No / Recently two alternative mechanisms have been put forward for the inhibition of tyrosinase by 6R-l-erythro 5,6,7,8-tetrahydrobiopterin (6BH4). Initially allosteric uncompetitive inhibition was demonstrated due to 1:1 binding of 10¿6 M 6BH4 to a specific domain 28 amino acids away from the CuA active site of the enzyme. Alternatively it was then shown that 10¿3 M 6BH4 inhibit the reaction by the reduction of the product dopaquinone back to l-dopa. In the study presented herein we have used two structural analogues of 6BH4 (i.e., 6,7-(R,S)-dimethyl tetrahydrobiopterin and 6-(R,S)-tetrahydromonapterin) confirming classical uncompetitive inhibition due to specific binding of the pyrimidine ring of the pterin moiety to the regulatory domain on tyrosinase. Under these conditions there was no reduction of l-dopaquinone back to l-dopa by both cofactor analogues. Inhibition of tyrosinase by 6BH4 occurs in the concentration range of 10¿6 M after preactivation with l-tyrosine and this mechanism uncouples the enzyme reaction producing H2O2 from O2. Moreover, a direct oxidation of 6BH4 to 7,8-dihydrobiopterin by tyrosinase in the absence of the substrate l-tyrosine was demonstrated. The enzyme was activated by low concentrations of H2O2 (<0.3 × 10¿3 M), but deactivated at concentrations in the range 0.5¿5.0 × 10¿3 M. In summary, our results confirm a major role for 6BH4 in the regulation of human pigmentation.

No / The pro-opiomelanocortin (POMC)-derived peptides, -melanocyte-stimulating hormone, and adrenocorticotropic hormone, are important mediators of human skin pigmentation via action at the melanocortin-1 receptor. Recent data suggests that such a regulatory role also exists for the endogenous opiate, -endorphin (-END). A role for this -END in the regulation of follicular pigmentation, however, has not been determined. This study was designed to examine the involvement of the -END/-opiate receptor system in human follicular melanocyte biology. We employed RT-PCR, and immunohisto/cytochemistry and immunoelectron microscopy using -END and -opiate receptor specific antibodies and a functional role for -END was assessed by direct stimulation with the peptide. This study has demonstrated that human hair follicle melanocytes (HFM) express mRNA for the -opiate receptor and POMC. Furthermore, -END and its high affinity -opiate receptor are expressed at the protein level in glycoprotein100-positive follicular melanocytes and as a function of their anatomic location and differentiation status during the hair growth cycle. Functional studies revealed that -END is a modifier of HFM phenotype via its ability to upregulate melanogenesis, dendricity, and proliferation. These findings suggest a new regulatory role for -END in human HFM biology, providing a new research direction into the fundamental regulation of human hair pigmentation.

No / The skin pigment melanin is produced in melanocytes in highly specialized organelles known as melanosomes. Melanosomes are related to the organelles of the endosomal/lysosomal pathway and can have a low internal pH. In the present study we have shown that melanin synthesis in human pigment cell lysates is maximal at pH 6.8. We therefore investigated the role of intramelanosomal pH as a possible control mechanism for melanogenesis. To do this we examined the effect of neutralizing melanosomal pH on tyrosinase activity and melanogenesis in 11 human melanocyte cultures and in 3 melanoma lines. All melanocyte cultures (9 of 9) from Caucasian skin as well as two melanomacell lines with comparable melanogenic activity showed rapid (within 24 h) increases in melanogenesis in response to neutralization of melanosomal pH. Chemical analysis of total melanin indicated a preferential increase in eumelanin production. Electron microscopy revealed an accumulation of melanin and increased maturation of melanosomes in response to pH neutralization. In summary, our findings show that: (i) near neutral melanosomal pH is optimal for human tyrosinase activity and melanogenesis; (ii) melanin production in Caucasian melanocytes is suppressed by low melanosomal pH; (iii) the ratio of eumelanin/phaeomelanin production and maturation rate of melanosomes can be regulated by melanosomal pH. We conclude that melanosomal pH is an essential factor which regulates multiple stages of melanin production. Furthermore, since we have recently identified that pink locus product (P protein) mediates neutralization of melanosomal pH, we propose that P protein is a key control point for skin pigmentation. We would further propose that the wide variations in both constitutive and facultative skin pigmentation seen in the human population could be associated with the high degree of P-locus polymorphism.

The melanocortin-1 receptor (MC1R) is a G-protein coupled receptor (GPCR) expressed on the surface of the melanocyte. MC1R activation after UV exposure results in the production of the dark eumelanin pigment and the tanning process in humans, providing protection from UV induced DNA damage. MC1R activation has also recently been linked to DNA repair. The MC1R gene is highly polymorphic in Caucasian populations with a number of MC1R variant alleles associated with red hair, fair skin, poor tanning and increased risk of melanoma and non-melanoma skin cancer. These MC1R variant receptors were thought to be loss of function, however the type of defect and the extent of the loss of function for individual variants was relatively unknown before the commencement of this PhD project. Many GPCR mutant proteins are intracellularly retained, resulting in a loss of signalling ability. To determine if this was the case for MC1R variant receptors, the localisation of the wild type and variant MC1R protein was investigated using immunofluorescence and radio-ligand binding on transfected melanocytic cells as well as primary melanocyte strains. For the first time, several MC1R variants including V60L, R151C, I155T, R160W and R163Q, were shown to have reduced cell surface expression compared to wild type MC1R. cAMP assays were used to determine the signalling ability of activated wild type and variant MC1R, importantly, variant receptors with reduced cell surface expression showed corresponding impairment in cAMP signalling. In contrast, the R142H and D294H variants, which have normal cell surface expression but significantly impaired cAMP signalling, are thought to have a defect in G-protein coupling. Some MC1R variants were found to have dominant negative activity on the wild type receptor in co-expression studies, this result may explain the MC1R heterozygote effect on human pigmentation phenotypes. This dominant negative effect resulted in either reduced wild type cell surface expression or reduced G-protein coupling and may be mediated by receptor dimerisation. In order to validate the in vitro studies, comparison of variant receptor characteristics with skin and hair colour data of individuals both homozygous and heterozygous for MC1R variant alleles was performed. This revealed parallels between variant MC1R cell surface expression, functional ability, dominant negative activity and the strength of the effects of variant alleles on human pigmentation. From the in vitro functional studies, it was clear that most variant receptors retained some signaling ability, although the relative abilities varied. An important unanswered question in the literature was whether the phenotype of carriers of the high penetrance MC1R variant alleles was actually representative of complete loss of function for MC1R. Due to the rarity of MC1R null alleles they had only previously been found in the heterozygous state, however we described the phenotype of one individual compound heterozygous for two frameshift mutations resulting in an individual unable to produce any functional MC1R protein. Phenotypic analysis indicated that red hair and fair skin is found in the absence of MC1R. Finally, preliminary studies using low temperature, chemical or pharmacological chaperones indicated that the cell surface expression of some MC1R variants could be rescued in cell transfection experiments. This resulted in a restoration of signaling ability after stimulation with agonist. These studies into the localization and function of MC1R variants have contributed to a greater understanding of the molecular mechanism underlying the association of MC1R with skin cancer risk phenotypes, and may lead to future drug based therapies that are able to rescue the function of MC1R variants that are intracellularly retained.

Dans l’épiderme, les mélanocytes participent à la protection de la peau contre les rayons ionisants du soleil en synthétisant un pigment, la mélanine, dans des compartiments apparentés aux lysosomes appelés melanosomes. La mélanogenèse est un processus séquentiel initié par la production de fibres amyloïdes dont la composante principale est la protéine PMEL. Ces fibres séquestrent la mélanine et permettent l’élimination d’intermédiaires toxiques produits lors de sa synthèse. La mélanogenèse et le phénotype pigmenté sont affectés lorsque le processus de formation des fibres est altéré. Les fibres résultent du clivage de PMEL dans les endosomes précurseurs des mélanosomes mais les protéases impliquées dans ce processus restent peu ou pas caractérisées. Afin de mieux comprendre les mécanismes de formation des fibres amyloïdes dérivées de PMEL, j’ai étudié le rôle de deux protéases : les Bêta-sécrétases BACE1 et BACE2. En combinant des techniques de biochimie, d’immunocytochimie et d’imagerie photonique et électronique, j’ai montré que la perte de l’expression de Bace2 in vivo (souris KO BACE2) ou sa déplétion (siRNA) dans une lignée de mélanocytes inhibent le clivage amyloïdogénique de PMEL et affectent à la fois la formation de fibres de PMEL dans les mélanosomes et la pigmentation. J’ai pu notamment reproduire in vitro le clivage spécifique de PMEL en utilisant une forme recombinante de BACE2. En parallèle, j’ai également étudié le rôle de BACE1 dans la mélanogenèse. Mes résultats indiquent que BACE1, bien que n’étant pas impliquée dans le clivage de PMEL, régulerait la maturation des mélanosomes précoces in vivo et in cellulo, en modulant les contacts entre mélanosomes et réticulum endoplasmique (RE). Dans les mélanocytes, BACE1 est présente dans le RE et interagit avec des protéines impliquées dans les contacts RE-endosomes. Ces contacts seraient cruciaux pour le transfert de molécules nécessaires à la maturation des mélanosomes. L’ensemble de ces résultats démontre un rôle pour chacune des Bêta-sécrétases dans le processus de mélanogenèse, levant le voile sur des processus clés liés à la biogenèse des mélanosomes. Par ailleurs, les fibres de PMEL constituant le modèle le plus abouti de l’amyloïdogenèse physiologique chez les mammifères, ces études pourraient à plus long terme aider à la compréhension de la formation des fibres amyloïdes pathologiques ; notamment dans la maladie d’Alzheimer où l’amyloïdogenèse d’APP est très similaire à celle de PMEL. / In the epidermis, melanocytes synthetize a pigment called melanin, in lysosome-related-organelles called melanosomes, in order to protect the skin against the ionizing radiations of the sun. Melanogenesis is a sequential process initiated by the formation of amyloid fibrils whose principal component is the protein PMEL. Those fibrils sequester the melanin pigment and allow the removal of toxic intermediates formed during its synthesis. Melanogenesis and the pigmented phenotype are affected when the process of fibrils formation is altered. Fibrils come from the processing of PMEL in endosome precursors of melanosomes but the proteases implicated in this process are not well characterized. In order to better understand the mechanisms implicated in the formation of the PMEL amyloid fibrils, I studied the role of two proteases: the Beta-secretases BACE1 and BACE2. Using a combination of biochemical, immunocytochemical methods and photonic and electronic imaging, I have shown that the loss of Bace2 expression in vivo (BACE2 KO mice) or its depletion (siRNA), in a melanocyte cell line, inhibit the amyloidogenic processing of PMEL and affect both the formation of the PMEL fibrils in melanosomes and pigmentation. I could reproduce in vitro the specific cleavage of PMEL by using a recombinant form of BACE2. In parallel, I have also studied the role of BACE1 in melanogenesis. My results indicate that BACE1, even though it is not implicated in PMEL processing, could regulate the maturation of early melanosomes in vivo and in cellulo, by modulating the contacts between melanosomes and endoplasmic reticulum (ER). In melanocytes, BACE1 is present in the ER and interacts with proteins implicated in ER-endosomes contacts. Those contacts would be crucial for the transfer of molecules that are necessary for melanosome maturation. All together those results demonstrate the role of both Beta-secretases in melanogenesis, and reveal key processes involved in melanosome biogenesis. Moreover, because PMEL fibrils are the most completed model of physiological amyloidogenesis in mammals, theses studies could help in the future the understanding of the formation of pathological amyloid fibrils; in particular in the Alzheimer’s disease where the amyloidogenesis of APP is very similar to the one of PMEL.

No description available.

Ultraviolet radiation (UVR) stimulates melanogenesis in melanocytes, primarily via release of alpha-melanocyte stimulating hormone from keratinocytes. UVR also induced an inflammatory response in the skin in which Interferon-gamma (IFNγ) cytokine plays an important orchestrating role. Here we report that recombinant IFNγ induces a temporal increase of melanogenesis in mouse melanoma cells. IFNγ elevates expression of microphthalmia-associated transcription factor (Mitf), which is the master regulator of melanogenesis by initiating transcription of melanogenic enzymes, tyrosinase (Tyr), tyrosinese-related protein 1 (Tyrp1) and dopachrome tautomerase (Dct). Interestingly, tyrosinase protein, but not mRNA expression, accumulated in response to IFNγ treatment and was consistent with tyrosinase activity. In addition, glycosidase digestion showed that IFNγ induced ER-resistant, fully mature tyrosinase via post-transcriptional mechanisms, rather than increased de novo synthesis or early processing in the ER. Most strikingly, IFNγ mediated alkalization of melanosomes by elevating Oca2 expression, which leads to facilitate melanosome maturation and sequential accumulation of mature tyrosianse. Both Jak1/Jak2 inhibitor Ruxolitinib and knockout of Stat1 mediated by CRISPR-CAS9 blocked the IFNγ-induced Mitf, tyrosinase, Oca2 expressions and melanin biosynthesis. Our data reveals that IFNγ-Jak1/2-Stat1 axis regulates melanogenesis by inducing maturation of melanosomes and accumulation of mature tyrosinase via post-translational mechanisms. CTLA4 is a cell surface receptor on T cells that functions as an immune checkpoint molecule to enforce tolerance to cognate antigens. Anti-CTLA4 immunotherapy is highly effective at reactivating T cell responses against melanoma, which is postulated to be due to targeting CTLA4 on T cells. Here we report that CTLA4 is also highly expressed by most human melanoma cell lines, as well as in normal human melanocytes. Interferon-gamma (IFNγ) signaling activated the expression of the human CTLA4 gene in a melanocyte and melanoma cell-specific manner. Mechanistically, IFNγ activated CTLA4 expression through JAK1/2-dependent phosphorylation of STAT1, which bound a specific gamma-activated sequence (GAS) site on the CTLA4 promoter, thereby licensing CBP/p300-mediated histone acetylation and local chromatin opening. In melanoma cell lines, elevated baseline expression relied upon constitutive activation of the MAPK pathway. Notably, RNA-seq analyses of melanoma specimens obtained from patients who had received anti-CTLA4 immunotherapy (ipilimumab) showed upregulation of an IFNγ -response gene expression signature, including CTLA4 itself, which correlated significantly with durable response. We also show that ectopic expression of Ctla4 in mouse melanoma cells promotes tumor growth in immunocompetent mice. Ctla4-enhanced melanomagenesis is blocked in immunodeficient NSG mice. In addition, ligation of CD86 (one of Ctla4 ligands) in T cells inhibits CD8 T cells proliferation in vitro. Expression of Ctla4 in melanoma cells are resistant to CD8 T cell cytoxicity in vitro. Our data demonstrates and highlights the novel and unrecognized functions of CTLA4 in melanoma cells that aids their survival, immunoevasion and tumorigenic capabilities. Taken together, these findings have potential implications for the conventional and prototypical roles of the IFNγ signaling pathway and CTLA4 in tumor immunosurveillance and tumor immunoevasion. More importantly, our results raise the possibility that CTLA4 targeting on melanoma cells may contribute to the clinical immunobiology of anti-CTLA4 responses. / Biomedical Sciences

The metabolism of tyrosine is essential for many critical biochemical events including catecholamine synthesis, melanogenesis and insect cuticle sclerotization. These pathways are highly regulated in both insects and mammals by many well-characterized enzymes including dopa decarboxylase and tyrosine hydroxylase. On the other hand, there are still many enzymes involved in these processes that we know very little about. Dopachrome tautomerase (DCT), dopachrome conversion enzyme (DCE) and α-methyldopa resistant protein (AMD) fall into the category of the less characterized enzymes. Dopachrome is a pivotal intermediate in melanogenesis. Mammalian DCT and insect DCE both use dopachrome as a substrate. DCE catalyzes a decarboxylative structural rearrangement of dopachrome to 5,6-dihydroxyindole (DHI), whereas DCT mediates the isomerization/tautomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA). DHI is oxidized easily, leading to the production of melanin, as well as reactive oxygen species (ROS). DHICA is less reactive, relative to DHI, and consequently produces less toxic byproducts during melanogenesis; therefore DCT plays an important role in detoxification of DHI and ROS. Purification and MS analysis of DCE and DCT determined that N-glycosylation is a primary post-translational modification. Q-TOF mass spectrometry was used to determine N-glycosylation patterns from Aedes aegypti DCE and MALDI-TOF/TOF was used to determine multiple glycosylation sites in DCT. N-glycosylation is critical for the folding and trafficking of secreted proteins in the endomembrane system. The analysis of glycosylation sites in DCE and DCT therefore is essential toward achieving a comprehensive understanding of their structure and function. Like DCT, AMD also plays a protective role. The AMD protein was originally identified in Drosophila mutants hypersensitive to α-methyldopa, an inhibitor of dopa decarboxylase (DDC). Production of dopamine by DDC is critical for developing insects because dopamine conjugates are used as crosslinking agents for cuticle sclerotization. Although there has been much discussion into the function of AMD, what exactly this protein does has been unknown. AMD shares 48% sequence identity with DDC, however we have found that AMD is an enzyme, which possesses a different catalytic activity. GC-MS analysis of AMD enzymatic reaction components revealed that AMD catalyzes the oxidative decarboxylation of L-DOPA to DOPAL, and also the oxidative decarboxlation of α-methyldopa to 3,4-dihydroxyphenylacetone. In summary, multiple N-glycosylation sites were characterized in DCT and DCE, furthermore a new protein function has been demonstrated for AMD. These experiments were performed using classical biochemistry techniques in combination with mass spectrometry. / Ph. D.

No / Human skin is a local source of corticotropin-releasing hormone (CRH) and expresses CRH and CRH receptors (CRH-R) at mRNA and protein levels. Epidermal melanocytes respond to CRH by induction of cAMP with up-regulation of pro-opiomelanocortin gene expression and subsequent production of adrenocorticotropin hormone. However, the role of CRH/CRH-R in melanocyte biology is complicated by the significant heterogeneity of cutaneous melanocyte subpopulations, from continuously active and UV-responsive melanocytes in epidermis to UV nonresponsive, hair growth cycle-coupled melanogenesis in hair follicles. In the present study we report that normal human scalp hair follicle melanocytes express CRH at the mRNA level. Furthermore, CRH, urocortin and CRH-R 1 and 2 were differentially expressed in follicular melanocytes, fibroblasts, and keratinocytes depending on anatomic location and differentiation status in situ and in vitro. Stimulation of follicular melanocytes with CRH and CRH peptides, modified for selectivity for CRH-R1 and/or CRH-R2, variably induced cell melanogenesis, dendricity, and proliferation. CRH-peptides also stimulated the expression and activity of Tyrosinase, and expression of Tyrosinase-related protein-1 and-2. However, a modified urocortin peptide highly selective for CRH-R2 down-regulated melanocyte differentiation phenotype. This study indicates that CRH peptides can differentially influence hair follicle melanocyte behavior not only via CRH-R1 signaling but also by complex cross-talk between CRH-R1 and CRH-R2.¿Kauser, S., Slominski, A., Wei, E. T., Tobin, D. J. Modulation of the human hair follicle pigmentary unit by corticotropin-releasing hormone and urocortin peptides.

No / Erythema occurs in human skin following excessive exposure to ultraviolet radiation (UVR), and this is in part mediated by the vasodilator prostaglandin E2 (PGE2). While keratinocytes are a major source of this pro-inflammatory eicosanoid, epidermal melanocytes (EM) also express some of the cellular machinery required for PGE2 production. The primary aim of this study is to determine whether EM can produce PGE2 and so potentially also contribute to UVR-induced skin inflammation. Furthermore, we investigate the likely pathway by which this PGE2 production is achieved and investigate whether PGE2 production by EM is correlated with melanogenic capacity. Primary cultures of EM were established from nine normal healthy individuals with skin phototype-1 (n=4) and 4 (n=5), and PGE2 production and melanogenic status were assessed. EM produced PGE2 under baseline conditions and this was increased further upon stimulation with arachidonic acid. Moreover, EM expressed cytoplasmic phospholipase A2, cyclooxygenase-1 and cytoplasmic prostaglandin E synthase. However, no EM culture expressed cyclooxygenase-2 under baseline conditions or following arachidonic acid, UVB- or H2O2 treatments. PGE2 production in response to UVB was highly variable in EM cultures derived from different donors but when pooled for skin phototype exhibited a positive correlation only with SPT-1 derived EM. Interestingly, PGE2 production by EM in response to UVB showed no correlation with baseline levels of melanin, tyrosinase expression/activity or tyrosinase-related protein-1 expression. However, there was an apparent negative correlation with baseline expression of dopachrome tautomerase (DCT), a melanogenic enzyme with reported anti-oxidant potential. These findings suggest that EM have the potential to contribute to UVR-induced erythema via PGE2 production, but that this response may be more related to oxidative stress than to their melanogenesis status. / The Wellcome Trust