scholarly journals Selenoprotein P Regulates Synaptic Zinc and Reduces Tau Phosphorylation

2021 ◽  
Vol 8 ◽  
Author(s):  
Arlene C. P. Kiyohara ◽  
Daniel J. Torres ◽  
Ayaka Hagiwara ◽  
Jenna Pak ◽  
Rachel H. L. H. Rueli ◽  
...  
Keyword(s):  
Metal Binding ◽  
Cultured Cells ◽  
Hippocampal Slices ◽  
Biological Significance ◽  
Tau Phosphorylation ◽  
Mass Spectrometry Analysis ◽  
Selenoprotein P ◽  
Normal Brain ◽  
Spectrometry Analysis ◽  
Extracellular Transport

Selenoprotein P (SELENOP1) is a selenium-rich antioxidant protein involved in extracellular transport of selenium (Se). SELENOP1 also has metal binding properties. The trace element Zinc (Zn2+) is a neuromodulator that can be released from synaptic terminals in the brain, primarily from a subset of glutamatergic terminals. Both Zn2+ and Se are necessary for normal brain function. Although these ions can bind together with high affinity, the biological significance of an interaction of SELENOP1 with Zn2+ has not been investigated. We examined changes in brain Zn2+ in SELENOP1 knockout (KO) animals. Timm-Danscher and N-(6-methoxy-8-quinolyl)-p-toluenesulphonamide (TSQ) staining revealed increased levels of intracellular Zn2+ in the SELENOP1−/− hippocampus compared to wildtype (WT) mice. Mass spectrometry analysis of frozen whole brain samples demonstrated that total Zn2+ was not increased in the SELENOP1−/− mice, suggesting only local changes in Zn2+ distribution. Unexpectedly, live Zn2+ imaging of hippocampal slices with a selective extracellular fluorescent Zn2+ indicator (FluoZin-3) showed that SELENOP1−/− mice have impaired Zn2+ release in response to KCl-induced neuron depolarization. The zinc/metal storage protein metallothionein 3 (MT-3) was increased in SELENOP1−/− hippocampus relative to wildtype, possibly in response to an elevated Zn2+ content. We found that depriving cultured cells of selenium resulted in increased intracellular Zn2+, as did inhibition of selenoprotein GPX4 but not GPX1, suggesting the increased Zn2+ in SELENOP1−/− mice is due to a downregulation of antioxidant selenoproteins and subsequent release of Zn2+ from intracellular stores. Surprisingly, we found increased tau phosphorylation in the hippocampus of SELENOP1−/− mice, possibly resulting from intracellular zinc changes. Our findings reveal important roles for SELENOP1 in the maintenance of synaptic Zn2+ physiology and preventing tau hyperphosphorylation.

scholarly journals Re-Discovery of Giardiavirus: Genomic and Functional Analysis of Viruses from Giardia duodenalis Isolates

Biomedicines ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 654
Author(s):  
Gianluca Marucci ◽  
Ilaria Zullino ◽  
Lucia Bertuccini ◽  
Serena Camerini ◽  
Serena Cecchetti ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Diarrheal Disease ◽  
Current Knowledge ◽  
Biological Significance ◽  
Giardia Duodenalis ◽  
Protozoan Parasite ◽  
Protein Translation ◽  
Mass Spectrometry Analysis ◽  
Animal Origin ◽  
Spectrometry Analysis

Giardiasis, caused by the protozoan parasite Giardia duodenalis, is an intestinal diarrheal disease affecting almost one billion people worldwide. A small endosymbiotic dsRNA viruses, G. lamblia virus (GLV), genus Giardiavirus, family Totiviridae, might inhabit human and animal isolates of G. duodenalis. Three GLV genomes have been sequenced so far, and only one was intensively studied; moreover, a positive correlation between GLV and parasite virulence is yet to be proved. To understand the biological significance of GLV infection in Giardia, the characterization of several GLV strains from naturally infected G. duodenalis isolates is necessary. Here we report high-throughput sequencing of four GLVs strains, from Giardia isolates of human and animal origin. We also report on a new, unclassified viral sequence (designed GdRV-2), unrelated to Giardiavirus, encoding and expressing for a single large protein with an RdRp domain homologous to Totiviridae and Botybirnaviridae. The result of our sequencing and proteomic analyses challenge the current knowledge on GLV and strongly suggest that viral capsid protein translation unusually starts with a proline and that translation of the RNA-dependent RNA polymerase (RdRp) occurs via a +1/−2 ribosomal frameshift mechanism. Nucleotide polymorphism, confirmed by mass-spectrometry analysis, was also observed among and between GLV strains. Phylogenetic analysis indicated the occurrence of at least two GLV subtypes which display different phenotypes and transmissibility in experimental infections of a GLV naïve Giardia isolate.

scholarly journals Functional Characterization of Sesquiterpene Synthase fromPolygonum minus

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Su-Fang Ee ◽  
Zeti-Azura Mohamed-Hussein ◽  
Roohaida Othman ◽  
Noor Azmi Shaharuddin ◽  
Ismanizan Ismail ◽  
...  
Keyword(s):  
Transgenic Plants ◽  
Metal Binding ◽  
Functional Characterization ◽  
Biological Properties ◽  
Mass Spectrometry Analysis ◽  
Gas Chromatography Mass Spectrometry ◽  
Sesquiterpene Synthase ◽  
Conserved Motifs ◽  
Spectrometry Analysis ◽  
Metal Binding Domain

Polygonum minusis an aromatic plant, which contains high abundance of terpenoids, especially the sesquiterpenes C15H24. Sesquiterpenes were believed to contribute to the many useful biological properties in plants. This study aimed to functionally characterize a full length sesquiterpene synthase gene fromP. minus.P. minussesquiterpene synthase (PmSTS) has a complete open reading frame (ORF) of 1689 base pairs encoding a 562 amino acid protein. Similar to other sesquiterpene synthases, PmSTS has two large domains: the N-terminal domain and the C-terminal metal-binding domain. It also consists of three conserved motifs: the DDXXD, NSE/DTE, and RXR. A three-dimensional protein model for PmSTS built clearly distinguished the two main domains, where conserved motifs were highlighted. We also constructed a phylogenetic tree, which showed that PmSTS belongs to the angiosperm sesquiterpene synthase subfamily Tps-a. To examine the function ofPmSTS, we expressed this gene inArabidopsis thaliana. Two transgenic lines, designated asOE3andOE7, were further characterized, both molecularly and functionally. The transgenic plants demonstrated smaller basal rosette leaves, shorter and fewer flowering stems, and fewer seeds compared to wild type plants. Gas chromatography-mass spectrometry analysis of the transgenic plants showed that PmSTS was responsible for the production ofβ-sesquiphellandrene.

scholarly journals Hunting monolignol transporters: membrane proteomics and biochemical transport assays with membrane vesicles of Norway spruce

2020 ◽  
Vol 71 (20) ◽  
pp. 6379-6395 ◽  
Author(s):  
Enni Väisänen ◽  
Junko Takahashi ◽  
Ogonna Obudulu ◽  
Joakim Bygdell ◽  
Pirkko Karhunen ◽  
...  
Keyword(s):  
Norway Spruce ◽  
Cultured Cells ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Monolignol Biosynthesis ◽  
Nicotiana Tabacum L ◽  
Km Value ◽  
Mfs Transporters

Abstract Both the mechanisms of monolignol transport and the transported form of monolignols in developing xylem of trees are unknown. We tested the hypothesis of an active, plasma membrane-localized transport of monolignol monomers, dimers, and/or glucosidic forms with membrane vesicles prepared from developing xylem and lignin-forming tissue-cultured cells of Norway spruce (Picea abies L. Karst.), as well as from control materials, comprising non-lignifying Norway spruce phloem and tobacco (Nicotiana tabacum L.) BY-2 cells. Xylem and BY-2 vesicles transported both coniferin and p-coumaryl alcohol glucoside, but inhibitor assays suggested that this transport was through the tonoplast. Membrane vesicles prepared from lignin-forming spruce cells showed coniferin transport, but the Km value for coniferin was much higher than those of xylem and BY-2 cells. Liquid chromatography-mass spectrometry analysis of membrane proteins isolated from spruce developing xylem, phloem, and lignin-forming cultured cells revealed multiple transporters. These were compared with a transporter gene set obtained by a correlation analysis with a selected set of spruce monolignol biosynthesis genes. Biochemical membrane vesicle assays showed no support for ABC-transporter-mediated monolignol transport but point to a role for secondary active transporters (such as MFS or MATE transporters). In contrast, proteomic and co-expression analyses suggested a role for ABC transporters and MFS transporters.

scholarly journals Localization of Drosophila CENP-A to non-centromeric sites depends on the NuRD complex

2019 ◽  
Author(s):  
Engin Demirdizen ◽  
Matthias Spiller-Becker ◽  
Arion Förtsch ◽  
Alexander Wilhelm ◽  
Samuel Corless ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Cultured Cells ◽  
Mass Spectrometry Analysis ◽  
Nurd Complex ◽  
Centromeric Chromatin ◽  
Spectrometry Analysis ◽  
Centromere Function ◽  
Histone H3 Variant ◽  
Associated Proteins ◽  
Specific Association

Abstract Centromere function requires the presence of the histone H3 variant CENP-A in most eukaryotes. The precise localization and protein amount of CENP-A are crucial for correct chromosome segregation, and misregulation can lead to aneuploidy. To characterize the loading of CENP-A to non-centromeric chromatin, we utilized different truncation- and localization-deficient CENP-A mutant constructs in Drosophila melanogaster cultured cells, and show that the N-terminus of Drosophila melanogaster CENP-A is required for nuclear localization and protein stability, and that CENP-A associated proteins, rather than CENP-A itself, determine its localization. Co-expression of mutant CENP-A with its loading factor CAL1 leads to exclusive centromere loading of CENP-A whereas co-expression with the histone-binding protein RbAp48 leads to exclusive non-centromeric CENP-A incorporation. Mass spectrometry analysis of non-centromeric CENP-A interacting partners identified the RbAp48-containing NuRD chromatin remodeling complex. Further analysis confirmed that NuRD is required for ectopic CENP-A incorporation, and RbAp48 and MTA1-like subunits of NuRD together with the N-terminal tail of CENP-A mediate the interaction. In summary, our data show that Drosophila CENP-A has no intrinsic specificity for centromeric chromatin and utilizes separate loading mechanisms for its incorporation into centromeric and ectopic sites. This suggests that the specific association and availability of CENP-A interacting factors are the major determinants of CENP-A loading specificity.

scholarly journals Ripply2 recruits proteasome complex for Tbx6 degradation to define segment border during murine somitogenesis

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Wei Zhao ◽  
Masayuki Oginuma ◽  
Rieko Ajima ◽  
Makoto Kiso ◽  
Akemi Okubo ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Es Cells ◽  
Mass Spectrometry Analysis ◽  
Expression Domain ◽  
Spectrometry Analysis ◽  
T Box ◽  
Induction System ◽  
Segmentation Boundary ◽  
The Stability

The metameric structure in vertebrates is based on the periodic formation of somites from the anterior end of the presomitic mesoderm (PSM). The segmentation boundary is defined by the Tbx6 expression domain, whose anterior limit is determined by Tbx6 protein destabilization via Ripply2. However, the molecular mechanism of this process is poorly understood. Here, we show that Ripply2 directly binds to Tbx6 in cultured cells without changing the stability of Tbx6, indicating an unknown mechanism for Tbx6 degradation in vivo. We succeeded in reproducing in vivo events using a mouse ES induction system, in which Tbx6 degradation occurred via Ripply2. Mass spectrometry analysis of the PSM-fated ES cells revealed that proteasomes are major components of the Ripply2-binding complex, suggesting that recruitment of a protein-degradation-complex is a pivotal function of Ripply2. Finally, we identified a motif in the T-box, which is required for Tbx6 degradation independent of binding with Ripply2 in vivo.

scholarly journals MomL, a Novel Marine-DerivedN-Acyl Homoserine Lactonase from Muricauda olearia

2014 ◽  
Vol 81 (2) ◽  
pp. 774-782 ◽  
Author(s):  
Kaihao Tang ◽  
Ying Su ◽  
Gilles Brackman ◽  
Fangyuan Cui ◽  
Yunhui Zhang ◽  
...  
Keyword(s):  
Metal Binding ◽  
Catalytic Efficiency ◽  
Quorum Quenching ◽  
Site Directed Mutagenesis ◽  
Mass Spectrometry Analysis ◽  
Infection Model ◽  
Liquid Chromatography Mass Spectrometry ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Virulence Factor Production

ABSTRACTGram-negative bacteria useN-acyl homoserine lactones (AHLs) as quorum sensing (QS) signaling molecules for interspecies communication, and AHL-dependent QS is related with virulence factor production in many bacterial pathogens. Quorum quenching, the enzymatic degradation of the signaling molecule, would attenuate virulence rather than kill the pathogens, and thereby reduce the potential for evolution of drug resistance. In a previous study, we showed thatMuricauda oleariaTh120, belonging to the classFlavobacteriia, has strong AHL degradative activity. In this study, an AHL lactonase (designated MomL), which could degrade both short- and long-chain AHLs with or without a substitution ofoxo-group at the C-3 position, was identified from Th120. Liquid chromatography-mass spectrometry analysis demonstrated that MomL functions as an AHL lactonase catalyzing AHL degradation through lactone hydrolysis. MomL is an AHL lactonase belonging to the metallo-β-lactamase superfamily that harbors an N-terminal signal peptide. The overall catalytic efficiency of MomL for C6-HSL is ∼2.9 × 105s−1M−1. Metal analysis and site-directed mutagenesis showed that, compared to AiiA, MomL has a different metal-binding capability and requires the histidine and aspartic acid residues for activity, while it shares the “HXHXDH” motif with other AHL lactonases belonging to the metallo-β-lactamase superfamily. This suggests that MomL is a representative of a novel type of secretory AHL lactonase. Furthermore, MomL significantly attenuated the virulence ofPseudomonas aeruginosain aCaenorhabditis elegansinfection model, which suggests that MomL has the potential to be used as a therapeutic agent.

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