Functional divergence in the role of N-linked glycosylation in smoothened signaling

The G protein-coupled receptor (GPCR) Smoothened (Smo) is the requisite signal transducer of the evolutionarily conserved Hedgehog (Hh) pathway. Although aspects of Smo signaling are conserved from Drosophila to vertebrates, significant differences have evolved. These include changes in its active sub-cellular localization, and the ability of vertebrate Smo to induce distinct G protein-dependent and independent signals in response to ligand. Whereas the canonical Smo signal to Gli transcriptional effectors occurs in a G protein-independent manner, its non-canonical signal employs Gαi. Whether vertebrate Smo can selectively bias its signal between these routes is not yet known. N-linked glycosylation is a post-translational modification that can influence GPCR trafficking, ligand responsiveness and signal output. Smo proteins in Drosophila and vertebrate systems harbor N-linked glycans, but their role in Smo signaling has not been established. Herein, we present a comprehensive analysis of Drosophila and murine Smo glycosylation that supports a functional divergence in the contribution of N-linked glycans to signaling. Of the seven predicted glycan acceptor sites in Drosophila Smo, one is essential. Loss of N-glycosylation at this site disrupted Smo trafficking and attenuated its signaling capability. In stark contrast, we found that all four predicted N-glycosylation sites on murine Smo were dispensable for proper trafficking, agonist binding and canonical signal induction. However, the under-glycosylated protein was compromised in its ability to induce a non-canonical signal through Gαi, providing for the first time evidence that Smo can bias its signal and that a post-translational modification can impact this process. As such, we postulate a profound shift in N-glycan function from affecting Smo ER exit in flies to influencing its signal output in mice

Recurrent Die-Offs of Adult Coho Salmon Returning to Spawn in Puget Sound Lowland Urban Streams

Several Seattle-area streams in Puget Sound were the focus of habitat restoration projects in the 1990s. Post-project effectiveness monitoring surveys revealed anomalous behaviors among adult coho salmon returning to spawn in restored reaches. These included erratic surface swimming, gaping, fin splaying, and loss of orientation and equilibrium. Affected fish died within hours, and female carcasses generally showed high rates (>90%) of egg retention. Beginning in the fall of 2002, systematic spawner surveys were conducted to 1) assess the severity of the adult die-offs, 2) compare spawner mortality in urban vs. non-urban streams, and 3) identify water quality and spawner condition factors that might be associated with the recurrent fish kills. The forensic investigation focused on conventional water quality parameters (e.g., dissolved oxygen, temperature, ammonia), fish condition, pathogen exposure and disease status, and exposures to metals, polycyclic aromatic hydrocarbons, and current use pesticides. Daily surveys of a representative urban stream (Longfellow Creek) from 2002–2009 revealed premature spawner mortality rates that ranged from 60–100% of each fall run. The comparable rate in a non-urban stream was <1% (Fortson Creek, surveyed in 2002). Conventional water quality, pesticide exposure, disease, and spawner condition showed no relationship to the syndrome. Coho salmon did show evidence of exposure to metals and petroleum hydrocarbons, both of which commonly originate from motor vehicles in urban landscapes. The weight of evidence suggests that freshwater-transitional coho are particularly vulnerable to an as-yet unidentified toxic contaminant (or contaminant mixture) in urban runoff. Stormwater may therefore place important constraints on efforts to conserve and recover coho populations in urban and urbanizing watersheds throughout the western United States

Membrane Cholesterol Regulates Lysosome-Plasma Membrane Fusion Events and Modulates Trypanosoma cruzi Invasion of Host Cells

Trypanosoma cruzi, is the etiological agent of a neglected tropical malady known as Chagas' disease, which affects about 8 million people in Latin America. 30–40% of affected individuals develop a symptomatic chronic infection, with cardiomyopathy being the most prevalent condition. T. cruzi utilizes an interesting strategy for entering cells: T. cruzi enhances intracellular calcium levels, which in turn trigger the exocytosis of lysosomal contents. Lysosomes then donate their membrane for the formation of the parasitophorous vacuole. Membrane rafts, cholesterol-enriched microdomains in the host cell plasma membrane, have also been implicated in T. cruzi invasion process. Since both plasma membrane and lysosomes collaborate in parasite invasion, we decided to study the importance of these membrane domains for lysosomal recruitment and fusion during T. cruzi invasion into host cells. Our results show that drug dependent depletion of plasma membrane cholesterol changes raft organization and induces excessive lysosome exocytosis in the earlier stages of treatment, leading to a depletion of lysosomes near the cell cortex, which in turn compromises T. cruzi invasion. Based on these results, we propose that cholesterol depletion leads to unregulated exocytic events of pre-docked lysosomes, reducing lysosome availability at the cell cortex and consequently compromising T. cruzi infection

In vivo analysis of compound activity and mechanism of action using epistasis in Drosophila

The recent establishment of high-throughput methods for culturing Drosophila provided a unique ability to screen compound libraries against complex disease phenotypes in the context of whole animals. However, as compound studies in Drosophila have been limited so far, the degree of conservation of compound activity between Drosophila and vertebrates or the effectiveness of feeding as a compound delivery system is not well known. Our comprehensive in vivo analysis of 27 small molecules targeting seven signaling pathways in Drosophila revealed a high degree of conservation of compound activity between Drosophila and vertebrates. We also investigated the mechanism of action of AY9944, one of the Hh pathway antagonists that we identified in our compound feeding experiments. Our epistasis analysis of AY9944 provided novel insights into AY9944’s mechanism of action and revealed a novel role for cholesterol transport in Hh signal transduction

Smoothened Signaling Through a G-Protein Effector Network

Disruption of the Hedgehog (HH) signaling pathway underlies an increasingly large array of different human tumors. Consistent with the important role HH plays in cancer, the tumor burden of patients treated with a novel HH inhibitor was ­dramatically reduced in a recent clinical trial. This drug binds directly to and antagonizes activity of the seven-transmembrane protein Smoothened (SMO), attenuating downstream signaling events that remain largely unknown. While functional studies of SMO signaling have provided a basic roadmap of information flow following ligand stimulation, the exact routes of signaling, and how they communicate with each other and with other signaling pathways, are not well characterized. We recently demonstrated that one route of SMO-mediated signal transduction involves activation of the heterotrimeric guanine nucleotide binding protein (G-protein) Gαi, suggesting that SMO can signal, at least in part, as a canonical G-protein-coupled receptor (GPCR). In this chapter, we discuss structural, functional, and mechanistic aspects of SMO signaling that relate to its function as a GPCR, and provide insight into how G-protein-dependent signaling might impact HH pathway activity