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Influenza hemagglutinin epitope (YPYDVPDYA) antibody

RRID:AB_2314672

PARP1-dependent recruitment of the FBXL10-RNF68-RNF2 ubiquitin ligase to sites of DNA damage controls H2A.Z loading.

  • Rona G
  • Elife
  • 2018 Jul 9

Literature context:


Abstract:

The mammalian FBXL10-RNF68-RNF2 ubiquitin ligase complex (FRRUC) mono-ubiquitylates H2A at Lys119 to repress transcription in unstressed cells. We found that the FRRUC is rapidly and transiently recruited to sites of DNA damage in a PARP1- and TIMELESS-dependent manner to promote mono-ubiquitylation of H2A at Lys119, a local decrease of H2A levels, and an increase of H2A.Z incorporation. Both the FRRUC and H2A.Z promote transcriptional repression, double strand break signaling, and homologous recombination repair (HRR). All these events require both the presence and activity of the FRRUC. Moreover, the FRRUC and its activity are required for the proper recruitment of BMI1-RNF2 and MEL18-RNF2, two other ubiquitin ligases that mono-ubiquitylate Lys119 in H2A upon genotoxic stress. Notably, whereas H2A.Z is not required for H2A mono-ubiquitylation, impairment of the latter results in the inhibition of H2A.Z incorporation. We propose that the recruitment of the FRRUC represents an early and critical regulatory step in HRR.

Funding information:
  • American Cancer Society - ACS 130304-RSG-16-241-01-DMC()
  • National Institutes of Health - R01- GM057691()
  • National Institutes of Health - R01-CA076584()
  • National Institutes of Health - R01-GM057587()
  • National Institutes of Health - R21-CA187612()
  • NIEHS NIH HHS - ES07784(United States)
  • NIGMS NIH HHS - R01 GM057587()
  • The Rosztoczy Foundation - Fellowship()

PTPσ drives excitatory presynaptic assembly via various extracellular and intracellular mechanisms.

  • Han KA
  • J. Neurosci.
  • 2018 Jun 22

Literature context:


Abstract:

Leukocyte common antigen-receptor protein tyrosine phosphatases (LAR-RPTPs) are hub proteins that organize excitatory and inhibitory synapse development through binding to various extracellular ligands. Here, we report that knockdown (KD) of the LAR-RPTP family member PTPσ reduced excitatory synapse number and transmission in cultured rat hippocampal neurons, whereas KD of PTPδ produced comparable decreases at inhibitory synapses, in both cases without altering expression levels of interacting proteins. An extensive series of rescue experiments revealed that extracellular interactions of PTPσ with Slitrks are important for excitatory synapse development. These experiments further showed that the intracellular D2 domain of PTPσ is required for induction of heterologous synapse formation by Slitrk1 or TrkC, suggesting that interaction of LAR-RPTPs with distinct intracellular presynaptic proteins drives presynaptic machinery assembly. Consistent with this, double-KD of liprin-α2 and -α3 or KD of PTPσ substrates (N-cadherin and p250RhoGAP) in neurons inhibited Slitrk6-induced, PTPσ-mediated heterologous synapse formation activity. We propose a synaptogenesis model in presynaptic neurons involving LAR-RPTP-organized retrograde signaling cascades, in which both extracellular and intracellular mechanisms are critical in orchestrating distinct synapse types.SIGNIFICANCE STATEMENTIn this study, we sought to test the unproven hypothesis that PTPσ and PTPδ are required for excitatory and inhibitory synapse formation/transmission, respectively, in cultured hippocampal neurons, using knockdown-based loss-of-function analyses. We further performed extensive structure-function analyses, focusing on PTPσ-mediated actions, to address the mechanisms of presynaptic assembly at excitatory synaptic sites. Utilizing interdisciplinary approaches, we systematically applied varied set of PTPσ deletion variants, point mutants, and splice variants to demonstrate that both extracellular and intracellular mechanisms are involved in organizing presynaptic assembly. Strikingly, extracellular interactions of PTPσ with heparan sulfates and Slitrks, intracellular interactions of PTPσ with liprin-α and its associated proteins through the D2 domain, as well as distinct substrates are all critical.

Funding information:
  • NIDCR NIH HHS - DE14036(United States)

mTORC1 Promotes Metabolic Reprogramming by the Suppression of GSK3-Dependent Foxk1 Phosphorylation.

  • He L
  • Mol. Cell
  • 2018 Jun 7

Literature context:


Abstract:

The mammalian Target of Rapamycin Complex 1 (mTORC1)-signaling system plays a critical role in the maintenance of cellular homeostasis by sensing and integrating multiple extracellular and intracellular cues. Therefore, uncovering the effectors of mTORC1 signaling is pivotal to understanding its pathophysiological effects. Here we report that the transcription factor forkhead/winged helix family k1 (Foxk1) is a mediator of mTORC1-regulated gene expression. Surprisingly, Foxk1 phosphorylation is increased upon mTORC1 suppression, which elicits a 14-3-3 interaction, a reduction of DNA binding, and nuclear exclusion. Mechanistically, this occurs by mTORC1-dependent suppression of nuclear signaling by the Foxk1 kinase, Gsk3. This pathway then regulates the expression of multiple genes associated with glycolysis and downstream anabolic pathways directly modulated by Foxk1 and/or by Foxk1-regulated expression of Hif-1α. Thus, Foxk1 mediates mTORC1-driven metabolic rewiring, and it is likely to be critical for metabolic diseases where improper mTORC1 signaling plays an important role.

Funding information:
  • NHLBI NIH HHS - R01 HL121266()
  • NIGMS NIH HHS - P41 GM103533(United States)
  • NIGMS NIH HHS - R01 GM051405()

TU-Tagging: A Method for Identifying Layer-Enriched Neuronal Genes in Developing Mouse Visual Cortex.

  • Tomorsky J
  • eNeuro
  • 2018 Jun 14

Literature context:


Abstract:

Thiouracil (TU)-tagging is an intersectional method for covalently labeling newly transcribed RNAs within specific cell types. Cell type specificity is generated through targeted transgenic expression of the enzyme uracil phosphoribosyl transferase (UPRT); temporal specificity is generated through a pulse of the modified uracil analog 4TU. This technique has been applied in mouse using a Cre-dependent UPRT transgene, CA>GFPstop>HA-UPRT, to profile RNAs in endothelial cells, but it remained untested whether 4TU can cross the blood-brain barrier (BBB) or whether this transgene can be used to purify neuronal RNAs. Here, we crossed the CA>GFPstop>HA-UPRT transgenic mouse to a Sepw1-cre line to express UPRT in layer 2/3 of visual cortex or to an Nr5a1-cre line to express UPRT in layer 4 of visual cortex. We purified thiol-tagged mRNA from both genotypes at postnatal day (P)12, as well as from wild-type (WT) mice not expressing UPRT (background control). We found that a comparison of Sepw1-purified RNA to WT or Nr5a1-purified RNA allowed us to identify genes enriched in layer 2/3 of visual cortex. Here, we show that Cre-dependent UPRT expression can be used to purify cell type-specific mRNA from the intact mouse brain and provide the first evidence that 4TU can cross the BBB to label RNA in vivo.

Funding information:
  • Howard Hughes Medical Institute - (United States)

The HIV-1 Tat protein recruits a ubiquitin ligase to reorganize the 7SK snRNP for transcriptional activation.

  • Faust TB
  • Elife
  • 2018 May 30

Literature context:


Abstract:

The HIV-1 Tat protein hijacks P-TEFb kinase to activate paused RNA polymerase II (RNAP II) at the viral promoter. Tat binds additional host factors, but it is unclear how they regulate RNAP II elongation. Here, we identify the cytoplasmic ubiquitin ligase UBE2O as critical for Tat transcriptional activity. Tat hijacks UBE2O to ubiquitinate the P-TEFb kinase inhibitor HEXIM1 of the 7SK snRNP, a fraction of which also resides in the cytoplasm bound to P-TEFb. HEXIM1 ubiquitination sequesters it in the cytoplasm and releases P-TEFb from the inhibitory 7SK complex. Free P-TEFb then becomes enriched in chromatin, a process that is also stimulated by treating cells with a CDK9 inhibitor. Finally, we demonstrate that UBE2O is critical for P-TEFb recruitment to the HIV-1 promoter. Together, the data support a unique model of elongation control where non-degradative ubiquitination of nuclear and cytoplasmic 7SK snRNP pools increases P-TEFb levels for transcriptional activation.

Funding information:
  • National Institute of Allergy and Infectious Diseases - R01AI114362()
  • National Institute of Allergy and Infectious Diseases - RO1AI114362()
  • National Institute of General Medical Sciences - P50GM082250()
  • NHLBI NIH HHS - R15 HL071526(United States)
  • NIAID NIH HHS - P30 AI027763()
  • Welch Foundation - I-1782()

RIM C2B Domains Target Presynaptic Active Zone Functions to PIP2-Containing Membranes.

  • de Jong APH
  • Neuron
  • 2018 Apr 18

Literature context:


Abstract:

Rapid and efficient synaptic vesicle fusion requires a pool of primed vesicles, the nearby tethering of Ca2+ channels, and the presence of the phospholipid PIP2 in the target membrane. Although the presynaptic active zone mediates the first two requirements, it is unclear how fusion is targeted to membranes with high PIP2 content. Here we find that the C2B domain of the active zone scaffold RIM is critical for action potential-triggered fusion. Remarkably, the known RIM functions in vesicle priming and Ca2+ influx do not require RIM C2B domains. Instead, biophysical experiments reveal that RIM C2 domains, which lack Ca2+ binding, specifically bind to PIP2. Mutational analyses establish that PIP2 binding to RIM C2B and its tethering to the other RIM domains are crucial for efficient exocytosis. We propose that RIM C2B domains are constitutive PIP2-binding modules that couple mechanisms for vesicle priming and Ca2+ channel tethering to PIP2-containing target membranes.

Funding information:
  • NCRR NIH HHS - S10 RR026461()
  • NIH HHS - S10 OD018027()
  • NIMH NIH HHS - R01 MH113349()
  • NINDS NIH HHS - P30 NS072030()
  • NINDS NIH HHS - R01 NS083898()
  • NINDS NIH HHS - R35 NS097333()
  • Wellcome Trust - GM043375(United Kingdom)

Proteasome storage granules protect proteasomes from autophagic degradation upon carbon starvation.

  • Marshall RS
  • Elife
  • 2018 Apr 6

Literature context:


Abstract:

26S proteasome abundance is tightly regulated at multiple levels, including the elimination of excess or inactive particles by autophagy. In yeast, this proteaphagy occurs upon nitrogen starvation but not carbon starvation, which instead stimulates the rapid sequestration of proteasomes into cytoplasmic puncta termed proteasome storage granules (PSGs). Here, we show that PSGs help protect proteasomes from autophagic degradation. Both the core protease and regulatory particle sub-complexes are sequestered separately into PSGs via pathways dependent on the accessory proteins Blm10 and Spg5, respectively. Modulating PSG formation, either by perturbing cellular energy status or pH, or by genetically eliminating factors required for granule assembly, not only influences the rate of proteasome degradation, but also impacts cell viability upon recovery from carbon starvation. PSG formation and concomitant protection against proteaphagy also occurs in Arabidopsis, suggesting that PSGs represent an evolutionarily conserved cache of proteasomes that can be rapidly re-mobilized based on energy availability.

Funding information:
  • National Institutes of Health - R01-GM124452-01A1()
  • National Science Foundation - IOS-1329956()
  • NIDCR NIH HHS - DE020817(United States)
  • NIGMS NIH HHS - R01 GM124452()
  • U.S. Department of Energy - DE-FG02-88ER13968()

An RNAi screen of Rho signalling networks identifies RhoH as a regulator of Rac1 in prostate cancer cell migration.

  • Tajadura-Ortega V
  • BMC Biol.
  • 2018 Mar 6

Literature context:


Abstract:

BACKGROUND: Cell migration is essential for development and tissue repair, but it also contributes to disease. Rho GTPases regulate cell migration, but a comprehensive analysis of how each Rho signalling component affects migration has not been carried out. RESULTS: Through an RNA interference screen, and using a prostate cancer cell line, we find that approximately 25% of Rho network components alter migration. Some genes enhance migration while others decrease basal and/or hepatocyte growth factor-stimulated migration. Surprisingly, we identify RhoH as a screen hit. RhoH expression is normally restricted to haematopoietic cells, but we find it is expressed in multiple epithelial cancer cell lines. High RhoH expression in samples from prostate cancer patients correlates with earlier relapse. RhoH depletion reduces cell speed and persistence and decreases migratory polarity. Rac1 activity normally localizes to the front of migrating cells at areas of dynamic membrane movement, but in RhoH-depleted cells active Rac1 is localised around the whole cell periphery and associated with membrane regions that are not extending or retracting. RhoH interacts with Rac1 and with several p21-activated kinases (PAKs), which are Rac effectors. Similar to RhoH depletion, PAK2 depletion increases cell spread area and reduces cell migration. In addition, RhoH depletion reduces lamellipodium extension induced by PAK2 overexpression. CONCLUSIONS: We describe a novel role for RhoH in prostate cancer cell migration. We propose that RhoH promotes cell migration by coupling Rac1 activity and PAK2 to membrane protrusion. Our results also suggest that RhoH expression levels correlate with prostate cancer progression.

Funding information:
  • Cancer Research UK - C41786/A132()
  • Cancer Research UK - C6220/A8833()
  • Cancer Research UK - C6620/A15961()
  • National Institutes of Health - PO1-GM103723()
  • National Institutes of Health - R01-GM079271()
  • National Institutes of Health - U01-EB018816()
  • NCI NIH HHS - R01 CA133966-04(United States)

Npas4 Is a Critical Regulator of Learning-Induced Plasticity at Mossy Fiber-CA3 Synapses during Contextual Memory Formation.

  • Weng FJ
  • Neuron
  • 2018 Mar 7

Literature context:


Abstract:

Synaptic connections between hippocampal mossy fibers (MFs) and CA3 pyramidal neurons are essential for contextual memory encoding, but the molecular mechanisms regulating MF-CA3 synapses during memory formation and the exact nature of this regulation are poorly understood. Here we report that the activity-dependent transcription factor Npas4 selectively regulates the structure and strength of MF-CA3 synapses by restricting the number of their functional synaptic contacts without affecting the other synaptic inputs onto CA3 pyramidal neurons. Using an activity-dependent reporter, we identified CA3 pyramidal cells that were activated by contextual learning and found that MF inputs on these cells were selectively strengthened. Deletion of Npas4 prevented both contextual memory formation and this learning-induced synaptic modification. We further show that Npas4 regulates MF-CA3 synapses by controlling the expression of the polo-like kinase Plk2. Thus, Npas4 is a critical regulator of experience-dependent, structural, and functional plasticity at MF-CA3 synapses during contextual memory formation.

Funding information:
  • FIC NIH HHS - 1R03TW008208(United States)
  • NIDA NIH HHS - R01 DA017392()
  • NIDCD NIH HHS - R01 DC014701()
  • NIMH NIH HHS - R01 MH081935()
  • NIMH NIH HHS - R01 MH091220()
  • NINDS NIH HHS - R21 NS088412()

Identification of a Botulinum Neurotoxin-like Toxin in a Commensal Strain of Enterococcus faecium.

  • Zhang S
  • Cell Host Microbe
  • 2018 Feb 14

Literature context:


Abstract:

Botulinum neurotoxins (BoNTs), produced by various Clostridium strains, are a family of potent bacterial toxins and potential bioterrorism agents. Here we report that an Enterococcus faecium strain isolated from cow feces carries a BoNT-like toxin, designated BoNT/En. It cleaves both VAMP2 and SNAP-25, proteins that mediate synaptic vesicle exocytosis in neurons, at sites distinct from known BoNT cleavage sites on these two proteins. Comparative genomic analysis determines that the E. faecium strain carrying BoNT/En is a commensal type and that the BoNT/En gene is located within a typical BoNT gene cluster on a 206 kb putatively conjugative plasmid. Although the host species targeted by BoNT/En remains to be determined, these findings establish an extended member of BoNTs and demonstrate the capability of E. faecium, a commensal organism ubiquitous in humans and animals and a leading cause of hospital-acquired multi-drug-resistant (MDR) infections, to horizontally acquire, and possibly disseminate, a unique BoNT gene cluster.

Context-dependent deposition and regulation of mRNAs in P-bodies.

  • Wang C
  • Elife
  • 2018 Jan 3

Literature context:


Abstract:

Cells respond to stress by remodeling their transcriptome through transcription and degradation. Xrn1p-dependent degradation in P-bodies is the most prevalent decay pathway, yet, P-bodies may facilitate not only decay, but also act as a storage compartment. However, which and how mRNAs are selected into different degradation pathways and what determines the fate of any given mRNA in P-bodies remain largely unknown. We devised a new method to identify both common and stress-specific mRNA subsets associated with P-bodies. mRNAs targeted for degradation to P-bodies, decayed with different kinetics. Moreover, the localization of a specific set of mRNAs to P-bodies under glucose deprivation was obligatory to prevent decay. Depending on its client mRNA, the RNA-binding protein Puf5p either promoted or inhibited decay. Furthermore, the Puf5p-dependent storage of a subset of mRNAs in P-bodies under glucose starvation may be beneficial with respect to chronological lifespan.

Funding information:
  • NIDDK NIH HHS - DK089016(United States)

Molecular basis of fatty acid taste in Drosophila.

  • Ahn JE
  • Elife
  • 2017 Dec 12

Literature context:


Abstract:

Behavioral studies have established that Drosophila appetitive taste responses towards fatty acids are mediated by sweet sensing Gustatory Receptor Neurons (GRNs). Here we show that sweet GRN activation requires the function of the Ionotropic Receptor genes IR25a, IR76b and IR56d. The former two IR genes are expressed in several neurons per sensillum, while IR56d expression is restricted to sweet GRNs. Importantly, loss of appetitive behavioral responses to fatty acids in IR25a and IR76b mutant flies can be completely rescued by expression of respective transgenes in sweet GRNs. Interestingly, appetitive behavioral responses of wild type flies to hexanoic acid reach a plateau at ~1%, but decrease with higher concentration, a property mediated through IR25a/IR76b independent activation of bitter GRNs. With our previous report on sour taste, our studies suggest that IR-based receptors mediate different taste qualities through cell-type specific IR subunits.

Funding information:
  • NIAAA NIH HHS - T32 AA007463(United States)
  • NIDCD NIH HHS - R01 DC013967()

Post-transcriptional Regulation of De Novo Lipogenesis by mTORC1-S6K1-SRPK2 Signaling.

  • Lee G
  • Cell
  • 2017 Dec 14

Literature context:


Abstract:

mTORC1 is a signal integrator and master regulator of cellular anabolic processes linked to cell growth and survival. Here, we demonstrate that mTORC1 promotes lipid biogenesis via SRPK2, a key regulator of RNA-binding SR proteins. mTORC1-activated S6K1 phosphorylates SRPK2 at Ser494, which primes Ser497 phosphorylation by CK1. These phosphorylation events promote SRPK2 nuclear translocation and phosphorylation of SR proteins. Genome-wide transcriptome analysis reveals that lipid biosynthetic enzymes are among the downstream targets of mTORC1-SRPK2 signaling. Mechanistically, SRPK2 promotes SR protein binding to U1-70K to induce splicing of lipogenic pre-mRNAs. Inhibition of this signaling pathway leads to intron retention of lipogenic genes, which triggers nonsense-mediated mRNA decay. Genetic or pharmacological inhibition of SRPK2 blunts de novo lipid synthesis, thereby suppressing cell growth. These results thus reveal a novel role of mTORC1-SRPK2 signaling in post-transcriptional regulation of lipid metabolism and demonstrate that SRPK2 is a potential therapeutic target for mTORC1-driven metabolic disorders.

Funding information:
  • NHLBI NIH HHS - R01 HL074894(United States)

Transsynaptic Mapping of Second-Order Taste Neurons in Flies by trans-Tango.

  • Talay M
  • Neuron
  • 2017 Nov 15

Literature context:


Abstract:

Mapping neural circuits across defined synapses is essential for understanding brain function. Here we describe trans-Tango, a technique for anterograde transsynaptic circuit tracing and manipulation. At the core of trans-Tango is a synthetic signaling pathway that is introduced into all neurons in the animal. This pathway converts receptor activation at the cell surface into reporter expression through site-specific proteolysis. Specific labeling is achieved by presenting a tethered ligand at the synapses of genetically defined neurons, thereby activating the pathway in their postsynaptic partners and providing genetic access to these neurons. We first validated trans-Tango in the Drosophila olfactory system and then implemented it in the gustatory system, where projections beyond the first-order receptor neurons are not fully characterized. We identified putative second-order neurons within the sweet circuit that include projection neurons targeting known neuromodulation centers in the brain. These experiments establish trans-Tango as a flexible platform for transsynaptic circuit analysis.

Funding information:
  • NIDCD NIH HHS - R01 DC013561()
  • NIDCD NIH HHS - R21 DC014333()
  • NIMH NIH HHS - R01 MH086920()
  • NIMH NIH HHS - R01 MH105368()

Autophagosomal Content Profiling Reveals an LC3C-Dependent Piecemeal Mitophagy Pathway.

  • Le Guerroué F
  • Mol. Cell
  • 2017 Nov 16

Literature context:


Abstract:

Autophagy allows the degradation of cytosolic endogenous and exogenous material in the lysosome. Substrates are engulfed by double-membrane vesicles, coined autophagosomes, which subsequently fuse with lysosomes. Depending on the involvement of specific receptor proteins, autophagy occurs in a selective or nonselective manner. While this process is well understood at the level of bulky cargo such as mitochondria and bacteria, we know very little about individual proteins and protein complexes that are engulfed and degraded by autophagy. In contrast to the critical role of autophagy in balancing proteostasis, our current knowledge of the autophagic degradome is very limited. Here, we combined proximity labeling with quantitative proteomics to systematically map the protein inventory of autophagosomes. Using this strategy, we uncovered a basal, housekeeping mitophagy pathway that involves piecemeal degradation of mitochondrial proteins in a LC3C- and p62-dependent manner and contributes to mitochondrial homeostasis maintenance when cells rely on oxidative phosphorylation.

Funding information:
  • NIDA NIH HHS - K02 DA026990(United States)

NOTCH2 Hajdu-Cheney Mutations Escape SCFFBW7-Dependent Proteolysis to Promote Osteoporosis.

  • Fukushima H
  • Mol. Cell
  • 2017 Nov 16

Literature context:


Abstract:

Hajdu-Cheney syndrome (HCS), a rare autosomal disorder caused by heterozygous mutations in NOTCH2, is clinically characterized by acro-osteolysis, severe osteoporosis, short stature, neurological symptoms, cardiovascular defects, and polycystic kidneys. Recent studies identified that aberrant NOTCH2 signaling and consequent osteoclast hyperactivity are closely associated with the bone-related disorder pathogenesis, but the exact molecular mechanisms remain unclear. Here, we demonstrate that sustained osteoclast activity is largely due to accumulation of NOTCH2 carrying a truncated C terminus that escapes FBW7-mediated ubiquitination and degradation. Mice with osteoclast-specific Fbw7 ablation revealed osteoporotic phenotypes reminiscent of HCS, due to elevated Notch2 signaling. Importantly, administration of Notch inhibitors in Fbw7 conditional knockout mice alleviated progressive bone resorption. These findings highlight the molecular basis of HCS pathogenesis and provide clinical insights into potential targeted therapeutic strategies for skeletal disorders associated with the aberrant FBW7/NOTCH2 pathway as observed in patients with HCS.

Funding information:
  • NIMH NIH HHS - R01 MH086920(United States)

Ionotropic Receptors Mediate Drosophila Oviposition Preference through Sour Gustatory Receptor Neurons.

  • Chen Y
  • Curr. Biol.
  • 2017 Sep 25

Literature context:


Abstract:

Carboxylic acids are present in many foods, being especially abundant in fruits. Yet, relatively little is known about how acids are detected by gustatory systems and whether they have a potential role in nutrition or provide other health benefits. Here we identify sour gustatory receptor neurons (GRNs) in tarsal taste sensilla of Drosophila melanogaster. We find that most tarsal sensilla harbor a sour GRN that is specifically activated by carboxylic and mineral acids but does not respond to sweet- and bitter-tasting chemicals or salt. One pair of taste sensilla features two GRNs that respond only to a subset of carboxylic acids and high concentrations of salt. All sour GRNs prominently express two Ionotropic Receptor (IR) genes, IR76b and IR25a, and we show that both these genes are necessary for the detection of acids. Furthermore, we establish that IR25a and IR76b are essential in sour GRNs of females for oviposition preference on acid-containing food. Our investigations reveal that acids activate a unique set of taste cells largely dedicated to sour taste, and they indicate that both pH/proton concentration and the structure of carboxylic acids contribute to sour GRN activation. Together, our studies provide new insights into the cellular and molecular basis of sour taste.

Funding information:
  • NIDCD NIH HHS - R01 DC005606()
  • NIDCD NIH HHS - R01 DC013967()

Activity-Dependent Gating of Parvalbumin Interneuron Function by the Perineuronal Net Protein Brevican.

  • Favuzzi E
  • Neuron
  • 2017 Aug 2

Literature context:


Abstract:

Activity-dependent neuronal plasticity is a fundamental mechanism through which the nervous system adapts to sensory experience. Several lines of evidence suggest that parvalbumin (PV+) interneurons are essential in this process, but the molecular mechanisms underlying the influence of experience on interneuron plasticity remain poorly understood. Perineuronal nets (PNNs) enwrapping PV+ cells are long-standing candidates for playing such a role, yet their precise contribution has remained elusive. We show that the PNN protein Brevican is a critical regulator of interneuron plasticity. We find that Brevican simultaneously controls cellular and synaptic forms of plasticity in PV+ cells by regulating the localization of potassium channels and AMPA receptors, respectively. By modulating Brevican levels, experience introduces precise molecular and cellular modifications in PV+ cells that are required for learning and memory. These findings uncover a molecular program through which a PNN protein facilitates appropriate behavioral responses to experience by dynamically gating PV+ interneuron function.

Assembly rules for GABAA receptor complexes in the brain.

  • Martenson JS
  • Elife
  • 2017 Aug 17

Literature context:


Abstract:

GABAA receptor (GABAAR) pentamers are assembled from a pool of 19 subunits, and variety in subunit combinations diversifies GABAAR functions to tune brain activity. Pentamers with distinct subunit compositions localize differentially at synaptic and non-synaptic sites to mediate phasic and tonic inhibition, respectively. Despite multitudes of theoretical permutations, limited subunit combinations have been identified in the brain. Currently, no molecular model exists for combinatorial GABAAR assembly in vivo. Here, we reveal assembly rules of native GABAAR complexes that explain GABAAR subunit subcellular distributions using mice and Xenopus laevis oocytes. First, α subunits possess intrinsic signals to segregate into distinct pentamers. Second, γ2 is essential for GABAAR assembly with Neuroligin-2 (NL2) and GARLHs, which localize GABAARs at synapses. Third, δ suppresses α6 synaptic localization by preventing assembly with GARLHs/NL2. These findings establish the first molecular model for combinatorial GABAAR assembly in vivo and reveal an assembly pathway regulating GABAAR synaptic localization.

Funding information:
  • NIMH NIH HHS - R01 MH077939()
  • NIMH NIH HHS - U01 MH104984()

Discovery and characterization of two novel CB1 receptor splice variants with modified N-termini in mouse.

  • Ruehle S
  • J. Neurochem.
  • 2017 Aug 13

Literature context:


Abstract:

Numerous studies have been carried out in the mouse model, investigating the role of the cannabinoid receptor type 1 (CB1). However, mouse CB1 (mCB1) receptor differs from human CB1 (hCB1) receptor in 13 amino acid residues. Two splice variants, hCB1a and hCB1b, diverging in their amino-termini, have been reported to be unique for hCB1 and, via different signaling properties, contribute to CB1 receptor physiology and pathophysiology. We hypothesized that splice variants also exist for the mCB1 receptor and have different signaling properties. On murine hippocampal cDNA, we identified two novel mCB1 receptor splice variants generated by splicing of introns with 117 bp and 186 bp in the N-terminal domain, corresponding to deletions of 39 or 62 amino acids, respectively. The mRNAs for the splice variants mCB1a and mCB1b are expressed at low levels in different brain regions. Western blot analysis of protein extracts from stably transfected HEK293 cells indicates a strongly reduced glycosylation because of the absence of two glycosylation sites in mCB1b. On-cell western analysis in these stable lines revealed increased internalization of mCB1a and mCB1b upon stimulation with the agonist WIN55,212-2 as compared to mCB1. Results also point toward an increased affinity to SR141716 for mCB1a, as well as slightly enhanced inhibition of neurotransmission compared to mCB1. In mCB1b, agonist-induced MAPK phosphorylation was decreased compared to mCB1 and mCB1a. Identification of mouse CB1 receptor splice variants may help to explain differences found between human and mouse endocannabinoid systems and improve the understanding of CB1 receptor signaling and trafficking in different species.

Funding information:
  • NIDA NIH HHS - K05 DA021696()
  • NIDA NIH HHS - P01 DA009158()
  • NIDA NIH HHS - R01 DA011322()

The complex of TRIP-Br1 and XIAP ubiquitinates and degrades multiple adenylyl cyclase isoforms.

  • Hu W
  • Elife
  • 2017 Jun 28

Literature context:


Abstract:

Adenylyl cyclases (ACs) generate cAMP, a second messenger of utmost importance that regulates a vast array of biological processes in all kingdoms of life. However, almost nothing is known about how AC activity is regulated through protein degradation mediated by ubiquitination or other mechanisms. Here, we show that transcriptional regulator interacting with the PHD-bromodomain 1 (TRIP-Br1, Sertad1), a newly identified protein with poorly characterized functions, acts as an adaptor that bridges the interaction of multiple AC isoforms with X-linked inhibitor of apoptosis protein (XIAP), a RING-domain E3 ubiquitin ligase. XIAP ubiquitinates a highly conserved Lys residue in AC isoforms and thereby accelerates the endocytosis and degradation of multiple AC isoforms in human cell lines and mice. XIAP/TRIP-Br1-mediated degradation of ACs forms part of a negative-feedback loop that controls the homeostasis of cAMP signaling in mice. Our findings reveal a previously unrecognized mechanism for degrading multiple AC isoforms and modulating the homeostasis of cAMP signaling.

Structural Insights into Modulation of Neurexin-Neuroligin Trans-synaptic Adhesion by MDGA1/Neuroligin-2 Complex.

  • Kim JA
  • Neuron
  • 2017 Jun 21

Literature context:


Abstract:

Membrane-associated mucin domain-containing glycosylphosphatidylinositol anchor proteins (MDGAs) bind directly to neuroligin-1 (NL1) and neuroligin-2 (NL2), thereby respectively regulating excitatory and inhibitory synapse development. However, the mechanisms by which MDGAs modulate NL activity to specify development of the two synapse types remain unclear. Here, we determined the crystal structures of human NL2/MDGA1 Ig1-3 complex, revealing their stable 2:2 arrangement with three interaction interfaces. Cell-based assays using structure-guided, site-directed MDGA1 mutants showed that all three contact patches were required for the MDGA's negative regulation of NL2-mediated synaptogenic activity. Furthermore, MDGA1 competed with neurexins for NL2 via its Ig1 domain. The binding affinities of both MDGA1 and MDGA2 for NL1 and NL2 were similar, consistent with the structural prediction of similar binding interfaces. However, MDGA1 selectively associated with NL2, but not NL1, in vivo. These findings collectively provide structural insights into the mechanism by which MDGAs negatively modulate synapse development governed by NLs/neurexins.

LARP1 functions as a molecular switch for mTORC1-mediated translation of an essential class of mRNAs.

  • Hong S
  • Elife
  • 2017 Jun 26

Literature context:


Abstract:

The RNA binding protein, LARP1, has been proposed to function downstream of mTORC1 to regulate the translation of 5'TOP mRNAs such as those encoding ribosome proteins (RP). However, the roles of LARP1 in the translation of 5'TOP mRNAs are controversial and its regulatory roles in mTORC1-mediated translation remain unclear. Here we show that LARP1 is a direct substrate of mTORC1 and Akt/S6K1. Deep sequencing of LARP1-bound mRNAs reveal that non-phosphorylated LARP1 interacts with both 5' and 3'UTRs of RP mRNAs and inhibits their translation. Importantly, phosphorylation of LARP1 by mTORC1 and Akt/S6K1 dissociates it from 5'UTRs and relieves its inhibitory activity on RP mRNA translation. Concomitantly, phosphorylated LARP1 scaffolds mTORC1 on the 3'UTRs of translationally-competent RP mRNAs to facilitate mTORC1-dependent induction of translation initiation. Thus, in response to cellular mTOR activity, LARP1 serves as a phosphorylation-sensitive molecular switch for turning off or on RP mRNA translation and subsequent ribosome biogenesis.

Funding information:
  • NIDDK NIH HHS - R01 DK083491()
  • NIGMS NIH HHS - R01 GM088565()
  • NIGMS NIH HHS - R01 GM110019()

The Mammalian Ribo-interactome Reveals Ribosome Functional Diversity and Heterogeneity.

  • Simsek D
  • Cell
  • 2017 Jun 1

Literature context:


Abstract:

During eukaryotic evolution, ribosomes have considerably increased in size, forming a surface-exposed ribosomal RNA (rRNA) shell of unknown function, which may create an interface for yet uncharacterized interacting proteins. To investigate such protein interactions, we establish a ribosome affinity purification method that unexpectedly identifies hundreds of ribosome-associated proteins (RAPs) from categories including metabolism and cell cycle, as well as RNA- and protein-modifying enzymes that functionally diversify mammalian ribosomes. By further characterizing RAPs, we discover the presence of ufmylation, a metazoan-specific post-translational modification (PTM), on ribosomes and define its direct substrates. Moreover, we show that the metabolic enzyme, pyruvate kinase muscle (PKM), interacts with sub-pools of endoplasmic reticulum (ER)-associated ribosomes, exerting a non-canonical function as an RNA-binding protein in the translation of ER-destined mRNAs. Therefore, RAPs interconnect one of life's most ancient molecular machines with diverse cellular processes, providing an additional layer of regulatory potential to protein expression.

Funding information:
  • NICHD NIH HHS - R21 HD086730()
  • NIH HHS - DP2 OD008509()

CRISPR/Cas9 Screens Reveal Epstein-Barr Virus-Transformed B Cell Host Dependency Factors.

  • Ma Y
  • Cell Host Microbe
  • 2017 May 10

Literature context:


Abstract:

Epstein-Barr virus (EBV) causes endemic Burkitt lymphoma (BL) and immunosuppression-related lymphomas. These B cell malignancies arise by distinct transformation pathways and have divergent viral and host expression programs. To identify host dependency factors resulting from these EBV+, B cell-transformed cell states, we performed parallel genome-wide CRISPR/Cas9 loss-of-function screens in BL and lymphoblastoid cell lines (LCLs). These highlighted 57 BL and 87 LCL genes uniquely important for their growth and survival. LCL hits were enriched for EBV-induced genes, including viral super-enhancer targets. Our systematic approach uncovered key mechanisms by which EBV oncoproteins activate the PI3K/AKT pathway and evade tumor suppressor responses. LMP1-induced cFLIP was found to be critical for LCL defense against TNFα-mediated programmed cell death, whereas EBV-induced BATF/IRF4 were critical for BIM suppression and MYC induction in LCLs. Finally, EBV super-enhancer-targeted IRF2 protected LCLs against Blimp1-mediated tumor suppression. Our results identify viral transformation-driven synthetic lethal targets for therapeutic intervention.

Funding information:
  • NIAID NIH HHS - R01 AI123420()

RasGRP3 Mediates MAPK Pathway Activation in GNAQ Mutant Uveal Melanoma.

  • Chen X
  • Cancer Cell
  • 2017 May 8

Literature context:


Abstract:

Constitutive activation of Gαq signaling by mutations in GNAQ or GNA11 occurs in over 80% of uveal melanomas (UMs) and activates MAPK. Protein kinase C (PKC) has been implicated as a link, but the mechanistic details remained unclear. We identified PKC δ and ɛ as required and sufficient to activate MAPK in GNAQ mutant melanomas. MAPK activation depends on Ras and is caused by RasGRP3, which is significantly and selectively overexpressed in response to GNAQ/11 mutation in UM. RasGRP3 activation occurs via PKC δ- and ɛ-dependent phosphorylation and PKC-independent, DAG-mediated membrane recruitment, possibly explaining the limited effect of PKC inhibitors to durably suppress MAPK in UM. The findings nominate RasGRP3 as a therapeutic target for cancers driven by oncogenic GNAQ/11.

Funding information:
  • NCI NIH HHS - R01 CA142873()
  • NCI NIH HHS - U54 CA143874()
  • NIAID NIH HHS - P01 AI091580()

Aging Triggers Cytoplasmic Depletion and Nuclear Translocation of the E3 Ligase Mahogunin: A Function for Ubiquitin in Neuronal Survival.

  • Benvegnù S
  • Mol. Cell
  • 2017 May 4

Literature context:


Abstract:

A decline in proteasome function is causally connected to neuronal aging and aging-associated neuropathologies. By using hippocampal neurons in culture and in vivo, we show that aging triggers a reduction and a cytoplasm-to-nucleus redistribution of the E3 ubiquitin ligase mahogunin (MGRN1). Proteasome impairment induces MGRN1 monoubiquitination, the key post-translational modification for its nuclear entry. One potential mechanism for MGRN1 monoubiquitination is via progressive deubiquitination at the proteasome of polyubiquitinated MGRN1. Once in the nucleus, MGRN1 potentiates the transcriptional cellular response to proteotoxic stress. Inhibition of MGRN1 impairs ATF3-mediated neuronal responsiveness to proteosomal stress and increases neuronal stress, while increasing MGRN1 ameliorates signs of neuronal aging, including cognitive performance in old animals. Our results imply that, among others, the strength of neuronal survival in a proteasomal deterioration background, like during aging, depends on the fine-tuning of ubiquitination-deubiquitination.

Co-option of an endogenous retrovirus envelope for host defense in hominid ancestors.

  • Blanco-Melo D
  • Elife
  • 2017 Apr 11

Literature context:


Abstract:

Endogenous retroviral sequences provide a molecular fossil record of ancient infections whose analysis might illuminate mechanisms of viral extinction. A close relative of gammaretroviruses, HERV-T, circulated in primates for ~25 million years (MY) before apparent extinction within the past ~8 MY. Construction of a near-complete catalog of HERV-T fossils in primate genomes allowed us to estimate a ~32 MY old ancestral sequence and reconstruct a functional envelope protein (ancHTenv) that could support infection of a pseudotyped modern gammaretrovirus. Using ancHTenv, we identify monocarboxylate transporter-1 (MCT-1) as a receptor used by HERV-T for attachment and infection. A single HERV-T provirus in hominid genomes includes an env gene (hsaHTenv) that has been uniquely preserved. This apparently exapted HERV-T env could not support virion infection but could block ancHTenv mediated infection, by causing MCT-1 depletion from cell surfaces. Thus, hsaHTenv may have contributed to HERV-T extinction, and could also potentially regulate cellular metabolism.

A Compendium of RNA-Binding Proteins that Regulate MicroRNA Biogenesis.

  • Treiber T
  • Mol. Cell
  • 2017 Apr 20

Literature context:


Abstract:

During microRNA (miRNA) biogenesis, two endonucleolytic reactions convert stem-loop-structured precursors into mature miRNAs. These processing steps can be posttranscriptionally regulated by RNA-binding proteins (RBPs). Here, we have used a proteomics-based pull-down approach to map and characterize the interactome of a multitude of pre-miRNAs. We identify ∼180 RBPs that interact specifically with distinct pre-miRNAs. For functional validation, we combined RNAi and CRISPR/Cas-mediated knockout experiments to analyze RBP-dependent changes in miRNA levels. Indeed, a large number of the investigated candidates, including splicing factors and other mRNA processing proteins, have effects on miRNA processing. As an example, we show that TRIM71/LIN41 is a potent regulator of miR-29a processing and its inactivation directly affects miR-29a targets. We provide an extended database of RBPs that interact with pre-miRNAs in extracts of different cell types, highlighting a widespread layer of co- and posttranscriptional regulation of miRNA biogenesis.

Initiation of Quality Control during Poly(A) Translation Requires Site-Specific Ribosome Ubiquitination.

  • Juszkiewicz S
  • Mol. Cell
  • 2017 Feb 16

Literature context:


Abstract:

Diverse cellular stressors have been observed to trigger site-specific ubiquitination on several ribosomal proteins. However, the ubiquitin ligases, biochemical consequences, and physiologic pathways linked to these modifications are not known. Here, we show in mammalian cells that the ubiquitin ligase ZNF598 is required for ribosomes to terminally stall during translation of poly(A) sequences. ZNF598-mediated stalling initiated the ribosome-associated quality control (RQC) pathway for degradation of nascent truncated proteins. Biochemical ubiquitination reactions identified two sites of mono-ubiquitination on the 40S protein eS10 as the primary ribosomal target of ZNF598. Cells lacking ZNF598 activity or containing ubiquitination-resistant eS10 ribosomes failed to stall efficiently on poly(A) sequences. In the absence of stalling, read-through of poly(A) produces a poly-lysine tag, which might alter the localization and solubility of the associated protein. Thus, ribosome ubiquitination can modulate translation elongation and impacts co-translational quality control to minimize production of aberrant proteins.

Funding information:
  • Medical Research Council - MC_UP_A022_1007()

Multiplex image-based autophagy RNAi screening identifies SMCR8 as ULK1 kinase activity and gene expression regulator.

  • Jung J
  • Elife
  • 2017 Feb 14

Literature context:


Abstract:

Autophagy is an intracellular recycling and degradation pathway that depends on membrane trafficking. Rab GTPases are central for autophagy but their regulation especially through the activity of Rab GEFs remains largely elusive. We employed a RNAi screen simultaneously monitoring different populations of autophagosomes and identified 34 out of 186 Rab GTPase, GAP and GEF family members as potential autophagy regulators, amongst them SMCR8. SMCR8 uses overlapping binding regions to associate with C9ORF72 or with a C9ORF72-ULK1 kinase complex holo-assembly, which function in maturation and formation of autophagosomes, respectively. While focusing on the role of SMCR8 during autophagy initiation, we found that kinase activity and gene expression of ULK1 are increased upon SMCR8 depletion. The latter phenotype involved association of SMCR8 with the ULK1 gene locus. Global mRNA expression analysis revealed that SMCR8 regulates transcription of several other autophagy genes including WIPI2. Collectively, we established SMCR8 as multifaceted negative autophagy regulator.

ZNF598 and RACK1 Regulate Mammalian Ribosome-Associated Quality Control Function by Mediating Regulatory 40S Ribosomal Ubiquitylation.

  • Sundaramoorthy E
  • Mol. Cell
  • 2017 Feb 16

Literature context:


Abstract:

Ribosomes that experience terminal stalls during translation are resolved by ribosome-associated quality control (QC) pathways that oversee mRNA and nascent chain destruction and recycle ribosomal subunits. The proximal factors that sense stalled ribosomes and initiate mammalian ribosome-associated QC events remain undefined. We demonstrate that the ZNF598 ubiquitin ligase and the 40S ribosomal protein RACK1 help to resolve poly(A)-induced stalled ribosomes. They accomplish this by regulating distinct and overlapping regulatory 40S ribosomal ubiquitylation events. ZNF598 primarily mediates regulatory ubiquitylation of RPS10 and RPS20, whereas RACK1 regulates RPS2, RPS3, and RPS20 ubiquitylation. Gain or loss of ZNF598 function or mutations that block RPS10 or RPS20 ubiquitylation result in defective resolution of stalled ribosomes and subsequent readthrough of poly(A)-containing stall sequences. Together, our results indicate that ZNF598, RACK1, and 40S regulatory ubiquitylation plays a pivotal role in mammalian ribosome-associated QC pathways.

Funding information:
  • NIGMS NIH HHS - DP2 GM119132()
  • NIGMS NIH HHS - P50 GM085764()

Myofiber-specific TEAD1 overexpression drives satellite cell hyperplasia and counters pathological effects of dystrophin deficiency.

  • Southard S
  • Elife
  • 2016 Oct 11

Literature context:


Abstract:

When unperturbed, somatic stem cells are poised to affect immediate tissue restoration upon trauma. Yet, little is known regarding the mechanistic basis controlling initial and homeostatic 'scaling' of stem cell pool sizes relative to their target tissues for effective regeneration. Here, we show that TEAD1-expressing skeletal muscle of transgenic mice features a dramatic hyperplasia of muscle stem cells (i.e. satellite cells, SCs) but surprisingly without affecting muscle tissue size. Super-numeral SCs attain a 'normal' quiescent state, accelerate regeneration, and maintain regenerative capacity over several injury-induced regeneration bouts. In dystrophic muscle, the TEAD1 transgene also ameliorated the pathology. We further demonstrate that hyperplastic SCs accumulate non-cell-autonomously via signal(s) from the TEAD1-expressing myofiber, suggesting that myofiber-specific TEAD1 overexpression activates a physiological signaling pathway(s) that determines initial and homeostatic SC pool size. We propose that TEAD1 and its downstream effectors are medically relevant targets for enhancing muscle regeneration and ameliorating muscle pathology.

Rickettsia Sca4 Reduces Vinculin-Mediated Intercellular Tension to Promote Spread.

  • Lamason RL
  • Cell
  • 2016 Oct 20

Literature context:


Abstract:

Spotted fever group (SFG) rickettsiae are human pathogens that infect cells in the vasculature. They disseminate through host tissues by a process of cell-to-cell spread that involves protrusion formation, engulfment, and vacuolar escape. Other bacterial pathogens rely on actin-based motility to provide a physical force for spread. Here, we show that SFG species Rickettsia parkeri typically lack actin tails during spread and instead manipulate host intercellular tension and mechanotransduction to promote spread. Using transposon mutagenesis, we identified surface cell antigen 4 (Sca4) as a secreted effector of spread that specifically promotes protrusion engulfment. Sca4 interacts with the cell-adhesion protein vinculin and blocks association with vinculin's binding partner, α-catenin. Using traction and monolayer stress microscopy, we show that Sca4 reduces vinculin-dependent mechanotransduction at cell-cell junctions. Our results suggest that Sca4 relieves intercellular tension to promote protrusion engulfment, which represents a distinctive strategy for manipulating cytoskeletal force generation to enable spread.

Chemogenetic Activation of an Extinction Neural Circuit Reduces Cue-Induced Reinstatement of Cocaine Seeking.

  • Augur IF
  • J. Neurosci.
  • 2016 Sep 28

Literature context:


Abstract:

The ventromedial prefrontal cortex (vmPFC) has been shown to negatively regulate cocaine-seeking behavior, but the precise conditions by which vmPFC activity can be exploited to reduce cocaine relapse are currently unknown. We used viral-mediated gene transfer of designer receptors (DREADDs) to activate vmPFC neurons and examine the consequences on cocaine seeking in a rat self-administration model of relapse. Activation of vmPFC neurons with the Gq-DREADD reduced reinstatement of cocaine seeking elicited by cocaine-associated cues, but not by cocaine itself. We used a retro-DREADD approach to confine the Gq-DREADD to vmPFC neurons that project to the medial nucleus accumbens shell, confirming that these neurons are responsible for the decreased cue-induced reinstatement of cocaine seeking. The effects of vmPFC activation on cue-induced reinstatement depended on prior extinction training, consistent with the reported role of this structure in extinction memory. These data help define the conditions under which chemogenetic activation of extinction neural circuits can be exploited to reduce relapse triggered by reminder cues. SIGNIFICANCE STATEMENT: The ventromedial prefrontal cortex (vmPFC) projection to the nucleus accumbens shell is important for extinction of cocaine seeking, but its anatomical proximity to the relapse-promoting projection from the dorsomedial prefrontal cortex to the nucleus accumbens core makes it difficult to selectively enhance neuronal activity in one pathway or the other using traditional pharmacotherapy (e.g., systemically administered drugs). Viral-mediated gene delivery of an activating Gq-DREADD to vmPFC and/or vmPFC projections to the nucleus accumbens shell allows the chemogenetic exploitation of this extinction neural circuit to reduce cocaine seeking and was particularly effective against relapse triggered by cocaine reminder cues.

Corticothalamic Projection Neuron Development beyond Subtype Specification: Fog2 and Intersectional Controls Regulate Intraclass Neuronal Diversity.

  • Galazo MJ
  • Neuron
  • 2016 Jul 6

Literature context:


Abstract:

Corticothalamic projection neurons (CThPN) are a diverse set of neurons, critical for function of the neocortex. CThPN development and diversity need to be precisely regulated, but little is known about molecular controls over their differentiation and functional specialization, critically limiting understanding of cortical development and complexity. We report the identification of a set of genes that both define CThPN and likely control their differentiation, diversity, and function. We selected the CThPN-specific transcriptional coregulator Fog2 for functional analysis. We identify that Fog2 controls CThPN molecular differentiation, axonal targeting, and diversity, in part by regulating the expression level of Ctip2 by CThPN, via combinatorial interactions with other molecular controls. Loss of Fog2 specifically disrupts differentiation of subsets of CThPN specialized in motor function, indicating that Fog2 coordinates subtype and functional-area differentiation. These results confirm that we identified key controls over CThPN development and identify Fog2 as a critical control over CThPN diversity.

Spatial control of translation repression and polarized growth by conserved NDR kinase Orb6 and RNA-binding protein Sts5.

  • Nuñez I
  • Elife
  • 2016 Jul 30

Literature context:


Abstract:

RNA-binding proteins contribute to the formation of ribonucleoprotein (RNP) granules by phase transition, but regulatory mechanisms are not fully understood. Conserved fission yeast NDR (Nuclear Dbf2-Related) kinase Orb6 governs cell morphogenesis in part by spatially controlling Cdc42 GTPase. Here we describe a novel, independent function for Orb6 kinase in negatively regulating the recruitment of RNA-binding protein Sts5 into RNPs to promote polarized cell growth. We find that Orb6 kinase inhibits Sts5 recruitment into granules, its association with processing (P) bodies, and degradation of Sts5-bound mRNAs by promoting Sts5 interaction with 14-3-3 protein Rad24. Many Sts5-bound mRNAs encode essential factors for polarized cell growth, and Orb6 kinase spatially and temporally controls the extent of Sts5 granule formation. Disruption of this control system affects cell morphology and alters the pattern of polarized cell growth, revealing a role for Orb6 kinase in the spatial control of translational repression that enables normal cell morphogenesis.

Funding information:
  • NIMH NIH HHS - T32 MH067631(United States)

The PTTG1-binding factor (PBF/PTTG1IP) regulates p53 activity in thyroid cells.

  • Read ML
  • Endocrinology
  • 2014 May 19

Literature context:


Abstract:

The PTTG1-binding factor (PBF/PTTG1IP) has an emerging repertoire of roles, especially in thyroid biology, and functions as a protooncogene. High PBF expression is independently associated with poor prognosis and lower disease-specific survival in human thyroid cancer. However, the precise role of PBF in thyroid tumorigenesis is unclear. Here, we present extensive evidence demonstrating that PBF is a novel regulator of p53, a tumor suppressor protein with a key role in maintaining genetic stability, which is infrequently mutated in differentiated thyroid cancer. By coimmunoprecipitation and proximity-ligation assays, we show that PBF binds specifically to p53 in thyroid cells and significantly represses transactivation of responsive promoters. Further, we identify that PBF decreases p53 stability by enhancing ubiquitination, which appears dependent on the E3 ligase activity of Mdm2. Impaired p53 function was evident in a transgenic mouse model with thyroid-specific PBF overexpression (transgenic PBF mice), which had significantly increased genetic instability as indicated by fluorescent inter simple sequence repeat-PCR analysis. Consistent with this, approximately 40% of all DNA repair genes examined were repressed in transgenic PBF primary cultures, including genes with critical roles in maintaining genomic integrity such as Mgmt, Rad51, and Xrcc3. Our data also revealed that PBF induction resulted in up-regulation of the E2 enzyme Rad6 in murine thyrocytes and was associated with Rad6 expression in human thyroid tumors. Overall, this work provides novel insights into the role of the protooncogene PBF as a negative regulator of p53 function in thyroid tumorigenesis, in which PBF is generally overexpressed and p53 mutations are rare compared with other tumor types.

Funding information:
  • NIMH NIH HHS - R01 MH073991(United States)

A map of octopaminergic neurons in the Drosophila brain.

  • Busch S
  • J. Comp. Neurol.
  • 2009 Apr 20

Literature context:


Abstract:

The biogenic amine octopamine modulates diverse behaviors in invertebrates. At the single neuron level, the mode of action is well understood in the peripheral nervous system owing to its simple structure and accessibility. For elucidating the role of individual octopaminergic neurons in the modulation of complex behaviors, a detailed analysis of the connectivity in the central nervous system is required. Here we present a comprehensive anatomical map of candidate octopaminergic neurons in the adult Drosophila brain: including the supra- and subesophageal ganglia. Application of the Flp-out technique enabled visualization of 27 types of individual octopaminergic neurons. Based on their morphology and distribution of genetic markers, we found that most octopaminergic neurons project to multiple brain structures with a clear separation of dendritic and presynaptic regions. Whereas their major dendrites are confined to specific brain regions, each cell type targets different, yet defined, neuropils distributed throughout the central nervous system. This would allow them to constitute combinatorial modules assigned to the modulation of distinct neuronal processes. The map may provide an anatomical framework for the functional constitution of the octopaminergic system. It also serves as a model for the single-cell organization of a particular neurotransmitter in the brain.