A multidisciplinary approach to understanding the vertebrate Hedgehog signaling pathway
The Hedgehog pathway has critical role in many developmental processes, ranging from patterning of the central nervous system to limb and kidney forr .. ation. In adults, Hedgehog signaling plays an essential role in the maintenance of stem cells in various tissues. Additionally, Hedgehog signaling is involved in cancers that account for approximately 25% of all cancer deaths. The secreted protein Hedgehog is covalently attached to cholesterol, a unique modification essential for function. On the surface of the responding cell, Hedgehog binds its receptor, initiating a chain of events that ultimately activate the transcription factors Gli, which mediate Hedgehog transcriptional responses in vertebrates. In the absence of Hedgehog, Gli is proteolyzed to a shorter form that has dominant-negative activity. Hedgehog pathway activation blocks Gli proteolysis, resulting in the accumulation of full-length Gli and activation of target gene transcription.
Our current understanding of Hh signaling comes from genetics and cell culture experiments. In spite of the power of these approaches, many steps of the pathway are poorly understood mechanistically. How is cholesterol attached to Hedgehog in cells? Are other proteins regulated by cholesterol modification? How is Gli proteolyzed in the resting state of the Hedgehog pathway? How does Hedgehog signaling block Gli proteolysis? How can we inhibit Hedgehog signaling and block cell proliferation in cancer? It is apparent that answering these questions would be greatly aided by the ability to reconstitute in vitro the biochemical reactions involved in Hedgehog signaling. With this goal in mind, we have recently begun developing cell-free systems, which together with in vivo approaches will address these critical questions in Hedgehog signaling. Using vertebrate cell extracts, we discovered evidence of a factor that greatly stimulates Hedgehog cholesterol modification. We first propose to identify this factor and elucidate its role in Hedgehog signaling. We also developed a novel, cell-free system that recapitulates the proteolytic processing of vertebrate Gli. Using this system, we next propose to determine the molecular mechanisms that govern Gli proteolysis and its regulation by signaling through the Hedgehog pathway. finally, we propose expression cloning and chemical biology strategies to identify and study other vertebrate proteins modified with cholesterol. These studies will advance our mechanistic understanding of Hedgehog signaling, of how cells become malignant due to Hedgehog activation, and will be used in the future to discover novel small molecule inhibitors of Hedgehog signaling.
Arnold O. Beckman exemplifies the meaning of the word humanitarian. Combined with his unwavering enthusiasm for life, his keen sense of humor and his strong moral and ethical principles, he is a national icon.