scholarly journals SARS-CoV-2 viroporin triggers the NLRP3 inflammatory pathway

2020 ◽  
Author(s):  
Huanzhou Xu ◽  
Siddhi A. Chitre ◽  
Ibukun A. Akinyemi ◽  
Julia C. Loeb ◽  
John A. Lednicky ◽  
...  
Keyword(s):  
Amino Acid ◽  
Inflammatory Response ◽  
Inflammasome Activation ◽  
Potassium Ions ◽  
Cytokine Storm ◽  
Amino Acid Residues ◽  
Virus Release ◽  
Prominent Feature ◽  
A Cell ◽  
Virus Isolates

AbstractCytokine storm resulting from a heightened inflammatory response is a prominent feature of severe COVID-19 disease. This inflammatory response results from assembly/activation of a cell-intrinsic defense platform known as the inflammasome. We report that the SARS-CoV-2 viroporin encoded by ORF3a activates the NLRP3 inflammasome, the most promiscuous of known inflammasomes. ORF3a triggers IL-1β expression via NFκB, thus priming the inflammasome while also activating it via ASC-dependent and -independent modes. ORF3a-mediated inflammasome activation requires efflux of potassium ions and oligomerization between NEK7 and NLRP3. With the selective NLRP3 inhibitor MCC950 able to block ORF3a-mediated inflammasome activation and key ORF3a residues needed for virus release and inflammasome activation conserved in SARS-CoV-2 isolates across continents, ORF3a and NLRP3 present prime targets for intervention.SummaryDevelopment of anti-SARS-CoV-2 therapies is aimed predominantly at blocking infection or halting virus replication. Yet, the inflammatory response is a significant contributor towards disease, especially in those severely affected. In a pared-down system, we investigate the influence of ORF3a, an essential SARS-CoV-2 protein, on the inflammatory machinery and find that it activates NLRP3, the most prominent inflammasome by causing potassium loss across the cell membrane. We also define key amino acid residues on ORF3a needed to activate the inflammatory response, and likely to facilitate virus release, and find that they are conserved in virus isolates across continents. These findings reveal ORF3a and NLRP3 to be attractive targets for therapy.

scholarly journals Regulation of N-linked core glycosylation: use of a site-directed mutagenesis approach to identify Asn-Xaa-Ser/Thr sequons that are poor oligosaccharide acceptors

1997 ◽  
Vol 323 (2) ◽  
pp. 415-419 ◽  
Author(s):  
Lakshmi KASTURI ◽  
Hegang CHEN ◽  
Susan H. SHAKIN-ESHLEMAN
Keyword(s):  
Amino Acid ◽  
Amino Acid Residue ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Free Translation ◽  
A Cell ◽  
And Function ◽  
A Site ◽  
The Impact

N-linked glycosylation can profoundly affect protein expression and function. N-linked glycosylation usually occurs at the sequon Asn-Xaa-Ser/Thr, where Xaa is any amino acid residue except Pro. However, many Asn-Xaa-Ser/Thr sequons are glycosylated inefficiently or not at all for reasons that are poorly understood. We have used a site-directed mutagenesis approach to examine how the Xaa and hydroxy (Ser/Thr) amino acid residues in sequons influence core-glycosylation efficiency. We recently demonstrated that certain Xaa amino acids inhibit core glycosylation of the sequon, Asn37-Xaa-Ser, in rabies virus glycoprotein (RGP). Here we examine the impact of different Xaa residues on core-glycosylation efficiency when the Ser residue in this sequon is replaced with Thr. The core-glycosylation efficiencies of RGP variants with different Asn37-Xaa-Ser/Thr sequons were compared by using a cell-free translation/glycosylation system. Using this approach we confirm that four Asn-Xaa-Ser sequons are poor oligosaccharide acceptors: Asn-Trp-Ser, Asn-Asp-Ser, Asn-Glu-Ser and Asn-Leu-Ser. In contrast, Asn-Xaa-Thr sequons are efficiently glycosylated, even when Xaa = Trp, Asp, Glu or Leu. A comparison of the glycosylation status of Asn-Xaa-Ser and Asn-Xaa-Thr sequons in other glycoproteins confirms that sequons with Xaa = Trp, Asp, Glu or Leu are rarely glycosylated when Ser is the hydroxy amino acid residue, and that these sequons are unlikely to serve as glycosylation sites when introduced into proteins by site-directed mutagenesis.

A Mutation in the  Subunit of the Platelet Integrin IIbβ3 Identifies a Novel Region Important for Ligand Binding

Blood ◽  
1999 ◽  
Vol 93 (3) ◽  
pp. 918-924 ◽  
Author(s):  
Eileen Collins Tozer ◽  
Elizabeth K. Baker ◽  
Mark H. Ginsberg ◽  
Joseph C. Loftus
Keyword(s):  
Amino Acid ◽  
Ligand Binding ◽  
Single Amino Acid ◽  
Chimeric Receptor ◽  
Amino Acid Residues ◽  
Genetic Approach ◽  
Specific Amino Acid ◽  
Transfected Cells ◽  
Binding Function ◽  
A Cell

Abstract An unbiased genetic approach was used to identify a specific amino acid residue in the IIb subunit important for the ligand binding function of the integrin IIbβ. Chemically mutagenized cells were selected by flow cytometry based on their inability to bind the ligand mimetic antibody PAC1 and a cell line containing a single amino acid substitution in IIb at position 224 (D→V) was identified. Although well expressed on the surface of transfected cells, IIbD224Vβ3 as well as IIbD224Aβ3 did not bind IIbβ3-specific ligands or a RGD peptide, a ligand shared in common with vβ3. Insertion of exon 5 of IIb, residues G193-W235, into the backbone of the v subunit did not enable the chimeric receptor to bind IIbβ3-specific ligands. However, the chimeric receptor was still capable of binding to a RGD affinity matrix. IIbD224 is not well conserved among other integrin  subunits and is located in a region of significant variability. In addition, amino acid D224 lies within a predicted loop of the recently proposed β-propeller model for integrin  subunits and is adjacent to a loop containing amino acid residues previously implicated in receptor function. These data support a role for this region in ligand binding function of the IIbβ3 receptor.

scholarly journals Mitochondrial ROS promote macrophage pyroptosis by inducing GSDMD oxidation

2019 ◽  
Vol 11 (12) ◽  
pp. 1069-1082 ◽  
Author(s):  
Yufang Wang ◽  
Peiliang Shi ◽  
Qin Chen ◽  
Zan Huang ◽  
Dayuan Zou ◽  
...  
Keyword(s):  
Amino Acid ◽  
Nlrp3 Inflammasome ◽  
De Novo ◽  
Oxidative Modification ◽  
Ros Generation ◽  
Inflammasome Activation ◽  
Amino Acid Residues ◽  
Caspase 1 ◽  
Hydrogen Peroxide Treatment ◽  
Mitochondrial Ros

Abstract Disrupted mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) generation are often associated with macrophage pyroptosis. It remains unclear how these forms of mitochondrial dysfunction relate to inflammasome activation and gasdermin-D (Gsdmd) cleavage, two central steps of the pyroptotic process. Here, we also found MMP collapse and ROS generation induced by Nlrp3 inflammasome activation as previous studies reported. The elimination of ROS alleviated the cleavage of Gsdmd, suggesting that Gsdmd cleavage occurs downstream of ROS release. Consistent with this result, hydrogen peroxide treatment augmented the cleavage of Gsdmd by caspase-1. Indeed, four amino acid residues of Gsdmd were oxidized under oxidative stress in macrophages. The efficiency of Gsdmd cleavage by inflammatory caspase-1 was dramatically reduced when oxidative modification was blocked by mutation of these amino acid residues. These results demonstrate that Gsdmd oxidation serves as a de novo mechanism by which mitochondrial ROS promote Nlrp3 inflammasome-dependent pyroptotic cell death.

A Mutation in the  Subunit of the Platelet Integrin IIbβ3 Identifies a Novel Region Important for Ligand Binding

Blood ◽  
1999 ◽  
Vol 93 (3) ◽  
pp. 918-924 ◽  
Author(s):  
Eileen Collins Tozer ◽  
Elizabeth K. Baker ◽  
Mark H. Ginsberg ◽  
Joseph C. Loftus
Keyword(s):  
Amino Acid ◽  
Ligand Binding ◽  
Single Amino Acid ◽  
Chimeric Receptor ◽  
Amino Acid Residues ◽  
Genetic Approach ◽  
Specific Amino Acid ◽  
Transfected Cells ◽  
Binding Function ◽  
A Cell

An unbiased genetic approach was used to identify a specific amino acid residue in the IIb subunit important for the ligand binding function of the integrin IIbβ. Chemically mutagenized cells were selected by flow cytometry based on their inability to bind the ligand mimetic antibody PAC1 and a cell line containing a single amino acid substitution in IIb at position 224 (D→V) was identified. Although well expressed on the surface of transfected cells, IIbD224Vβ3 as well as IIbD224Aβ3 did not bind IIbβ3-specific ligands or a RGD peptide, a ligand shared in common with vβ3. Insertion of exon 5 of IIb, residues G193-W235, into the backbone of the v subunit did not enable the chimeric receptor to bind IIbβ3-specific ligands. However, the chimeric receptor was still capable of binding to a RGD affinity matrix. IIbD224 is not well conserved among other integrin  subunits and is located in a region of significant variability. In addition, amino acid D224 lies within a predicted loop of the recently proposed β-propeller model for integrin  subunits and is adjacent to a loop containing amino acid residues previously implicated in receptor function. These data support a role for this region in ligand binding function of the IIbβ3 receptor.

scholarly journals Full Genomic Sequences of H5N1 Highly Pathogenic Avian Influenza Virus in Human Autopsy Specimens Reveal Genetic Variability and Adaptive Changes for Growth in MDCK Cell Cultures

2021 ◽  
Vol 2021 ◽  
pp. 1-13
Author(s):  
Kantima Sangsiriwut ◽  
Pirom Noisumdaeng ◽  
Mongkol Uiprasertkul ◽  
Jarunee Prasertsopon ◽  
Sunchai Payungporn ◽  
...  
Keyword(s):  
Amino Acid ◽  
Avian Influenza ◽  
Highly Pathogenic Avian Influenza ◽  
Mdck Cells ◽  
Amino Acid Residues ◽  
Highly Pathogenic ◽  
Viral Genomes ◽  
Pathogenic Avian Influenza ◽  
Autopsy Specimens ◽  
Virus Isolates

The entire H5N1 highly pathogenic avian influenza viral genomes were identified in the frozen autopsy specimens: the trachea, lung, colon, and intestinal feces from a patient who died of the disease in 2006. Phylogenetic analysis of the viral genomes showed that these viruses belonged to clade 1 and were the reassortants generated from the reassortment of the viruses within the same clade. The sequencing data from the autopsy specimens revealed at least 8 quasispecies of the H5N1 viruses across all 4 specimen types. These sequences were compared to those derived from the virus isolates grown in Madin Darby canine kidney (MDCK) cells. The virus isolates from the trachea, lung, and fecal specimens showed 27 nucleotide substitutions, leading to the changes of 18 amino acid residues. However, there was no change in the amino acid residues that determined the viral virulence. The changes were more commonly observed in the lung, particularly in the HA and NA genes. Our study suggested that the adaptation changes for the viral fitness to survive in a new host species (MDCK cells) might involve many genes, for example, the amino acid substitution 177G or 177W adjacent to the receptor-binding residues in the HA1 globular head and the substitution M315I in PB2. However, a mutation changes near the receptor binding domain may play an important role in determining the cell tropism and is needed to be further explored.

scholarly journals Identification of conformational neutralizing epitopes on the capsid protein of canine calicivirus

2001 ◽  
Vol 82 (7) ◽  
pp. 1695-1702 ◽  
Author(s):  
Yuichi Matsuura ◽  
Yukinobu Tohya ◽  
Masami Mochizuki ◽  
Kozo Takase ◽  
Takaaki Sugimura
Keyword(s):  
Amino Acid ◽  
Capsid Protein ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Amino Acid Residues ◽  
Conformational Epitopes ◽  
A Cell ◽  
Neutralizing Epitopes

Two neutralizing monoclonal antibodies (MAbs) against canine calicivirus (CaCV), which has a distinct antigenicity from feline calicivirus (FCV), were obtained. Both MAbs recognized conformational epitopes on the capsid protein of CaCV and were used to identify these epitopes. Neutralization-resistant variants of CaCV were selected in the presence of individual MAbs in a cell culture. Cross-neutralization tests using the variants indicated that the MAbs recognized functionally independent epitopes on the capsid protein. Recombinantly expressed ORF2 products (capsid precursors) of the variants showed no reactivity to the MAbs used for the selection, suggesting that the resistance was induced by a failing in binding of the MAbs to the variant capsid proteins. Several nucleotide changes resulting in amino acid substitutions in the capsid protein were found by sequence analysis. Reactivities of the MAbs to the revertant ORF2 products produced from each variant ORF2 by site-directed mutagenesis identified a single amino acid substitution in each variant capsid protein responsible for the failure of MAb binding. The amino acid residues related to forming the conformational neutralizing epitopes were located in regions equivalent to the 5′ and 3′ hypervariable regions of the FCV capsid protein, where antigenic sites were demonstrated in previous studies. The recombinant ORF2 products expressed in bacteria failed to induce neutralizing antibody, suggesting that neutralizing antibodies were only generated when properly folded capsid protein was used as an antigen. In CaCV, the conformational epitopes may play a more important role in neutralization than do linear epitopes.

scholarly journals Insights into polymer versus oligosaccharide synthesis: mutagenesis and mechanistic studies of a novel levansucrase from Bacillus megaterium

2007 ◽  
Vol 407 (2) ◽  
pp. 189-198 ◽  
Author(s):  
Arne Homann ◽  
Rebekka Biedendieck ◽  
Sven Götze ◽  
Dieter Jahn ◽  
Jürgen Seibel
Keyword(s):  
Amino Acid ◽  
Bacillus Megaterium ◽  
Structural Information ◽  
Oligosaccharide Synthesis ◽  
Amino Acid Residues ◽  
Sequence Alignments ◽  
Specific Amino Acid ◽  
Product Specificity ◽  
Structural Differences ◽  
A Cell

A novel levansucrase was identified in the supernatant of a cell culture of Bacillus megaterium DSM319. In order to test for the contribution of specific amino acid residues to levansucrase catalysis, the wild-type enzyme along with 16 variants based on sequence alignments and structural information were heterologously produced in Escherichia coli. The purified enzymes were characterized kinetically and the product spectrum of each variant was determined. Comparison of the X-ray structures of the levansucrases from Gram-positive Bacillus subtilis and Gram-negative Gluconacetobacter diazotrophicus in conjunction with the corresponding product spectra identified crucial amino acid residues responsible for product specificity and catalysis. Highly conserved regions such as the previously described RDP and DXXER motifs were identified as being important. Two crucial structural differences localized at amino acid residues Arg370 and Asn252 were of high relevance in polymer compared with oligosaccharide synthesis.

scholarly journals The N-terminal domain of antithrombin-III is essential for heparin binding and complex-formation with, but not cleavage by, α-thrombin

1992 ◽  
Vol 282 (2) ◽  
pp. 345-351 ◽  
Author(s):  
R C Austin ◽  
W P Sheffield ◽  
R A Rachubinski ◽  
M A Blajchman
Keyword(s):  
Amino Acid ◽  
Complex Formation ◽  
Antithrombin Iii ◽  
Amino Acid Residues ◽  
Heparin Binding ◽  
Free System ◽  
Cell Free System ◽  
Terminal Domain ◽  
At Iii ◽  
A Cell

Normal and mutant forms of human antithrombin-III (AT-III) were synthesized in a cell-free system in order to identify putative functional domains required for heparin binding and complex-formation with alpha-thrombin. Heparin-Sepharose chromatography resulted in the elution of approx. 70% of cell-free-derived normal AT-III-(1-432)-polypeptide as a peak between 0.2 M- and 0.7 M-NaCl. The cell-free-derived normal AT-III also reacted with alpha-thrombin. Approx. 15% of this AT-III formed covalent complexes with alpha-thrombin in 2 min. Unfractionated heparin accelerated the rate of formation of such complexes. Two truncated forms of AT-III (amino acid residues 219-432 and 251-432), containing only the putative thrombin-binding domain, were synthesized independently in this cell-free system. These truncated AT-III polypeptides did not bind heparin and were unable to form stable covalent complexes with alpha-thrombin. However, both of these AT-III polypeptides were cleaved by alpha-thrombin, presumably at the reactive centre Arg-393-Ser-394. The formation of the disulphide bond between Cys-247 and Cys-430 in AT-III-(219-432)-polypeptide had no effect on the results obtained. Mutations in full-length AT-III at Cys-430 had no effect on the ability of AT-III to bind heparin. There was, however, a slight decrease in the formation of stable inhibitory complexes with alpha-thrombin. A cell-free-derived AT-III mutant, devoid of amino acid residues 41-49, which comprise heparin-binding region 1 of AT-III, had slightly decreased heparin binding compared with cell-free-derived normal AT-III-(1-432)-polypeptide. This mutant AT-III polypeptide was unable, however, to form a stable complex with alpha-thrombin. We conclude therefore that the N-terminal domain of AT-III is essential for both heparin binding and complex-formation with alpha-thrombin, but not for the cleavage of AT-III at its reactive centre by alpha-thrombin.

scholarly journals Methyl-CpG-Binding Protein MeCP2 Represses Sp1-Activated Transcription of the Human Leukosialin Gene When the Promoter Is Methylated

1998 ◽  
Vol 18 (9) ◽  
pp. 5492-5499 ◽  
Author(s):  
Shinichi Kudo
Keyword(s):  
Dna Methylation ◽  
Amino Acid ◽  
Transcriptional Repression ◽  
Cell Lineage ◽  
Amino Acid Residues ◽  
A Cell ◽  
Differentiation Stage ◽  
Genomic Methylation ◽  
Protein Mecp2

ABSTRACT Human leukosialin (CD43) is expressed in a cell lineage-specific as well as a differentiation stage-specific fashion. The leukosialin promoter, made up of an Sp1 binding site and a sequence similar to that of an initiator, possesses high transcriptional potential. Previous data have demonstrated that the leukosialin gene is down-regulated in nonproducing cells by DNA methylation. In this paper the repressive mechanism of DNA methylation in expression systems is reported. In vitro DNA methylation with SssI (CpG) methylase of leukosialin-chloramphenicol acetyltransferase (CAT) constructs drastically reduced transcriptional activities in stable transfection systems with the human HeLa and Jurkat cell lines. On the other hand, the transcriptional repression by in vitro methylation was less pronounced in Drosophila melanogaster cells, which lack genomic methylation. In these cells, Sp1 could transactivate equally well both the unmethylated and methylated leukosialin promoter. In order to test whether one of the methyl-CpG-binding proteins, MeCP2, is responsible for transcriptional repression of the leukosialin gene, I isolated the human MeCP2 cDNA (encoding 486 amino acid residues) and expressed it in Drosophila cells. I found that MeCP2 substantially inhibited Sp1-activated transcription when the leukosialin promoter was methylated. The level of repression was directly proportional to the amount of MeCP2 expression vector transfected. Analysis of C-terminal deletion mutants of MeCP2 showed that repressive activity of Sp1 transactivation is localized to the N-terminal region consisting of amino acid residues 1 to 193, which encompass the methyl-binding domain. These results suggest that interference with Sp1 transactivation by MeCP2 is an important factor in the down-regulation of leukosialin gene expression by DNA methylation.

scholarly journals Peptides from a mycobacillin-synthesizing cell-free system

1972 ◽  
Vol 128 (1) ◽  
pp. 47-52 ◽  
Author(s):  
S. Sengupta ◽  
S. K. Bose
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cyclic Peptide ◽  
Single Amino Acid ◽  
Amino Acid Residues ◽  
Ph Optimum ◽  
Free System ◽  
Cell Free System ◽  
A Cell ◽  
The Given

In a cell-free system from Bacillus subtilis B3, ATP–Pi exchange was catalysed by l-proline at a pH optimum of 7.2. Further stimulation by component amino acids of mycobacillin was inhibited by deprivation from the synthesizing system of even a single amino acid occurring at any point of the cyclic peptide. This inhibition, however, decreased with the distance in the molecule of the given amino acid from l-proline. Peptides containing respectively two, three, four, five and six amino acids were isolated from the mycobacillin-synthesizing system by an amino acid-deprivation technique. The amino acid composition of these peptides and also their N- and C-terminal amino acid residues were the same as those of peptides that would be obtained if mycobacillin synthesis occurred starting from l-proline and was interrupted at various points along the polypeptide chain.

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