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2016 Scientific Report

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KARSTEN MELCHER, PH.D. Dr. Melcher earned his Master’s degree in biology and his Ph.D. degree in biochemistry from the Eberhard Karls Universität in Tübingen, Germany. He was recruited to VARI in 2007, and in 2013 he was promoted to Associate Professor. STAFF STEPHANIE GRANT, M.P.A. XIN GU, M.S. JIYUAN KE, PH.D. AMANDA KOVACH, B.S. EDWARD ZHOU, PH.D. RESEARCH INTERESTS Our laboratory studies the structure and function of proteins that have central roles in cellular signaling. To do so, we employ X-ray crystallography in combination with biochemical and cellular methods to identify structural mechanisms of signaling at high resolution. STUDENT CHRISTIAN CAVACECE In addition to their fundamental physiological roles, most signaling proteins are also important targets of therapeutic drugs. Determination of the three-dimensional structures of protein–drug complexes at atomic resolution allows a detailed understanding of how a drug binds its target and modifies its activity. This knowledge allows the rational design of new and better drugs against diseases such as cancer, diabetes, and neurological disorders. Three areas of focus in the lab are the adenosine monophosphate (AMP)–activated protein kinase (AMPK); the receptors and key signaling proteins for a plant hormone, abscisic acid (ABA); and the folate receptors. AMP-activated protein kinase (AMPK) Cells use ATP to drive cellular processes such as muscle contraction, cell growth, and neuronal excitation. AMPK is a three-subunit protein kinase that functions as an energy sensor and regulator of homeostasis in human cells. Its kinase activity, triggered by energy stress (i.e., a drop in the ratio of ATP to AMP/ADP), activates ATP-generating pathways and reduces energy-consuming metabolic pathways and cell proliferation. To adjust energy balance, AMPK regulates • almost all cellular metabolic processes (activation of ATP-generating pathways such as glucose and fatty acid uptake and catabolism, and inhibition of energy-consuming pathways such as the synthesis of glycogen, fatty acids, cholesterol, proteins, and ribosomal RNA); 16 Van Andel Research Institute | Scientific Report

• whole-body energy balance (appetite regulation in the hypothalamus via leptin, adiponectin, ghrelin, and cannabinoids); and • many nonmetabolic processes (cell growth and proliferation, mitochondrial homeostasis, autophagy, aging, neuronal activity, and cell polarity). Because of its central roles in the uptake and metabolism of glucose and fatty acids, AMPK is an important pharmacological target for treating diabetes and obesity. Moreover, AMPK activation restrains the growth and metabolism of tumor cells and has thus become an exciting new target for cancer therapy. In this project we strive to determine the structural mechanisms of AMPK regulation by direct binding of AMP, ADP, ATP, drugs, and glycogen, in order to provide a structural framework for the rational design of new therapeutic AMPK modulators. Abscisic acid Abscisic acid (ABA) is an ancient signaling molecule found in plants, fungi, and metazoans ranging from sponges to humans. In plants, ABA is an essential hormone and is also the central regulator protecting plants against abiotic stresses such as drought, cold, and high salinity. These stresses—most prominently, the scarcity of fresh water—are major limiting factors in crop production and therefore major contributors to malnutrition. Malnutrition affects an estimated one billion people and contributes to more than 50% of human disease worldwide, including cancer and infectious diseases. We have determined the structure of ABA receptors in their free state and while bound to ABA. Using computational receptor-docking experiments, we have identified and verified synthetic small-molecule receptor activators as new chemical scaffolds toward the development of new, environmentally friendly, and affordable compounds that will protect plants against abiotic stresses. We have also identified the structural mechanism of the core ABA signaling pathway, which will allow modulation of this pathway through genetic engineering of crop plants. Folate receptors Folic acid and its derivatives are one-carbon donors required for the synthesis of DNA. Rapidly dividing cells such as cancer cells require rapid DNA synthesis and are therefore selectively dependent on high folate levels. This vulnerability has been therapeutically exploited since the 1940s, when toxic folate analogs (antifolates) were used as the first chemotherapeutic agents. However, current antifolates have severe side effects such as immunosuppression, nausea, and hair loss, because they also kill nonmalignant proliferative cells. Cells can take up folates in two main ways: by a ubiquitous, high-capacity, low-affinity uptake system known as RFC (reduced folate carrier) and by folate receptors. The latter are cysteine-rich cell surface glycoproteins that allow high-affinity uptake of folates by endocytosis but do not take up the current antifolate drugs. While folate receptors are expressed at very low levels in most tissues, they are “hijacked” and expressed at high levels in numerous cancers. This selective expression has been therapeutically and diagnostically exploited by administering antibodies against folate receptor α, folate-based imaging agents, and folate-conjugated drugs and toxins. We expect that antifolates that can be taken up by folate receptors but not by the RFC would have greatly reduced side effects. We have determined the structure of folate receptor α in complex with folic acid. The structure, validated by systematic mutations of pocket residues and quantitative folic acid binding assays, has provided a detailed map of the extensive interactions between folic acid and FRα. It provides a structural framework for the design of new antifolates that are selectively taken up by folate receptors. Our short-term goal is to determine the structures of novel, preclinical chemotherapeutic antifolates, bound to folate receptors and bound to the folate-metabolizing enzymes they inhibit, as a step toward designing antifolates that selectively target cancer cells. RECENT PUBLICATIONS Kang, Yanyong, X. Edward Zhou, Xiang Gao, Yuanzheng He, Wei Liu, Andrii Ishchenko, Anton Barty, Thomas A. White, Oleksandr Yefanov, et al. 2015. Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser. Nature 523(7562): 561–567. Ke, Jiyuan, Honglei Ma, Xin Gu, Adam Thelen, Joseph S. Brunzelle, Jiayang Li, H. Eric Xu, and Karsten Melcher. 2015. Structural basis for recognition of diverse transcriptional repressors by the TOPLESS family of corepressors. Science Advances 1: 21500107. CENTER FOR CANCER AND CELL BIOLOGY 17

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