Faculty Bibliography

A triazine-based combinatorial library of small molecules was screened in zebrafish to identify compounds that produced interesting phenotypes. One compound (of 1536 screened) induced a dramatic increase in the pigmentation of early stage zebrafish embryos. This compound, PPA, was also found to increase pigmentation in cultured mammalian melanocytes. The cellular target was identified as the mitochondrial F1F0-ATP synthase (ATPase) by affinity chromatography. Oligomycin, a small molecule known to inhibit the mitochondrial ATPase, competed with PPA for its cellular target in melanocytes. In addition, PPA was shown to alter the membrane potential of mitochondria, consistent with inhibition of the mitochondrial ATPase. Thus, PPA has been successfully used as a chemical probe in a forward chemical genetic approach to establish a link between the phenotype and the protein. The results attest to the power of screening small molecule libraries in zebrafish as a means of identifying mammalian targets and suggest the mitochondrial ATPase as a target for modulating pigmentation in both melanocytes and melanoma cells

Summary Tyrosinase, the rate-limiting enzyme of melanin synthesis, is a di-copper metalloprotein that catalyzes the conversion of l-tyrosine to l-DOPAquinone. Phenylthiourea (PTU) is a well-known inhibitor of tyrosinase and melanin synthesis and is known to interact with sweet potato catechol oxidase, an enzyme possessing copper binding domain homology to tyrosinase. While PTU is frequently used to induce hypopigmentation in biological systems, little is known about its effects on tyrosinase and other melanogenic proteins. We have found that PTU induces degradation of tyrosinase but not of other melanogenic proteins including the tyrosinase-related metalloproteins tyrosinase-related protein (Tyrp)1 and Tyrp2. Using pulse-chase analysis coupled with glycosidase digestion, we observed that tyrosinase degradation occurs following complete maturation of the protein and that degradation was reversed by cysteine protease inhibitor E64 but not proteasome inhibitor N-acetyl-l-leucinyl-l-leucinyl-l-norleucinal. We conclude that PTU specifically induces tyrosinse degradation following Golgi maturation. Our data suggest that in addition to well-known ER-directed quality control, tyrosinase is also subject to post-Golgi quality control

Bioactive compounds can be used to selectively modulate gene function. We utilized a chemical genetic approach to dissect the mammalian pigmentation pathway and identify protein regulators. We screened a tagged library of 1170 small molecules in a cell-based assay and discovered a class of pigment-enhancing chemicals. From this class we characterized the small molecule melanogenin. Using melanogenin bound to an affinity matrix and amino acid sequencing, we identified the mitochondrial protein, prohibitin, as an intracellular binding target. Studies employing siRNA demonstrate that prohibitin is required for melanogenin to exert its propigmentary effects and reveal an unsuspected functional role for this protein in melanin induction. This represents a mechanism by which propigmentary signals are transduced and ultimately provides a potential target for the treatment of pigmentary disorders

With the advent of new biological agents, interest in the treatment of psoriasis has been renewed. Vitamin A and its derivatives (retinoids) have been used successfully in the treatment of psoriasis for over 30 years. In this paper, data on the efficacy and safety of oral retinoids for the treatment of various forms of psoriasis is reviewed. Studies have shown that retinoids are particularly effective in the treatment of pustular and palmoplantar psoriasis. When used in conjunction with ultraviolet therapy, retinoids appear to have a synergistic effect and can be used safely as long-term maintenance therapy. The most common side effects of oral retinoids are usually modest, treatable or reversible, and predominantly affect the liver, musculoskeletal and neurological systems. Potential teratogenicity remains the primary concern with use in women. Oral retinoids appear to be well tolerated in paediatric and HIV-infected patients

Mating in haploid Saccharomyces cerevisiae occurs after activation of the pheromone response pathway. Biochemical components of this pathway are involved in other yeast signal transduction networks. To understand more about the coordination between signaling pathways, we used a 'chemical genetic' approach, searching for compounds that would activate the pheromone-responsive gene FUS1 and RLM1, a reporter for the cell integrity pathway. We found that catecholamines (l-3,4-hydroxyphenylalanine [l-dopa], dopamine, adrenaline, and noradrenaline) elevate FUS1 and RLM1 transcription. N-Acetyl-cysteine, a powerful antioxidant in yeast, completely reversed this effect, suggesting that FUS1 and RLM1 activation in response to catecholamines is a result of oxidative stress. The oxidant hydrogen peroxide also was found to activate transcription of an RLM1 reporter. Further genetic analysis combined with immunoblotting revealed that Kss1, one of the mating mitogen-activated protein kinases (MAPKs), and Mpk1, an MAPK of the cell integrity pathway, participated in l-dopa-induced stimulation of FUS1 and RLM1 transcription. We also report that Mpk1 and Hog1, the high osmolarity MAPK, were phosphorylated upon induction by hydrogen peroxide. Together, our results demonstrate that cells respond to oxidative stress via different signal transduction machinery dependent upon the nature of the oxidant

The albino mouse was already known in ancient times and was apparently selectively bred in Egypt, China, and Japan. Thus, it is not surprising that the c or albino locus (now the Tyr locus) was among the first used to demonstrate Mendelian inheritance in mammals at the dawn of the past century. This locus is now known to encode tyrosinase, the rate-limiting enzyme in the production of melanin pigment, and the molecular basis of the albino ( Tyr(c)) mutation is known. Here we describe the congenic series of Tyr-locus alleles, from wild type to null ( albino). We compare eye and skin pigmentation phenotypes and the genetic lesions that cause each. We suggest that this panel of congenic mutants contains rich, untapped resources for the study of many questions of basic cell biological interest

An association exists among neurofibromatosis 1 (NF1), juvenile xanthogranulomas (JXGs), and juvenile myelomonocytic leukemia (JMML). The authors describe a patient with the triple association of JXG, NF1, and JMML initially presenting with features of hemophagocytic lymphohistiocytosis (HLH). An 18-month old boy had multiple cutaneous and gastrointestinal JXG and NF1. At 3 years of age he developed anemia, thrombocytopenia, and hepatosplenomegaly. A bone marrow biopsy revealed features of HLH. Despite chemotherapy, he went on to develop JMML, which proved fatal

A triazine-based combinatorial library of small molecules was screened in albino murine melanocytes to identify compounds that induce pigmentation. Six compounds (of 1536 screened) produced at least 3-fold increases in pigmentation. Immunohistochemical studies demonstrated that the compounds conferred correct routing of the mistrafficked enzyme tyrosinase, which is critical to normal melanogenesis. Affinity matrices of the immobilized compounds allowed the cellular target to be identified as the mitochondrial F1F0-ATP synthase. Oligomycin and aurovertin B, small molecules known to inhibit the mitochondrial ATP synthase, were shown to compete with the triazine-based compounds for their cellular target in albino melanocytes and confer similar effects on pigmentation and tyrosinase rerouting. This is the first demonstration of the mitochondrial ATP synthase as a potential therapeutic target for restoring pigmentation in albino melanocytes