An RNAi screen identifies genes that regulate GABA synapses

GABA synapses play a critical role in many aspects of circuit development and function. For example, conditions that perturb GABA transmission have been implicated in epilepsy. To identify genes that regulate GABA transmission, we performed an RNAi screen for genes whose inactivation increases the activity of C. elegans body muscles, which receive direct input from GABAergic motor neurons. We identified 90 genes, 21 of which were previously implicated in seizure syndromes, suggesting that this screen has effectively identified candidate genes for epilepsy. Electrophysiological recordings and imaging of excitatory and inhibitory synapses indicate that several genes alter muscle activity by selectively regulating GABA transmission. In particular, we identify two humoral pathways and several protein kinases that modulate GABA transmission but have little effect on excitatory transmission at cholinergic neuromuscular junctions. Our data suggest these conserved genes are components of signaling pathways that regulate GABA transmission and consequently may play a role in epilepsy and other cognitive or psychiatric disorders.     

Dual-specificity phosphatases regulate mitogen-activated protein kinase signaling in adipocytes in response to inflammatory stress

Obesity is a strong predictor of heart disease, insulin resistance, and type II diabetes. Chronic, low-grade inflammation links obesity and insulin resistance through mitogen-activated protein kinase (MAPK) signaling pathways. Upstream kinases activate MAPK signaling, while MAPK-specific dual-specificity phosphatases (DUSPs) act as key modulators and controllers of MAPK deactivation (i.e. dephosphorylation). Using tumor necrosis factor α (TNFα) in 3 T3-L1 adipocytes as a model of inflammation, we report that TNFα-mediated induction of Dusp1, Dusp8 and Dusp16 modulated the transient regulation of MAPK (i.e., ERK, JNK, and p38) phosphorylation and subsequent inflammatory gene expression. All three MAPKs examined were phosphorylated in preadipocytes and adipocytes in response to TNFα, where signaling magnitude and duration were phenotype-specific. Moreover, TNFα increased mRNA abundance of DUSPs in preadipocytes and adipocytes in a phenotype-specific manner, concomitant with dephosphorylation of MAPKs. RNA interference (RNAi)-mediated knockdown of Dusp1, Dusp8 and Dusp16 increased signaling magnitude and duration of ERK, JNK, and p38 that subsequently resulted in significant increases in MAPK-dependent inflammatory gene expression of MCP-1, IL-6, and Cox-2 in response to TNFα. This study highlights important roles for DUSPs as integral components of MAPK signaling and adipocyte inflammatory gene expression.     

A genetic screen to identify sequences that mediate protein oligomerization in Escherichia coli

Many proteins assemble as oligomeric complexes and in several cases a distinct domain mediates the interaction between the subunits. The identification of new oligomerization modules is relevant to comprehend both the architecture and the evolution of protein sequences and also for protein engineering applications. Using the bacteriophage lambda repressor dimerization assay, we searched Escherichia coli genomic libraries for sequences able to mediate protein oligomerization in vivo. We identified short peptides that can substitute very effectively the dimerizing domain of the repressor. Most of these peptides belong to open reading frames that are normally not expressed in the bacterial cell.     

Genome-wide screen identifies signaling pathways that regulate autophagy during Caenorhabditis elegans development

The mechanisms that coordinate the regulation of autophagy with developmental signaling during multicellular organism development remain largely unknown. Here, we show that impaired function of ribosomal protein RPL-43 causes an accumulation of SQST-1 aggregates in the larval intestine, which are removed upon autophagy induction. Using this model to screen for autophagy regulators, we identify 139 genes that promote autophagy activity upon inactivation. Various signaling pathways, including Sma/Mab TGF-β signaling, lin-35/Rb signaling, the XBP-1-mediated ER stress response, and the ATFS-1-mediated mitochondrial stress response, regulate the expression of autophagy genes independently of the TFEB homolog HLH-30. Our study thus provides a framework for understanding the role of signaling pathways in regulating autophagy under physiological conditions.Subject Categories: Autophagy & Cell Death; Signal Transduction; Membrane & Intracellular Transport     

Protein phosphatases regulate DNA-dependent protein kinase activity

DNA-dependent protein kinase (DNA-PK) is a complex of DNA-PK catalytic subunit (DNA-PKcs) and the DNA end-binding Ku70/Ku80 heterodimer. DNA-PK is required for DNA double strand break repair by the process of nonhomologous end joining. Nonhomologous end joining is a major mechanism for the repair of DNA double strand breaks in mammalian cells. As such, DNA-PK plays essential roles in the cellular response to ionizing radiation and in V(D)J recombination. In vitro, DNA-PK undergoes phosphorylation of all three protein subunits (DNA-PK catalytic subunit, Ku70 and Ku80) and phosphorylation correlates with inactivation of the serine/threonine protein kinase activity of DNA-PK. Here we show that phosphorylation-induced loss of the protein kinase activity of DNA-PK is restored by the addition of the purified catalytic subunit of either protein phosphatase 1 or protein phosphatase 2A (PP2A) and that this reactivation is blocked by the potent protein phosphatase inhibitor, microcystin. We also show that treating human lymphoblastoid cells with either okadaic acid or fostriecin, at PP2A-selective concentrations, causes a 50-60% decrease in DNA-PK protein kinase activity, although the protein phosphatase 1 activity in these cells was unaffected. In vivo phosphorylation of DNA-PKcs, Ku70, and Ku80 was observed when cells were labeled with [(32)P]inorganic phosphate in the presence of the protein phosphatase inhibitor, okadaic acid. Together, our data suggest that reversible protein phosphorylation is an important mechanism for the regulation of DNA-PK protein kinase activity and that the protein phosphatase responsible for reactivation in vivo is a PP2A-like enzyme.     

Development of a modified in-gel assay to identify protein tyrosine phosphatases that are oxidized and inactivated in vivo

Protein tyrosine phosphatases (PTPs) comprise a superfamily of enzymes that control a diverse array of signal transduction pathways. However, the function and regulation of many of these enzymes remain undefined. Previous studies have shown that the optimal tyrosine phosphorylation response to various exogenous stimuli requires the production of reactive oxygen species (ROS). It has been proposed that ROS might transiently inactivate inhibitory PTPs, thus facilitating tyrosine phosphorylation-dependent signaling. Interestingly, the unique chemistry of the invariant, active site Cys residue located in the signature motif renders it highly susceptible to oxidation, leading to the inactivation of PTPs. We have developed a novel strategy to identify those PTPs that are oxidized and therefore, inactivated in response to extracellular stimuli. Iodoacetic acid (IAA) was used to alkylate selectively the thiolate anion of the active site Cys in the reduced PTPs. In contrast, any PTPs in which the active site Cys had been oxidized in response to the stimulus were resistant to alkylation. Following this key step to differentiate between the two pools of PTPs, the oxidized phosphatases were reduced back to the active state during the process of a standard in-gel PTP activity assay. This novel technique revealed, for the first time, that multiple cellular PTPs were indeed oxidized and inactivated in response to exogenous hydrogen peroxide. We have used this technique extensively to show that the ligand-stimulated production of intracellular hydrogen peroxide reversibly regulates the activity of specific PTPs in vivo. By defining the precise PTP targets of intracellular oxidants, the mechanistic details of signal transduction can be delineated. Due to the potential use of this method in finding the molecular targets of intracellular oxidants in diverse signaling pathways, we describe here the theoretical background and the detailed protocols of the modified in-gel PTP assay.     

Inositol hexakisphosphate and sulfonylureas regulate beta-cell protein phosphatases

In human type 2 diabetes, loss of glucose-stimulated insulin exocytosis from the pancreatic beta-cell is an early pathogenetic event. Mechanisms controlling insulin exocytosis are, however, not fully understood. We show here that inositol hexakisphosphate (InsP(6)), whose concentration transiently increases upon glucose stimulation, dose-dependently and differentially inhibits enzyme activities of ser/thr protein phosphatases in physiologically relevant concentrations. None of the hypoglycemic sulfonylureas tested affected protein phosphatase-1 or -2A activity at clinically relevant concentrations in these cells. Thus, an increase in cellular phosphorylation state, through inhibition of protein dephosphorylation by InsP(6), may be a novel regulatory mechanism linking glucose-stimulated polyphosphoinositide formation to insulin exocytosis in insulin-secreting cells.     

Sensitized RNAi screen of human kinases and phosphatases identifies new regulators of apoptosis and chemoresistance

Evasion from apoptosis is a hallmark of cancer, and recent success using targeted therapeutics underscores the importance of identifying anti-apoptotic survival pathways. Here we utilize RNA interference (RNAi) to systematically screen the kinase and phosphatase component of the human genome. In addition to known kinases, we identified several new survival kinases. Interestingly, numerous phosphatases and associated regulatory subunits contribute to cell survival, revealing a previously unrecognized general role for phosphatases as negative regulators of apoptosis. We also identified a subset of phosphatases with tumour-suppressor-like activity. Finally, RNAi targeting of specific protein kinases sensitizes resistant cells to chemotherapeutic agents. The development of inhibitors that target these kinases or phosphatases may lead to new anti-cancer strategies.     

cAMP-PKA signaling to the mitochondria: protein scaffolds, mRNA and phosphatases

Energy metabolism and, specifically, the coupling of mitochondria to growth and survival is controlled by the cAMP-PKA pathway in yeast. In higher eukaryotes, cAMP signaling originating at the plasma membrane is distributed to different subcellular districts by cAMP waves received by PKA bound to PKA anchor proteins (AKAPs) tethered to these compartments. This review focuses on the subgroup of AKAPs that anchor PKA to the mitochondrial outer membrane (mtAKAPs). Only PKA anchored to mtAKAPs can efficiently transmit cAMP signals to mitochondria. mtAKAP complexes are remarkably heterogeneous. In addition to PKA regulatory subunits, they may include mRNAs, tyrosine phosphatase(s) and tyrosine kinase(s). Selective regulation of these components by cAMP-PKA integrates various signal transduction pathways and can determine which subcellular compartment receives the signal. Unveiling the interactions among the components of these large complexes will shed light on how cAMP and PKA regulate vital mitochondrial processes.     

Intracellular screen to identify metagenomic clones that induce or inhibit a quorum-sensing biosensor

The goal of this study was to design and evaluate a rapid screen to identify metagenomic clones that produce biologically active small molecules. We built metagenomic libraries with DNA from soil on the floodplain of the Tanana River in Alaska. We extracted DNA directly from the soil and cloned it into fosmid and bacterial artificial chromosome vectors, constructing eight metagenomic libraries that contain 53,000 clones with inserts ranging from 1 to 190 kb. To identify clones of interest, we designed a high throughput "intracellular" screen, designated METREX, in which metagenomic DNA is in a host cell containing a biosensor for compounds that induce bacterial quorum sensing. If the metagenomic clone produces a quorum-sensing inducer, the cell produces green fluorescent protein (GFP) and can be identified by fluorescence microscopy or captured by fluorescence-activated cell sorting. Our initial screen identified 11 clones that induce and two that inhibit expression of GFP. The intracellular screen detected quorum-sensing inducers among metagenomic clones that a traditional overlay screen would not. One inducing clone carries a LuxI homologue that directs the synthesis of an N-acyl homoserine lactone quorum-sensing signal molecule. The LuxI homologue has 62% amino acid sequence identity to its closest match in GenBank, AmfI from Pseudomonas fluorescens, and is on a 78-kb insert that contains 67 open reading frames. Another inducing clone carries a gene with homology to homocitrate synthase. Our results demonstrate the power of an intracellular screen to identify functionally active clones and biologically active small molecules in metagenomic libraries.     

Genome-wide RNAi screen and in vivo protein aggregation reporters identify degradation of damaged proteins as an essential hypertonic stress response

The damaging effects of hypertonic stress on cellular proteins are poorly defined, and almost nothing is known about the pathways that detect and repair hypertonicity-induced protein damage. To begin addressing these problems, we screened approximately 19,000 Caenorhabditis elegans genes by RNA interference (RNAi) feeding and identified 40 that are essential for survival during acute hypertonic stress. Half (20 of 40) of these genes encode proteins that function to detect, transport, and degrade damaged proteins, including components of the ubiquitin-proteasome system, endosomal sorting complexes, and lysosomes. High-molecular-weight ubiquitin conjugates increase during hypertonic stress, suggesting a global change in the ubiquitinylation state of endogenous proteins. Using a polyglutamine-containing fluorescent reporter, we demonstrate that cell shrinkage induces rapid protein aggregation in vivo and that many of the genes that are essential for survival during hypertonic stress function to prevent accumulation of aggregated proteins. High levels of urea, a strong protein denaturant, do not cause aggregation, suggesting that factors such as macromolecular crowding also contribute to protein aggregate formation during cell shrinkage. Acclimation of C. elegans to mild hypertonicity dramatically increases the osmotic threshold for protein aggregation, demonstrating that protein aggregation-inhibiting pathways are activated by osmotic stress. Our studies demonstrate that hypertonic stress induces protein damage in vivo and that detection and degradation of damaged proteins are essential mechanisms for survival under hypertonic conditions.     

Agents that stabilize mutated von Hippel-Lindau (VHL) protein: results of a high-throughput screen to identify compounds that modulate VHL proteostasis

Von Hippel-Lindau (VHL) disease is an autosomal dominant disorder that affects multiple organs. Treatment is mainly surgical, and effective systemic therapies are needed. We developed a cell-based screening tool to identify compounds that stabilize or upregulate full-length, point-mutated VHL protein. The 786-0 cell line was infected with full-length W117A-mutated VHL linked to a C-terminal Venus fluorescent protein. This VHL-W117A-Venus line was used to screen the Prestwick drug library and was tested against proteasome inhibitors MG132 and bortezomib. Western blot validation and evaluation of functional readouts, including hypoxia-inducible factor 2α (HIF2α) and glucose transporter 1 (Glut1) levels, were performed. We found that bortezomib, MG132, and the Prestwick compounds 8-azaguanine, thiostrepton, and thioguanosine upregulated VHL-W117A-Venus in 786-0 cells. 8-Azaguanine downregulated HIF2α levels and was augmented by the presence of VHL W117A. VHL p30 band intensities varied as a function of compound used, suggesting alternate posttranslational processing. Nuclear-cytoplasmic localization of VHL-W117A-Venus varied among the different compounds. In conclusion, a 786-0 cell line containing VHL-W117A-Venus was successfully used to identify compounds that upregulate VHL levels, with differential effect on VHL intracellular localization and posttranslational processing. Further screening efforts will broaden the number of pharmacophores available to develop therapeutic agents that will upregulate and refunctionalize mutated VHL.     

Genome-wide screen for Mycobacterium tuberculosis genes that regulate host immunity

In spite of its highly immunogenic properties, Mycobacterium tuberculosis (Mtb) establishes persistent infection in otherwise healthy individuals, making it one of the most widespread and deadly human pathogens. Mtb's prolonged survival may reflect production of microbial factors that prevent even more vigorous immunity (quantitative effect) or that divert the immune response to a non-sterilizing mode (qualitative effect). Disruption of Mtb genes has produced a list of several dozen candidate immunomodulatory factors. Here we used robotic fluorescence microscopy to screen 10,100 loss-of-function transposon mutants of Mtb for their impact on the expression of promoter-reporter constructs for 12 host immune response genes in a mouse macrophage cell line. The screen identified 364 candidate immunoregulatory genes. To illustrate the utility of the candidate list, we confirmed the impact of 35 Mtb mutant strains on expression of endogenous immune response genes in primary macrophages. Detailed analysis focused on a strain of Mtb in which a transposon disrupts Rv0431, a gene encoding a conserved protein of unknown function. This mutant elicited much more macrophage TNFα, IL-12p40 and IL-6 in vitro than wild type Mtb, and was attenuated in the mouse. The mutant list provides a platform for exploring the immunobiology of tuberculosis, for example, by combining immunoregulatory mutations in a candidate vaccine strain.     

Receptor protein tyrosine phosphatases regulate neural development and axon guidance

The regulation of tyrosine phosphorylation is recognized as an important developmental mechanism. Both addition and removal of phosphate moieties on tyrosine residues are tightly regulated during development. Originally, most attention focused on the role of tyrosine kinases during development, but more recently, the developmental importance of tyrosine phosphatases has been gaining interest. Receptor protein tyrosine phosphatases (RPTPs) are of particular interest to developmental biologists because the extracellular domains of RPTPs are similar to those of cell adhesion molecules (CAMs). This suggests that RPTPs may have functions in development similar to CAMs. This review focuses on the role of RPTPs in development of the nervous system in processes such as axon guidance, synapse formation, and neural tissue morphogenesis.