Literature context: COR Biosciences Cat# 926-68070, RRID:AB_10956588 IRDye 800CW Goat anti-Rabbit Ig
Neuronal activity regulates the transcription and translation of the immediate-early gene Arc/Arg3.1, a key mediator of synaptic plasticity. Proteasome-dependent degradation of Arc tightly limits its temporal expression, yet the significance of this regulation remains unknown. We disrupted the temporal control of Arc degradation by creating an Arc knockin mouse (ArcKR) where the predominant Arc ubiquitination sites were mutated. ArcKR mice had intact spatial learning but showed specific deficits in selecting an optimal strategy during reversal learning. This cognitive inflexibility was coupled to changes in Arc mRNA and protein expression resulting in a reduced threshold to induce mGluR-LTD and enhanced mGluR-LTD amplitude. These findings show that the abnormal persistence of Arc protein limits the dynamic range of Arc signaling pathways specifically during reversal learning. Our work illuminates how the precise temporal control of activity-dependent molecules, such as Arc, regulates synaptic plasticity and is crucial for cognition.
Literature context: I-COR BioScience Cat#926-68070; RRID:AB_10956588 Bacterial and Virus Strains
Noncoding mutations in cancer genomes are frequent but challenging to interpret. PVT1 encodes an oncogenic lncRNA, but recurrent translocations and deletions in human cancers suggest alternative mechanisms. Here, we show that the PVT1 promoter has a tumor-suppressor function that is independent of PVT1 lncRNA. CRISPR interference of PVT1 promoter enhances breast cancer cell competition and growth in vivo. The promoters of the PVT1 and the MYC oncogenes, located 55 kb apart on chromosome 8q24, compete for engagement with four intragenic enhancers in the PVT1 locus, thereby allowing the PVT1 promoter to regulate pause release of MYC transcription. PVT1 undergoes developmentally regulated monoallelic expression, and the PVT1 promoter inhibits MYC expression only from the same chromosome via promoter competition. Cancer genome sequencing identifies recurrent mutations encompassing the human PVT1 promoter, and genome editing verified that PVT1 promoter mutation promotes cancer cell growth. These results highlight regulatory sequences of lncRNA genes as potential disease-associated DNA elements.
Literature context: COR Biosciences Cat# 926-68070; RRID:AB_10956588 IRDyeÂ® 800CW Goat anti-Rabbit I
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.
Literature context: LI-COR, Inc 926-68070 RRID:AB_10956588 Goat anti-mouse HRP conjugated
Malate dehydrogenases (Mdhs) reversibly convert malate to oxaloacetate and serve as important enzymes in several metabolic pathways. In the yeast Saccharomyces cerevisiae there are three Mdh isozymes, localized to different compartments in the cell. In order to identify specifically the Mdh2 isozyme, GenScript USA produced three different antibodies that we further tested by western blot. All three antibodies recognized the S. cerevisiae Mdh2 with different background and specificity properties. One of the antibodies had a relatively low background and high specificity and thus can be used for specific identification of Mdh2 in various experimental settings.
Literature context: t#926-68070; RRID:AB_10956588 Chemicals, Peptides, and Recomb
Val-boroPro (PT-100, Talabostat) induces powerful anti-tumor immune responses in syngeneic cancer models, but its mechanism of action has not yet been established. Val-boroPro is a non-selective inhibitor of post-proline-cleaving serine proteases, and the inhibition of the highly related cytosolic serine proteases Dpp8 and Dpp9 (Dpp8/9) by Val-boroPro was recently demonstrated to trigger an immunostimulatory form of programmed cell death known as pyroptosis selectively in monocytes and macrophages. Here we show that Dpp8/9 inhibition activates the inflammasome sensor protein Nlrp1b, which in turn activates pro-caspase-1 to mediate pyroptosis. This work reveals a previously unrecognized mechanism for activating an innate immune pattern recognition receptor and suggests that Dpp8/9 serve as an intracellular checkpoint to restrain Nlrp1b and the innate immune system.
Literature context: H + L), 0.1 mg Licor 926-68070; RRID:AB_10956588 Deposited Data
Coenzyme Q (CoQ) is a redox-active lipid required for mitochondrial oxidative phosphorylation (OxPhos). How CoQ biosynthesis is coordinated with the biogenesis of OxPhos protein complexes is unclear. Here, we show that the Saccharomyces cerevisiae RNA-binding protein (RBP) Puf3p regulates CoQ biosynthesis. To establish the mechanism for this regulation, we employed a multi-omic strategy to identify mRNAs that not only bind Puf3p but also are regulated by Puf3p in vivo. The CoQ biosynthesis enzyme Coq5p is a critical Puf3p target: Puf3p regulates the abundance of Coq5p and prevents its detrimental hyperaccumulation, thereby enabling efficient CoQ production. More broadly, Puf3p represses a specific set of proteins involved in mitochondrial protein import, translation, and OxPhos complex assembly (pathways essential to prime mitochondrial biogenesis). Our data reveal a mechanism for post-transcriptionally coordinating CoQ production with OxPhos biogenesis, and they demonstrate the power of multi-omics for defining genuine targets of RBPs.
Literature context: :25,000 LI-COR; cat# 926-68,070 RRID:AB_10956588
No treatment is available for patients with spinal cord injury (SCI). Patients often arrive to the hospital hours after SCI suggesting the need of a therapy that can be used on a clinically relevant window. Previous studies showed that Tamoxifen (TAM) treatment 24h after SCI benefits locomotor recovery in female rats. Tamoxifen exerts beneficial effects in male and female rodents but a gap of knowledge exists on: the therapeutic window of TAM, the spatio-temporal mechanisms activated and if this response is sexually dimorphic. We hypothesized that TAM will favor locomotor recovery when administered up-to 24h after SCI in male Sprague-Dawley rats. Rats received a thoracic (T10) contusion using the MACSIS impactor followed by placebo or TAM (15mg/21days) pellets in a therapeutic window of 0, 6, 12, or 24h. Animals were sacrificed at 2, 7, 14, 28 or 35days post injury (DPI) to study the molecular and cellular changes in the acute and chronic stages. Immediate or delayed therapy (t=6h) improved locomotor function, increased white matter spared tissue, and neuronal survival. TAM reduced reactive gliosis during chronic stages and increased the expression of Olig-2. A significant difference was observed in estrogen receptor alpha between male and female rodents from 2 to 28 DPI suggesting a sexually dimorphic characteristic that could be related to the behavioral differences observed in the therapeutic window of TAM. This study supports the use of TAM in the SCI setting due to its neuroprotective effects but with a significant sexually dimorphic therapeutic window.
Literature context: se 680RD IRDye LiCOR 926-68070; RRID:AB_10956588 Goat Anti-Mouse 800CW IRDye LiC
Histone deacetylase (HDAC) catalytic activity is regulated by formation of co-regulator complexes and post-translational modification. Whether these mechanisms are transformed in cancer and how this affects the binding and selectivity of HDAC inhibitors (HDACis) is unclear. In this study, we developed a method that identified a 3- to 16-fold increase in HDACi selectivity for HDAC3 in triple-negative breast cancer (TNBC) cells in comparison with luminal subtypes that was not predicted by current practice measurements with recombinant proteins. We found this increase was caused by c-Jun N-terminal kinase (JNK) phosphorylation of HDAC3, was independent of HDAC3 complex composition or subcellular localization, and was associated with a 5-fold increase in HDAC3 enzymatic activity. This study points to HDAC3 and the JNK axes as targets in TNBC, highlights how HDAC phosphorylation affects HDACi binding and selectivity, and outlines a method to identify changes in individual HDAC isoforms catalytic activity, applicable to any disease state.
Literature context: 0; RRID:AB_10956588) for 1 hr at RT, and again rins
Adhesion-type G protein-coupled receptors (aGPCRs), a large molecule family with over 30 members in humans, operate in organ development, brain function and govern immunological responses. Correspondingly, this receptor family is linked to a multitude of diverse human diseases. aGPCRs have been suggested to possess mechanosensory properties, though their mechanism of action is fully unknown. Here we show that the Drosophila aGPCR Latrophilin/dCIRL acts in mechanosensory neurons by modulating ionotropic receptor currents, the initiating step of cellular mechanosensation. This process depends on the length of the extended ectodomain and the tethered agonist of the receptor, but not on its autoproteolysis, a characteristic biochemical feature of the aGPCR family. Intracellularly, dCIRL quenches cAMP levels upon mechanical activation thereby specifically increasing the mechanosensitivity of neurons. These results provide direct evidence that the aGPCR dCIRL acts as a molecular sensor and signal transducer that detects and converts mechanical stimuli into a metabotropic response.
Literature context: 26-68070; RRID:AB_10956588 Anti-phosp
In this study, we show that evolutionarily conserved chromosome loop anchors bound by CCCTC-binding factor (CTCF) and cohesin are vulnerable to DNA double strand breaks (DSBs) mediated by topoisomerase 2B (TOP2B). Polymorphisms in the genome that redistribute CTCF/cohesin occupancy rewire DNA cleavage sites to novel loop anchors. While transcription- and replication-coupled genomic rearrangements have been well documented, we demonstrate that DSBs formed at loop anchors are largely transcription-, replication-, and cell-type-independent. DSBs are continuously formed throughout interphase, are enriched on both sides of strong topological domain borders, and frequently occur at breakpoint clusters commonly translocated in cancer. Thus, loop anchors serve as fragile sites that generate DSBs and chromosomal rearrangements. VIDEO ABSTRACT.
Literature context: 26-68070; RRID:AB_10956588 Biological
Processive elongation of RNA Polymerase II from a proximal promoter paused state is a rate-limiting event in human gene control. A small number of regulatory factors influence transcription elongation on a global scale. Prior research using small-molecule BET bromodomain inhibitors, such as JQ1, linked BRD4 to context-specific elongation at a limited number of genes associated with massive enhancer regions. Here, the mechanistic characterization of an optimized chemical degrader of BET bromodomain proteins, dBET6, led to the unexpected identification of BET proteins as master regulators of global transcription elongation. In contrast to the selective effect of bromodomain inhibition on transcription, BET degradation prompts a collapse of global elongation that phenocopies CDK9 inhibition. Notably, BRD4 loss does not directly affect CDK9 localization. These studies, performed in translational models of T cell leukemia, establish a mechanism-based rationale for the development of BET bromodomain degradation as cancer therapy.
Literature context: , 1:8000, RRID:AB_10956588; anti-rabb
Traditionally, human primary visual cortex (V1) has been thought to mature within the first few years of life, based on anatomical studies of synapse formation, and establishment of intracortical and intercortical connections. Human vision, however, develops well beyond the first few years. Previously, we found prolonged development of some GABAergic proteins in human V1 (Pinto et al., 2010). Yet as >80% of synapses in V1 are excitatory, it remains unanswered whether the majority of synapses regulating experience-dependent plasticity and receptive field properties develop late, like their inhibitory counterparts. To address this question, we used Western blotting of postmortem tissue from human V1 (12 female, 18 male) covering a range of ages. Then we quantified a set of postsynaptic glutamatergic proteins (PSD-95, GluA2, GluN1, GluN2A, GluN2B), calculated indices for functional pairs that are developmentally regulated (GluA2:GluN1; GluN2A:GluN2B), and determined interindividual variability. We found early loss of GluN1, prolonged development of PSD-95 and GluA2 into late childhood, protracted development of GluN2A until ∼40 years, and dramatic loss of GluN2A in aging. The GluA2:GluN1 index switched at ∼1 year, but the GluN2A:GluN2B index continued to shift until ∼40 year before changing back to GluN2B in aging. We also identified young childhood as a stage of heightened interindividual variability. The changes show that human V1 develops gradually through a series of five orchestrated stages, making it likely that V1 participates in visual development and plasticity across the lifespan.SIGNIFICANCE STATEMENT Anatomical structure of human V1 appears to mature early, but vision changes across the lifespan. This discrepancy has fostered two hypotheses: either other aspects of V1 continue changing, or later changes in visual perception depend on extrastriate areas. Previously, we showed that some GABAergic synaptic proteins change across the lifespan, but most synapses in V1 are excitatory leaving unanswered how they change. So we studied expression of glutamatergic proteins in human V1 to determine their development. Here we report prolonged maturation of glutamatergic proteins, with five stages that map onto life-long changes in human visual perception. Thus, the apparent discrepancy between development of structure and function may be explained by life-long synaptic changes in human V1.
Literature context: 32211IRDye 680RD anti-mouseLICOR926-68070Chemicals, Peptides, and Recombi
Pyroptosis is a lytic form of programmed cell death mediated by the inflammatory caspase-1, -4, and -5. We recently discovered that small-molecule inhibitors of the serine peptidases DPP8 and DPP9 (DPP8/9) induce pro-caspase-1-dependent pyroptosis in monocytes and macrophages. Notably, DPP8/9 inhibitors, unlike microbial agents, absolutely require caspase-1 to induce cell death. Therefore, DPP8/9 inhibitors are useful probes to study caspase-1 in cells. Here, we show that, in the absence of the pyroptosis-mediating substrate gasdermin D (GSDMD), caspase-1 activates caspase-3 and -7 and induces apoptosis, demonstrating that GSDMD is the only caspase-1 substrate that induces pyroptosis. Conversely, we found that, during apoptosis, caspase-3/-7 specifically block pyroptosis by cleaving GSDMD at a distinct site from the inflammatory caspases that inactivates the protein. Overall, this work reveals bidirectional crosstalk between apoptosis and pyroptosis in monocytes and macrophages, further illuminating the complex interplay between cell death pathways in the innate immune system.
Literature context: 926-68070 RRID:AB-10956588), IRDye 68
Diencephalic defects underlie an array of neurological diseases. Previous studies have suggested that retinoic acid (RA) signaling is involved in diencephalic development at late stages of embryonic development, but its roles and mechanisms of action during early neural development are still unclear. Here we demonstrate that mice lacking enzymatic activity of the acetyltransferase GCN5 ((Gcn5hat/hat )), which were previously characterized with respect to their exencephalic phenotype, exhibit significant diencephalic expansion, decreased diencephalic RA signaling, and increased diencephalic WNT and SHH signaling. Using a variety of molecular biology techniques in both cultured neuroepithelial cells treated with a GCN5 inhibitor and forebrain tissue from (Gcn5hat/hat ) embryos, we demonstrate that GCN5, RARα/γ, and the poorly characterized protein TACC1 form a complex in the nucleus that binds specific retinoic acid response elements in the absence of RA. Furthermore, RA triggers GCN5-mediated acetylation of TACC1, which results in dissociation of TACC1 from retinoic acid response elements and leads to transcriptional activation of RA target genes. Intriguingly, RA signaling defects caused by in vitro inhibition of GCN5 can be rescued through RA-dependent mechanisms that require RARβ. Last, we demonstrate that the diencephalic expansion and transcriptional defects seen in (Gcn5hat/hat ) mutants can be rescued with gestational RA supplementation, supporting a direct link between GCN5, TACC1, and RA signaling in the developing diencephalon. Together, our studies identify a novel, nonhistone substrate for GCN5 whose modification regulates a previously undescribed, tissue-specific mechanism of RA signaling that is required to restrict diencephalic size during early forebrain development.SIGNIFICANCE STATEMENT Changes in diencephalic size and shape, as well as SNPs associated with retinoic acid (RA) signaling-associated genes, have been linked to neuropsychiatric disorders. However, the mechanisms that regulate diencephalic morphogenesis and the involvement of RA signaling in this process are poorly understood. Here we demonstrate a novel role of the acetyltransferase GCN5 in a previously undescribed mechanism of RA signaling in the developing forebrain that is required to maintain the appropriate size of the diencephalon. Together, our experiments identify a novel nonhistone substrate of GCN5, highlight an essential role for both GCN5 and RA signaling in early diencephalic development, and elucidate a novel molecular regulatory mechanism for RA signaling that is specific to the developing forebrain.
Literature context: 92; NIH AIDS Reagent Program3537goat anti-mouse-680Li-Cor925-68070goat anti-rabbit-
The magnitude of the 2013-2016 Ebola virus disease (EVD) epidemic enabled an unprecedented number of viral mutations to occur over successive human-to-human transmission events, increasing the probability that adaptation to the human host occurred during the outbreak. We investigated one nonsynonymous mutation, Ebola virus (EBOV) glycoprotein (GP) mutant A82V, for its effect on viral infectivity. This mutation, located at the NPC1-binding site on EBOV GP, occurred early in the 2013-2016 outbreak and rose to high frequency. We found that GP-A82V had heightened ability to infect primate cells, including human dendritic cells. The increased infectivity was restricted to cells that have primate-specific NPC1 sequences at the EBOV interface, suggesting that this mutation was indeed an adaptation to the human host. GP-A82V was associated with increased mortality, consistent with the hypothesis that the heightened intrinsic infectivity of GP-A82V contributed to disease severity during the EVD epidemic.
Literature context: 0RD Goat anti-Mouse IgG, Cat. # 926â€“68070) were used for band detection.
Gap junctions are membrane specialization domains identified in most tissue types where cells abut each other. The connexin channels found in these membrane domains are conduits for direct cell-to-cell transfer of ions and molecules. Connexin43 (Cx43) is the most ubiquitous connexin, with critical roles in heart, skin, and brain. Several studies described the interaction between Cx43 and the cytoskeleton involving the actin binding proteins Zonula occludens (ZO-1) and drebrin, as well as with tubulin. However, a direct interaction has not been identified between drebrin and Cx43. In this study, co-IP and NMR experiments were used to demonstrate that the Cx43-CT directly interacts with the highly conserved N-terminus region of drebrin. Three Cx43-CT areas were found to be involved in drebrin binding, with residues 264-275 being critical for the interaction. Mimicking Src phosphorylation within this region (Y265) significantly disrupted the interaction between the Cx43-CT and drebrin. Immunofluorescence showed colocalization of Cx43, drebrin, and F-actin in astrocytes and Vero cells membrane, indicating that Cx43 forms a submembrane protein complex with cytoskeletal and scaffolding proteins. The co-IP data suggest that Cx43 indirectly interacts with F-actin through drebrin. Along with the known interaction of the Cx43-CT with ZO-1 and tubulin, the data presented here for drebrin indicate non-overlapping and separated binding sites for all three proteins for which simultaneous binding could be important in regulating cytoskeleton rearrangements, especially for neuronal migration during brain development.
Literature context: 926-68070 RRID:AB_10956588) or IRDye
The degradation of damaged proteins is an important vital function especially during aging and stress. The ubiquitin proteasome system is one of the major cellular machineries for protein degradation. Health and longevity are associated with high proteasome activity. To demonstrate such a role in aging of Podospora anserina, we first analyzed the transcript and protein abundance of selected proteasome components in wild-type cultures of different age. No significant differences were observed. Next, in order to increase the overall proteasome abundance we generated strains overexpressing the catalytic proteasome subunits PaPRE2 and PaPRE3. Although transcript levels were strongly increased, no substantial effect on the abundance of the corresponding proteins was observed. Finally, the analysis of the P. anserina strains expressing the sequence coding for the CL1 degron fused to the Gfp gene revealed no evidence for degradation of the GFP-CL1 fusion protein by the proteasome. Instead, our results demonstrate the degradation of the CL1-degron sequence via autophagy, indicating that basal autophagy appears to be a very effective protein quality control pathway in P. anserina.
Literature context: 26-68070, RRID:AB_10956588), and IRDy
Local regulation of protein synthesis allows a neuron to rapidly alter the proteome in response to synaptic signals, an essential mechanism in synaptic plasticity that is altered in many neurological diseases. Synthesis of many synaptic proteins is under local control and much of this regulation occurs through structures termed RNA granules. KIS is a protein kinase that associates with stathmin, a modulator of the tubulin cytoskeleton. Furthermore, KIS is found in RNA granules and stimulates translation driven by the β-actin 3'UTR in neurites. Here we explore the physiological and molecular mechanisms underlying the action of KIS on hippocampal synaptic plasticity in mice. KIS downregulation compromises spine development, alters actin dynamics, and reduces postsynaptic responsiveness. The absence of KIS results in a significant decrease of protein levels of PSD-95, a postsynaptic scaffolding protein, and the AMPAR subunits GluR1 and GluR2 in a CPEB3-dependent manner. Underlying its role in spine maturation, KIS is able to suppress the spine developmental defects caused by CPEB3 overexpression. Moreover, either by direct or indirect mechanisms, KIS counteracts the inhibitory activity of CPEB3 on the GluR2 3'UTR at both mRNA translation and polyadenylation levels. Our study provides insights into the mechanisms that mediate dendritic spine morphogenesis and functional synaptic maturation, and suggests KIS as a link regulating spine cytoskeleton and postsynaptic activity in memory formation.
Aldosterone synthesis is initiated upon the transport of cholesterol from the outer to the inner mitochondrial membrane, where the cholesterol is hydrolyzed to pregnenolone. This process is the rate-limiting step in acute aldosterone production and is mediated by the steroidogenic acute regulatory (StAR) protein. We have previously shown that angiotensin II (AngII) activation of the serine/threonine protein kinase D (PKD) promotes acute aldosterone production in bovine adrenal glomerulosa cells, but the mechanism remains unclear. Thus, the purpose of this study was to determine the downstream signaling effectors of AngII-stimulated PKD activity. Our results demonstrate that overexpression of the constitutively active serine-to-glutamate PKD mutant enhances, whereas the dominant-negative serine-to-alanine PKD mutant inhibits, AngII-induced StAR mRNA expression relative to the vector control. PKD has been shown to phosphorylate members of the activating transcription factor (ATF)/cAMP response element binding protein (CREB) family of leucine zipper transcription factors, which have been shown previously to bind the StAR proximal promoter and induce StAR mRNA expression. In primary glomerulosa cells, AngII induces ATF-2 and CREB phosphorylation in a time-dependent manner. Furthermore, overexpression of the constitutively active PKD mutant enhances the AngII-elicited phosphorylation of ATF-2 and CREB, and the dominant-negative mutant inhibits this response. Furthermore, the constitutively active PKD mutant increases the binding of phosphorylated CREB to the StAR promoter. Thus, these data provide insight into the previously reported role of PKD in AngII-induced acute aldosterone production, providing a mechanism by which PKD may be mediating steroidogenesis in primary bovine adrenal glomerulosa cells.
The Developmental Origins of Health and Disease hypothesis holds that alterations to homeostasis during critical periods of development can predispose individuals to adult-onset chronic diseases such as diabetes and metabolic syndrome. It remains controversial whether preimplantation embryo manipulation, clinically used to treat patients with infertility, disturbs homeostasis and affects long-term growth and metabolism. To address this controversy, we have assessed the effects of in vitro fertilization (IVF) on postnatal physiology in mice. We demonstrate that IVF and embryo culture, even under conditions considered optimal for mouse embryo culture, alter postnatal growth trajectory, fat accumulation, and glucose metabolism in adult mice. Unbiased metabolic profiling in serum and microarray analysis of pancreatic islets and insulin sensitive tissues (liver, skeletal muscle, and adipose tissue) revealed broad changes in metabolic homeostasis, characterized by systemic oxidative stress and mitochondrial dysfunction. Adopting a candidate approach, we identify thioredoxin-interacting protein (TXNIP), a key molecule involved in integrating cellular nutritional and oxidative states with metabolic response, as a marker for preimplantation stress and demonstrate tissue-specific epigenetic and transcriptional TXNIP misregulation in selected adult tissues. Importantly, dysregulation of TXNIP expression is associated with enrichment for H4 acetylation at the Txnip promoter that persists from the blastocyst stage through adulthood in adipose tissue. Our data support the vulnerability of preimplantation embryos to environmental disturbance and demonstrate that conception by IVF can reprogram metabolic homeostasis through metabolic, transcriptional, and epigenetic mechanisms with lasting effects for adult growth and fitness. This study has wide clinical relevance and underscores the importance of continued follow-up of IVF-conceived offspring.