ABSTRACT
Here, we present evidence that caveolae-mediated endocytosis using LDLR is the pathway for SARS-CoV-2 virus internalization in the ocular cell line ARPE-19. Firstly, we found that, while Angiotensin-converting enzyme 2 (ACE2) is expressed in these cells, blocking ACE2 by antibody treatment did not prevent infection by SARS-CoV-2 spike pseudovirions, nor did antibody blockade of extracellular vimentin and other cholesterol-rich lipid raft proteins. Next, we implicated the role of cholesterol homeostasis in infection by showing that incubating cells with different cyclodextrins and oxysterol 25-hydroxycholesterol (25-HC) inhibits pseudovirion infection of ARPE-19. However, the effect of 25-HC is likely not via cholesterol biosynthesis, as incubation with lovastatin did not appreciably affect infection. Additionally, is it not likely to be an agonistic effect of 25-HC on LXR receptors, as the LXR agonist GW3965 had no significant effect on infection of ARPE-19 cells at up to 5 µM GW3965. We probed the role of endocytic pathways but determined that clathrin-dependent and flotillin-dependent rafts were not involved. Furthermore, 20 µM chlorpromazine, an inhibitor of clathrin-mediated endocytosis (CME), also had little effect. In contrast, anti-dynamin I/II antibodies blocked the entry of SARS-CoV-2 spike pseudovirions, as did dynasore, a noncompetitive inhibitor of dynamin GTPase activity. Additionally, anti-caveolin-1 antibodies significantly blocked spike pseudotyped lentiviral infection of ARPE-19. However, nystatin, a classic inhibitor of caveolae-dependent endocytosis, did not affect infection while indomethacin inhibited only at 10 µM at the 48 h time point. Finally, we found that anti-LDLR antibodies block pseudovirion infection to a similar degree as anti-caveolin-1 and anti-dynamin I/II antibodies, while transfection with LDLR-specific siRNA led to a decrease in spike pseudotyped lentiviral infection, compared to scrambled control siRNAs. Thus, we conclude that SARS-CoV-2 spike pseudovirion infection in ARPE-19 cells is a dynamin-dependent process that is primarily mediated by LDLR.
Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Humans , Angiotensin-Converting Enzyme 2/pharmacology , Cholesterol/metabolism , Clathrin/metabolism , Dynamin II , Lipoproteins, LDL/pharmacology , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/pharmacology , Virus InternalizationABSTRACT
The visual cycle is a complex biological process that involves the sequential action of proteins in the retinal pigment epithelial (RPE) cells and photoreceptors to modify and shuttle visual retinoids. A majority of the visual cycle proteins are membrane proteins, either integral or peripheral membrane proteins. Despite significant progress in understanding their physiological function, very limited structural information is available for the visual cycle proteins. Moreover, the mechanism of membrane interaction is not yet clear in all cases. Here, we demonstrate the presence of an amphipathic helix in selected RPE visual cycle proteins, using in silico tools, and highlight their role in membrane association and function.
Subject(s)
Retinal Pigment Epithelium , Retinoids , Carrier Proteins/metabolism , Eye Proteins/metabolism , Membrane Proteins/metabolism , cis-trans-IsomerasesABSTRACT
Proximity ligation assay (PLA) is an immunofluorescence assay, which determines in situ interaction of two biomolecules present within 40 nm close proximity. Here, we describe a modification of PLA for visual detection of in situ protein interactions with nascent RNA in a single cell (IPNR-PLA). In IPNR-PLA, nascent RNA is labeled by incorporating 5-fluorouridine (FU), a uridine nucleotide analogue, followed by covalent cross-linking of the interacting partners in proximity to newly synthesized RNA. By using combination of anti-BrdU antibody, which specifically binds to FU, and primary antibody against a protein of interest, the IPNR reaction results in fluorescent puncta as a positive signal, only if the candidate proteins are in proximity to nascent RNA. We have validated this method by demonstrating known CDK9 and elongating RNA pol II interaction with nascent RNA. Finally, we used this method to test for the presence of DNA double strand breaks as well as Poly (ADP-ribose) polymerase 1 (PARP1), an RNA binding protein, in the vicinity of nascent RNA in cancer cells. The capability of performing parallel IF labeling and quantifiable multiparameter measurements within heterogeneous cell populations makes IPNR-PLA very attractive for use in biological studies. Overall, we have developed the IPNR-PLA method for analysis of protein association with nascent RNA with single-cell resolution, which is highly sensitive, quantitative, efficient, and requires little starting experimental material.
Subject(s)
Antibodies , RNA , Animals , RNA/metabolism , Antibodies/chemistry , Antibodies/metabolism , RNA-Binding Proteins , Mammals/metabolismABSTRACT
New targeted therapy for triple negative breast cancer (TNBC) is an urgent need, as advanced disease responds poorly to conventional chemotherapy. Genomic and proteomic studies are currently investigating new genes and proteins as promising therapeutic targets. One of such therapeutic targets is a cell cycle regulatory kinase; Maternal Embryonic Leucine Zipper Kinase (MELK), overexpressed in TNBC and correlated with cancer development. We performed molecular docking for virtual screening of chemical libraries (phytochemicals/synthetic drugs) against MELK protein structure and identified 8 phytoconstituents (isoxanthorin, emodin, gamma-coniceine, quercetin, tenuazonic acid, isoliquiritigenin, kaempferol, and Nobiletin) and 8 synthetic drugs (tetrahydrofolic acid, alfuzosin, lansoprazole, ketorolac, ketoprofen, variolin B, orantinib, and firestein) as potential hits interacting with the active site residues of MELK based on bound poses, hydrogen bond, hydrophobic interactions and MM/GBSA binding free energies. ADME and drug-likeness prediction further identified few hits with high drug-likeness properties and were further tested for anti-tumorigenic potential. Two phytochemicals isoliquiritigenin and emodin demonstrated growth inhibitory effects on TNBC MDA-MB-231 cells while much lower effect was observed on non-tumorigenic MCF-10A mammary epithelial cells. Treatment with both molecules downregulated MELK expression, induced cell cycle arrest, accumulated DNA damage and enhanced apoptosis. The study identified isoliquiritigenin and emodin as potential MELK inhibitors and provides a basis for subsequent experimental validation and drug development against cancer.
Subject(s)
Emodin , Triple Negative Breast Neoplasms , Humans , Triple Negative Breast Neoplasms/drug therapy , Protein Serine-Threonine Kinases/metabolism , Small Molecule Libraries/pharmacology , Molecular Docking Simulation , Emodin/pharmacology , Proteomics , Cell Proliferation , Early Detection of Cancer , Cell Line, TumorABSTRACT
The Development of reliable and field-compatible detection methods is essential to monitoring and controlling the spread of any global pandemic. We herein report a novel anti-RNA:DNA hybrid (anti-RDH) antibody-based biosensor for visual, colorimetric lateral flow assay, using gold nanoparticles, coupled with transcription-mediated-isothermal-RNA-amplification (TMIRA) for specific and sensitive detection of viral RNA. We have demonstrated its utility for SARS-CoV-2 RNA detection. This technique, which we have named RDH-LFA (anti-RNA:DNA hybrid antibody-based lateral flow assay), exploits anti-RDH antibody for immunocapture of viral RNA hybridized with specific DNA probes in lateral flow assay. This method uses biotinylated-oligonucleotides (DNAB) specific to SARS-CoV-2 RNA (vRNA) to generate a vRNA-DNAB hybrid. The biotin-tagged vRNA-DNAB hybrid molecules bind to streptavidin conjugated with gold nanoparticles. This hybrid complex is trapped by the anti-RDH antibody immobilized on the nitrocellulose membrane resulting in pink color signal leading to visual naked-eye detection in 1â minute. Combining RDH-LFA with isothermal RNA amplification (TMIRA) significantly improves the sensitivity (LOD:10â copies/µl) with a total turnaround time of an hour. More importantly, RDH-LFA coupled with the TMIRA method showed 96.6% sensitivity and 100% specificity for clinical samples when compared to a commercial gold standard reverse-transcription quantitative polymerase-chain-reaction assay. Thus, the present study reports a rapid, sensitive, specific, and simple method for visual detection of viral RNA, which can be used at the point-of-care without requiring sophisticated instrumentation.
ABSTRACT
Bismuth vanadate (BiVO4) is a promising photoactive material for the design of photoelectrochemical (PEC) analytical devices for the non-enzymatic detection of glucose. In this work, un-doped and La/Ce/Zr doped BiVO4 photo anodes were developed by spray pyrolysis coating to generate unique 2D hierarchical architectures using the facile ultrasonic spray coating technique without any complex pre or post-treatment. The influence of different dopants on the morphology and photoelectrochemical activity of BiVO4 coatings was investigated. X-ray diffraction, scanning electron microscopy, UV-vis optical absorbance, and positron annihilation techniques were used to evaluate the structure, defects, and optical properties of BiVO4 films. DFT simulation confirmed the Zr doping induced band gap reduction in the BiVO4 lattice. The Zr doping on the Bi site in BiVO4 lattice provided significantly low Bi and V-based defect density and a higher bulk diffusion length of charge pairs (4 times that of pristine) as well as charge transfer efficiency and this led to the foremost photocurrent for water splitting. The Zr-doped BiVO4 photo anode showed remarkable sensitivity in glucose sensing. The sensitivity and limit of detection of the Zr-doped BiVO4 PEC device towards glucose were 0.14 mA cm-2 mM-1 and 1.22 µM, respectively, in the concentration range of 1-7 mM. The system showed sensitive detection of glucose in blood serum. This is the first time that a 2D morphology electrode design consisting of Zr-doped BiVO4, which leads to exceptionally high sensitivity for glucose sensing, has been reported.
Subject(s)
Glucose , Serum , Vanadates , DiffusionABSTRACT
RPE65 retinol isomerase is an indispensable player in the visual cycle between the vertebrate retina and RPE. Although membrane association is critical for RPE65 function, its mechanism is not clear. Residues 107-125 are believed to interact with membranes but are unresolved in all RPE65 crystal structures, whereas palmitoylation at C112 also plays a role. We report the mechanism of membrane recognition and binding by RPE65. Binding of aa107-125 synthetic peptide with membrane-mimicking micellar surfaces induces transition from unstructured loop to amphipathic α-helical (AH) structure but this transition is automatic in the C112-palmitoylated peptide. We demonstrate that the AH significantly affects palmitoylation level, membrane association, and isomerization activity of RPE65. Furthermore, aa107-125 functions as a membrane sensor and the AH as a membrane-targeting motif. Molecular dynamic simulations clearly show AH-membrane insertion, supporting our experimental findings. Collectively, these studies allow us to propose a working model for RPE65-membrane binding, and to provide a novel role for cysteine palmitoylation.
Subject(s)
Cysteine , Eye Proteins , Carrier Proteins/metabolism , Cysteine/metabolism , Eye Proteins/chemistry , Eye Proteins/metabolism , Lipoylation , Protein Conformation, alpha-Helical , cis-trans-IsomerasesABSTRACT
Understanding the mechanism of fate decision and lineage commitment is the key step for developing novel stem cell applications in therapeutics. This process is coordinately regulated through systematic epigenetic reprogramming and concomitant changes in the transcriptional landscape of the stem cells. One of the bromo- and extra-terminal domain (BET) family member proteins, bromodomain protein 4 (BRD4), performs the role of epigenetic reader and modulates gene expression by recruiting other transcription factors and directly regulating RNA polymerase II elongation. Controlled gene regulation is the critical step in maintenance of stem cell potency and dysregulation may lead to tumor formation. As a key transcriptional factor and epigenetic regulator, BRD4 contributes to stem cell maintenance in several ways. Being a druggable target, BRD4 is an attractive candidate for exploiting its potential in stem cell therapeutics. Therefore, it is crucial to elucidate how BRD4, through its interplay with pluripotency transcriptional regulators, control lineage commitment in stem cells. Here, we systemically review the role of BRD4 in complex gene regulatory network during three specific states of stem cell transitions: cell differentiation, cell reprogramming and transdifferentiation. A thorough understanding of BRD4 mediated epigenetic regulation in the maintenance of stem cell potency will be helpful to strategically control stem cell fates in regenerative medicine.
Subject(s)
Nuclear Proteins , Transcription Factors , Cell Cycle Proteins/metabolism , Cell Differentiation/genetics , Embryonic Stem Cells/metabolism , Epigenesis, Genetic , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
The SARS-CoV-2 Spike glycoprotein (S protein) acquired a unique new 4 amino acid -PRRA- insertion sequence at amino acid residues (aa) 681-684 that forms a new furin cleavage site in S protein as well as several new glycosylation sites. We studied various statistical properties of the -PRRA- insertion at the RNA level (CCUCGGCGGGCA). The nucleotide composition and codon usage of this sequence are different from the rest of the SARS-CoV-2 genome. One of such features is two tandem CGG codons, although the CGG codon is the rarest codon in the SARS-CoV-2 genome. This suggests that the insertion sequence could cause ribosome pausing as the result of these rare codons. Due to population variants, the Nextstrain divergence measure of the CCU codon is extremely large. We cannot exclude that this divergence might affect host immune responses/effectiveness of SARS-CoV-2 vaccines, possibilities awaiting further investigation. Our experimental studies show that the expression level of original RNA sequence "wildtype" spike protein is much lower than for codon-optimized spike protein in all studied cell lines. Interestingly, the original spike sequence produces a higher titer of pseudoviral particles and a higher level of infection. Further mutagenesis experiments suggest that this dual-effect insert, comprised of a combination of overlapping translation pausing and furin sites, has allowed SARS-CoV-2 to infect its new host (human) more readily. This underlines the importance of ribosome pausing to allow efficient regulation of protein expression and also of cotranslational subdomain folding.
Subject(s)
RNA, Viral/metabolism , Ribosomes/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/genetics , Animals , Base Sequence , COS Cells , COVID-19/pathology , COVID-19/virology , Chlorocebus aethiops , Codon Usage , HEK293 Cells , Humans , Mutagenesis , SARS-CoV-2/isolation & purification , Sequence Alignment , Spike Glycoprotein, Coronavirus/metabolismABSTRACT
We report the development of an ultrasensitive label-free DNA biosensor device with fully integrated standalone carbon nanotube (CNT) aerogel electrode. The multi-directional tenuous network of clustered CNT embedding into the CNT aerogel electrode demonstrates linear ohmic and near isotropic electrical properties, thereby providing high sensitivity for nucleic acid detection. Using this device, the target DNA hybridization is detected by a quantifiable change in the electrochemical impedance, with a distinct response to the single-stranded probe alone or double-stranded target-probe complex. The target DNA is specifically detected with limit of detection (LoD) of 1 pM with a turnaround time of less than 20 min, which is unprecedented for a miniaturized CNT aerogel sensor and impedance spectroscopy without an intermediate DNA amplification step. Moreover, this system is able to differentiate between the closely related target sequences by the distinct impedance response rendering it highly specific. To the best of our knowledge, this is the first report showing the use of standalone bare CNT aerogel electrode without any substrate support, coupled with electrochemical impedance spectroscopy, for the detection of DNA hybridization. Altogether, the results show that our system is fast, sensitive and specific for label-free rapid direct DNA detection, promising a novel avenue for bio-sensing.
Subject(s)
Biosensing Techniques , Nanotubes, Carbon , DNA/genetics , Electrochemical Techniques , Limit of Detection , Nucleic Acid HybridizationABSTRACT
An extensive body of work has documented the antioxidant role of xanthophylls (lutein and zeaxanthin) in human health and specifically how they provide photoprotection in human vision. More recently, evidence is emerging for the transcriptional regulation of antioxidant response by lutein/lutein cleavage products, similar to the role of ß-carotene cleavage products in the modulation of retinoic acid receptors. Supplementation with xanthophylls also provides additional benefits for the prevention of age-related macular degeneration (AMD) and attenuation of Alzheimer's disease symptoms. Mammalian ß-carotene oxygenase 2 (BCO2) asymmetrically cleaves xanthophylls as well as ß-carotene in vitro. We recently demonstrated that mouse BCO2 (mBCO2) is a functionally palmitoylated enzyme and that it loses palmitoylation when cells are treated with ß-carotene. The mouse enzyme is the easiest model to study mammalian BCO2 because it has only one isoform, unlike human BCO2 with several major isoforms with various properties. Here, we used the same acyl-RAC methodology and confocal microscopy to elucidate palmitoylation and localization status of mBCO2 in the presence of xanthophylls. We created large unilamellar vesicle-based nanocarriers for the successful delivery of xanthophylls into cells. We demonstrate here that, upon treatment with low micromolar concentration of lutein (0.15 µM), mBCO2 is depalmitoylated and shows partial nuclear localization (38.00 ± 0.04%), while treatment with zeaxanthin (0.45 µM) and violaxanthin (0.6 µM) induces depalmitoylation and protein translocation from mitochondria to a lesser degree (20.00 ± 0.01% and 35.00 ± 0.02%, respectively). Such a difference in the behavior of mBCO2 toward various xanthophylls and its translocation into the nucleus in the presence of various xanthophylls suggests a possible mechanism for transport of lutein/lutein cleavage products to the nucleus to affect transcriptional regulation.
ABSTRACT
Cationic liposomes have become an attractive tool to deliver genes and interfering RNA into cells. Herein, we report the application of spontaneously formed cationic vesicles in mixtures of lecithin and cationic amphiphiles for efficient transfection of plasmid DNA and siRNA into cells. The average hydrodynamic diameter of the phospholipid vesicles was modulated by changing the ratio of dihexadecyldimethylammonium bromide (DDAB) to phospholipid in the vesicles. The vesicles were characterized by dynamic light scattering, ζ potential, and small-angle X-ray scattering. Depending on the ratio of DDAB to phospholipid, the average size of the vesicles can be varied in the range of 150-300 nm with a ζ potential of +40 mV. The ability of these cationic vesicles to form lipoplexes upon binding with pDNA is demonstrated by ζ potential, isothermal titration calorimetry, gel retardation, and DNase I digestion assay. The enthalpy of binding between pDNA and cationic liposome was found to be -5.7 (±0.8) kJ/mol. The cellular uptake studies of lipoplexes observed by fluorescence microscopy confirmed good transfection efficiency of DDAB liposomes in MCF-7 and HeLa cells. The fluorescent imaging analysis showed effective gene delivery and expression of green fluorescent protein. In addition, the formulation has demonstrated an ability to deliver small interfering RNA (siBRD4) for efficient gene silencing as seen by a significant decrease in BRD4 protein level in siBRD4-treated cells. Comparison of the transfection efficiency of different formulations suggests that DDAB-rich mixed phospholipid vesicles with size <200 nm are better than large size vesicles for improved endocytosis and gene expression.
Subject(s)
Lecithins , Liposomes , Cations/chemistry , Cell Cycle Proteins/genetics , DNA/genetics , HeLa Cells , Humans , Liposomes/chemistry , Nuclear Proteins/genetics , Plasmids/genetics , Quaternary Ammonium Compounds , RNA, Small Interfering/genetics , Transcription Factors/genetics , TransfectionABSTRACT
AIMS: The aim of our study is to understand the biophysical traits that govern the stability and folding of Synechocystis hemoglobin, a unique cyanobacterial globin that displays unusual traits not observed in any of the other globins discovered so far. BACKGROUND: For the past few decades, classical hemoglobins such as vertebrate hemoglobin and myoglobin have been extensively studied to unravel the stability and folding mechanisms of hemoglobins. However, the expanding wealth of hemoglobins identified in all life forms with novel properties, like heme coordination chemistry and globin fold, have added complexity and challenges to the understanding of hemoglobin stability, which has not been adequately addressed. Here, we explored the unique truncated and hexacoordinate hemoglobin from the freshwater cyanobacterium Synechocystis sp. PCC 6803 known as "Synechocystis hemoglobin (SynHb)". The "three histidines" linkages to heme are novel to this cyanobacterial hemoglobin. OBJECTIVE: Mutational studies were employed to decipher the residues within the heme pocket that dictate the stability and folding of SynHb. METHODS: Site-directed mutants of SynHb were generated and analyzed using a repertoire of spectroscopic and calorimetric tools. RESULTS: The results revealed that the heme was stably associated to the protein under all denaturing conditions with His117 playing the anchoring role. The studies also highlighted the possibility of existence of a "molten globule" like intermediate at acidic pH in this exceptionally thermostable globin. His117 and other key residues in the heme pocket play an indispensable role in imparting significant polypeptide stability. CONCLUSION: Synechocystis hemoglobin presents an important model system for investigations of protein folding and stability in general. The heme pocket residues influenced the folding and stability of SynHb in a very subtle and specific manner and may have been optimized to make this Hb the most stable known as of date. Other: The knowledge gained hereby about the influence of heme pocket amino acid side chains on stability and expression is currently being utilized to improve the stability of recombinant human Hbs for efficient use as oxygen delivery vehicles.
Subject(s)
Hemoglobins/chemistry , Hemoglobins/metabolism , Histidine/chemistry , Histidine/metabolism , Protein Binding , Synechocystis/metabolism , Hemoglobins/genetics , Histidine/genetics , Humans , Models, Molecular , Mutation , Protein Conformation , Protein Folding , Protein Stability , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
One popular and relevant proposed function for cyanobacterial hemoglobin (Synechocystis Hb) is anaerobic nitrite reductase in vivo. During such reduction reactions, the hexacoordinated heme iron atom of SynHb is oxidized from the ferrous (Fe+2) to ferric (Fe+3) state and prevent damage by limiting the concentration of toxic metabolites such as nitrite. In order to perform these functions in vivo, there must be a mechanism that converts inactive Fe+3-SynHb back to the active Fe+2-SynHb to accomplish the nitrite reductase function. Here, we report a cognate reductase protein for Synechocystis hemoglobin which can reduce the Fe+3-SynHb to Fe+2-SynHb, thus lending a support to the proposed nitrite reductase function. This reductase is also able to reduce pentacoordinate Hbs such as myoglobin but with lower affinity compared to hexacoordinate SynHb. Insilico model of reductase protein-cyanobacterial hemoglobin complex revealed that the heme active site of Hb faces the catalytic center of the reductase protein and several amino acids in the interface interacts non-covalently thus favoring their interaction. Overall, our in vitro study provides the basic foundation for the understanding of the specific molecular mechanism of action and interaction of the SynHb reductase protein, which need to be investigated in further detail.
Subject(s)
Bacterial Proteins/chemistry , Hemoglobins/chemistry , Models, Molecular , Oxidoreductases/chemistry , Synechocystis/enzymology , Bacterial Proteins/genetics , Oxidoreductases/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Synechocystis/geneticsABSTRACT
The protooncogene MYC regulates a variety of cellular processes, including proliferation and metabolism. Maintaining MYC at homeostatic levels is critical to normal cell function; overexpression drives many cancers. MYC stability is regulated through phosphorylation: phosphorylation at Thr58 signals degradation while Ser62 phosphorylation leads to its stabilization and functional activation. The bromodomain protein 4 (BRD4) is a transcriptional and epigenetic regulator with intrinsic kinase and histone acetyltransferase (HAT) activities that activates transcription of key protooncogenes, including MYC We report that BRD4 phosphorylates MYC at Thr58, leading to MYC ubiquitination and degradation, thereby regulating MYC target genes. Importantly, BRD4 degradation, but not inhibition, results in increased levels of MYC protein. Conversely, MYC inhibits BRD4's HAT activity, suggesting that MYC regulates its own transcription by limiting BRD4-mediated chromatin remodeling of its locus. The MYC stabilizing kinase, ERK1, regulates MYC levels directly and indirectly by inhibiting BRD4 kinase activity. These findings demonstrate that BRD4 negatively regulates MYC levels, which is counteracted by ERK1 activation.
Subject(s)
Cell Cycle Proteins/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Transcription Factors/metabolism , Acetylation , Cell Nucleus/metabolism , Chromatin/metabolism , Dipeptides/pharmacology , Gene Expression Regulation/drug effects , HeLa Cells , Heterocyclic Compounds, 3-Ring/pharmacology , Histones/metabolism , Humans , Mitogen-Activated Protein Kinase 3/metabolism , Phosphorylation , Protein Binding , Protein Stability/drug effects , Proto-Oncogene Proteins c-myc/genetics , UbiquitinationABSTRACT
Abundant in nature, carotenoids are a class of fat-soluble pigments with a polyene tetraterpenoid structure. They possess antioxidant properties and their consumption leads to certain health benefits in humans. Carotenoid cleavage oxygenases (CCOs) are a superfamily of enzymes which oxidatively cleave carotenoids and they are present in all kingdoms of life. Complexity of CCO evolution is high. For example, in this study we serendipitously found a new family of eukaryotic CCOs, the apocarotenoid oxygenase-like (ACOL) family. This family has several members in animal genomes and lacks the animal-specific amino acid motif PDPCK. This motif is likely to be associated with palmitoylation of some animal CCOs. We recently demonstrated that two mammalian members of the carotenoid oxygenase family retinal pigment epithelial-specific 65 kDa protein (RPE65) and beta-carotene oxygenase 2 (BCO2) are palmitoylated proteins. Here we used the acyl-resin-assisted capture (acyl-RAC) method to demonstrate protein palmitoylation and immunochemistry to localize mouse BCO2 (mBCO2) in COS7 cell line in the absence and presence of its substrate ß-carotene. We demonstrate that mBCO2 palmitoylation depends on the evolutionarily conserved motif PDPCK and that metazoan family members lacking the motif (Lancelet beta-carotene oxygenase-like protein (BCOL) and Acropora ACOL) are not palmitoylated. Additionally, we observed that the palmitoylation status of mBCO2 and its membrane association depend on the presence of its substrate ß-carotene. Based on our results we conclude that most metazoan carotenoid oxygenases retain the evolutionarily conserved palmitoylation PDPCK motif to target proteins to internal membranes depending on substrate status. Exceptions are in the secreted BCOL subfamily and the strictly cytosolic ancient ACOL subfamily of carotenoid oxygenases.
Subject(s)
Oxygenases/chemistry , Animals , Carotenoids/chemistry , Dioxygenases/metabolism , Fatty Acids, Monounsaturated/chemistry , Fluorescent Antibody Technique , Humans , Mice , Multigene Family , Mutation , Oxygenases/genetics , Phylogeny , Protein Transport , Substrate SpecificityABSTRACT
The carotenoids are terpenoid fat-soluble pigments produced by plants, algae, and several bacteria and fungi. They are ubiquitous components of animal diets. Carotenoid cleavage oxygenase (CCO) superfamily members are involved in carotenoid metabolism and are present in all kingdoms of life. Throughout the animal kingdom, carotenoid oxygenases are widely distributed and they are completely absent only in two unicellular organisms, Monosiga and Leishmania. Mammals have three paralogs 15,15'-ß-carotene oxygenase (BCO1), 9',10'-ß-carotene oxygenase (BCO2) and RPE65. The first two enzymes are classical carotenoid oxygenases: they cleave carboncarbon double bonds and incorporate two atoms of oxygen in the substrate at the site of cleavage. The third, RPE65, is an unusual family member, it is the retinoid isomerohydrolase in the visual cycle that converts all-trans-retinyl ester into 11-cis-retinol. Here we discuss evolutionary aspects of the carotenoid cleavage oxygenase superfamily and their enzymology to deduce what insight we can obtain from their evolutionary conservation.
Subject(s)
Dioxygenases/genetics , Evolution, Molecular , beta-Carotene 15,15'-Monooxygenase/genetics , cis-trans-Isomerases/genetics , Animals , Carotenoids/metabolism , Lipid Metabolism/genetics , Mammals/genetics , Oxygenases/classification , Oxygenases/geneticsABSTRACT
RPE65, the retinal pigment epithelium (RPE) smooth endoplasmic reticulum (sER) membrane-associated retinoid isomerase, plays an indispensable role in sustaining visual function in vertebrates. An important aspect which has attracted considerable attention is the posttranslational modification by S-palmitoylation of RPE65. Some studies show that RPE65 is a palmitoylated protein, but others deny that conclusion. While it is considered to be mainly responsible for RPE65's membrane association, we still lack conclusive evidence about RPE65 palmitoylation. In this review, we provide an overview of the history and current understanding of RPE65 palmitoylation.
Subject(s)
Eye Proteins/chemistry , Lipids/chemistry , Lipoylation , Protein Processing, Post-Translational , Retinal Pigment Epithelium/enzymology , cis-trans-Isomerases/chemistry , Animals , Endoplasmic Reticulum , HumansABSTRACT
The bromodomain protein 4 (BRD4) is an atypical kinase and histone acetyl transferase (HAT) that binds to acetylated histones and contributes to chromatin remodeling and early transcriptional elongation. During transcription, BRD4 travels with the elongation complex. Since most alternative splicing events take place co-transcriptionally, we asked if BRD4 plays a role in regulating alternative splicing. We report that distinct patterns of alternative splicing are associated with a conditional deletion of BRD4 during thymocyte differentiation in vivo. Similarly, the depletion of BRD4 in T cell acute lymphoblastic leukemia (T-ALL) cells alters patterns of splicing. Most alternatively spliced events affected by BRD4 are exon skipping. Importantly, BRD4 interacts with components of the splicing machinery, as assessed by both immunoprecipitation (IP) and proximity ligation assays (PLAs), and co-localizes on chromatin with the splicing regulator, FUS. We propose that BRD4 contributes to patterns of alternative splicing through its interaction with the splicing machinery during transcription elongation.