Elena Oancea - US grants

PKC gamma is one of the four members of the conventional PKC family, which are regulated by calcium and diacylglycerol. Unlike any other members of the family, PKC gamma has an unique expression pattern, being expressed predominantly in brain and spinal cord. Recently PKC gamma was also found in heart. The experiments proposed here are designed to answer two main questions. The first is what allows PKC gamma to respond to some signals but not others by phosphorylating unique substrates in response to each signal. Our working model is that PKC gamma is spatially confined in both its' states, active and inactive. Spatial localization is achieved by binding to particular proteins and the affinity of the binding is regulated by upstream signals. To test this hypothesis I will first identify binding proteins/substrates for the active and inactive PKC gamma by using the yeast two hybrid approach. I will then test the interaction in the native tissue and determine the factors that modulate the interaction. The second question is what are the physiological signals that activate PKC gamma in excitable cells. Is it activated by calcium influx through ion channels during elevated electrical activity of the neurons or is it exclusively regulated through metahotropic receptors? To answer these questions I will first design novel fluorescent indicators which will allow me to detect the activation of PKC gamma in a single cell assay. Using these indicators I will determine the spatial and temporal aspects of PKC gamma activation in excitable cells in response to a variety of stimuli.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Malignant melanoma is the deadliest form of skin cancer that continues to increase in incidence at a rate of 3% per year. Melastatin (TRPM1) was discovered as a gene down-regulated in highly metastatic tumors in mice and humans, suggesting that TRPM1 expression may be an indicator of melanoma aggressiveness. This finding raised the interesting possibility that TrpM1 might be a tumor suppressor, an unprecedented function for an ion channel. Despite being the founding member of the TrpM family of ion channels, very little is known about the molecular properties and cellular functions of TrpM1. The goal of the proposed research is understanding the function of TrpM1 and the connection between TrpM1 and melanoma. Determining the molecular mechanisms by which TrpM1 is involved in normal and malignant cells could lead to significant progress in the diagnostic and treatment of melanoma. To understand the role of TrpM1 in cellular function, it is critical to characterize its sub-cellular localization and cellular dynamic. To express fluorescently tagged TRPM1 in primary human melanocytes and melanoma cells we will use a lenti-viral system. We will then employ confocal imaging, total internal reflection fluorescence (TIRF) and electron microscopy (EM) to determine the intracellular versus plasma membrane distribution of TrpM1, the dynamic properties of the intracellular structures containing TRPM1 and their identity. The results of the proposed experiments will represent a significant step towards understanding the function of TrpM1 in melanocytes.

PROJECT SUMMARY Skin, the largest organ of the human body, is constantly exposed to solar ultraviolet radiation (UVR), a powerful environmental carcinogen. Unlike the skin of other mammals, human skin responds to UVR by increased pigmentation resulting from melanin production in epidermal melanocytes and its transfer to keratinocytes. Melanin protects the nuclear DNA of melanocytes and keratinocytes by directly absorbing UVR and neutralizing free radicals, thus preventing genetic damage and cancer formation. How does human skin detect and respond to UVR? UVR at the earth's surface consists of 95% long wavelength UVR (UVA) and 5% short wavelength UVR (UVB). Although the mechanism by which UVB causes DNA damage and results in delayed melanin synthesis more than a day after exposure is well characterized, the effects of UVA are poorly understood. High UVA doses cause oxidative damage, but no specific pathway for detecting physiological doses of UVA has been characterized. Our preliminary results describe a novel UVA-activated signaling pathway in melanocytes that is retinal-dependent and G-protein coupled. Activation of this pathway by physiological doses of UVA leads to the activation of transient receptor potential A1 (TRPA1) ion channels, a rapid increase in intracellular Ca2+, and increased melanin content on a rapid time scale (beginning about an hour after UVA exposure). The goal of this proposal is to characterize the molecular steps by which UVA causes a Ca2+ response (Aim 1) and an increase in cellular melanin concentration (Aim 2). Using live-cell fluorescence imaging, electrophysiology, and biochemistry, we will identify in Aim1 the G protein pathway activated by UVA, characterize the cellular messengers and determine the mechanisms that lead to a rapid and transient Ca2+ response. In Aim 2 we will test the hypothesis that UVA-mediated cellular depolarization is required for a sustained Ca2+ response and persistent PKC activation, which leads to early melanin production. In Aim 3 we will test the hypothesis that UVA and UVB signaling pathways, which are simultaneously activated by solar UVR, interact synergistically to regulate melanin production and pigmentation. We will use mice with humanized skin to investigate the interaction between the UVA and UVB activated pathways and characterize the molecular mechanisms mediating this interaction. The proposed studies will identify and characterize a novel UVA phototransduction pathway and determine its function in skin, and will significantly advance our understanding of melanocyte function and of the skin's response to solar UVR.

PROJECT SUMMARY Melanosomes are unique lysosome-related organelles in skin, hair, and eye melanocytes and pigment epithelia of the retina, iris and ciliary body of the eye, in which melanins - the main pigments in mammals - are synthesized and stored. Genetic defects in melanosome components or biogenetic machinery result in albinism, characterized by hypopigmentation, impairments in vision, and increased susceptibility to skin and eye cancers. Some of the genes that are defective in various forms of oculocutaneous albinism (OCA), including OCA2 and SLC45A2, encode transmembrane proteins that regulate the ionic environment of melanosomes or melanosome precursors. For example, we recently showed that OCA2 functions as a chloride channel that neutralizes melanosome pH, thereby activating melanin synthesis, whereas two-pore channel 2 (TPC2) ? the first identified melanosomal cation channel ? negatively regulates pigmentation. Despite these advances, our understanding of ion transport across the melanosome membrane and how ion flux regulates pigmentation is rudimentary. In particular, it is not known how SLC45A2 regulates pigmentation, or how genetic variation in SLC45A2 interferes with pigment production. While melanocytes lacking SLC45A2 or OCA2 share some characteristics such as impaired in vivo activity of a key melanogenic enzyme, tyrosinase, how these two proteins influence the tyrosinase activity cooperatively or separately remains elusive. Finally, how TPC2 ? a nonselective, sodium and calcium permeable channel ? influences melanogenesis is completely unknown. The goal of this proposal is to answer critical questions regarding SLC45A2, OCA2 and TPC2 function and to dissect the molecular pathways that allow these proteins, directly or indirectly, to control the ionic milieu within melanosomes and melanin synthesis. Based on solid preliminary data, we will test that: (1) OCA2 and SLC45A2 each function to increase the luminal pH of melanosomes, but at distinct stages of maturation; (2) SLC45A2 functions directly on melanosomes from a specific microdomain and that assembly into the microdomain is disrupted in hypopigmentation-associated SLC45A2 variants; and (3) TPC2 functions as part of a multi-protein complex that mediates tyrosine transport across the melanosome membrane. Broader impact: These studies will have a broad impact on understanding the mechanisms that regulate skin and eye pigmentation, will advance our understanding of how ion transport across melanosomal membranes is critical for melanosomal function, will uncover mechanisms underlying pigmentation disorders, and will set a precedent for understanding ion transport control in other lysosome-related organelles. Relevance to public health: Mutations in the genes encoding several proteins involved in ion transport across melanosomal membranes cause albinism with pigmentation defects, impaired vision, and increased susceptibility of the skin and eye to cancer. Our studies will elucidate the molecular mechanisms by which these proteins affect melanogenesis and how patient mutations result in pigmentation and vision defects.

? DESCRIPTION (provided by applicant): Predoctoral Training Program in Trans-Disciplinary Pharmacological Sciences This is the resubmission of an application to continue the existing Predoctoral Training Program in Trans- Disciplinary Pharmacological Sciences (T32) within the Molecular Pharmacology and Physiology (MPP) Graduate Program at Brown University. In its first cycle (2010-2015), the training grant funded 13 predoctoral trainees. This renewal application requests funding for 4 predoctoral trainees per year for 5 years and is intended to fund trainees in their second and third years of study. The training program is designed to produce graduates capable of establishing outstanding independent research in the interdisciplinary fields contributing to modern pharmacological sciences. The T32 already has produced several major improvements in this small, developing graduate program, with positive effects extending to the Division of Biology & Medicine and the University. The training program has 33 outstanding faculty trainers drawn from several departments at Brown University and its Warren Alpert Medical School. The research productivity, funding and mentoring records of the training faculty are strong, and there are many collaborative interactions that benefit the trainees. The research areas of trainers within the program fall into five broad categories: 1) molecular structure and its role in disease; 2) neuropharmacology and neural circuit function; 3) receptor and channel pharmacology, physiology and signal transduction; 4) translational and clinical applications; and 5) chemical biology and its applications. Trainees acquire career skills and proficiency in the areas of pharmacology through coursework, lab rotations, and many mechanisms for scientific interaction with each other and with faculty trainers, as well as with scientists from outside Brown. There is extensive advising and evaluation by the Graduate Program Director, Training Grant PI, Graduate Program Committee, Thesis Advisors and Thesis Committees, as well as by Brown's Office of Graduate and Postdoctoral Studies. Trainees also are exposed to a variety of career paths through programs sponsored within and outside the MPP graduate program. We have almost no attrition, and our graduates have outstanding career outcomes. The program strives to recruit and retain students of all ethnicities and socioeconomic backgrounds, and has great success to-date -- the program typically consists of ~40% underrepresented minorities, including African-American, Hispanic and Native American. Continued funding of the Predoctoral Training Program in Trans-Disciplinary Pharmacological Sciences will allow us to continue to design new methods with the goal of graduating outstanding members of the scientific community with the skills essential to developing new drugs and therapeutics.

The Graduate Program in Molecular Pharmacology & Physiology is a small and very diverse program with a strong set of courses and activities, as well as extensive mentoring mechanisms. Leadership of the program is by a group of three accomplished MPIs with complementary strengths and expertise that guarantee effective oversight of the program. The request is for 4 T32 slots per year; the total number of students in the program is 20. This training program in Interdisciplinary Training in Pharmacological Sciences will replace our current NIGMS pharmacology T32, ending in June of 2021. The training in our program focuses on fundamental principles of pharmacology, rigor and reproducibility, and cutting-edge quantitative methods. Our trainees participate in many activities that promote interaction, collaboration and professional development. Here we propose a number of new initiatives in the curriculum, professional development, program oversight and recruitment. A recent innovative initiative we are developing in the program is the establishment of summer internships at Pfizer pharmaceutical company labs; these internships will give our trainees the unique opportunity to experience first-hand the scientific environment in a pharmaceutical company and make informed decisions for their future careers. Our current admissions and recruitment practices have yielded a high percentage of students from disadvantaged and underrepresented groups (in the last 5 years, 47% underrepresented minorities plus 7% disabled), and there has been 100% retention of these students in the program during the last 15 years (retention for the program in general is 95%). The career outcomes of the students are excellent, with 52% of students staying in academia (25% faculty, 27% currently postdocs) often at top institutions, about 25% as scientists in pharmaceutical or biotechnology companies, and the remainder in a variety of science and medicine- related careers. Thus, our program has been training a highly diverse group of students who have become successful in different areas of science; the new initiatives proposed in our T32 application will further enhance our unique program and improve the quality of the training.