2011 — 2015 |
Salic, Adrian |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mechanisms of Vertebrate Hedgehog Signaling
DESCRIPTION (provided by applicant): The Hedgehog (Hh) signaling pathway has critical roles in embryonic development, adult stem cell maintenance and in many cancers. In vertebrates, Hh signaling is initiated at the primary cilium, from where the signal is relayed to the cytoplasm and then nucleus, to ultimately control expression of specific target genes, mediated by the Gli transcription factors. In the absence of stimulation, Gli proteins are kept off by at least three inhibitory mechanisms: 1) direct binding of Suppressor of Fused (SuFu);2) partial degradation to repressor forms and 3) inhibition by protein kinase A (PKA). When cells receive an Hh signal, these inhibitions are overcome, allowing Gli activation. In spite of the importance of Hh signaling, we still do not understand many of the critical mechanisms involved in inhibiting and in activating Gli proteins, in the resting and stimulated states of the Hh pathway, respectively. We discovered that Hh stimulation recruits SuFu-Gli complexes to primary cilia and causes their dissociation, resulting in Gli activation;we found that this process is antagonized by PKA. This provided a novel mechanism for activation of Gli proteins and for the inhibitory effect of PKA. We also discovered that inhibition of the proteasome potently blocks Gli activity, suggesting that Gli turnover and transcriptional activation are intimately coupled. We propose to use a combination of biochemistry and cell biology, to elucidate the mechanism of the following critical events in vertebrate Hh signaling: A) How does Hh signaling activate Gli and inhibit SuFu? B) What is the function of SuFu-Gli recruitment to cilia in Hh signaling, and how does PKA block SuFu-Gli recruitment to cilia? C) How does inhibition of the proteasome block transcriptional activation by Gli proteins and how is the partial proteolysis of Gli proteins regulated? These studies are important for the following reasons: 1) They will elucidate basic mechanisms that control the critical Gli proteins, thus advancing our understanding of Hh signaling;2) They will identify novel targets for Hh inhibition in cancer;and 3) Our finding that proteasome inhibitors potently block Hh signaling could have immediate therapeutic implications in cancer, particularly since the proteasome inhibitor bortezomib is an FDA-approved drug for the treatment of multiple myeloma. PUBLIC HEALTH RELEVANCE: Cell-cell communication through the Hedgehog signaling pathway is critical for normal embryonic development and for the maintenance of adult stem cells, while excessive Hedgehog signaling is involved in a large number of cancers. In spite of its importance, many of the molecular mechanisms involved in Hedgehog signal transduction are not well understood. Deciphering them will have a broad impact on human health, by advancing our understanding of cancer pathogenesis, by contributing to improved cancer diagnostics and by identifying novel targets for drug therapy in cancer.
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2014 — 2017 |
Salic, Adrian |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Novel Mechanisms For Oxysterols in Cell-Cell Signaling
DESCRIPTION (provided by applicant): Oxysterols are molecules that cells synthesize from cholesterol, which affect many biological processes such as metabolism, cell-cell signaling and cellular migration. Surprisingly, in spite of their potent activities, we understand only poorly th roles that oxysterols play, and how they act to accomplish various effects on cells. Oxysterols have been implicated in Hedgehog signaling, a cell-cell signaling pathway essential for embryonic development, for adult stem cell maintenance, and deeply involved in many human cancers. It is known that oxysterols bind and activate a membrane protein, Smoothened, which is critical for triggering Hedgehog signal transduction. However, many critical aspects of the role of oxysterols in Hedgehog signaling have remained obscure: we do not know if oxysterols are required for normal Hedgehog signaling, how they are regulated, and whether blocking oxysterols might be a good way to inhibit Hedgehog signaling in cancer. Additionally, there is a need for new and powerful chemical approaches to visualize and assay oxysterols in vivo; such methods would greatly aid our understanding of their role in Hedgehog signaling and beyond. We developed novel chemicals, called azasterols, which block binding of oxysterols to Smoothened; as a result, Hedgehog signaling is inhibited, which shows that antagonizing oxysterols is an effective strategy to inhibit the Hedgehog pathway. Using azasterol and oxysterol chemical probes, we pinpointed where oxysterols bind to Smoothened, which allowed us to build Smoothened mutants that no longer respond to oxysterols. Interestingly, we discovered that these Smoothened mutants cannot signal properly; this showed that oxysterols are required for normal Hedgehog signaling. Finally, we have synthesized and characterized novel chemical probes that allow us to visualize and assay oxysterols in cells, with better sensitivity and specificity than before. We plan to use these probes to better understand the role of oxysterols in Hedgehog signaling. We propose to use a combination of chemical biology, biochemistry and cell biology, to accomplish the following aims: A) To elucidate how Hedgehog signaling regulates oxysterols, with the aid of our novel oxysterol probes B) To determine precisely which oxysterols are involved in Hedgehog signaling C) To discover how oxysterols involved in Hedgehog signaling are synthesized in cells These studies are important for the following reasons: 1) They will advance our understanding of Hedgehog signaling, by elucidating the critical role of oxysterols; 2) They will identify novel targets for blocking Hedgehog signaling in cancer, based on oxysterol inhibition; and 3) Our novel chemical probes will be broadly applicable to study oxysterol mechanisms in health and in disease, will provide diagnostic tools for sterol disturbances and will help identify small molecule inhibitors of sterol function.
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2018 — 2021 |
Salic, Adrian |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Sending and Receiving Hedgehog Signals
Project summary The Hedgehog cell-cell signaling pathway is essential for embryonic development, for adult stem cell maintenance, and is deeply involved in human cancer and birth defects. The pathway is activated by the Hedgehog ligand, a secreted protein uniquely modified with two lipids, which are both essential for function: a fatty acid and cholesterol. From the producing cell, the Hedgehog ligand spreads to distant cells, on the surface of which it binds its receptor, a tumor suppressor membrane protein called Patched, thus triggering a specific set of cellular responses. A puzzling aspect of the Hedgehog ligand is that it spreads and signals at a distance, in spite of the fact that its lipid modifications make it stick strongly to membranes. Many critical aspects of the Hedgehog ligand remain obscure: we do not know how it is released from cells, how it is able to diffuse outside the cell, and how it is delivered to responding cells. We recently discovered that Hedgehog ligand release is accomplished by two synergistic interactions involving its cholesterol modification: one with the membrane protein Dispatched, and the second with the secreted protein Scube. Using novel chemical probes, we found that Dispatched and Scube recognize cholesterol differently, suggesting that the Hedgehog ligand is handed off from Dispatched to Scube. We also discovered that Scube is important for ligand reception, via Cdon and Boc, two proteins critical for Hedgehog signaling; this suggested a novel mechanism for Hedgehog ligand delivery. Finally, we found that Gas1, another protein important for Hedgehog ligand reception, interacts with the ligand in a unique way, dependent on its fatty acid modification; this indicated that Gas1 uses a mechanism distinct from Cdon and Boc. We propose to use a combination of biochemistry, cell biology, and chemical biology to accomplish the following aims: A) To elucidate precisely how the Hedgehog ligand is released form the membrane of producing cells B) To determine how Scube delivers the Hedgehog ligand to responding cells via Cdon and Boc C) To elucidate how Gas1 participates in Hedgehog ligand reception These studies are important to undertake for the following reasons: 1) They will advance our understanding of the Hedgehog pathway, by elucidating the route taken by the Hedgehog ligand during the signaling process; 2) They will identify novel targets for blocking Hedgehog signaling in cancer, based on the mechanisms of Hedgehog ligand release and delivery; and 3) Our novel chemical probes will be broadly applicable to study cholesterol and fatty acids in health and in disease.
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2020 — 2021 |
Salic, Adrian |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Transducing Hedgehog Signals Across the Plasma Membrane
Project summary The Hedgehog (Hh) signaling pathway is essential for embryogenesis, for adult stem cell maintenance, and is involved in birth defects and cancer. In the absence of stimulation, the tumor suppressor membrane protein, Patched1 (Ptch1), inhibits the seven-spanner oncoprotein Smoothened (Smo), thus inhibiting Hh signaling. The pathway is activated by the Sonic Hedgehog ligand (Shh), which binds and inhibits Ptch1, allowing Smo to become active and to trigger downstream signaling events. In spite of the critical importance of Ptch1 and Smo, their molecular mechanisms remain obscure: it is unknown how Ptch1 inhibits Smo, how the Shh inhibits Ptch1, how Smo is activated, and how it relays signals downstream. We recently discovered that cholesterol is the long-sought endogenous Smo activator, and that Ptch1 controls Smo via cholesterol. We solved X-ray structures of active Smo, which suggested a mechanism for activation by cholesterol. The structures also explained the hyperactivity of a classical oncogenic Smo mutant and pointed to a portion in Smo likely involved in downstream signaling. In preliminary work, we discovered rapid cholesterol transfer from Smo to Ptch1, suggesting a novel mechanism for Smo inhibition by Ptch1. This cholesterol transfer is blocked by Shh, via the novel palmitate-dependent interaction between Shh and Ptch1, which we previously discovered, suggesting a simple mechanism for Hh pathway activation. We propose to use biochemistry, chemical, cell and structural biology to accomplish the following aims: A) To determine how the Ptch1 inhibits Smo, and how the Hh ligand inhibits Ptch1, to trigger Hh signaling B) To determine precisely how cholesterol activates Smo C) To elucidate how Smo relays Hh signals to the cytoplasm These studies are important for the following reasons: 1) They will advance our understanding of the Hh pathway, by deciphering critical signaling mechanisms; 2) They will clarify how Smo and Ptch1 mutations cause cancer; 3) They will identify novel targets for blocking oncogenic Hh signaling, based on the mechanisms of Smo and Ptch1; and 4) The novel lipid probes that we developed for studying Shh and Ptch1 will be broadly applicable beyond the Hh pathway.
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