Purification of the full-length, membrane-associated form of the antiviral enzyme viperin utilizing nanodiscs.
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ABSTRACT:
SUBMITTER: Patel AM
PROVIDER: S-EPMC9279394 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Many enveloped viruses bud from cholesterol-rich lipid rafts on the cell membrane. Depleting cellular cholesterol impedes this process and results in viral particles with reduced viability. Viperin (Virus Inhibitory Protein, Endoplasmic Reticulum-associated, Interferon iNducible) is an endoplasmic reticulum membrane-associated enzyme that exerts broad-ranging antiviral effects, including inhibiting the budding of some enveloped viruses. However, the relationship between viperin expression and the retarded budding of virus particles from lipid rafts on the cell membrane is unclear. Here, we investigated the effect of viperin expression on cholesterol biosynthesis using transiently expressed genes in the human cell line human embryonic kidney 293T (HEK293T). We found that viperin expression reduces cholesterol levels by 20% to 30% in these cells. Following this observation, a proteomic screen of the viperin interactome identified several cholesterol biosynthetic enzymes among the top hits, including lanosterol synthase (LS) and squalene monooxygenase (SM), which are enzymes that catalyze key steps in establishing the sterol carbon skeleton. Coimmunoprecipitation experiments confirmed that viperin, LS, and SM form a complex at the endoplasmic reticulum membrane. While coexpression of viperin was found to significantly inhibit the specific activity of LS in HEK293T cell lysates, coexpression of viperin had no effect on the specific activity of SM, although did reduce SM protein levels by approximately 30%. Despite these inhibitory effects, the coexpression of neither LS nor SM was able to reverse the viperin-induced depletion of cellular cholesterol levels, possibly because viperin is highly expressed in transfected HEK293T cells. Our results establish a link between viperin expression and downregulation of cholesterol biosynthesis that helps explain viperin's antiviral effects against enveloped viruses.
| S-EPMC8254119 | biostudies-literature
Project description:Viperin, an interferon-inducible antiviral protein, is shown to bind an iron-sulfur cluster, based on iron analysis as well as UV-Vis and electron paramagnetic resonance spectroscopic data. The reduced protein contains a [4Fe-4S](1+) cluster whose g-values are altered upon addition of S-adenosylmethionine (SAM), consistent with SAM coordination to the cluster. Incubation of reduced viperin with SAM results in reductive cleavage of SAM to produce 5'-deoxyadenosine (5'-dAdo), a reaction characteristic of the radical SAM superfamily. The 5'-dAdo cleavage product was identified by a combination of HPLC and mass spectrometry analysis.
| S-EPMC3149979 | biostudies-literature
Project description:Viperin is an IFN-inducible radical S-adenosylmethionine (SAM) enzyme that inhibits viral replication. We determined crystal structures of an anaerobically prepared fragment of mouse viperin (residues 45-362) complexed with S-adenosylhomocysteine (SAH) or 5'-deoxyadenosine (5'-dAdo) and l-methionine (l-Met). Viperin contains a partial (??)6-barrel fold with a disordered N-terminal extension (residues 45-74) and a partially ordered C-terminal extension (residues 285-362) that bridges the partial barrel to form an overall closed barrel structure. Cys84, Cys88, and Cys91 located after the first ?-strand bind a [4Fe-4S] cluster. The active site architecture of viperin with bound SAH (a SAM analog) or 5'-dAdo and l-Met (SAM cleavage products) is consistent with the canonical mechanism of 5'-deoxyadenosyl radical generation. The viperin structure, together with sequence alignments, suggests that vertebrate viperins are highly conserved and that fungi contain a viperin-like ortholog. Many bacteria and archaebacteria also express viperin-like enzymes with conserved active site residues. Structural alignments show that viperin is similar to several other radical SAM enzymes, including the molybdenum cofactor biosynthetic enzyme MoaA and the RNA methyltransferase RlmN, which methylates specific nucleotides in rRNA and tRNA. The viperin putative active site contains several conserved positively charged residues, and a portion of the active site shows structural similarity to the GTP-binding site of MoaA, suggesting that the viperin substrate may be a nucleoside triphosphate of some type.
| S-EPMC5495270 | biostudies-literature
Project description:Glycinergic synapses play a central role in motor control and pain processing in the central nervous system. Glycine receptors (GlyRs) are key players in mediating fast inhibitory neurotransmission at these synapses. While previous high-resolution structures have provided insights into the molecular architecture of GlyR, several mechanistic questions pertaining to channel function are still unanswered. Here, we present Cryo-EM structures of the full-length GlyR protein complex reconstituted into lipid nanodiscs that are captured in the unliganded (closed), glycine-bound (open and desensitized), and allosteric modulator-bound conformations. A comparison of these states reveals global conformational changes underlying GlyR channel gating and modulation. The functional state assignments were validated by molecular dynamics simulations, and the observed permeation events are in agreement with the anion selectivity and conductance of GlyR. These studies provide the structural basis for gating, ion selectivity, and single-channel conductance properties of GlyR in a lipid environment.
| S-EPMC7385131 | biostudies-literature
Project description:Ecarin is a metalloproteinase found in snake venom (SVMP) with an important role in coagulation and control of hemostasis. It can specifically produce active-thrombin from prethrombin-2 and does not differentiate between normal and abnormal prothrombin. It is used in diagnostic tests and to evaluate the treatment process of many diseases. There are many drawbacks associated with separating these compounds from snake venom. Therefore, in this study, full-length recombinant Ecarin (r-Ecarin) was cloned, expressed, and purified in eukaryotic host cells. To determine the most effective form of the enzyme, r-Ecarin was compared with the recombinant truncated form, which has only the metalloprotease domain of the protein (r-Ecamet) in terms of function and expression. Briefly, A DNA construct composed of sequence-encoding Ecarin was designed and cloned into pCAGGS expression vector and, subsequently, expressed in Chinese Hamster Ovary (CHO) cells. To identify the enzymatic activity of expressed protein, a bioactivity assay was performed. Blood coagulation time and expression levels of r-Ecarin and r-Ecamet proteins were compared. Also, a histopathological assessment was carried out on the liver of mice treated with these proteins. Comparison of r-Ecarin and r-Ecamet expression pattern demonstrated that full-length Ecarin expression has at least 2-fold higher expression in eukaryotic cells. Determination of r-Ecarin function proved that this protein is capable of prothrombin cleavage and producing thrombin. Comparison of PT test results between the r-Ecarin and r-Ecamet showed that there is a significant difference in the activity of the two enzymes and the full-length protein coagulates the blood in less time.
| S-EPMC9191215 | biostudies-literature
Project description:Viperin is a radical SAM enzyme that has been shown to possess antiviral activity against a broad spectrum of viruses; however, its molecular mechanism is unknown. We report here that recombinant fungal and archaeal viperin enzymes catalyze the addition of the 5'-deoxyadenosyl radical (5'-dA•) to the double bond of isopentenyl pyrophosphate (IPP), producing a new compound we named adenylated isopentyl pyrophosphate (AIPP). The reaction is specific for IPP, as other pyrophosphate compounds involved in the mevalonate biosynthetic pathway did not react with 5'-dA• Enzymatic reactions employing IPP derivatives as substrates revealed that any chemical change in IPP diminishes its ability to be an effective substrate of fungal viperin. Mutational studies disclosed that the hydroxyl group on the side chain of Tyr-245 in fungal viperin is the likely source of hydrogen in the last step of the radical addition, providing mechanistic insight into the radical reaction catalyzed by fungal viperin. Structure-based molecular dynamics (MD) simulations of viperin interacting with IPP revealed a good fit of the isopentenyl motif of IPP to the active site cavity of viperin, unraveling the molecular basis of substrate specificity of viperin for IPP. Collectively, our findings indicate that IPP is an effective substrate of fungal and archaeal viperin enzymes and provide critical insights into the reaction mechanism.
| S-EPMC6130951 | biostudies-literature
Project description:Fucosyltransferase III [galactoside 3(4)-L-fucosyltransferase; EC 2.4.1.65] (FT3) is a Golgi type II membrane protein that catalyses the synthesis of fucosylated Lewis motifs that are associated with cell-adhesion events and are differentially expressed during cell differentiation. In the present work, the full-length membrane bound form of FT3 has been expressed in baby hamster kidney cells. The enzyme has been found in the trans-Golgi and trans-Golgi network (TGN) of the transfected cells, where it appeared as monomers and dimers, but not as oligomers with high molecular masses. Therefore oligomerization is not the basis for correct localization of FT3 in the Golgi. The enzyme has been purified, with a final yield of 2% and a total purification of 2900-fold, by DEAE-Sepharose, SP-Sepharose, GDP-Fractogel and Superdex 200 chromatography. The purified enzyme showed a clear preference for the Gal beta 3GlcNAc motif in oligosaccharides conjugated with the hydrophobic tail (CH(2))(3)-NHCO-(CH(2))(5)-NH-biotin. Substitution of galactose with alpha 2-linked fucose or alpha 2,3-linked N-acetylneuraminic acid yielded a 1.9-fold increase or a 43% decrease in activity respectively. The enzyme showed no activity towards asialofetuin, a glycoprotein containing the Gal beta 3GlcNAc acceptor motif. Therefore it has been concluded that the membrane-bound form of FT3 is present in the Golgi and the TGN in an equilibrium of monomers<-->dimers, which might fucosylate glycans from glycolipids, but not from glycoproteins.
| S-EPMC1222010 | biostudies-other
Project description:The ribosome is a two-subunit, macromolecular machine composed of RNA and proteins that carries out the polymerization of α-amino acids into polypeptides. Efforts to engineer ribosomal RNA (rRNA) deepen our understanding of molecular translation and provide opportunities to expand the chemistry of life by creating ribosomes with altered properties. Toward these efforts, reverse transcription PCR (RT-PCR) of the entire 16S and 23S rRNAs, which make up the 30S small subunit and 50S large subunit, respectively, is important for isolating desired phenotypes. However, reverse transcription of rRNA is challenging due to extensive secondary structure and post-transcriptional modifications. One key challenge is that existing commercial kits for RT-PCR rely on reverse transcriptases that lack the extreme thermostability and processivity found in many commercial DNA polymerases, which can result in subpar performance on challenging templates. Here, we develop methods employing a synthetic thermostable reverse transcriptase (RTX) to enable and optimize RT-PCR of the complete Escherichia coli 16S and 23S rRNAs. We also characterize the error rate of RTX when traversing the various post-transcriptional modifications of the 23S rRNA. We anticipate that this work will facilitate efforts to study and characterize many naturally occurring long RNAs and to engineer the translation apparatus for synthetic biology.
| S-EPMC7772474 | biostudies-literature
Project description:Selenoprotein K (SelK) is a membrane protein residing in the endoplasmic reticulum. The function of SelK is mostly unknown; however, it has been shown to participate in anti-oxidant defense, calcium regulation and in the endoplasmic reticulum associated protein degradation (ERAD) pathway. In order to study the function of SelK and the role of selenocysteine in catalysis, we have tested heterologous expression of human SelK in E. coli. Consequently, we have developed an over-expression strategy that exploits the maltose binding protein as a fusion partner to stabilize and solubilize SelK. The fusion partner can be cleaved from SelK in the presence of a variety of detergents compatible with structural characterization and the protein purified to homogeneity. SelK acquires a helical secondary structure in detergent micelles, even though it was predicted to be an intrinsically disordered protein due to its high percentage of polar residues. The same strategy was successfully applied to preparation of SelK binding partner - selenoprotein S (SelS). Hence, this heterologous expression and purification strategy can be applied to other members of the membrane enzyme family to which SelK belongs.
| S-EPMC3483405 | biostudies-literature
Project description:BackgroundAccurate identification of bacterial communities is crucial for research applications, diagnostics, and clinical interventions. Although 16S ribosomal RNA (rRNA) gene sequencing is a widely employed technique for bacterial taxonomic classification, it often results in misclassified or unclassified bacterial taxa. This study sought to refine the full-length 16S rRNA gene sequencing protocol using the MinION sequencer, focusing on the V1-V9 regions. Our methodological enquiry examined several factors, including the number of PCR amplification cycles, choice of primers and Taq polymerase, and specific sequence databases and workflows employed. We used a microbial standard comprising eight bacterial strains (five gram-positive and three gram-negative) in known proportions as a validation control.ResultsBased on the MinION protocol, we employed the microbial standard as the DNA template for the 16S rRNA gene amplicon sequencing procedure. Our analysis showed that an elevated number of PCR amplification cycles introduced PCR bias, and the selection of Taq polymerase and primer sets significantly affected the subsequent analysis. Bacterial identification at genus level demonstrated Pearson correlation coefficients ranging from 0.73 to 0.79 when assessed using BugSeq, Kraken-Silva and EPI2ME-16S workflows. Notably, the EPI2ME-16S workflow exhibited the highest Pearson correlation with the microbial standard, minimised misclassification, and increased alignment accuracy. At the species taxonomic level, the BugSeq workflow was superior, with a Pearson correlation coefficient of 0.92.ConclusionsThese findings emphasise the importance of careful selection of PCR settings and a well-structured analytical framework for 16S rRNA full-length gene sequencing. The results showed a robust correlation between the predicted and observed bacterial abundances at both the genus and species taxonomic levels, making these findings applicable across diverse research contexts and with clinical utility for reliable pathogen identification.
| S-EPMC10870487 | biostudies-literature