June 14 - 17, 2001, Minneapolis, Minnesota
Below are reports on various sessions of the PASPCR X Annual Meeting. I would like to thank those individuals who wrote the excellent reviews on the sessions that they attended as noted at each section. Both Dick King and myself hope that you enjoyed the meeting in Minneapolis. This meeting was filled with science covering a broad range of topics in the pigment field as y;ou can see in the report below.
Reports can be found for the following symposium
Symposium I: Comparative Biology and Evolution
Slide Session I: Pigmentation
Symposium II: New Approaches to the Pigment Cell
Slide session II: Cell biology
Symposium III: Intracellular Trafficking and Organelle Biogenesis
Slide Session V: Cutaneous Pathology and Vitiligo
Slide Session VI: Model Systems and Late Breaking Research
Symposium V: Phenoloxidases, Melanogenesis and Evolution
Slide Session VII: Gene Regulation
Symposium I: Comparative Biology and Evolution
By Nels Granholm
It was the express purpose of the Organizing Committee of the 10th Annual PASPCR conference to present provocative symposia in order to place the concepts of pigment cell biology into a broader context. Symposium I : Comparative Biology and Evolution advanced that goal.
Three symposium and four platform speakers provided an overview and current state of the art on how demographic, evolutionary, and genetic factors may have altered the sequence and/or incidence of polymorphisms within the human MC1R gene (Makova, Rees), the value of genetic analysis of skin and hair color in humans as evidence for the modus operandi of evolution in humans (Shriver), and current updates on the overall reliability of various methods to measure response to UV radiation in human skin (Tadokora), contributions of P gene and MC1R gene mutations in the etiology of OCA2 (Schmidt), absence of human agouti gene polymorphisms in previously identified MC1R variants (van Daal), and roles of various stimulatory agents (IBMX, bafilomycin) on the dynamics of tyrosinase synthesis and/or activation in human melanocytes as mediated by hydrogen exchanger/transporter mechanisms within melanosomes (Spaulding).
MC1R, one of a five-member family of G protein-coupled seven-repeat transmembrane receptor encoding genes, is indeed an intriguing gene, since it plays a central role in skin color variation in humans. To date, MCIR is about the only gene known that can provide at least a partial explanation for phenotypic variation in human pigmentation. GenBank comparisons, including nucleotide change estimates of synonymous (no change in the amino acid) and non-synonymous (changes in nucleotides leading to a different amino acid) substitutions, indicate that MC1R has evolved at a faster rate than other members of the melanocortin receptor family.
Point mutations that affect the functionality of proteins are generally disfavored by natural selection; one would assume that amino sequences of such critically important proteins would remain constant over time within and between species, like histones perhaps or like the amino acids at active sites of specific enzymes. However, point mutations affecting amino acids of proteins not as essential for protein function are not disfavored and may become fixed in species ancestors by random genetic drift. And some mutations may be favored by selection and are thus rapidly fixed in a species. If the rate of change of DNA or amino acids is constant or linear over time (a kind of molecular clock), then we can make assumptions about when groups or species diverged from one another based on the numbers of substitutions. Contributions by Drs. Makova, Rees, and Shriver helped us to gain a practical application and understanding of these and related principles as applied to evolutionary changes in gene (MC1R) sequences.
Much of the data on MC1R dealt with the role of selection on MC1R nucleotide differences (polymorphisms) between African, Asian, and European populations. Jon Rees looked particularly at the ORF of MC1R whereas Dr. Makova characterized 6.6 kb of the MC1R upstream of the ORF. So, what does this tell us about the polymorphisms of MC1R? We have data on polymorphisms of the expressed sequences of MC1R as well as 6.6 kb of DNA 'upstream' of the ORF.
Dr Makova drew the following conclusions: 1. The average nucleotide diversity (polymorphism) in the 6.6 kb upstream regions of human MC1R, exceptionally high when compared to other comparable gene sequences, may be due to high mutation rate, high recombination rate, and/or presence of Alu repeats, 2. As opposed to the coding region of MC1R, the promoter region is highly polymorphic in Africans when compared to Asians and Europeans, and this pattern is consistent with a population expansion in Africans, 3. Exhaustive analyses of patters of polymorphisms in 54 Asians, Africans, and Europeans suggest possible purifying selection acting within the middle and portions of the 5? subregions, diversifying selection in some sites, and possible relaxation of functional constraints, and 4. Dr. Makova also identified sites potentially important for MC1R promoter function.
Following Dr. Makova's analysis of the non-coding region of MC1R, Dr. Jon Rees summarized the current status of the MC1R coding region in his presentation, "The Importance of being Red". Sequence analysis of MC1R can enable us to determine the extent to which MC1R controls human pigmentation. It is of interest to know the number of MC1R functional variants, how those variants regulate phenotype, and in an evolutionary sense, what can MC1R tell us about evolution of pigmentation and about human evolution in general. Dr. Rees outlined a number of common sequence variants (codons 151, 160, 294, and 142) that account for about 80% of northern Europeans with red hair. MC1R also has an effect on cutaneous phenotypes, especially in response to UV irradiation; Dr. Rees reported a dosage effect of UV between wildtype, heterozygote, and compound heterozygote genotypes of MC1R. Experiments designed to relate MC1R genotype to precise pigmentation patterns in hair (balance of eu- and phaeomelanin) and skin response to UV allow Dr. Rees and coworkers to further our understanding of the relationship between MC1R sequence diversity and the actual physiology of the mutated receptors.
Dr. Mark Shriver outlined his recent work on the identification of genes contributing to pigmentation phenotypes in humans. As contrasted from the analysis of other complex multifactorial human disorders (e.g., obesity, diabetes, hypertension), Dr. Shriver believes that mammalian pigmentation genes may offer a more compelling experimental model to assess complex genetic interaction in the understanding of human pathology etiologies. For example many of the known pigmentation genes in mice and other mammals possess homologous sequences in humans that are responsible for human pathologies (e.g., albinism and Waardenberg Syndrome) that regulate in part the wide variation in human pigmentary phenotypes. By adopting methods like Mapping by Admixture Linkage Disequilibrium (MALD) to identify genes for polygenic traits, Dr. Shriver outlined current studies designed to detect and map pigmentation genes for skin color, hair color, eye color, and UV responsiveness.
Slide Session I: Pigmentation
By Nels Granholm
Dr. Tadokora and others presented data on the ability of normal human skin of various racial/ethnic groups to respond to UV exposure as determined by DNA damage/repair and melanin content as well as melanin synthesis. Their results suggest that both skin sensitivity as well as racial/ethnic origin are important determinants in response to UV and may be of value in predicting the risk of skin cancers.
An unusually pigmented human subject predicted to be a compound mutant at both P and MC1R loci (OCA2 plus unusual red hair) was discussed by Schmidt and others. Sequence analysis revealed a compound heterozygote for the P gene (N489D/W679C) and heterozygous at MC1R (R160W). This is the first published identification of a human OCA-like phenotype associated with red hair; the particular type of OCA for this subject should be distinguished from OCA3 (rufous/red OCA) due to the unique compound genotype.
Because of the existence of red-haired human phenotypes possessing wildtype MC1R sequences, additional loci besides MC1R may be directly involved in hair color. The agouti locus is a likely candidate due to its prominent role in coat color genetics of mice and other mammals. Drs. van Daal and Voisey undertook a study to analyze the entire agouti gene in humans (various racial/ethnic groups) for the presence of polymorphisms. Interestingly, following exhaustive analyses of agouti (ASP) sequences of subjects previously identified as positive for unusual MC1R variations, no polymorphisms were detected. Thus loci other than ASP and MC1Rmost likely participate in hair color in humans.
Tyrosinase activity in human melanosomes may be regulated in part by melanosomal pH. Data presented by Spaulding and others support a potentiating effect of IBMX and bafilomycin on the activation of preexisting tyrosinase rather than de novo tyrosinase synthesis. Regarding the mechanism, these authors also presented data suggesting that cAMP-elevating drugs like IBMX may be functioning in part via a hydrogen exchanger/transported within melanosomes
To summarize, analysis of MC1R is not only interesting in and of itself but also as a model for human evolution. Characterizations of MC1R DNA and various expressed mutant proteins provide valuable data on generation of pigmentation phenotypes, UV susceptibility, as well as physiologically significant ligand-receptor interactions at MC1R as well as other members of the melanocortin family. Secondly, as a determinant of human skin and hair color, MC1R may be a major model gene, along with a number of others, for the analysis of human evolution. Thanks to all speakers of this session for provocative discussions.
Symposium II: New Approaches to the Pigment Cell
By Jean Bolognia
Using Gene Expression Patterns to Characterize Biological Diversity
Charles P. Perou, PhD, UNC
The Role of Stem Cells in the Development of the Retina
Thomas Reh, PhD, Univ Washington
Modulation of Melanogenesis in vitro: Importance of Keratinocyte-Melanocyte Interactions
Rainer Schmidt, PhD, L?Oréal
Slide session II: Cell biology
By Jean Bolognia
The Pink-Eyed Dilution Protein Acts Early in Melanosome Biogenesis
P. Manga, K. Chen, S.J. Orlow, New York University SOM
Melanosome Mapping by Purification of Early Stage Melanosomes
T. Kushimoto, V. Basrur, J. Valencia, J. Matsunaga, W.D. Vieira, J. Muller, E. Appella, V.J. Hearing, NCI, NIH
Melanosome Transfer to Keratinocytes is Regulated by Surface Glycoproteins and Melanosome Distribution in Keratinocytes is Regulated by the Recipient Keratinocytes
R.E. Boissy, L. Minwalla, I.C. LePoole, R.R. Wickett: presented by R. Sarangarajan Univ of Cincinnati SOM
Induction of Melanogenesis and Cellular Signaling Pathways by Bicyclic Monoterpene Diols
D.A. Brown, J.W. Galvin, M.T. Canning, A.B. Brown, D.B. Yarosh, AGI Dermatics
Analysis of the Signaling Pathway and the DNA Damaging Effects of UVB on Human Melanocytes
E. Pereira, M.C. Scott, A.L. Kadekaro, R. Kavanagh, H.G. Shertzer, and Z.A. Abdel-Malek, Univ Cincinnati SOM
and
UVA Induces Oxidative Stress and Genotoxicity in Human Melanocytes
Z.A. Abdel-Malek, A.L. Kadekaro, M.C. Scott, E. Pereira, R. Kavanagh, H. Kanto, and H.G. Shertzer, Univ Cincinnati SOM
Symposium III: Intracellular Trafficking and Organelle Biogenesis
By Vijayasaradhi Setaluri
This symposium highlighted the recent advances in intracellular sorting of lysosomal and melanosomal proteins, biogenesis of melanosomes and their transport in melanocytes. The speakers illustrated how a wide range of experimental approaches, including mouse genetics and molecular genetic analysis of human pigmentation disorders, are helping us understand the intricate mechanisms of assembly and intracellular transport of organelles. Appropriately, the symposium opened with a talk by J. Bonifacino (NIH) on the molecular machinery for biogenesis of lysosomes, the most extensively studied organelles. Discussion of lysosome biogenesis is also relevant for pigment cell biology in light of many similarities between melanosomes and lysosomes. Bonifacino described the role of a new class of proteins known as Golgi-localized, gamma-ear containing, ARF-binding proteins (GGAs) in sorting of mannose 6-phosphate receptors (MPRs). Binding of the amino terminal VHS domains of clathrin-associated GGAs, specifically to the di-leucine sorting signal in the cytoplasmic tails of MPRs was shown to mediate sorting of MPRs from the trans-Golgi network (TGN) to endosomes. AP-1, the adaptor protein which was originally thought to mediate this sorting, is now relegated to a less important position in sorting MPRs and thereby lysosome biogenesis.
The ongoing debate over the relationship between lysosomes and melanosomes was addressed by Micheal Marks (Univ. Pennsylvania). Using immunogold electronmicroscopy as a principal tool, Marks suggested that in pigment cells, melanosomes represent a lineage of organelles distinct from conventional endosomes and lysosomes. The most significant findings described include the possible involvement of a Pmel17 enriched coated-endosome like structures in melanosome biogenesis, and the observation that melanosomal proteins are segregated from the late endocytic pathway.
While the early events in the biogenesis of melanosomes are still being worked out, much progress has been made in understanding the molecular mechanisms involved in the polarized transport of melanosomes toward keratinocytes. N. Jenkins (NCI) described the genetic approaches that led to the identification of melanophilin, a protein encoded by leaden (ln) gene. In ln mice melanin synthesis is normal but melanosome transport is impaired resulting in clumping of melanosomes, a phenotype similar to that found in dilute (d) and ashen (ash) mutant mice. Jenkins proposed that melanophilin, a novel Rab effector protein, functions as part of a transport complex with MyoVa and Rab27a proteins encoded, respectively, by d and ash loci. Data on candidate genes for dilute suppressor, a locus that suppresses ashen and leaden was also presented.
Understanding vesicular transport has implications for not only pigmentation but also other human disorders. Defects in vesicle formation and trafficking manifest as hypopigmentation and storage pool deficiencies. Whereas Griscelli syndrome and Chediak-Higashi syndrome result from vesicle trafficking, defects in vesicle formation appear to be responsible for Hermansky-Pudlak syndrome (HPS), a group of disorders characterized by oculocutaneous albinism and platelet storage pool deficiency. William Gahl (NIH) described the molecular characterization of HPS-1, HPS-2 and HPS-3 genes and a candidate HPS-4 gene. Among these, the function of only HPS-2 gene product, a subunit of adaptor complex (AP-3), in vesicle formation is understood.
It is becoming increasingly clear that exit of melanosomal proteins from the endoplasmic reticulum (ER) is a regulated event, and some pigmentary disorders are ER retention diseases. R. Halaban (Yale Univ.) presented data that suggests proper folding and exit of tyrosinase from the ER is induced by its substrates DOPA and tyrosinase. V. Hearing et al. (Toyofuku, NCI) analyzed intracellular processing of tyrosinase and TRP-1 in mouse melanocyte lines expressing mutant tyrosinase or mutant TRP-1, and proposed that OCA1 and OCA3 are ER retention diseases where mutation of one melanogenic protein affects the maturation and stability of others in the melanogenic pathway. S. Orlow and his coworkers (B. Shen, NYU) expressed wild type and mutant Oa1-GFP in heterologous COS cells and showed that Oa1 affects structure of late endosomes. Setaluri and coworkers (Wake Forest U.) presented data that suggests a role for GIPC, a PDZ-domain protein, in sorting and targeting of TRP-1.
Slide Session V: Cutaneous Pathology and Vitiligo
By Gisela F. Erf
Dr. G. Emilia Costin presented data on the assessment of drug-delivery systems using N-butyldeoxynojirimycin (NB-DJN) inhibition of tyrosinase as an end-point. NB-DJN is an inhibitor of ER ?-glucosidase known to inactivate tyrosinase in B16-F1 melanoma cells. However, high concentrations of NB-DJN (5 mM) were required to effectively inhibit tyrosinase, suggesting inefficient cellular uptake of NB-DJN. Cellular uptake of NB-DJN could be greatly increased by encapsulating NB-DJN in liposomes. The most effective delivery system for NB-DJN was found to be pH-sensitive liposomes, requiring 100 to 1000 times less NB-DJN for inhibition of tyrosinase activity. Empty pH-sensitive liposomes carriers did not affect tyrosinase activity and were not toxic to the cell. Hence, the pH-sensitive liposome is a highly efficient carrier for delivery of ER-targeted drugs.
Dr. Guido W. Swart presented data on novel cDNAs identified when comparing mRNA expression profiles at various stages of human melanocyte transformation. One of the transcripts picked up during RT PCR-based subtractive hybridization is pCMa1which was localized to the distal, telomere proximal region on the short arm of chromosome 11.p15.1-2. cDNA clone pCMa1 (0.45 kb) did not contain a unique long reading frame and Northern blot analyses revealed multiple complementary pCMa1 transcripts of different lengths. In situ hybridization with an arbitrarily defined minus strand cRNA probe known to bind to a 4.0 kb plus transcript of pCMa1 revealed differential expression of pCMa1depending on the stage of neoplastic progression of melanocytes. The level of plus transcripts was highest in melanocytic nevi (10/10), variable in primary melanoma lesions (5/6), and negative in normal skin melanocytes and most (3/4) metastatic melanoma. The transient expression of pCMa1 in the neoplastic progression of melanocytes suggests that pCMa1 is a molecular marker for early stages of melanocyte transformation.
Dr. Rangaprasad Sarangarajan reported data on the role of the pro- and anti-apoptotic proteins of the Bcl-2 family in melanocyte apoptosis induced by 4-tertiary butyl phenol (4-TBP) in the etiopathology of contact vitiligo. Expression of four members of the Bcl-2 family (i.e., Bcl-2, Bcl-x, Bax, and Mcl-1) in normal human melanocytes (NHM) cultured with or without 250 M 4-TBP was analyzed by flow cytometry and immunofluorescence techniques. Exposure of NHM to 4-TBP altered Bcl-2 expression whereas expression of the other apoptotic proteins was unchanged. Western blotting for tyrosinase, TRP-1 and Bcl-2 in NHM cells exposed to 4-TBP for 24, 48, and 72 h revealed no detectable change in all three proteins at 24 h, whereas decreased levels of Bcl-2 and tyrosinase were observed at later time points of 4-TBP exposure. Considering the anti-apoptotic role of Bcl-2, a minimal increase in Bcl-2 expression at 24 h may be an effort to protect the melanocyte from apoptosis induced by 4-TBP, whereas the drop in Bcl-2 after prolonged exposure to 4-TBP may promote cells to undergo apoptosis.
Ms. Xiaoli Wang presented two papers on the etiopathology of autoimmune vitiligo in Smyth line (SL) chickens. In her first presentation, she reported data on demonstrating the presence of interferon gamma (IFN) in the feather (the site of melanocyte destruction) of vitiliginous SL chickens using Northern blotting with an anti-sense chicken (ch)-INF-specific digoxigen-labeled riboprobe and immunoblotting with anti-chINF monoclonal antibodies. Using this approach, INF was detected in feathers of chickens with active vitiligo but not in chickens with stable vitiligo or chickens without vitiligo (non-vitiliginous SL chickens and normally pigmented parental Brown line controls). Based on flow cytometry, the INF producing cells in the feather included CD4+ lymphocytes. Taken together, these observations support a role of a Th1 dominated cell-mediated immune response in the loss of melanocytes in SL vitiligo.
In her second presentation, Ms. Xiaoli Wang showed data on in situ TUNEL and immunohistochemical staining of feather tissue obtained from SL chickens at various times prior to and throughout the development of visible vitiligo. In vitiliginous SL chickens, the numbers of apoptotic cells in the feather, especially in the epithelial barb ridge where melanocyte cell bodies are located, were higher than in non-vitiliginous SL and control chickens. The increased incidence of apoptosis was first observed at onset of vitiligo and was highest in active vitiligo, suggesting a close association between apoptosis and the disappearance of melanocytes. The number of CD8+ cells and MHC class II+ cell (including MART-1+ cells) increased two weeks prior to onset of vitiligo. Considering the temporal relationship and the close physical location between CD8+ feather infiltrating lymphocyte and TUNEL+ cells suggests that the apoptosis in vitiliginous feathers was induced by cytotoxic T cells.
Dr. Gisela Erf presented data on studies examining the role of turkey herpesvirus (HVT) in the expression of vitiligo in SL chickens. Using a time course approach, flow cytometry and virus reisolation techniques, it was found that HVT vaccination of SL chickens at hatch greatly increased the proportions of CD4+ splenocytes at 3 days of age and those of CD8+ splenocytes between 14 and 42 days of age. These changes in T cell profiles were suggestive of cell-mediated immune activity. Although HVT could be isolated from the thymus at 3, 6, and 9 days of age, the proportions among thymocyte populations were not affected by HVT. HVT did not affect lymphocyte profiles in the thymus and spleen of BL controls. However, HVT could be reisolated from thymus, spleen, bursa and blood at the same time points and at comparable amounts in both HVT-vaccinated SL and BL chickens. These observations suggest that SL chickens may have a heightened/inappropriate immune response to HVT that may play a role in triggering vitiligo.
Dr. Roger Bowers completed the session by presenting data from his research on other avian models for vitiligo, the Barred Plymouth Rock (BPR) and White Leghorn (WL) chickens. Premature death of melanocytes in BPR and WL chickens is due in part to low antioxidant superoxide dismutase (SOD) activity (50% and 75% of SOD activity in the wild type Jungle Fowl (JF), respectively). Molecular characterization studies of the CT Cu/Zn SOD gene, revealed 99% homology in cDNA sequence between the three types of chickens. A missense mutation observed in BPR may affect protein structure and, hence, SOD activity. SOD mRNA levels were lower in WL chickens compared to BPR and JF chickens, with SOD mRNA levels being highest in BPR chickens. It appears that the low SOD activity in WL chickens may be due to reduced transcription of the SOD gene. Whereas, the reduced BPR SOD activity must be due to post-transcriptional control of the SOD enzyme, because of the elevated levels of SOD mRNA in BPR compared to JF. SOD gene transcription may be increased in BPR in response to the reduced activity of SOD. Considering that WL chickens have both the barring gene (associated with a post-transcriptional decrease in SOD activity) and the dominant white gene (associated with reduced SOD gene transcription), Dr. Bowers suggested that this could explain why WL melanocytes are much more susceptible to premature cell death than BPR melanocytes.
Slide Session VI: Model Systems and Late Breaking Research
By William Oetting
The search for genes associated with albinism started in 1986 with the cloning of the tyrosinase gene, and has not shown any signs of ending. This session included two papers, one from Dr. Murray Brilliant, at the University of Arizona, and Dr. William Gahl, at the N.I.H. identifying two new genes in which mutations result in albinism in humans.
Dr Murray Brilliant presented a paper showing that the human homologue of the mouse underwhite locus, underwhite-dominant brown (UWdb) is responsible for a fourth type of albinism in humans, oculocutaneous albinism type 4 (OCA4). The UWdb phenotype looks like the pink-eyed dilution (p) locus in the mouse, the human homologue of which is associated with OCA2. This UWdb allele is dominant; the heterozygote is less hypopigmented than the homozygote. The gene was cloned from both the mouse (chromosome 15) and the human (chromosome 5p). The protein has 12 membrane spanning regions and appears to be a membrane spanning transport protein that has some homology to a plant proton-sucrose transporter. One possible role for the protein is to balance the osmolarity in either the melanosome or the melanocyte. The human gene has been tentatively termed membrane spanning transport protein-1 (MSTP1). The human sequence is highly conserved to the mouse coding sequence. Two individuals with albinism, with residual pigmentation, were identified as having mutations in MSTP1 locus. One individual was homozygous for a splice site acceptor at exon 2, and the second individual had an in-frame deletion, that was thought to alter the protein structure. Sequencing of the tyrosinase gene (OCA1) and the P gene (OCA2) in individuals with albinism has shown that a significant percentage have no identifiable mutations in either of these two genes, showing that other genes associated with albinism most likely exist. TYRP1 associated with OCA3 and now MSTP1, associated with OCA4, have provided an explanation for the albinism in these individuals. It could be that this is only the beginning of an expanding list of genes associated with albinism.
A long list of genes associated with albinism is highly evident in genes associated with the Hermansky-Pudlak Syndrome (HPS). To date, two genes, HPS1 and ADTB3A (HPS2) have been identified. Dr. William Gahl now reported a third locus associated with HPS, HPS3. Individuals with HPS have albinism, along with platelet storage pool deficiency resulting in a bleeding disorder, and in some cases lysosomal ceroid lipofuscinosis, pulmonary fibrosis and granulomatous colitis. This newly identified gene is part of the very interesting story of HPS in Puerto Rico. The initial HPS gene, HPS1, was identified in a founder population in the Northwest corner of Puerto Rico. Affected individuals all shared a 16 bp duplication in the HPS1 gene. Although this mutation explained the HPS in some individuals in Puerto Rico, it was also known that there was a population of individuals in Central Puerto Rico that had HPS but did not have this mutation, or any other mutation in the HPS1 gene. Analysis of these individuals showed that their HPS mapped to another location. For these individuals a candidate gene was identified and cloned. The gene contains 17 exons and the coding sequence coded for a protein containing 1004 amino acids (113.7 kDa). No homology was found to other proteins. The HPS3 protein contains a clathrin binding site, an ER retention signal and a dileucine motif associated with protein trafficking to the melanosome. In the HPS population in Central Puerto Rico, a 3,904 bp deletion, including exon 1, was found in this gene. This deletion was found to be flanked by Alu repeats, which may describe the mechanism for the deletion. The deletion was thought to occur about 5.3 generation ago, or about 110 to 120 years. There are 15 known mouse loci that present with a HPS like phenotype, yet this gene was not any of the known mouse HPS loci. The HPS3 mouse homologue is the subtle grey locus. There is every reason to assume that other individuals with HPS will have mutations in these other genes, making the work of understanding the molecular basis of HPS a continuing story.
Symposium V: Phenoloxidases, Melanogenesis and Evolution
By Vincent Hearing
This Symposium was obviously scheduled to see who really, really couldn?t get enough of pigmentation. It was held early on a Sunday morning, starting at 8 am on the final day of the meeting. Despite that, there was a good turnout and those who had the energy to attend were in for a treat. Prof. Heinz Decker (Univ of Mainz, Germany) was an invited lecturer who spoke on the structure, function and evolution of hemocyanin in the context of its relationship to tyrosinase. He notes that the enzymes tyrosinase, catechol oxidase and hemocyanin all share similar active sites (utilizing copper as the ligand), although their physiological functions are quite distinct. Tyrosinases in lower species (such as amphibians) are activated in vivo by proteolytic cleavage, which might open up substrate access to the catalytic site, and he made the interesting finding that if hemocyanin (typically found in arthropods) is subjected to similar proteolytic treatment in vitro, it shows a catechol oxidase activity reminiscent of that of lower forms of tyrosinase. Characterizing the structure of hemocyanin is an important model to understand the substrate active-site interactions of tyrosinases. Want more detail? Check out his recent reviews published in J Biol Chem (2001;276:15563-9) and Trends in Biochem Sci (2000;25:392-7). Prof. Manickam Sugumaran (Univ of Massachusetts, USA) moved up the evolutionary ladder to insects and discussed the role of phenol oxidases which play important roles in sclerotin formation, wound healing and defense reactions. It turns out that in addition to tyrosinase, insects also have a tyrosinase-related protein, called dopachrome isomerase. That latter enzyme is distinct from our favorite enzyme, dopachrome tautomerase, since the catalytic reaction in insects eliminates the carboxyl group rather than keeping it. Sugumaran's studies confirm the presence of a melanogenic complex between the phenol oxidase and dopachrome isomerase, and by forming that complex, the enzymes regulate each other's activity and control the levels of endogenous quinones produced. The complex is critically important for the defense strategies of insects. Need to know more? Check out his recent paper in Adv Exp Med Biol (2001;484:289098) or wait for any early issue of Pigment Cell Research next year in which Prof Sugumaran will review this field.
Slide Session VII: Gene Regulation
By Vincent Hearing
The morning Symposium then continued with 6 talks selected from the submitted abstracts. Dr. Brian Potterf discussed Sox10, a transcription factor that activates expression of Mitf, another transcription factor that we all know regulates at least some of the melanogenic genes. Both Sox10 and Mitf play important roles during melanoblast development, and then reprise their roles to regulate melanocyte differentiation in later stages of life. Potterf and colleagues examined the effects of mutations in Sox10 on the development of neural-crest derived melanocytes; their evidence suggests that Sox10 is an transcriptional activator of Dct expression, which is consistent with the early expression patterns of Dct in mouse embryos. Dr. Dong Fang then reported on his work on the regulation of expression of Tyrp1; they found an upstream enhancer element in the Tyrp1 promoter and their results suggest that transcription of Tyrp1 is regulated not only by the M-box, but also by 2 novel elements in the Tyrp1 promoter. The Tbx2 transcription factor may function as an inhibitor of Tyrp1 expression, perhaps by blocking the binding of Mitf to the M-box of the Tyrp1 promoter. This repressor site is not found in the tyrosinase promoter, which may explain the coordinated but sometimes distinct expression patterns of those 2 genes. Dr. James Lister then discussed the duplicate Mitf genes that are found in zebrafish. There is a redundancy in their patterns of expression and they are expressed differentially in neural crest derived melanocytes and in the retinal pigment epithelium. One of them is similar to the mammalian 'A' form of Mitf, the other being similar to the ?M? form. These genes probably original from a single Mitf ancestor via duplication. Dr. Thomas Hornyak then discussed his work with Mash1 (a neurogenic transcription factor); it is a bHLH transcription factor that is involved in regulating neural crest development. Mash1 was found to negatively regulate Dct transcription. Expression of Mash1 in transgenic mice (regulated by the Dct promoter) led to the development of fewer neural crest-derived melanoblasts, although those did successfully localize in the hair follicles of adult mice. Dr. S. Shriram then presented the results of a study characterizing various mutations of the tyrosinase gene that are found in oculocutaneous albinism, but interestingly, focusing on cases of OCA1 in which no mutation has been identified, or only 1 allele is affected. They found (in a limited number of cases so far) that expression of only 1 allele (the known mutant) was found in the heterozygous cases, perhaps because of a mutation in the promoter region of the other allele which abrogates transcription of that other allele (which may encode a wild-type tyrosinase enzyme). It will be interesting to see how common this phenomenon is seen in such recessive diseases. And finally, Dr. Caroline LePoole presented a paper on the regulation of gp100 (sometimes called Pmel17, silver and/or HMB45) transcription. This completed the circuit of the known melanosomal-specific genes discussed in the Symposium. LePoole found that the gp100 promoter has a predicted upstream region (~ 1 kB upstream) that might bind CDK-2, and 3 upstream (~ 0.5 kB) potential E-box sites. However, Mitf was unable to activate the gp100 promoter. The sum of their results suggest that CDK2 is a negative regulatory element of gp100 expression and that Mitf is not involved in the regulation of expression of gp100.
In sum, this was an exciting session that showed the distinct and independent regulation of 4 genes which encode melanosome-specific proteins (tyrosinase, Tyrp1, Dct and gp100). It is clear that all 4 genes are regulated by positive and by negative regulatory factors in distinctive and complex patterns.
This page was modified last on December 7, 2001.