.1037-7 T > A:p.G346Efs*11. The results showed that the compound heterozygous variants c.107G > C:p.C36S and c.1037-7 T > A:p.G346Efs*11 of TYR gene were the pathogenic variants for this OCA1 family.

Enhancing the thermostability of Streptomyces cyaneofuscatus strain Ms1 tyrosinase by multi-factors rational design and molecular dynamics simulations.

This study presents a multi-factor rational design strategy combined with molecular dynamics simulation to improve the thermostability of Streptomyces cyaneofuscatus strain Ms1 tyrosinase. Candidate mutation sites were identified using Discovery Studio and FoldX software, and the double mutant G124W/G137W was obtained. The mutant was heterogeneously expressed in Escherichia coli strain Rosetta2 (DE3), and its thermostability was verified. Results indicate that the rational design method, combined with molecular dynamics simulation and protein energy calculation, improved the enzyme's thermostability more accurately and effectively. The double mutant G124W/G137W had an optimum temperature of 60°C, about 5.0°C higher than that of the wild-type TYRwt, and its activity was 171.06% higher than the wild-type TYRwt. Its thermostability was enhanced, 42.78% higher than the wild-type at 50°C. These findings suggest that the rational design strategy applied in this study can facilitate the application of industrial enzymes in the pharmaceutical industry.

Raspberry ketone glucoside suppresses melanin synthesis through IL6/JAK1/STAT3 signal pathway.

OBJECTIVE: This study aimed to evaluate the anti-melanogenic activity of raspberry ketone glucoside (RKG) and further explore the specific molecular mechanisms by which RKG affects melanogenesis. MATERIALS AND METHODS: The B16F10 cells model, the mushroom tyrosinase model and the zebrafish model were used to assess the whitening activity of RKG. We subsequently identified possible pathways related to RKG inhibition of melanogenesis by RNA-seq analysis and qRT-PCR on the zebrafish model, and further explored the effects of key genes on the pathway on the melanogenic effect of RKG by using related pathway inhibitors and Tg [mpeg: EGFP] transgenic zebrafish line. RESULTS: RKG could noticeably inhibit melanogenesis in B16F10 cells in vitro and on zebrafish in vivo. The RNA-Seq analysis and the qRT-PCR in zebrafish embryos indicated that the inhibition of melanogenesis by RKG could be achieved by activating JAK1/STAT3 signal pathway and inhibiting the expression levels of the MITFa, TYR, TYRP1a genes directly associated with melanogenesis. The inhibitor tests revealed that the inhibitory effect of the RKG on melanogenesis was restored by the IL6, JAK1/2, and STAT3 inhibitors, specifically STAT3 inhibitor. We further examine the relationship between the JAK1/STAT3 signal pathway and the MITFa. The achieved results indicate that the RKG could activate the zebrafish macrophages via the JAK1, but the inhibition of macrophage activation by loganin did not affect the anti-pigmentation effect of the RKG. CONCLUSIONS: RKG showed remarkable whitening activity on both B16F10 cells in vitro and zebrafish model in vivo. Furthermore, RKG could inhibit melanogenesis by activating the IL6/JAK1/STAT3 pathway, inhibiting the transcriptional activity of MITFa, and its downstream expression levels of the TYR and TYRP1a genes.

Pigmentation and TYRP1 expression are mediated by zinc through the early secretory pathway-resident ZNT proteins.

Tyrosinase (TYR) and tyrosinase-related proteins 1 and 2 (TYRP1 and TYRP2) are essential for pigmentation. They are generally classified as type-3 copper proteins, with binuclear copper active sites. Although there is experimental evidence for a copper cofactor in TYR, delivered via the copper transporter, ATP7A, the presence of copper in TYRP1 and TYRP2 has not been demonstrated. Here, we report that the expression and function of TYRP1 requires zinc, mediated by ZNT5-ZNT6 heterodimers (ZNT5-6) or ZNT7-ZNT7 homodimers (ZNT7). Loss of ZNT5-6 and ZNT7 function results in hypopigmentation in medaka fish and human melanoma cells, and is accompanied by immature melanosomes and reduced melanin content, as observed in TYRP1 dysfunction. The requirement of ZNT5-6 and ZNT7 for TYRP1 expression is conserved in human, mouse, and chicken orthologs. Our results provide novel insights into the pigmentation process and address questions regarding metalation in tyrosinase protein family.

Proprotein Convertase Furin Regulates Melanogenesis via the Notch Signaling Pathway.

BACKGROUND: Furin is a calcium-dependent serine protease found in almost all mammals. It plays an important role in embryogenesis, tissue homeostasis, tumors pathogenesis, viral infectious diseases, and neurodegenerative diseases. However, whether furin directly regulates melanin synthesis and transport has rarely been evaluated yet. The present study aimed to investigate furin potential function and mechanisms in melanogenesis. METHODS: Short hairpin RNAs targeting furin gene (sh-furin RNAs) were used to inhibit furin gene expression in human melanoma cell line MNT-1 cells. Then, intracellular melanin content was measured using a sodium hydroxide method. Extracellular melanin content was measured determining cell culture medium absorbance at 450 nm. Levodopa (L-DOPA) oxidation rate was measured to assess the tyrosinase activity. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting (WB) were performed to measure melanogenesis-related genes and Notch pathway-related genes expression levels. Human primary melanocytes (MCs) were extracted from foreskin tissues and were stimulated with a furin inhibitor. Then, the extracellular and intracellular melanin content, tyrosinase activity and molecules related to melanogenesis and the Notch pathway expression were measured in MCs with or without a furin inhibitor. Additionally, morpholino technology was used to inhibit furin in zebrafish. Zebrafish pigmentary phenotypes in the control group and furin inhibition group were observed with a stereo microscope. Then, MCs number in the tail and head of the zebrafish were counted using Image J software (version 1.53t, National Institute of Health, Bethesda, MD, USA). Meanwhile, melanin content, tyrosinase activity, and molecules related to melanogenesis and the Notch pathway expression levels were measured. Subsequently, valproic acid (VPA), a Notch pathway agonist, was used in MNT-1 melanoma cells treated with or without sh-furin lentiviral vectors for rescue experiments. RESULTS: Furin inhibition enhanced intracellular and extracellular melanin content, and cellular tyrosinase activity in MNT-1 cells and MCs. Additionally, furin inhibition increased melanin synthesis-associated and transport-associated proteins expression levels while inhibiting Notch pathway-relevant proteins. After using VPA to activate the Notch pathway in MNT-1 cells transfected with a sh-furin RNA, the biological effects resulting from furin knockdown were reversed. In addition, the results of in vivo experiments using morpholino to knock down furin gene in zebrafish further confirmed that furin knockdown regulated melanogenesis and impaired the Notch pathway. CONCLUSIONS: This study clarified that furin affected the synthesis and transport of melanin via Notch pathway. Notch pathway may be a potential therapeutic target for pigmented skin diseases.