Aaron H. Nile

Mapping the Cellular Response to Small Molecules Using Chemogenomic Fitness Signatures

Yeasty HIPHOP In order to identify how chemical compounds target genes and affect the physiology of the cell, tests of the perturbations that occur when treated with a range of pharmacological chemicals are required. By examining the haploinsufficiency profiling (HIP) and homozygous profiling (HOP) chemogenomic platforms, Lee et al. (p. 208) analyzed the response of yeast to thousands of different small molecules, with genetic, proteomic, and bioinformatic analyses. Over 300 compounds were identified that targeted 121 genes within 45 cellular response signature networks. These networks were used to extrapolate the likely effects of related chemicals, their impact upon genetic pathways, and to identify putative gene functions. Guilt by association helps identify the chemogenomic signatures of compounds targeting yeast genes. Genome-wide characterization of the in vivo cellular response to perturbation is fundamental to understanding how cells survive stress. Identifying the proteins and pathways perturbed by small molecules affects biology and medicine by revealing the mechanisms of drug action. We used a yeast chemogenomics platform that quantifies the requirement for each gene for resistance to a compound in vivo to profile 3250 small molecules in a systematic and unbiased manner. We identified 317 compounds that specifically perturb the function of 121 genes and characterized the mechanism of specific compounds. Global analysis revealed that the cellular response to small molecules is limited and described by a network of 45 major chemogenomic signatures. Our results provide a resource for the discovery of functional interactions among genes, chemicals, and biological processes.

Roles of Formin Nodes and Myosin Motor Activity in Mid1p-dependent Contractile-Ring Assembly during Fission Yeast Cytokinesis

Two prevailing models have emerged to explain the mechanism of contractile-ring assembly during cytokinesis in the fission yeast Schizosaccharomyces pombe: the spot/leading cable model and the search, capture, pull, and release (SCPR) model. We tested some of the basic assumptions of the two models. Monte Carlo simulations of the SCPR model require that the formin Cdc12p is present in >30 nodes from which actin filaments are nucleated and captured by myosin-II in neighboring nodes. The force produced by myosin motors pulls the nodes together to form a compact contractile ring. Live microscopy of cells expressing Cdc12p fluorescent fusion proteins shows for the first time that Cdc12p localizes to a broad band of 30-50 dynamic nodes, where actin filaments are nucleated in random directions. The proposed progenitor spot, essential for the spot/leading cable model, usually disappears without nucleating actin filaments. alpha-Actinin ain1 deletion cells form a normal contractile ring through nodes in the absence of the spot. Myosin motor activity is required to condense the nodes into a contractile ring, based on slower or absent node condensation in myo2-E1 and UCS rng3-65 mutants. Taken together, these data provide strong support for the SCPR model of contractile-ring formation in cytokinesis.

A phosphatidylinositol transfer protein integrates phosphoinositide signaling with lipid droplet metabolism to regulate a developmental program of nutrient stress–induced membrane biogenesis

The Sec14-like phosphatidylinositol transfer protein Sfh3 associates with bulk LDs in vegetative cells but targets to a neutral lipid hydrolase-rich LD pool during sporulation. Sfh3 inhibits LD utilization by a PtdIns-4-phosphate–dependent mechanism, and this inhibition prevents prospore membrane biogenesis in sporulating cells.

Mammalian diseases of phosphatidylinositol transfer proteins and their homologs.

Abstract Inositol and phosphoinositide signaling pathways represent major regulatory systems in eukaryotes. The physiological importance of these pathways is amply demonstrated by the variety of diseases that involve derangements in individual steps in inositide and phosphoinositide production and degradation. These diseases include numerous cancers, lipodystrophies and neurological syndromes. Phosphatidylinositol transfer proteins (PITPs) are emerging as fascinating regulators of phosphoinositide metabolism. Recent advances identify PITPs (and PITP-like proteins) to be coincidence detectors, which spatially and temporally coordinate the activities of diverse aspects of the cellular lipid metabolome with phosphoinositide signaling. These insights are providing new ideas regarding mechanisms of inherited mammalian diseases associated with derangements in the activities of PITPs and PITP-like proteins.

Fatty acylation of Wnt proteins

Wnt proteins are critical regulators of signaling networks during embryonic development and in adult tissue homeostasis. The generation of active Wnt proteins requires their regulated secretion into the extracellular space. Once secreted, Wnts signal through the cell surface via receptor binding on Wnt-receiving cells, a mechanism that is prevalent in stem cell and cancer biology. Important to both Wnt secretion and receptor recognition is their post-translational fatty acylation. In this Perspective, we highlight progress in elucidating the biochemistry of Wnt fatty acylation and provide a molecular view on the enzymology of substrate recognition and catalysis, with a focus on the Wnt O-acyltransferase porcupine. Special emphasis is given to Wnt fatty acid biosynthesis, Wnt-porcupine interactions, clinical mutations of porcupine and emerging therapeutics for perturbing Wnt fatty acylation in cancer. Finally, we discuss models for the functional role of the unsaturated fatty acyl chain in mediating lipid-protein interactions and in Wnt trafficking.

Sec14-nodulin proteins and the patterning of phosphoinositide landmarks for developmental control of membrane morphogenesis

A Sec14-nodulin protein model is used to identify the nodulin domain as a novel phosphoinositide effector module with a role in controlling lateral organization of phosphoinositide. The domain organization of Sec14-nodulin proteins suggests a versatile principle for the bit mapping of membrane surfaces into high-definition lipid-signaling screens.

PITPs as targets for selectively interfering with phosphoinositide signaling in cells

Sec14-like phosphatidylinositol transfer proteins (PITPs) integrate diverse territories of intracellular lipid metabolism with stimulated phosphatidylinositol-4-phosphate production, and are discriminating portals for interrogating phosphoinositide signaling. Yet, neither Sec14-like PITPs, nor PITPs in general, have been exploited as targets for chemical inhibition for such purposes. Herein, we validate the first small molecule inhibitors (SMIs) of the yeast PITP Sec14. These SMIs are nitrophenyl(4-(2-methoxyphenyl)piperazin-1-yl)methanones (NPPMs), and are effective inhibitors in vitro and in vivo. We further establish Sec14 is the sole essential NPPM target in yeast, that NPPMs exhibit exquisite targeting specificities for Sec14 (relative to related Sec14-like PITPs), propose a mechanism for how NPPMs exert their inhibitory effects, and demonstrate NPPMs exhibit exquisite pathway selectivity in inhibiting phosphoinositide signaling in cells. These data deliver proof-of-concept that PITP-directed SMIs offer new and generally applicable avenues for intervening with phosphoinositide signaling pathways with selectivities superior to those afforded by contemporary lipid kinase-directed strategies.

Thoughts on Sec14-like nanoreactors and phosphoinositide signaling

Phosphoinositides are essential signaling molecules in eukaryotic cells (Fruman et al., 1998; Strahl and Thorner, 2007). These lipids have both intrinsic signaling capabilities, and also serve as reservoirs for production of other second messengers. As general examples of the former case, phosphoinositides form discerning chemical platforms for spatial and temporal regulation of protein activities, and also serve as co-factors that allosterically regulate the activities of various enzymes and ion channels (McLaughlin and Murray, 2005). In the latter case, phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2) is a precursor for the lipid and soluble second messenger molecules diacylglycerol and inositol polyphosphates, respectively. The metabolic cycle for regenerating the phosphoinositides consumed during the course of signaling is faced with the problem of navigating the intracellular architecture of eukaryotic cells. Phosphatidylinositol (PtdIns), while a minor cellular phospholipid in many eukaryotes (including mammals), is the metabolic precursor for phosphoinositides. PtdIns biosynthesis is catalyzed by a single PtdIns synthase which utilizes inositol and cytidine-diphospho-diacylglycerol as substrates to produce PtdIns and cytidine-monophosphate. CDP-DAG is itself generated from phosphatidic acid (PtdOH) and cytidine-trisphosphate by the enzyme CDP-DAG synthase. Both PtdIns- and CDP-DAG-synthases are integral membrane proteins of the endoplasmic reticulum – a compartment physically separated from the major compartment of PtdIns-4,5-P2 signaling (i.e. the plasma membrane). In a prescient synthesis of existing data, Robert Michell (Birmingham, UK) posited nearly forty years ago that PtdIns-4,5-P2 hydrolysis at the plasma membrane by phospholipase C generates a soluble inositide (now known to be inositol-1,4,5-trisphosphate or IP3) which sets off a trailing wave of calcium signaling (Michell, 1975). This prediction was verified experimentally (Berridge and Irvine, 1984), and extended to diacylglycerol-stimulated signaling via protein kinases C (Nishizuka, 1984). Of relevance to this discussion, Michell also recognized that his hypothesis raised the question of how are phosphoinositides replenished at the plasma membrane in the face of robust PLC activity? The crux of the matter lies in the assumption that phosphoinositide resynthesis at the plasma membrane requires PtdIns resupply from the ER – that is, the compartment where PtdIns is synthesized. To this end, a cycle was proposed where, in the first stage, soluble lipid carriers ferry either DAG or PtdOH (produced by plasma membrane DAG kinases) from the plasma membrane back to the ER to fuel PtdIns synthesis. This newly synthesized PtdIns is subsequently transported from the ER to the plasma membrane by a second set of lipid carriers, the PtdIns transfer proteins (PITPs). Indeed, PITPs with the expected biochemical properties (classically with dual activities of PtdIns and phosphatidylcholine binding/transfer) have been identified and are highly conserved proteins (Phillips et al., 2006: Cockcroft and Carvou, 2007). It is a testament to the power of the Michell conjecture linking phosphoinositide signaling with PtdIns synthesis and transport that general interpretations of PITP cellular function still borrow directly from his hypothesis (Cockcroft and Carvou, 2007). Recent work demonstrates that PITPs are unlikely to be bona fide lipid carriers, however, and that these proteins play unanticipated and interesting roles in regulating PtdIns kinase activities (Schaaf et al., 2008; Bankaitis et al., 2010). Sec14, the major yeast PITP, is the founding member of the Sec14 superfamily, and this protein arguably represents the best understood of the PITPs ( Bankaitis et al., 1989; Bankaitis et al., 1990; Cleves et al., 1991a,b; Bankaitis et al., 2010). Current thought describes Sec14 as a regulated scaffold, or nanoreactor, that chaperones an interfacial presentation of PtdIns to PtdIns-kinases. This presentation function provides an essential level of control that stimulates the biologically inadequate activity of PtdIns-kinases on membrane-incorporated PtdIns substrate. Sec14-mediated PtdIns-presentation is cued by Sec14 binding to a second lipid ligand, and this concept describes how the diverse cohort of Sec14-like proteins integrates diverse channels of lipid metabolism with phosphoinositide signaling (Schaaf et al., 2008; Bankaitis et al., 2010). Herein, we describe our perspective regarding what is known about the mechanism of Sec14 function as a single molecule. We also, identify what we consider to be the key questions for future address. This work is not intended to serve as a comprehensive review of the topic, but describes our own perspective of how we see the problem. Biological Function of Sec14 The bulk membrane PtdIns content of Saccharomyces cerevisiae is high, ca. 20-25 mol% of total glycerophospholipid (Strahl and Thorner, 2007). This value is in contrast to PtdIns representing a considerably more minor constituent of mammalian cells (ca. 5% of total glycerophospholipid). Given this natural surfeit of PtdIns, it is not at all clear why yeast would require a PITP-driven plasma membrane PtdIns supply pathway. Yet, Sec14 is essential for cell viability, even under conditions where PtdIns constitutes a remarkable 40 mol% of total membrane phospholipid (Cleves et al., 1991b). The available data, culled largely from studies of ‘bypass Sec14’mutants, argue that the essential biological function for Sec14 is to coordinate lipid metabolism with membrane trafficking in trans-Golgi network (TGN) and endosomal compartments (Cleves et al., 1989; Bankaitis et al., 1990; Cleves et al., 1991a,b; Fang et al., 1996; Li et al., 2002; Mousley et al., 2008). The key lipids involved are PtdCho, PtdIns-4-phosphate (the product of PtdIns 4-OH kinase-mediated activity) and diacylglycerol (Kearns et al., 1997; Hama et al., 1999; Rivas et al., 1999; Bankaitits et al., 2010; Figure 1). Current thought identifies the key regulatory axis as the coordination of PtdCho biosynthesis via the CDP-choline pathway with Sec14-mediated stimulation of PtdIns 4-OH kinase activity (Figure 1). Figure 1 Sec14 coordinates lipid metabolism with membrane trafficking. A lipid-regulated vesicle formation pathway is shown. This pathway features diacylglycerol (DAG) and PtdIns-4-P as pro-exocytotic lipids, and its activity is sensitive to flux through the DAG-consuming ... How Sec14 binds phospholipids The question of how Sec14 binds PtdCho and PtdIns was solved by structural studies of the yeast Sec14 homolog Sfh1 in complex with various phospholipid species – including PtdIns and PtdCho (Schaaf et al., 2008). The Sfh1 structural information translates directly to Sec14, and permits formulation of several important conclusions. First, and perhaps most remarkably, Sec14 binds the PtdCho and PtdIns headgroups at physically distinct sites. While the PtdCho headgroup and glycerol backbone moieties are buried deep within the protein’s interior, the corresponding regions of bound PtdIns are positioned much closer to the protein’s surface. Second, rational mutagenesis studies demonstrate PtdCho-binding and PtdIns-binding are both individually required for the biological function of Sec14. A Sec14 molecule must harbor the capacity to bind/exchange both PtdIns- and PtdCho in order to be a biologically active protein that stimulates PtdIns 4-OH kinase activity. What these data demonstrate is that stimulation of lipid kinase activity by Sec14 requires the PITP to undergo heterotypic exchange reactions (e.g. PtdIns for PtdCho or vice versa), and that homotypic exchange reactions (e.g. PtdIns for PtdIns) do not effect a suitable presentation of PtdIns to the PtdIns 4-OH kinase (Figure 1). Shaaf et al (2008) propose a kinetic trap model to account for these results, and an essential component of this hypothesis is that PtdIns and PtdCho traverse different trajectories during lipid exchange into and from the Sec14 hydrophobic pocket. The model also predicts slower kinetics for PtdCho exchange relative to the kinetics of PtdIns exchange. Another remarkable feature of Sec14/Sfh1 is that minimal conformational adjustments are required for these proteins to bind PtdCho vs PtdIns. Internal H2O flux is the foundation of this particular structural property, and internal H2O also plays an essential role in the conformational dynamics and the energetics associated with lipid exchange (see below). For example, five ordered H2O molecules occupy the ‘empty’ phosphoinositol binding cleft when Sec14/Sfh1 is bound to PtdCho. Reciprocally, two ordered H2O molecules fill the vacant phosphocholine-binding space in the PtdIns-bound protein. The selectivity for PtdIns vs PtdCho is estimated to be small in energetic terms, and H2O rearrangements are sufficient to negotiate the energy barriers that confront heterotypic phospholipid exchange reactions.

Sec14-like phosphatidylinositol-transfer proteins and diversification of phosphoinositide signalling outcomes.

The physiological functions of phosphatidylinositol (PtdIns)-transfer proteins (PITPs)/phosphatidylcholine (PtdCho)-transfer proteins are poorly characterized, even though these proteins are conserved throughout the eukaryotic kingdom. Much of the progress in elucidating PITP functions has come from exploitation of genetically tractable model organisms, but the mechanisms for how PITPs execute their biological activities remain unclear. Structural and molecular dynamics approaches are filling in the details for how these proteins actually work as molecules. In the present paper, we discuss our recent work with Sec14-like PITPs and describe how PITPs integrate diverse territories of the lipid metabolome with phosphoinositide signalling.