Benjamin R. Myers

FKBP12-rapamycin-associated protein associates with mitochondria and senses osmotic stress via mitochondrial dysfunction

FKBP12-rapamycin associated protein (FRAP, also known as mTOR or RAFT) is the founding member of the phosphatidylinositol kinase-related kinase family and functions as a sensor of physiological signals that regulate cell growth. Signals integrated by FRAP include nutrients, cAMP levels, and osmotic stress, and cellular processes affected by FRAP include transcription, translation, and autophagy. The mechanisms underlying the integration of such diverse signals by FRAP are largely unknown. Recently, FRAP has been reported to be regulated by mitochondrial dysfunction and depletion of ATP levels. Here we show that exposure of cells to hyperosmotic conditions (and to glucose-deficient growth medium) results in rapid and reversible dissipation of the mitochondrial proton gradient. These results suggest that the ability of FRAP to mediate osmotic stress response (and glucose deprivation response) is by means of an intermediate mitochondrial dysfunction. We also show that in addition to cytosolic FRAP a large portion of FRAP associates with the mitochondrial outer membrane. The results support the existence of a stress-sensing module consisting of mitochondria and mitochondrial outer membrane-associated FRAP. This module allows the cell to integrate a variety of stress signals that affect mitochondrial function and regulate a growth checkpoint involving p70 S6 kinase.

Zebrafish TRPA1 channels are required for chemosensation but not for thermosensation or mechanosensory hair cell function

Transient receptor potential (TRP) ion channels have been implicated in detecting chemical, thermal, and mechanical stimuli in organisms ranging from mammals to Caenorhabditis elegans. It is well established that TRPA1 detects and mediates behavioral responses to chemical irritants. However, the role of TRPA1 in detecting thermal and mechanical stimuli is controversial. To further clarify the functions of TRPA1 channels in vertebrates, we analyzed their roles in zebrafish. The two zebrafish TRPA1 paralogs are expressed in sensory neurons and are activated by several chemical irritants in vitro. High-throughput behavioral analyses of trpa1a and trpa1b mutant larvae indicate that TRPA1b is necessary for behavioral responses to these chemical irritants. However, TRPA1 paralogs are not required for behavioral responses to temperature changes or for mechanosensory hair cell function in the inner ear or lateral line. These results support a role for zebrafish TRPA1 in chemical but not thermal or mechanical sensing, and establish a high-throughput system to identify genes and small molecules that modulate chemosensation, thermosensation, and mechanosensation.

A Yeast Genetic Screen Reveals a Critical Role for the Pore Helix Domain in TRP Channel Gating

TRP cation channels function as cellular sensors in uni- and multicellular eukaryotes. Despite intensive study, the mechanisms of TRP channel activation by chemical or physical stimuli remain poorly understood. To identify amino acid residues crucial for TRP channel gating, we developed an unbiased, high-throughput genetic screen in yeast that uncovered rare, constitutively active mutants of the capsaicin receptor, TRPV1. We show that mutations within the pore helix domain dramatically increase basal channel activity and responsiveness to chemical and thermal stimuli. Mutation of corresponding residues within two related TRPV channels leads to comparable effects on their activation properties. Our data suggest that conformational changes in the outer pore region are critical for determining the balance between open and closed states, providing evidence for a general role for this domain in TRP channel activation.

Hedgehog pathway modulation by multiple lipid binding sites on the smoothened effector of signal response

Hedgehog (Hh) signaling during development and in postembryonic tissues requires activation of the 7TM oncoprotein Smoothened (Smo) by mechanisms that may involve endogenous lipidic modulators. Exogenous Smo ligands previously identified include the plant sterol cyclopamine (and its therapeutically useful synthetic mimics) and hydroxylated cholesterol derivatives (oxysterols); Smo is also highly sensitive to cellular sterol levels. The relationships between these effects are unclear because the relevant Smo structural determinants are unknown. We identify the conserved extracellular cysteine-rich domain (CRD) as the site of action for oxysterols on Smo, involving residues structurally analogous to those contacting the Wnt lipid adduct in the homologous Frizzled CRD; this modulatory effect is distinct from that of cyclopamine mimics, from Hh-mediated regulation, and from the permissive action of cellular sterol pools. These results imply that Hh pathway activity is sensitive to lipid binding at several Smo sites, suggesting mechanisms for tuning by multiple physiological inputs.

Multiple Unbiased Prospective Screens Identify TRP Channels and Their Conserved Gating Elements

It is now clear that transient receptor potential (TRP) channels are sensors of temperature, mechanical force, noxious chemicals, and G protein–coupled receptor–mediated signal transduction. Sequence and topological analyses group TRPs with other tetrameric cation channels (Yu and Catterall, 2004). Each subunit bears extensive N- and C-terminal cytoplasmic domains and an S1-S2-S3-S4-S5-P-S6 motif, where Ss are segments of transmembrane α helices and P is the “pore” that houses the ion filter. Although there are no crystal structures of TRPs at this writing, they are expected to be grossly similar to those of K+ channels (Doyle et al., 1998; Long et al., 2007). Indeed, recent electron cryomicroscopy of a TRP channel (TRPV1) at 19-A resolution is consistent with a tetrameric structure having a compact transmembrane core and a large cytoplasmic domain in the form of a “hanging gondola” (Moiseenkova-Bell et al., 2008). Various aspects of TRP channel research have been reviewed elsewhere (Ramsey et al., 2006; Nilius et al., 2007; Venkatachalam and Montell, 2007). These reviews delve deeply into each of the animal TRP subtypes with regard to their expression, activation, regulation, and medical implications. Here, we intend only to complement these reviews, and in so doing we draw attention to the presence of TRPs beyond animal species, the commonalities among TRP subfamilies, the role of prospective searches in founding these subfamilies, and the continued use of prospective methods in dissecting TRP channels. We find especially striking the repeated identifications of a site at which mutations lead to constitutive channel activity in different TRP subtypes and wish to highlight these forward genetic results.

TRP Channel Structural Biology: New Roles for an Old Fold

The capsaicin receptor, TRPV1, contributes to thermal and chemical sensitivity of primary afferent neurons of the pain pathway, but many aspects of its regulation remain elusive. In this issue of Neuron, Lishko et al. describe a high-resolution structure of a TRPV1 domain, providing insight into the molecular basis of channel modulation while revealing new functions for a widely expressed protein interaction fold.

Rapid, direct activity assays for Smoothened reveal Hedgehog pathway regulation by membrane cholesterol and extracellular sodium

Significance The Hedgehog pathway is critical in development and disease, but how cells respond to the secreted Hedgehog signal remains mysterious. A key step involves the regulation of the seven-transmembrane oncoprotein Smoothened by the 12-pass transporter-like Hedgehog receptor Patched1. We investigate the model that Patched1 is an ion-driven transporter of an endogenous lipidic Smoothened ligand. Whereas Patched–Smoothened regulation has traditionally been studied through indirect, downstream pathway readouts, we developed rapid, direct functional assays to dissect this step in simplified cell-based and in vitro systems. Cholesterol, a major membrane lipid, constitutively activates purified Smoothened by engaging its membrane-spanning region. Patched1 activity depends on extracellular Na+, suggesting that transmembrane Na+ gradients, universal among metazoans, might power Patched1 transporter-like activity in Smoothened regulation. Hedgehog signaling specifies tissue patterning and renewal, and pathway components are commonly mutated in certain malignancies. Although central to ensuring appropriate pathway activity in all Hedgehog-responsive cells, how the transporter-like receptor Patched1 regulates the seven-transmembrane protein Smoothened remains mysterious, partially due to limitations in existing tools and experimental systems. Here we employ direct, real-time, biochemical and physiology-based approaches to monitor Smoothened activity in cellular and in vitro contexts. Patched1–Smoothened coupling is rapid, dynamic, and can be recapitulated without cilium-specific proteins or lipids. By reconstituting purified Smoothened in vitro, we show that cholesterol within the bilayer is sufficient for constitutive Smoothened activation. Cholesterol effects occur independently of the lipid-binding Smoothened extracellular domain, a region that is dispensable for Patched1–Smoothened coupling. Finally, we show that Patched1 specifically requires extracellular Na+ to regulate Smoothened in our assays, raising the possibility that a Na+ gradient provides the energy source for Patched1 catalytic activity. Our work suggests a hypothesis wherein Patched1, chemiosmotically driven by the transmembrane Na+ gradient common to metazoans, regulates Smoothened by shielding its heptahelical domain from cholesterol, or by providing an inhibitor that overrides this cholesterol activation.

Abstract 3436: Ameloblastoma driver mutations revealed by next-generation sequencing of formalin-fixed paraffin-embedded specimens

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Rare cancer types are not only understudied, but are typically represented by formalin-fixed paraffin-embedded (FFPE) (rather than freshly-frozen) specimens that are suboptimal for genomic analysis. Ameloblastoma is one such rare tumor type, thought to arise from ameloblasts, the cells that deposit enamel during tooth development. Though typically benign, ameloblastomas are locally destructive to the jaw and face, and new non-surgical interventions are needed. To discover novel driver mutations and therapeutic targets, we optimized methods and performed whole-transcriptome sequencing and/or targeted exon sequencing (TruSeq Cancer Panel) of 8 FFPE cases. Identified mutations were verified, and then evaluated on a larger, independent set of 21 FFPE cases by PCR and Sanger sequencing. From the analysis, we identified recurrent somatic mutations in three key developmental or signaling pathways, including Hedgehog, fibroblast growth factor, and MAP kinase pathways. Functional interrogation of a novel Hedgehog pathway mutation confirmed increased basal pathway activity, and defined the response profile to various pharmacologic Hedgehog inhibitors. Together, our results define new ameloblastoma drivers and nominate new molecularly-directed therapies for this rare but disfiguring disease. More generally, our findings validate a robust approach for discovering driver mutations in rare cancers. Citation Format: Andrew C. McClary, Robert T. Sweeney, Jewison Biscocho, Benjamin R. Myers, Lila Neahring, Kevin A. Kwei, Kunbin Qu, Xue Gong, Tony Ng, Carol D. Jones, Sushama Varma, Justin I. Odegaard, Brian Rubin, Megan L. Troxell, Robert J. Pelham, James L. Zehnder, Philip A. Beachy, Jonathan R. Pollack, Robert B. West. Ameloblastoma driver mutations revealed by next-generation sequencing of formalin-fixed paraffin-embedded specimens. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3436. doi:10.1158/1538-7445.AM2014-3436