Arylalkylamine N-acyltransferase-like 22 (AANATL2) from Drosophila melanogaster was expressed and shown to catalyze the formation of long-chain N-acylserotonins and N-acydopamines. Subsequent identification of endogenous amounts of N-acylserotonins and colocalization of these fatty acid amides and AANATL2 transcripts gives supporting evidence that AANATL2 has a role in the biosynthetic formation of these important cell signalling lipids.
Arylalkylamine N-acyltransferase; Arylalkylamine N-acetyltransferase; N-acyldopamines; N-acylserotonins; Drosophila melanogaster; thorax-abdomen; steady-state kinetics
Neurodegenerative diseases are attributed to impairment of the ubiquitin-proteasome system (UPS). Oxidative stress has been considered a contributing factor in the pathology of impaired UPS by promoting protein misfolding and subsequent protein aggregate formation. Increasing evidence suggests that NADPH oxidase is a likely source of excessive oxidative stress in neurodegenerative disorders. However, the mechanism of activation and its role in impaired UPS is not understood. We show that activation of NADPH oxidase in a neuroblastoma cell line (SHSY-5Y) resulted in increased oxidative and nitrosative stress, elevated cytosolic calcium, ER-stress, impaired UPS, and apoptosis. Rac1 inhibition mitigated the oxidative/nitrosative stress, prevented calcium-dependent ER-stress, and partially rescued UPS function. These findings demonstrate that Rac1 and NADPH oxidase play an important role in rotenone neurotoxicity.
Neurodegenerative diseases; NADPH oxidase; Nox; Rotenone; Rac1; Impaired UPS
•eRNAs are expressed from enhancers and have been shown to regulate gene expression.•Expression of eRNAs is widespread upon activation of the innate immune response.•We show that the NF-κB signalling pathway regulates LPS-induced eRNAs.•Expression of individual eRNAs is also dependent on ERK-1/2 and p38.
Recent studies have indicated that non-coding RNAs transcribed from enhancer regions are important regulators of enhancer function and gene expression. In this report, we have characterised the expression of six enhancer RNAs (eRNAs) induced in human monocytic THP1 cells following activation of the innate immune response by lipopolysaccharide (LPS). Specifically, we have demonstrated that LPS-induced expression of individual eRNAs is mediated through divergent intracellular signalling pathways that includes NF-κB and the mitogen activated protein kinases, extracellular regulated kinase-1/2 and p38.
Enhancer RNA; Long non-coding RNA; NF-κB; Lipopolysaccharide; Monocyte; ERK and p38
Dyslipidemia and insulin resistance are commonly associated with catabolic or lipodystrophic conditions (such as cancer and sepsis) and with pathological states of nutritional overload (such as obesity-related type 2 diabetes). Two common features of these metabolic disorders are adipose tissue dysfunction and elevated levels of tumour necrosis factor-alpha (TNF-α). Herein, we review the multiple actions of this pro-inflammatory adipokine on adipose tissue biology. These include inhibition of carbohydrate metabolism, lipogenesis, adipogenesis and thermogenesis and stimulation of lipolysis. TNF-α can also impact the endocrine functions of adipose tissue. Taken together, TNF-α contributes to metabolic dysregulation by impairing both adipose tissue function and its ability to store excess fuel. The molecular mechanisms that underlie these actions are discussed.
Obesity; Type 2 diabetes; Metabolic syndrome; Insulin resistance; Dyslipidemia; TNF signalling; Lipid metabolism; Antiadipogenesis
The translation machinery is the engine of life. Extracting the cytoplasmic milieu from a cell affords a lysate capable of producing proteins in concentrations reaching tens of micromolar. Such lysates, derivable from a variety of cells, allow the facile addition and subtraction of components that are directly or indirectly related to the translation machinery and/or the over-expressed protein. The flexible nature of such cell-free expression systems, when coupled with high throughput monitoring, can be especially suitable for protein engineering studies, allowing one to bypass multiple steps typically required using conventional in vivo protein expression.
Cell-free protein synthesis; transcription; translation; high throughput
The behavioral effects of a selective A3 adenosine receptor agonist 3-IB-MECA (N6-(3-iodobenzyl)-5′-N-methylcarboxamidoadenosine) in mice and the localization of radioligand binding sites in mouse brain were examined. Low levels of A3 adenosine receptors were detected in various regions of the mouse brain (hippocampus, cortex, cerebellum, striatum), using a radioiodinated, high-affinity Aragonist radioligand [125I]AB-MECA (N6-(3-iodo-4-aminobenzyl)-5′-N-methylcarboxamidoadenosine). Scatchard analysis in the cerebellum showed that the Kd value for binding to A3 receptors was 1.39 ± 0.04 nM with a Bmax of 14.8 ± 2.1 fmol/mg protein. 3-IB-MECA at 0.1 mg/kg i.p. was a locomotor depressant with> 50% reduction in activity. Although selective A1 or A2a antagonists reversed locomotor depression elicited by selective A1 or A2a agonists, respectively, the behavioral depressant effects of 3-IB-MECA were unaffected. 3-IB-MECA also caused scratching in mice, which was prevented by coadministration of the histamine antagonist cyproheptadine. The demonstration of a marked behavioral effect of A3 receptor activation suggests that the A3 receptor represents a potential new therapeutic target.
Adenosine receptor; Xanthine; Locomotor activity; Histamine; Radioligand binding
Chemically functionalized congeners of N6-phenyladenosine and I ,3-dipropyl-8-phenylxanthine have been covalently coupled to fatty acids, diglyceridcs, and a phospholipid. The lipid-drug conjugates inhibit R-[3H]-phenylisopropyladenosinc binding to A1-adcnosine receptors in rat cerebral cortex membranes. A xanthinepbosphatidylethanolaminc conjugate bound with a K1 value of 19 nM. Various xanthine esters of low potency are potential prod rugs. Amides of an adenosine amine congener (ADA C) with 18-carbon fatty acids exhibited Ki values at A1-adenosine receptors of 70 pM, representing a 130-fold enhancement over the affinity of the corresponding acetyl amide. The very high affinity of adenosine-lipid conjugates may be due to stabilization of these adducts in the phospholipid microenvironment of the receptor protein.
Lipid; Adenosine receptor; Xanthine; Adenosine derivative; Lipid-drug conjugate; Prodrug
Research following introduction of the MDCK model system to study epithelial polarity (1978) led to an initial paradigm that posited independent roles of the trans Golgi network (TGN) and recycling endosomes (RE) in the generation of, respectively, biosynthetic and recycling routes of plasma membrane (PM) proteins to apical and basolateral PM domains. This model dominated the field for 20 years. However, studies over the past decade and the discovery of the involvement of clathrin and clathrin adaptors in protein trafficking to the basolateral PM has led to a new paradigm. TGN and RE are now believed to cooperate closely in both biosynthetic and recycling trafficking routes. Here, we critically review these recent advances and the questions that remain unanswered.
Epithelial polarity; Sorting; Clathrin; Adaptor; Endosome
•Little is known of the kinetics of interactions between GPCRs and their signalling partners.•NTS1 binds Gαi1 and Gαs with affinities of 15 ± 6 nM and 31 ± 18 nM (SE), respectively.•This SPR assay may be applicable to multiple partners in the signalling cascade.•We provide the first direct evidence for GPCR-G protein coupling in nanodiscs.
Neurotensin receptor type 1 (NTS1) is a G protein-coupled receptor (GPCR) that affects cellular responses by initiating a cascade of interactions through G proteins. The kinetic details for these interactions are not well-known. Here, NTS1-nanodisc-Gαs and Gαi1 interactions were studied. The binding affinities of Gαi1 and Gαs to NTS1 were directly measured by surface plasmon resonance (SPR) and determined to be 15 ± 6 nM and 31 ± 18 nM, respectively. This SPR configuration permits the kinetics of early events in signalling pathways to be explored and can be used to initiate descriptions of the GPCR interactome.
AC, adenylate cyclase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; CHS, cholesteryl hemisuccinate; DDM, dodecyl-β-d-maltoside; EDTA, ethylenediamine tetraacetic acid; FLAG-NTS1, FLAG-tagged NTS1; GDP, guanosine diphosphate; GTP, guanosine triphosphate; GPCR, G protein-coupled receptor; IPTG, isopropyl β-d-1-thiogalactopyranoside; NTS1, neurotensin receptor type 1; PI3K, phosphoinositide 3-kinase; PLC, phospholipase C; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPE, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol); RU, response units; SCK, single cycle kinetics; G protein-coupled receptor; Electron microscopy; Surface plasmon resonance; Nanodisc; G protein
•A. nidulans has a GBF/Gea and a BIG/Sec7 subfamily Golgi Arf1-GEFs, both essential.•The late Golgi Arf1-GEF mutant hypB5 conditionally blocks secretion.•Residue substitution in the early Golgi Arf1-GEF GeaA suppresses hypB5 and hypBΔ.•The mutation alters a GBF/Gea amino acid motif and shifts GeaA localization.•GeaA1 alone satisfies the eukaryotic requirement for two Golgi Arf1 GEFs.
Golgi Arf1-guanine nucleotide exchange factors (GEFs) belong to two subfamilies: GBF/Gea and BIG/Sec7. Both are conserved across eukaryotes, but the physiological role of each is not well understood. Aspergillus nidulans has a single member of the early Golgi GBF/Gea-subfamily, geaA, and the late Golgi BIG/Sec7-subfamily, hypB. Both geaA and hypB are essential. hypB5 conditionally blocks secretion. We sought extragenic hypB5 suppressors and obtained geaA1. geaA1 results in Tyr1022Cys within a conserved GBF/Gea-specific S(Y/W/F)(L/I) motif in GeaA. This mutation alters GeaA localization. Remarkably, geaA1 suppresses hypBΔ, indicating that a single mutant Golgi Arf1-GEF suffices for growth.
Arf GTPases, ADP-ribosylation-factor GTPases; BFA, brefeldin A; BIG, brefeldin A-inhibited guanine nucleotide-exchange; GBF, Golgi-specific brefeldin A resistance factor; GEF, Guanine nucleotide exchange factor; PHOSBP, pleckstrin homology domain of the oxysterol binding protein; Sec7d, Sec7 domain; Golgi Arf1-GEFs; GBF/Gea-subfamily; BIG/Sec7-subfamily; Fungal secretion
Structured RNA elements within messenger RNA often direct or modulate the cellular production of active proteins. As reviewed here, RNA structures have been discovered that govern nearly every step in protein production: mRNA production and stability; translation initiation, elongation, and termination; protein folding; and cellular localization. Regulatory RNA elements are common within RNAs from every domain of life. This growing body of RNA-mediated mechanisms continues to reveal new ways in which mRNA structure regulates translation. We integrate examples from several different classes of RNA structure-mediated regulation to present a global perspective that suggests that the secondary and tertiary structure of RNA ultimately constitutes an additional level of the genetic code that both guides and regulates protein biosynthesis.
Neuronal nitric oxide synthase μ (nNOSμ) contains 34 additional residues in an autoregulatory element compared to nNOSα. Cytochrome c and flavin reductions in the absence of calmodulin (CaM) were faster in nNOSμ than nNOSα, while rates in the presence of CaM were smaller. The magnitude of stimulation by CaM is thus notably lower in nNOSμ. No difference in NO production was observed, while electron transfer between the FMN and heme moieties and formation of an inhibitory ferrous-nitrosyl complex were slower in nNOSμ. Thus, the insert affects electron transfer rates, modulation of electron flow by CaM, and heme-nitrosyl complex formation.
neuronal nitric oxide synthase; calmodulin; electron transfer; reductase; flavoproteins; heme
Transforming growth factor β (TGF-β) signaling is important for many biological processes. Although the sequential events of this cascade are known, the dynamics remain speculative. Here, live-cell single-molecule total internal reflection fluorescence microscopy was used to monitor the dynamics of SMAD4, a TGF-β downstream effector, in MDA-MB-231 breast cancer cells. Contrary to previous belief, SMAD4 was detectable at the cytoplasmic membrane, displaying two subpopulations with different membrane docking behaviors. These subpopulations were regulated by clathrin and caveolin-1, and had opposing roles in the nuclear shuttling of SMAD4 and the subsequent transcriptional regulation of genes associated with cell migration. The notion that membrane-docking behaviors of downstream molecules could predict the cellular response to growth factors may revolutionize the way we view cell signaling.
The human BAP1 deubiquitinating enzyme is a chromatin-bound transcriptional regulator and tumor suppressor. BAP1 functions in suppressing cell proliferation, yet its role in the DNA damage response pathway is less understood. In this study we characterized DNA damage-induced phosphorylation of BAP1 at Serine 592 (pS592) and the cellular outcomes of this modification. In contrast to the majority of BAP1, pS592-BAP1 is predominantly dissociated from chromatin. These findings support a model whereby stress induced phosphorylation functions to displace BAP1 from specific promoters. We hypothesize that this regulates the transcription of a subset of genes involved in the response to DNA damage.
BAP1; Deubiquitinating enzyme; DNA Damage; Phosphorylation; Ubiquitin
To investigate the effects of histone methyltransferase ESET (also known as SETDB1) on bone metabolism, we analyzed osteoblasts and osteoclasts in ESET knockout animals, and performed osteogenesis assays using ESET-null mesenchymal stem cells. We found that ESET deletion severely impairs osteoblast differentiation but has no effect on osteoclastogenesis, that co-transfection of ESET represses Runx2-mediated luciferase reporter while siRNA knockdown of ESET activates the luciferase reporter in mesenchymal cells, and that ESET is required for postnatal expression of Indian hedgehog protein in the growth plate. As the bone phenotype in ESET-null mice is 100% penetrant, these results support ESET as a critical regulator of osteoblast differentiation during bone development.
G-protein-coupled receptor kinase 2 (GRK2) levels are elevated in inflammation but its role is not clear yet. Here we show that GRK2 expression is dependent on NFκB transcriptional activity. In macrophages, LPS induces GRK2 accumulation in mitochondria increasing biogenesis. The overexpression of the carboxy-terminal domain of GRK2 (βARK-ct), known to displace GRK2 from plasma membranes, in macrophages induces earlier localization of GRK2 in mitochondria in response to LPS leading to increased mt-DNA transcription, reduced ROS production and cytokines expression. Our study shows the relevance of GRK2 subcellular localization in macrophage’s biology and its potential therapeutic properties in inflammation.
GRK2; mitochondria; inflammation; subcellular localization; βARK-ct
StyA2B represents a new class of styrene monooxygenases that integrates flavin-reductase and styrene-epoxidase activities into a single polypeptide. This naturally-occurring fusion protein offers new avenues for studying and engineering biotechnologically relevant enantioselective biochemical epoxidation reactions. Stopped-flow kinetic studies of StyA2B reported here identify reaction intermediates similar to those reported for the separate reductase and epoxidase components of related two-component systems. Our studies identify substrate epoxidation and elimination of water from the FAD C(4a)-hydroxide as rate-limiting steps in the styrene epoxidation reaction. Efforts directed at accelerating these reaction steps are expected to greatly increase catalytic efficiency and the value of StyA2B as biocatalyst.
catalytic mechanism; flavoprotein; monooxygenase; Rhodococcus opacus 1CP; styrene epoxidation
Acrolein, a reactive aldehyde found in cigarette smoke, is thought to induce its biological effects primarily by irreversible adduction to cellular nucleophiles such as cysteine thiols. Here, we demonstrate that acrolein rapidly inactivates the seleno-enzyme thioredoxin reductase (TrxR) in human bronchiolar epithelial HBE1 cells, which recovered over 4-8 hrs by a mechanism depending on the presence of cellular GSH and thioredoxin 1 (Trx1), and corresponding with reversal of protein-acrolein adduction. Our findings indicate that acrolein-induced protein alkylation is not necessarily a feature of irreversible protein damage, but may reflect a reversible signaling mechanism that is regulated by GSH and Trx1.
cysteine; selenocysteine; Michael addition; thioredoxin reductase; GSH; thioredoxin