Five known melanosome-specific proteins serve as immune targets for melanoma, i.e. TYR, TRP1/gp75, TRP2/Dct, Pmel17/gp100 and MART1. Host cellular and humoral immune responses play important roles in autoimmune pigmentary diseases (e.g. vitiligo) but are usually unable to control the growth of primary melanomas or their metastases. We are attempting to identify novel melanocyte-specific proteins expressed by less differentiated melanocytic cells that should provide more immune targets for diagnosis and/or therapy of melanoma. We are also looking at approaches to regulating the expression of genes in melanocytic cells which modulate their growth, and are mapping loci that are closely linked to the growth and metastases of melanomas in various locations on the body. Molecular approaches to clone pigment-related genes have been highly successful, but despite that only 50% of the more than 200 known pigment-related genes have been cloned leaving many potential melanocyte-specific targets to be identified. Tissue microarray and proteomics approaches to define markers of melanoma progression have also identified useful tumor markers. We have used a more direct approach, i.e. to purify melanosomes and use in-gel digestion and mass spec microsequencing to identify their protein constituents. We identified 1,500 proteins in melanosomes of all stages, with 600 in any given stage. They include 16 homologous to mouse coat color genes and many associated with human pigmentary diseases. 100 proteins shared by all stages of melanosomes define the essential melanosome proteome. As a result, we successfully published online the first global proteome database, an early gold standard for future proteomics studies on melanosomes and LROs. This project is now focused on identifying novel and specific melanosomal proteins that may prove to be useful targets for the immunodiagnosis and/or immunotherapy of melanoma. We are examining differences in proteins expressed in various maturation stages of melanosomes produced by pigmented and/or by amelanotic melanoma cells. Special emphasis will be made to identify new markers potentially suitable to identify amelanotic melanomas, which are difficult to detect and are highly aggressive. Complete and accurate profiling of cellular organelle proteomes is important to understand detailed cellular processes at the organelle level. We performed an informatics analysis and compiled human organelle reference datasets from large-scale proteomic studies and protein databases for seven LROs, as well as for ER and mitochondria. It is not a trivial challenge to characterize the subcellular locations and functions of those newly identified proteins in melanosomes. It can be tedious and expensive to generate new probes and antibodies required for such studies. We have done so already for many of them (e.g. PEDF, SLC24A5, ABCB5, VAT1, flotillin, syntenin, FLJ20420 and oculospanin) and have validated their localization in melanosomes. Functional analysis is complete for the previously known catalytic and structural melanosomal proteins, but for newly discovered proteins, other molecular approaches, such as siRNA and/or transfection, will be used. Our research then focused on one of the most frequent causes for failure to detect highly aggressive melanomas, which is the lost expression of melanoma antigens. Using data from our proteomics database, we identified proteins involved in the polarized sorting of proteins confirming that melanocytes are polarized cells. Polarization is the capacity of cells to transport proteins to specific cell membrane regions using a set of highly specialized proteins. We demonstrated that melanoma cells lose expression of polarized elements which compromises expression of critical melanoma antigens such as Pmel17/gp100. Furthermore, our work revealed that melanoma cells have a reduced capacity to correctly sialylate nascent carbohydrate chains. This results in the formation of two Pmel17 proteins with different carbohydrate contents and apparently different functions: one involved in melanosome fibril formation and the other involved in a still unknown function that may involve antigen presentation. To demonstrate the existence of these two Pmel17 proteins, we designed and tested a new specific antibody (termed alphaPEP25h) against the core region of Pmel17 which can distinguish those forms. Interestingly, this novel data not only demonstrates the polarized nature of melanocytes but also explains the complex mechanisms involved in the expression of specific melanoma antigens. We are collaborating with Dr. Gottesman's MDR group to define yet another unexpected function of melanosomes. That project evolved from the specific expression of an MDR protein (ABCB5) in melanocytes and its apparent localization in melanosomes. MDR mechanisms underlying the intractability of melanomas to chemotherapy remain largely unknown but are a critical therapeutic challenge. We found that MDR at least in some melanomas involves the sequestration of cytotoxic drugs within subcellular organelles (including melanosomes), which significantly reduces their nuclear localization. The accumulation of cisplatin in melanosomes also remarkably modulates melanogenesis through a pronounced increase in TYR activity, an 8-fold increase in intracellular pigmentation and increased extracellular transport of melanosomes containing cisplatin. Thus, our study provides evidence that melanosomes contribute to the refractory nature of melanoma cells by sequestering cytotoxic drugs and increasing melanosome-mediated export of drugs. Preventing sequestration of cytotoxic drugs by inhibiting the functions of melanosomes may have great potential as an approach to improve the chemosensitivity of melanoma tumors. Further, we think it likely that ABCB5 plays an important role in normal melanosomes to ensure that cytotoxic intermediates of melanin synthesis remain within that organelle, which are eventually lost by desquamation or hair growth, and we are testing that hypothesis. Regarding our studies of pigmentary disorders, mutations in the TYR gene result in OCA1, the most dramatic and severe form of that disease, where little or no melanin is produced in the hair, skin or eyes. Mutations in the gene encoding TYRP1 affect its interactions with TYR, and result in the hypopigmented phenotype of OCA3. Mutations in Tyr or Tyrp1 elicit the retention of TYR in the ER and its degradation by proteasomes, revealing that OCA1 and OCA3 are in fact ER-processing diseases. OCA2 and OCA4 are relatively severe forms of OCA which arise from mutations in the P and MATP loci, respectively. Both encode highly similar proteins with 12 transmembrane motifs. We found that both OCA2 and OCA4 result from altered trafficking of TYR after processing in the ER and Golgi en route to melanosomes, which reduces pigmentation despite the presence of functionally active TYR. We are testing the hypothesis that P and MATP affect the pH of sorting vesicles leading to disrupted trafficking of TYR to melanosomes. Our studies on the disrupted TYR processing and sorting in OCA2 and in OCA4 melanocytes explains their hypopigmented phenotypes will provide new insights into the involvement of transporters in the normal physiology of melano [summary truncated at 7800 characters]
| Passeron, Thierry; Valencia, Julio C; Namiki, Takeshi et al. (2009) Upregulation of SOX9 inhibits the growth of human and mouse melanomas and restores their sensitivity to retinoic acid. J Clin Invest 119:954-63 |
| Namiki, Takeshi; Valencia, Julio C; Hall, Matthew D et al. (2008) A novel approach to enhance antibody sensitivity and specificity by peptide cross-linking. Anal Biochem 383:265-9 |
| Yamaguchi, Yuji; Passeron, Thierry; Hoashi, Toshihiko et al. (2008) Dickkopf 1 (DKK1) regulates skin pigmentation and thickness by affecting Wnt/beta-catenin signaling in keratinocytes. FASEB J 22:1009-20 |
| Hu, Zhang-Zhi; Valencia, Julio C; Huang, Hongzhan et al. (2007) Comparative Bioinformatics Analyses and Profiling of Lysosome-Related Organelle Proteomes. Int J Mass Spectrom 259:147-160 |
