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1.
Nat Commun ; 14(1): 2007, 2023 04 10.
Article in English | MEDLINE | ID: mdl-37037810

ABSTRACT

Viral tropism within the brain and the role(s) of vertebrate immune response to neurotropic flaviviruses infection is largely understudied. We combine multimodal imaging (cm-nm scale) with single nuclei RNA-sequencing to study Langat virus in wildtype and interferon alpha/beta receptor knockout (Ifnar-/-) mice to visualize viral pathogenesis and define molecular mechanisms. Whole brain viral infection is imaged by Optical Projection Tomography coregistered to ex vivo MRI. Infection is limited to grey matter of sensory systems in wildtype mice, but extends into white matter, meninges and choroid plexus in Ifnar-/- mice. Cells in wildtype display strong type I and II IFN responses, likely due to Ifnb expressing astrocytes, infiltration of macrophages and Ifng-expressing CD8+ NK cells, whereas in Ifnar-/-, the absence of this response contributes to a shift in cellular tropism towards non-activated resident microglia. Multimodal imaging-transcriptomics exemplifies a powerful way to characterize mechanisms of viral pathogenesis and tropism.


Subject(s)
Encephalitis Viruses, Tick-Borne , Interferon Type I , Ticks , Mice , Animals , Interferon Type I/metabolism , Neurons/metabolism , Mice, Knockout , Brain/diagnostic imaging , Brain/metabolism , Encephalitis Viruses, Tick-Borne/genetics , Encephalitis Viruses, Tick-Borne/metabolism , Receptor, Interferon alpha-beta/genetics , Receptor, Interferon alpha-beta/metabolism , Tropism , Ticks/metabolism , Mice, Inbred C57BL
2.
Methods Mol Biol ; 2652: 439-463, 2023.
Article in English | MEDLINE | ID: mdl-37093490

ABSTRACT

Cryogenic electron microscopy (cryo-EM) is constantly developing and growing as a major technique for structure determination of protein complexes. Here, we detail the first steps of any cryo-EM project: specimen preparation and data collection. Step by step, a list of material needed is provided and the sequence of actions to carry out is given. We hope that these protocols will be useful to all people getting started with cryo-EM.


Subject(s)
Proteins , Specimen Handling , Humans , Cryoelectron Microscopy/methods , Specimen Handling/methods
3.
FEBS Lett ; 596(9): 1203-1213, 2022 05.
Article in English | MEDLINE | ID: mdl-35434785

ABSTRACT

Nonstructural protein 1 (Nsp1) of SARS-CoV-2 inhibits host cell translation through an interaction between its C-terminal domain and the 40S ribosome. The N-terminal domain (NTD) of Nsp1 is a target of recurring deletions, some of which are associated with altered COVID-19 disease progression. Here, we characterize the efficiency of translational inhibition by clinically observed Nsp1 deletion variants. We show that a frequent deletion of residues 79-89 severely reduces the ability of Nsp1 to inhibit translation while not abrogating Nsp1 binding to the 40S. Notably, while the SARS-CoV-2 5' untranslated region enhances translation of mRNA, it does not protect from Nsp1-mediated inhibition. Finally, thermal stability measurements and structure predictions reveal a correlation between stability of the NTD and the efficiency of translation inhibition.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/genetics , Humans , Protein Biosynthesis , Ribosomes/genetics , Ribosomes/metabolism , SARS-CoV-2/genetics , Viral Nonstructural Proteins/metabolism
4.
Nat Chem Biol ; 16(7): 783-790, 2020 07.
Article in English | MEDLINE | ID: mdl-32393899

ABSTRACT

Leukotrienes (LT) are lipid mediators of the inflammatory response that are linked to asthma and atherosclerosis. LT biosynthesis is initiated by 5-lipoxygenase (5-LOX) with the assistance of the substrate-binding 5-LOX-activating protein at the nuclear membrane. Here, we contrast the structural and functional consequences of the binding of two natural product inhibitors of 5-LOX. The redox-type inhibitor nordihydroguaiaretic acid (NDGA) is lodged in the 5-LOX active site, now fully exposed by disordering of the helix that caps it in the apo-enzyme. In contrast, the allosteric inhibitor 3-acetyl-11-keto-beta-boswellic acid (AKBA) from frankincense wedges between the membrane-binding and catalytic domains of 5-LOX, some 30 Å from the catalytic iron. While enzyme inhibition by NDGA is robust, AKBA promotes a shift in the regiospecificity, evident in human embryonic kidney 293 cells and in primary immune cells expressing 5-LOX. Our results suggest a new approach to isoform-specific 5-LOX inhibitor development through exploitation of an allosteric site in 5-LOX.


Subject(s)
Arachidonate 5-Lipoxygenase/chemistry , Biological Products/chemistry , Lipoxygenase Inhibitors/chemistry , Masoprocol/chemistry , Triterpenes/chemistry , Allosteric Site , Arachidonate 5-Lipoxygenase/genetics , Arachidonate 5-Lipoxygenase/metabolism , Biological Products/metabolism , Catalytic Domain , Cloning, Molecular , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Hydroxyeicosatetraenoic Acids/chemistry , Hydroxyeicosatetraenoic Acids/metabolism , Leukotriene B4/chemistry , Leukotriene B4/metabolism , Lipoxygenase Inhibitors/metabolism , Masoprocol/metabolism , Models, Molecular , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Multimerization , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity , Triterpenes/metabolism
5.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1864(4): 543-551, 2019 04.
Article in English | MEDLINE | ID: mdl-30291962

ABSTRACT

Leukotrienes (LT) are lipid mediators of the inflammatory response that play key roles in diseases such as asthma and atherosclerosis. The precursor leukotriene A4 (LTA4) is synthesized from arachidonic acid (AA) by 5­lipoxygenase (5-LOX), a membrane-associated enzyme, with the help of 5­lipoxygenase-activating protein (FLAP), a nuclear transmembrane protein. In lipoxygenases the main chain carboxylate of the C-terminus is a ligand for the non-heme iron and thus part of the catalytic center. We investigated the role of a lysine-rich sequence (KKK653-655) 20 amino acids upstream of the C-terminus unique to 5-LOX that might displace the main-chain carboxylate in the iron coordination sphere. A 5-LOX mutant in which KKK653-655 is replaced by ENL was transfected into HEK293 cells in the absence and presence of FLAP. This mutant gave ~20-fold higher 5-LOX product levels in stimulated HEK cells relative to the wild-type 5-LOX. Co-expression of the enzymes with FLAP led to an equalization of 5-LOX products detected, with wild-type 5-LOX product levels increased and those from the mutant enzyme decreased. These data suggest that the KKK motif limits 5-LOX activity and that this attenuated activity must be compensated by the presence of FLAP as a partner protein for effective LT biosynthesis.


Subject(s)
5-Lipoxygenase-Activating Proteins/metabolism , Arachidonate 5-Lipoxygenase/chemistry , Arachidonate 5-Lipoxygenase/metabolism , Leukotriene A4/metabolism , Amino Acid Motifs , Arachidonate 5-Lipoxygenase/genetics , Arachidonic Acid/metabolism , Binding Sites , HEK293 Cells , Humans , Models, Molecular , Mutation , Protein Binding
6.
Arch Biochem Biophys ; 636: 100-109, 2017 12 15.
Article in English | MEDLINE | ID: mdl-29100983

ABSTRACT

Acetyl-CoA carboxylase (ACC) in bacteria is composed of three components: biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase. ACC catalyzes the first committed step in fatty acid synthesis: the carboxylation of acetyl-CoA to form malonyl-CoA via a two-step reaction. In the first half-reaction, biotin carboxylase catalyzes the ATP-dependent carboxylation of the vitamin biotin covalently linked to biotin carboxyl carrier protein. In the second half-reaction, the carboxyl group is transferred from biotin to acetyl-CoA by the enzyme carboxyltransferase, to form malonyl-CoA. In most Gram-negative and Gram-positive bacteria, the three components of ACC form a complex that requires communication for catalysis, and is subject to feedback inhibition by acylated-acyl carrier proteins. This study investigated the mechanism of inhibition of palmitoyl-acyl carrier protein (PACP) on ACC. Unexpectedly, ACC was found to exhibit a significant hysteresis, meaning ACC was subject to inhibition by PACP in a time dependent manner. Pull-down assays demonstrated PACP does not prevent formation of the multiprotein complex, while steady-state kinetic analyses showed PACP inhibited ACC activity allosterically. Structure-activity analyses revealed that the pantothenic acid moiety of PACP is responsible for the inhibition of ACC. This study provides the first evidence of the hysteretic nature of ACC.


Subject(s)
Acetyl-CoA Carboxylase/chemistry , Acyl Carrier Protein/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/chemistry , Acetyl-CoA Carboxylase/genetics , Acetyl-CoA Carboxylase/metabolism , Acyl Carrier Protein/genetics , Acyl Carrier Protein/metabolism , Allosteric Regulation , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism
7.
J Biol Chem ; 291(37): 19413-24, 2016 09 09.
Article in English | MEDLINE | ID: mdl-27435673

ABSTRACT

The enzyme encoded by the ALOX15B gene has been linked to the development of atherosclerotic plaques in humans and in a mouse model of hypercholesterolemia. In vitro, these enzymes, which share 78% sequence identity, generate distinct products from their substrate arachidonic acid: the human enzyme, a 15-S-hydroperoxy product; and the murine enzyme, an 8-S-product. We probed the activities of these enzymes with nanodiscs as membrane mimics to determine whether they can access substrate esterified in a bilayer and characterized their activities at the membrane interface. We observed that both enzymes transform phospholipid-esterified arachidonic acid to a 15-S-product. Moreover, when expressed in transfected HEK cells, both enzymes result in significant increases in the amounts of 15-hydroxyderivatives of eicosanoids detected. In addition, we show that 15-LOX-2 is distributed at the plasma membrane when the HEK293 cells are stimulated by the addition Ca(2+) ionophore and that cellular localization is dependent upon the presence of a putative membrane insertion loop. We also report that sequence differences between the human and mouse enzymes in this loop appear to confer distinct mechanisms of enzyme-membrane interaction for the homologues.


Subject(s)
Arachidonate 15-Lipoxygenase , Arachidonic Acids , Atherosclerosis , Cell Membrane , Animals , Arachidonate 15-Lipoxygenase/chemistry , Arachidonate 15-Lipoxygenase/genetics , Arachidonate 15-Lipoxygenase/metabolism , Arachidonic Acids/chemistry , Arachidonic Acids/genetics , Arachidonic Acids/metabolism , Atherosclerosis/enzymology , Atherosclerosis/genetics , Calcium Ionophores/pharmacology , Cell Membrane/chemistry , Cell Membrane/enzymology , Cell Membrane/genetics , Disease Models, Animal , HEK293 Cells , Humans , Mice , Protein Structure, Secondary , Protein Transport/drug effects
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