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 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. Molecular approaches to clone pigment-related genes have been highly successful, but despite that only 50% of the >120 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 are using a more direct approach, i.e. to purify melanosomes and use in-gel digestion and mass spec microsequencing to identify their protein constituents. We used differential and density gradient centrifugations along with free flow electrophoresis to purify melanosomes at various stages from pigmented and from unpigmented melanoma cells. We developed a novel method to remove melanin which allowed the proteomes of melanosomes at various stages of development to be analyzed. 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. Many proteins were shared with other cellular compartments, most notably with LROs, which validates their common evolutionary origin and provides valuable clues about their biogenesis. 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. Heterogeneous sources of human organelle proteins and their homologs from lower species were mapped to human UniProtKB protein entries based on ID and/or peptide mappings, followed by functional annotation and categorization using the iProXpress proteomic expression analysis system. The shared proteins of LROs indicate their dynamic and hybrid nature, while the unique proteins identified may represent additional candidate markers for LROs. That searchable database is now freely accessible online and it should prove to be a rich and essential resource for all scientists in the field to characterize melanosomes and other LROs. 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 approaches, such as siRNA and/or transfection, will be used to characterize them. Importantly, we have collaborated with Dr. Michael Gottesmans MultiDrug Resistance (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. We are continuing this project based on our successful online publication of the first global proteome database, an early gold standard for future proteomics studies on melanosomes and LROs. The expertise of our collaborators in chemistry and immunology (Dr. Ettore Appella, LCB), in proteomics and mass spectrometry (Dr. Donald Hunt, Univ VA) and in biostatistics (Dr. Cathy Wu, Georgetown Univ) combined with our own pigment cell biology expertise provides a multi-faceted approach for such analysis. 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 will examine differences in proteins expressed in various maturation stages of melanosomes produced by pigmented (MNT1) and/or by amelanotic (Sk-Mel28) melanoma cells. The rationale behind this is to identify proteins that are critical for melanosome biogenesis and/or function. Special emphasis will be made to identify new markers potentially suitable to identify amelanotic melanomas, which are difficult to detect and are highly aggressive. 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 hyp [summary truncated at 7800 characters]
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| 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 |
| 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 |
| 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 |
