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Anti-HA High Affinity; Rat monoclonal antibody (clone 3F10)

RRID:AB_10094468

Antibody ID

AB_390918

Target Antigen

HA High Affinity; Rat monoclonal antibody (clone 3F10)

Proper Citation

(Roche Cat# 11867423001, RRID:AB_390918)

Clonality

unknown

Comments

The following antibodies were determined to be duplicates and consolidated by curator on 3/2019: AB_390918, AB_10094468.

Host Organism

rat

Vendor

Roche

Ubiquitination of ABCE1 by NOT4 in Response to Mitochondrial Damage Links Co-translational Quality Control to PINK1-Directed Mitophagy.

  • Wu Z
  • Cell Metab.
  • 2018 Jul 3

Literature context:


Abstract:

Translation of mRNAs is tightly regulated and constantly surveyed for errors. Aberrant translation can trigger co-translational protein and RNA quality control processes, impairments of which cause neurodegeneration by still poorly understood mechanism(s). Here we show that quality control of translation of mitochondrial outer membrane (MOM)-localized mRNA intersects with the turnover of damaged mitochondria, both orchestrated by the mitochondrial kinase PINK1. Mitochondrial damage causes stalled translation of complex-I 30 kDa subunit (C-I30) mRNA on MOM, triggering the recruitment of co-translational quality control factors Pelo, ABCE1, and NOT4 to the ribosome/mRNA-ribonucleoprotein complex. Damage-induced ubiquitination of ABCE1 by NOT4 generates poly-ubiquitin signals that attract autophagy receptors to MOM to initiate mitophagy. In the Drosophila PINK1 model, these factors act synergistically to restore mitophagy and neuromuscular tissue integrity. Thus ribosome-associated co-translational quality control generates an early signal to trigger mitophagy. Our results have broad therapeutic implications for the understanding and treatment of neurodegenerative diseases.

Funding information:
  • NCI NIH HHS - CA 19014(United States)
  • NIMH NIH HHS - R01 MH080378()
  • NINDS NIH HHS - R01 NS083417()
  • NINDS NIH HHS - R01 NS084412()

A GABAergic Feedback Shapes Dopaminergic Input on the Drosophila Mushroom Body to Promote Appetitive Long-Term Memory.

  • Pavlowsky A
  • Curr. Biol.
  • 2018 Jun 4

Literature context:


Abstract:

Memory consolidation is a crucial step for long-term memory (LTM) storage. However, we still lack a clear picture of how memory consolidation is regulated at the neuronal circuit level. Here, we took advantage of the well-described anatomy of the Drosophila olfactory memory center, the mushroom body (MB), to address this question in the context of appetitive LTM. The MB lobes, which are made by the fascicled axons of the MB intrinsic neurons, are organized into discrete anatomical modules, each covered by the terminals of a defined type of dopaminergic neuron (DAN) and the dendrites of a corresponding type of MB output neuron (MBON). We previously revealed the essential role of one DAN, the MP1 neuron, in the formation of appetitive LTM. The MP1 neuron is anatomically matched to the GABAergic MBON MVP2, which has been attributed feedforward inhibitory functions recently. Here, we used behavior experiments and in vivo imaging to challenge the existence of MP1-MVP2 synapses and investigate their role in appetitive LTM consolidation. We show that MP1 and MVP2 neurons form an anatomically and functionally recurrent circuit, which features a feedback inhibition that regulates consolidation of appetitive memory. This circuit involves two opposite type 1 and type 2 dopamine receptors in MVP2 neurons and the metabotropic GABAB-R1 receptor in MP1 neurons. We propose that this dual-receptor feedback supports a bidirectional self-regulation of MP1 input to the MB. This mechanism displays striking similarities with the mammalian reward system, in which modulation of the dopaminergic signal is primarily assigned to inhibitory neurons.

Funding information:
  • NIAMS NIH HHS - P30 AR053503(United States)

FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex.

  • Künzel U
  • Elife
  • 2018 Jun 13

Literature context:


Abstract:

Many intercellular signals are synthesised as transmembrane precursors that are released by proteolytic cleavage ('shedding') from the cell surface. ADAM17, a membrane-tethered metalloprotease, is the primary shedding enzyme responsible for the release of the inflammatory cytokine TNFα and several EGF receptor ligands. ADAM17 exists in complex with the rhomboid-like iRhom proteins, which act as cofactors that regulate ADAM17 substrate shedding. Here we report that the poorly characterised FERM domain-containing protein FRMD8 is a new component of the iRhom2/ADAM17 sheddase complex. FRMD8 binds to the cytoplasmic N-terminus of iRhoms and is necessary to stabilise iRhoms and ADAM17 at the cell surface. In the absence of FRMD8, iRhom2 and ADAM17 are degraded via the endolysosomal pathway, resulting in the reduction of ADAM17-mediated shedding. We have confirmed the pathophysiological significance of FRMD8 in iPSC-derived human macrophages and mouse tissues, thus demonstrating its role in the regulated release of multiple cytokine and growth factor signals.

Funding information:
  • Boehringer Ingelheim Fonds - PhD Fellowship()
  • Horizon 2020 Framework Programme - 659166()
  • Medical Research Council - 1374214()
  • Medical Research Council - MC_EX_MR/N50192X/1()
  • NINDS NIH HHS - NS054042(United States)
  • Wellcome - 101035/Z/13/Z()
  • Wellcome - Oxford Wellcome Institutional Strategic Support Fund 121302()

The Monocot-Specific Receptor-like Kinase SDS2 Controls Cell Death and Immunity in Rice.

  • Fan J
  • Cell Host Microbe
  • 2018 Apr 11

Literature context:


Abstract:

Programmed cell death (PCD) plays critical roles in plant immunity but must be regulated to prevent excessive damage. The E3 ubiquitin ligase SPL11 negatively regulates PCD and immunity in plants. We show that SPL11 cell-death suppressor 2 (SDS2), an S-domain receptor-like kinase, positively regulates PCD and immunity in rice by engaging and regulating SPL11 and related kinases controlling defense responses. An sds2 mutant shows reduced immune responses and enhanced susceptibility to the blast fungus Magnaporthe oryzae. Conversely, SDS2 over-expression induces constitutive PCD accompanied by elevated immune responses and enhanced resistance to M. oryzae. SDS2 interacts with and phosphorylates SPL11, which in turn ubiquitinates SDS2, leading to its degradation. In addition, SDS2 interacts with related receptor-like cytoplasmic kinases, OsRLCK118/176, that positively regulate immunity by phosphorylating the NADPH oxidase OsRbohB to stimulate ROS production. Thus, a plasma membrane-resident protein complex consisting of SDS2, SPL11, and OsRLCK118/176 controls PCD and immunity in rice.

Funding information:
  • NCI NIH HHS - P30 CA016672(United States)

IRS-1 acts as an endocytic regulator of IGF-I receptor to facilitate sustained IGF signaling.

  • Yoneyama Y
  • Elife
  • 2018 Apr 11

Literature context:


Abstract:

Insulin-like growth factor-I receptor (IGF-IR) preferentially regulates the long-term IGF activities including growth and metabolism. Kinetics of ligand-dependent IGF-IR endocytosis determines how IGF induces such downstream signaling outputs. Here, we find that the insulin receptor substrate (IRS)-1 modulates how long ligand-activated IGF-IR remains at the cell surface before undergoing endocytosis in mammalian cells. IRS-1 interacts with the clathrin adaptor complex AP2. IRS-1, but not an AP2-binding-deficient mutant, delays AP2-mediated IGF-IR endocytosis after the ligand stimulation. Mechanistically, IRS-1 inhibits the recruitment of IGF-IR into clathrin-coated structures; for this reason, IGF-IR avoids rapid endocytosis and prolongs its activity on the cell surface. Accelerating IGF-IR endocytosis via IRS-1 depletion induces the shift from sustained to transient Akt activation and augments FoxO-mediated transcription. Our study establishes a new role for IRS-1 as an endocytic regulator of IGF-IR that ensures sustained IGF bioactivity, independent of its classic role as an adaptor in IGF-IR signaling.

Funding information:
  • Austrian Research Promotion Agency (FFG) - 850681()
  • Japan Agency for Medical Research and Development and Ministry of Education, Culture, Sports, Science, and Technology - Platform Project for Supporting in Drug Discovery and Life Scien()
  • Japan Society for the Promotion of Science - 15K18766()
  • Ministry of Education, Culture, Sports, Science, and Technology - The Targeted Proteins Research Program (TPRP)()
  • NIGMS NIH HHS - 2T32GM008646(United States)
  • University of Applied Sciences Upper Austria and the Center for Technological Innovation in Medicine (TIMed Center) - Project GlucoSTAR()

Dunce Phosphodiesterase Acts as a Checkpoint for Drosophila Long-Term Memory in a Pair of Serotonergic Neurons.

  • Scheunemann L
  • Neuron
  • 2018 Apr 18

Literature context:


Abstract:

A key function of the brain is to filter essential information and store it in the form of stable, long-term memory (LTM). We demonstrate here that the Dunce (Dnc) phosphodiesterase, an important enzyme that degrades cAMP, acts as a molecular switch that controls LTM formation in Drosophila. We show that, during LTM formation, Dnc is inhibited in the SPN, a pair of newly characterized serotonergic neurons, which stimulates the cAMP/PKA pathway. As a consequence, the SPN activates downstream dopaminergic neurons, opening the gate for LTM formation in the olfactory memory center, the mushroom body. Strikingly, transient inhibition of Dnc in the SPN by RNAi was sufficient to induce LTM formation with a training protocol that normally generates only short-lived memory. Thus, Dnc activity in the SPN acts as a memory checkpoint to guarantee that only the most relevant learned experiences are consolidated into stable memory.

Funding information:
  • NIDDK NIH HHS - U19 DK06234(United States)

Dynamics of PARKIN-Dependent Mitochondrial Ubiquitylation in Induced Neurons and Model Systems Revealed by Digital Snapshot Proteomics.

  • Ordureau A
  • Mol. Cell
  • 2018 Apr 19

Literature context:


Abstract:

Flux through kinase and ubiquitin-driven signaling systems depends on the modification kinetics, stoichiometry, primary site specificity, and target abundance within the pathway, yet we rarely understand these parameters and their spatial organization within cells. Here we develop temporal digital snapshots of ubiquitin signaling on the mitochondrial outer membrane in embryonic stem cell-derived neurons, and we model HeLa cell systems upon activation of the PINK1 kinase and PARKIN ubiquitin ligase by proteomic counting of ubiquitylation and phosphorylation events. We define the kinetics and site specificity of PARKIN-dependent target ubiquitylation, and we demonstrate the power of this approach to quantify pathway modulators and to mechanistically define the role of PARKIN UBL phosphorylation in pathway activation in induced neurons. Finally, through modulation of pS65-Ub on mitochondria, we demonstrate that Ub hyper-phosphorylation is inhibitory to mitophagy receptor recruitment, indicating that pS65-Ub stoichiometry in vivo is optimized to coordinate PARKIN recruitment via pS65-Ub and mitophagy receptors via unphosphorylated chains.

Funding information:
  • Biotechnology and Biological Sciences Research Council - BB/I004815/1(United Kingdom)
  • NIDDK NIH HHS - K01 DK098285()
  • NIGMS NIH HHS - R01 GM067945()

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)

METTL14 Inhibits Hematopoietic Stem/Progenitor Differentiation and Promotes Leukemogenesis via mRNA m6A Modification.

  • Weng H
  • Cell Stem Cell
  • 2018 Feb 1

Literature context:


Abstract:

N6-methyladenosine (m6A), the most prevalent internal modification in eukaryotic messenger RNAs (mRNAs), plays critical roles in many bioprocesses. However, its functions in normal and malignant hematopoiesis remain elusive. Here, we report that METTL14, a key component of the m6A methyltransferase complex, is highly expressed in normal hematopoietic stem/progenitor cells (HSPCs) and acute myeloid leukemia (AML) cells carrying t(11q23), t(15;17), or t(8;21) and is downregulated during myeloid differentiation. Silencing of METTL14 promotes terminal myeloid differentiation of normal HSPCs and AML cells and inhibits AML cell survival/proliferation. METTL14 is required for development and maintenance of AML and self-renewal of leukemia stem/initiation cells (LSCs/LICs). Mechanistically, METTL14 exerts its oncogenic role by regulating its mRNA targets (e.g., MYB and MYC) through m6A modification, while the protein itself is negatively regulated by SPI1. Collectively, our results reveal the SPI1-METTL14-MYB/MYC signaling axis in myelopoiesis and leukemogenesis and highlight the critical roles of METTL14 and m6A modification in normal and malignant hematopoiesis.

Funding information:
  • Howard Hughes Medical Institute - DP1 OD003644-05(United States)
  • NCI NIH HHS - R01 CA178454()
  • NCI NIH HHS - R01 CA182528()
  • NCI NIH HHS - R01 CA211614()
  • NCI NIH HHS - R01 CA214965()
  • NCI NIH HHS - R50 CA211404()
  • NHGRI NIH HHS - RM1 HG008935()
  • NIDDK NIH HHS - R01 DK107615()

RHOA G17V Induces T Follicular Helper Cell Specification and Promotes Lymphomagenesis.

  • Cortes JR
  • Cancer Cell
  • 2018 Feb 12

Literature context:


Abstract:

Angioimmunoblastic T cell lymphoma (AITL) is an aggressive tumor derived from malignant transformation of T follicular helper (Tfh) cells. AITL is characterized by loss-of-function mutations in Ten-Eleven Translocation 2 (TET2) epigenetic tumor suppressor and a highly recurrent mutation (p.Gly17Val) in the RHOA small GTPase. Yet, the specific role of RHOA G17V in AITL remains unknown. Expression of Rhoa G17V in CD4+ T cells induces Tfh cell specification; increased proliferation associated with inducible co-stimulator (ICOS) upregulation and increased phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase signaling. Moreover, RHOA G17V expression together with Tet2 loss resulted in development of AITL in mice. Importantly, Tet2-/-RHOA G17V tumor proliferation in vivo can be inhibited by ICOS/PI3K-specific blockade, supporting a driving role for ICOS signaling in Tfh cell transformation.

Funding information:
  • NCI NIH HHS - R01 CA197945()
  • NIDDK NIH HHS - R37 DK44746(United States)

CCPG1 Is a Non-canonical Autophagy Cargo Receptor Essential for ER-Phagy and Pancreatic ER Proteostasis.

  • Smith MD
  • Dev. Cell
  • 2018 Jan 22

Literature context:


Abstract:

Mechanisms of selective autophagy of the ER, known as ER-phagy, require molecular delineation, particularly in vivo. It is unclear how these events control ER proteostasis and cellular health. Here, we identify cell-cycle progression gene 1 (CCPG1), an ER-resident protein with no known physiological role, as a non-canonical cargo receptor that directly binds to core autophagy proteins via an LIR motif to mammalian ATG8 proteins and, independently and via a discrete motif, to FIP200. These interactions facilitate ER-phagy. The CCPG1 gene is inducible by the unfolded protein response and thus directly links ER stress to ER-phagy. In vivo, CCPG1 protects against ER luminal protein aggregation and consequent unfolded protein response hyperactivation and tissue injury of the exocrine pancreas. Thus, via identification of this autophagy protein, we describe an unexpected molecular mechanism of ER-phagy and provide evidence that this may be physiologically relevant in ER luminal proteostasis.

Funding information:
  • NICHD NIH HHS - T32 HD068256(United States)

Radial Glial Fibers Promote Neuronal Migration and Functional Recovery after Neonatal Brain Injury.

  • Jinnou H
  • Cell Stem Cell
  • 2018 Jan 4

Literature context:


Abstract:

Radial glia (RG) are embryonic neural stem cells (NSCs) that produce neuroblasts and provide fibers that act as a scaffold for neuroblast migration during embryonic development. Although they normally disappear soon after birth, here we found that RG fibers can persist in injured neonatal mouse brains and act as a scaffold for postnatal ventricular-subventricular zone (V-SVZ)-derived neuroblasts that migrate to the lesion site. This injury-induced maintenance of RG fibers has a limited time window during post-natal development and promotes directional saltatory movement of neuroblasts via N-cadherin-mediated cell-cell contacts that promote RhoA activation. Transplanting an N-cadherin-containing scaffold into injured neonatal brains likewise promotes migration and maturation of V-SVZ-derived neuroblasts, leading to functional improvements in impaired gait behaviors. Together these results suggest that RG fibers enable postnatal V-SVZ-derived neuroblasts to migrate toward sites of injury, thereby enhancing neuronal regeneration and functional recovery from neonatal brain injuries.

Funding information:
  • NIDDK NIH HHS - R01 DK082659(United States)

ORP-Mediated ER Contact with Endocytic Sites Facilitates Actin Polymerization.

  • Encinar Del Dedo J
  • Dev. Cell
  • 2017 Dec 4

Literature context:


Abstract:

Oxysterol binding protein-related proteins (ORPs) are conserved lipid binding polypeptides, enriched at ER contacts sites. ORPs promote non-vesicular lipid transport and work as lipid sensors in the context of many cellular tasks, but the determinants of their distinct localization and function are not understood. Here, we demonstrate that the yeast endocytic invaginations associate with the ER and that this association specifically requires the ORPs Osh2 and Osh3, which bridge the endocytic myosin-I Myo5 to the ER integral-membrane VAMP-associated protein (VAP) Scs2. Disruption of the ER contact with endocytic sites using ORP, VAP, myosin-I, or reticulon mutants delays and weakens actin polymerization and interferes with vesicle scission. Finally, we provide evidence suggesting that ORP-dependent sterol transfer facilitates actin polymerization at endocytic sites.

Funding information:
  • Intramural NIH HHS - 1-U54-AI-057153(United States)

Intracerebroventricular administration of Cystatin C ameliorates disease in SOD1-linked amyotrophic lateral sclerosis mice.

  • Watanabe S
  • J. Neurochem.
  • 2017 Dec 29

Literature context:


Abstract:

Cystatin C (CysC) is a major protein component of Bunina bodies, which are a pathological hallmark observed in the remaining motor neurons of patients with amyotrophic lateral sclerosis (ALS). Dominant mutations in the SOD1 gene, encoding Cu/Zn superoxide dismutase (SOD1), are causative for a subset of inherited ALS cases. Our previous study showed that CysC exerts a neuroprotective effect against mutant SOD1-mediated toxicity in vitro; however, in vivo evidence of the beneficial effects mediated by CysC remains obscure. Here we examined the therapeutic potential of recombinant human CysC in vivo using a mouse model of ALS in which the ALS-linked mutated SOD1 gene is expressed (SOD1G93A mice). Intracerebroventricular administration of CysC during the early symptomatic SOD1G93A mice extended their survival times. Administered CysC was predominantly distributed in ventral horn neurons including motor neurons, and induced autophagy through AMP-activated kinase activation to reduce the amount of insoluble mutant SOD1 species. Moreover, PGC-1α, a disease modifier of ALS, was restored by CysC through AMP-activated kinase activation. Finally, the administration of CysC also promoted aggregation of CysC in motor neurons, which is similar to Bunina bodies. Taken together, our findings suggest that CysC represents a promising therapeutic candidate for ALS.

Funding information:
  • NIAMS NIH HHS - R01-AR050478(United States)

Neuropilin-2/PlexinA3 Receptors Associate with GluA1 and Mediate Sema3F-Dependent Homeostatic Scaling in Cortical Neurons.

  • Wang Q
  • Neuron
  • 2017 Dec 6

Literature context:


Abstract:

Regulation of AMPA-type glutamate receptor (AMPAR) number at synapses is a major mechanism for controlling synaptic strength during homeostatic scaling in response to global changes in neural activity. We show that the secreted guidance cue semaphorin 3F (Sema3F) and its neuropilin-2 (Npn-2)/plexinA3 (PlexA3) holoreceptor mediate homeostatic plasticity in cortical neurons. Sema3F-Npn-2/PlexA3 signaling is essential for cell surface AMPAR homeostatic downscaling in response to an increase in neuronal activity, Npn-2 associates with AMPARs, and Sema3F regulates this interaction. Therefore, Sema3F-Npn-2/PlexA3 signaling controls both synapse development and synaptic plasticity.

Heterophilic Type II Cadherins Are Required for High-Magnitude Synaptic Potentiation in the Hippocampus.

  • Basu R
  • Neuron
  • 2017 Sep 27

Literature context:


Abstract:

Hippocampal CA3 neurons form synapses with CA1 neurons in two layers, stratum oriens (SO) and stratum radiatum (SR). Each layer develops unique synaptic properties but molecular mechanisms that mediate these differences are unknown. Here, we show that SO synapses normally have significantly more mushroom spines and higher-magnitude long-term potentiation (LTP) than SR synapses. Further, we discovered that these differences require the Type II classic cadherins, cadherins-6, -9, and -10. Though cadherins typically function via trans-cellular homophilic interactions, our results suggest presynaptic cadherin-9 binds postsynaptic cadherins-6 and -10 to regulate mushroom spine density and high-magnitude LTP in the SO layer. Loss of these cadherins has no effect on the lower-magnitude LTP typically observed in the SR layer, demonstrating that cadherins-6, -9, and -10 are gatekeepers for high-magnitude LTP. Thus, Type II cadherins may uniquely contribute to the specificity and strength of synaptic changes associated with learning and memory.

Funding information:
  • NEI NIH HHS - R01 EY022073()

Inter-Synaptic Lateral Diffusion of GABAA Receptors Shapes Inhibitory Synaptic Currents.

  • de Luca E
  • Neuron
  • 2017 Jul 5

Literature context:


Abstract:

The lateral mobility of neurotransmitter receptors has been shown to tune synaptic signals. Here we report that GABAA receptors (GABAARs) can diffuse between adjacent dendritic GABAergic synapses in long-living desensitized states, thus laterally spreading "activation memories" between inhibitory synapses. Glutamatergic activity limits this inter-synaptic diffusion by trapping GABAARs at excitatory synapses. This novel form of activity-dependent hetero-synaptic interplay is likely to modulate dendritic synaptic signaling.

Red Blood Cell Invasion by the Malaria Parasite Is Coordinated by the PfAP2-I Transcription Factor.

  • Santos JM
  • Cell Host Microbe
  • 2017 Jun 14

Literature context:


Abstract:

Obligate intracellular parasites must efficiently invade host cells in order to mature and be transmitted. For the malaria parasite Plasmodium falciparum, invasion of host red blood cells (RBCs) is essential. Here we describe a parasite-specific transcription factor PfAP2-I, belonging to the Apicomplexan AP2 (ApiAP2) family, that is responsible for regulating the expression of genes involved in RBC invasion. Our genome-wide analysis by ChIP-seq shows that PfAP2-I interacts with a specific DNA motif in the promoters of target genes. Although PfAP2-I contains three AP2 DNA-binding domains, only one is required for binding of the target genes during blood stage development. Furthermore, we find that PfAP2-I associates with several chromatin-associated proteins, including the Plasmodium bromodomain protein PfBDP1 and that complex formation is associated with transcriptional regulation. As a key regulator of red blood cell invasion, PfAP2-I represents a potential new antimalarial therapeutic target.

Funding information:
  • NIAID NIH HHS - R01 AI076276()
  • NIAID NIH HHS - R01 AI125565()
  • NIGMS NIH HHS - P50 GM071508()
  • NIGMS NIH HHS - R01 GM114141()

Pharmacogenetic variants in TPMT alter cellular responses to cisplatin in inner ear cell lines.

  • Bhavsar AP
  • PLoS ONE
  • 2017 Apr 13

Literature context:


Abstract:

Cisplatin is a highly-effective and widely-used chemotherapeutic agent that causes ototoxicity in many patients. Pharmacogenomic studies of key genes controlling drug biotransformation identified variants in thiopurine methyltransferase (TPMT) as predictors of cisplatin-induced ototoxicity, although the mechanistic basis of this interaction has not been reported. Expression constructs of TPMT*3A, *3B and *3C variants were generated and monitored in cultured cells. Cellular TPMT*3A levels were detected at >20-fold lower amounts than the wild type confirming the unstable nature of this variant. The expression of wild type TPMT (TPMT*1) in two murine ear cell lines, HEI-OC1 and UB/OC-1, significantly mitigated their susceptibility to cisplatin toxicity. Cisplatin treatment induced Tlr4 gene expression in HEI-OC1 cells and this response was blunted by the expression of wild type TPMT but not TPMT*3A. In line with the significant mitigation of TPMT*1-expressing cells to cisplatin cytotoxicity, these findings demonstrate a drug-gene interaction between increased TPMT activity and decreased susceptibility to cisplatin-induced toxicity of inner ear cells.

Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Cav2.1 (P/Q-Type) Calcium Channels.

  • Fu SJ
  • J. Neurosci.
  • 2017 Mar 1

Literature context:


Abstract:

Voltage-gated CaV2.1 channels comprise a pore-forming α1A subunit with auxiliary α2δ and β subunits. CaV2.1 channels play an essential role in regulating synaptic signaling. Mutations in the human gene encoding the CaV2.1 subunit are associated with the cerebellar disease episodic ataxia type 2 (EA2). Several EA2-causing mutants exhibit impaired protein stability and exert dominant-negative suppression of CaV2.1 wild-type (WT) protein expression via aberrant proteasomal degradation. Here, we set out to delineate the protein degradation mechanism of human CaV2.1 subunit by identifying RNF138, an E3 ubiquitin ligase, as a novel CaV2.1-binding partner. In neurons, RNF138 and CaV2.1 coexist in the same protein complex and display notable subcellular colocalization at presynaptic and postsynaptic regions. Overexpression of RNF138 promotes polyubiquitination and accelerates protein turnover of CaV2.1. Disrupting endogenous RNF138 function with a mutant (RNF138-H36E) or shRNA infection significantly upregulates the CaV2.1 protein level and enhances CaV2.1 protein stability. Disrupting endogenous RNF138 function also effectively rescues the defective protein expression of EA2 mutants, as well as fully reversing EA2 mutant-induced excessive proteasomal degradation of CaV2.1 WT subunits. RNF138-H36E coexpression only partially restores the dominant-negative effect of EA2 mutants on CaV2.1 WT functional expression, which can be attributed to defective membrane trafficking of CaV2.1 WT in the presence of EA2 mutants. We propose that RNF138 plays a critical role in the homeostatic regulation of CaV2.1 protein level and functional expression and that RNF138 serves as the primary E3 ubiquitin ligase promoting EA2-associated aberrant degradation of human CaV2.1 subunits.SIGNIFICANCE STATEMENT Loss-of-function mutations in the human CaV2.1 subunit are linked to episodic ataxia type 2 (EA2), a dominantly inherited disease characterized by paroxysmal attacks of ataxia and nystagmus. EA2-causing mutants may exert dominant-negative effects on the CaV2.1 wild-type subunit via aberrant proteasomal degradation. The molecular nature of the CaV2.1 ubiquitin-proteasome degradation pathway is currently unknown. The present study reports the first identification of an E3 ubiquitin ligase for CaV2.1, RNF138. CaV2.1 protein stability is dynamically regulated by RNF138 and auxiliary α2δ and β subunits. We provide a proof of concept that protecting the human CaV2.1 subunit from excessive proteasomal degradation with specific interruption of endogenous RNF138 function may partially contribute to the future development of a novel therapeutic strategy for EA2 patients.

Macrophage Transcriptional Profile Identifies Lipid Catabolic Pathways That Can Be Therapeutically Targeted after Spinal Cord Injury.

  • Zhu Y
  • J. Neurosci.
  • 2017 Mar 1

Literature context:


Abstract:

Although infiltrating macrophages influence many pathological processes after spinal cord injury (SCI), the intrinsic molecular mechanisms that regulate their function are poorly understood. A major hurdle has been dissecting macrophage-specific functions from those in other cell types as well as understanding how their functions change over time. Therefore, we used the RiboTag method to obtain macrophage-specific mRNA directly from the injured spinal cord in mice and performed RNA sequencing to investigate their transcriptional profile. Our data show that at 7 d after SCI, macrophages are best described as foam cells, with lipid catabolism representing the main biological process, and canonical nuclear receptor pathways as their potential mediators. Genetic deletion of a lipoprotein receptor, CD36, reduces macrophage lipid content and improves lesion size and locomotor recovery. Therefore, we report the first macrophage-specific transcriptional profile after SCI and highlight the lipid catabolic pathway as an important macrophage function that can be therapeutically targeted after SCI.SIGNIFICANCE STATEMENT The intrinsic molecular mechanisms that regulate macrophage function after spinal cord injury (SCI) are poorly understood. We obtained macrophage-specific mRNA directly from the injured spinal cord and performed RNA sequencing to investigate their transcriptional profile. Our data show that at 7 d after SCI, macrophages are best described as foam cells, with lipid catabolism representing the main biological process and canonical nuclear receptor pathways as their potential mediators. Genetic deletion of a lipoprotein receptor, CD36, reduces macrophage lipid content and improves lesion size and locomotor recovery. Therefore, we report the first macrophage-specific transcriptional profile after SCI and highlight the lipid catabolic pathway as an important macrophage function that can be therapeutically targeted after SCI.

Cell-Type-Specific Alternative Splicing Governs Cell Fate in the Developing Cerebral Cortex.

  • Zhang X
  • Cell
  • 2016 Aug 25

Literature context:


Abstract:

Alternative splicing is prevalent in the mammalian brain. To interrogate the functional role of alternative splicing in neural development, we analyzed purified neural progenitor cells (NPCs) and neurons from developing cerebral cortices, revealing hundreds of differentially spliced exons that preferentially alter key protein domains-especially in cytoskeletal proteins-and can harbor disease-causing mutations. We show that Ptbp1 and Rbfox proteins antagonistically govern the NPC-to-neuron transition by regulating neuron-specific exons. Whereas Ptbp1 maintains apical progenitors partly through suppressing a poison exon of Flna in NPCs, Rbfox proteins promote neuronal differentiation by switching Ninein from a centrosomal splice form in NPCs to a non-centrosomal isoform in neurons. We further uncover an intronic human mutation within a PTBP1-binding site that disrupts normal skipping of the FLNA poison exon in NPCs and causes a brain-specific malformation. Our study indicates that dynamic control of alternative splicing governs cell fate in cerebral cortical development.

Required enhancer-matrin-3 network interactions for a homeodomain transcription program.

  • Skowronska-Krawczyk D
  • Nature
  • 2014 Oct 9

Literature context:


Abstract:

Homeodomain proteins, described 30 years ago, exert essential roles in development as regulators of target gene expression; however, the molecular mechanisms underlying transcriptional activity of homeodomain factors remain poorly understood. Here investigation of a developmentally required POU-homeodomain transcription factor, Pit1 (also known as Pou1f1), has revealed that, unexpectedly, binding of Pit1-occupied enhancers to a nuclear matrin-3-rich network/architecture is a key event in effective activation of the Pit1-regulated enhancer/coding gene transcriptional program. Pit1 association with Satb1 (ref. 8) and β-catenin is required for this tethering event. A naturally occurring, dominant negative, point mutation in human PIT1(R271W), causing combined pituitary hormone deficiency, results in loss of Pit1 association with β-catenin and Satb1 and therefore the matrin-3-rich network, blocking Pit1-dependent enhancer/coding target gene activation. This defective activation can be rescued by artificial tethering of the mutant R271W Pit1 protein to the matrin-3 network, bypassing the pre-requisite association with β-catenin and Satb1 otherwise required. The matrin-3 network-tethered R271W Pit1 mutant, but not the untethered protein, restores Pit1-dependent activation of the enhancers and recruitment of co-activators, exemplified by p300, causing both enhancer RNA transcription and target gene activation. These studies have thus revealed an unanticipated homeodomain factor/β-catenin/Satb1-dependent localization of target gene regulatory enhancer regions to a subnuclear architectural structure that serves as an underlying mechanism by which an enhancer-bound homeodomain factor effectively activates developmental gene transcriptional programs.

Funding information:
  • NIGMS NIH HHS - R01 GM104047(United States)

GABAergic complex basket formations in the human neocortex.

  • Blazquez-Llorca L
  • J. Comp. Neurol.
  • 2010 Dec 15

Literature context:


Abstract:

Certain GABAergic interneurons in the cerebral cortex, basket cells, establish multiple connections with cell bodies that typically outline the somata and proximal dendrites of pyramidal cells. During studies into the distribution of the vesicular GABA transporter (VGAT) in the human cerebral cortex, we were struck by the presence of a very dense, pericellular arrangement of multiple VGAT-immunoreactive (-ir) terminals in certain cortical areas. We called these terminals "Complex basket formations" (Cbk-formations) to distinguish them from the simpler and more typical pericellular GABAergic innervations of most cortical neurons. Here we examined the distribution of these VGAT-ir Cbk-formations in various cortical areas, including the somatosensory (area 3b), visual (areas 17 and 18), motor (area 4), associative frontal (dorsolateral areas 9, 10, 45, 46, and orbital areas 11, 12, 13, 14, 47), associative temporal (areas 20, 21, 22, and 38), and limbic cingulate areas (areas 24, 32). Furthermore, we used dual or triple staining techniques to study the chemical nature of the innervated cells. We found that VGAT-ir Cbk-formations were most frequently found in area 4 followed by areas 3b, 13, and 18. In addition, they were mostly observed in layer III, except in area 17, where they were most dense in layer IV. We also found that 70% of the innervated neurons were pyramidal cells, while the remaining 30% were multipolar cells. Most of these multipolar cells expressed the calcium-binding protein parvalbumin and the lectin Vicia villosa agglutinin.

Funding information:
  • NCRR NIH HHS - R01RR025342(United States)
  • NEI NIH HHS - EY1765(United States)

Immunolocalization of NMDA receptor subunit NR3B in selected structures in the rat forebrain, cerebellum, and lumbar spinal cord.

  • Wee KS
  • J. Comp. Neurol.
  • 2008 Jul 1

Literature context:


Abstract:

N-methyl-D-aspartate (NMDA) receptors have been implicated in many neurological disorders. Although NMDA receptors are best known for their high calcium permeability, the recently discovered NR3 subunits, NR3A and NR3B, have been shown to reduce the calcium permeability of the NMDA receptor. Thus, NR3 subunits may be important players in modulating synaptic plasticity in neurons. Although NR3B expression in the rodent and human brain has been studied, little is known about its distribution in different cell types. Here we used immunolabeling with a specific NR3B antibody together with antibodies against established neurochemical markers to determine the cellular and subcellular localization of NR3B. The nucleus was concurrently stained with NR3B immunolabeling to show that NR3B is widely expressed by many cells in each brain region. Our findings indicate that NR3B is widely expressed in the structures examined in the rat forebrain (hippocampus, cerebral cortex, caudoputamen, and nucleus accumbens), cerebellum, and lumbar sections of the spinal cord. Within these regions NR3B was found to be expressed in all the substructures of the hippocampus (CA1, CA3, dentate gyrus), the various layers of the cerebral cortex, projection neurons and interneurons of the striatum, different cell types of the cerebellum, and motor neurons of the spinal cord. Furthermore, when stained with NR1-the obligatory subunit responsible for forming functional NMDA receptors-the distribution of NR3B appears to be as ubiquitous as NR1. Taken together, our data suggest that there may be a population of NR3B-containing NMDA receptors conferring new functional roles in the mammalian central nervous system.

Funding information:
  • NIDCR NIH HHS - DE-015576(United States)