Publications by authors named "Norah A Alturki"

5 Publications

  • Page 1 of 1

Tristetraprolin regulates necroptosis during tonic Toll-like receptor 4 (TLR4) signaling in murine macrophages.

J Biol Chem 2020 04 24;295(14):4661-4672. Epub 2020 Feb 24.

Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada

The necrosome is a protein complex required for signaling in cells that results in necroptosis, which is also dependent on tumor necrosis factor receptor (TNF-R) signaling. TNFα promotes necroptosis, and its expression is facilitated by mitogen-activated protein (MAP) kinase-activated protein kinase 2 (MK2) but is inhibited by the RNA-binding protein tristetraprolin (TTP, encoded by the gene). We have stimulated murine macrophages from WT, , , , , and mice with graded doses of lipopolysaccharide (LPS) and various inhibitors to evaluate the role of various genes in Toll-like receptor 4 (TLR4)-induced necroptosis. Necrosome signaling, cytokine production, and cell death were evaluated by immunoblotting, ELISA, and cell death assays, respectively. We observed that during TLR4 signaling, necrosome activation is mediated through the adaptor proteins MyD88 and TRIF, and this is inhibited by MK2. In the absence of MK2-mediated necrosome activation, lipopolysaccharide-induced TNFα expression was drastically reduced, but MK2-deficient cells became highly sensitive to necroptosis even at low TNFα levels. In contrast, during tonic TLR4 signaling, WT cells did not undergo necroptosis, even when MK2 was disabled. Of note, necroptosis occurred only in the absence of TTP and was mediated by the expression of TNFα and activation of JUN N-terminal kinase (JNK). These results reveal that TTP plays an important role in inhibiting TNFα/JNK-induced necrosome signaling and resultant cytotoxicity.
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http://dx.doi.org/10.1074/jbc.RA119.011633DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7135981PMC
April 2020

Differentiated macrophages acquire a pro-inflammatory and cell death-resistant phenotype due to increasing XIAP and p38-mediated inhibition of RipK1.

J Biol Chem 2018 07 13;293(30):11913-11927. Epub 2018 Jun 13.

From the Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario K1H 8M8, Canada,

Monocytes differentiate into macrophages, which deactivate invading pathogens. Macrophages can be resistant to cell death mechanisms in some situations, and the mechanisms involved are not clear. Here, using mouse immune cells, we investigated whether the differentiation of macrophages affects their susceptibility to cell death by the ripoptosome/necrosome pathways. We show that treatment of macrophages with a mimetic of second mitochondrial activator of caspases (SMAC) resulted in ripoptosome-driven cell death that specifically depended on tumor necrosis factor α (TNFα) expression and the receptor-interacting serine/threonine protein kinase 1 (RipK1)-RipK3-caspase-8 interaction in activated and cycling macrophages. Differentiation of macrophages increased the expression of pro-inflammatory cytokines but reduced RipK1-dependent cell death and the RipK3-caspase-8 interaction. The expression of the anti-apoptotic mediators, X-linked inhibitor of apoptosis protein (XIAP) and caspase-like apoptosis regulatory protein (cFLIP), also increased in differentiated macrophages, which inhibited caspase activation. The resistance to cell death was abrogated in XIAP-deficient macrophages. However, even in the presence of increased XIAP expression, inhibition of the mitogen-activated protein kinase (MAPK) p38 and MAPK-activated protein kinase 2 (MK2) made differentiated macrophages susceptible to cell death. These results suggest that the p38/MK2 pathway overrides apoptosis inhibition by XIAP and that acquisition of resistance to cell death by increased expression of XIAP and cFLIP may allow inflammatory macrophages to participate in pathogen control for a longer duration.
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http://dx.doi.org/10.1074/jbc.RA118.003614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6066316PMC
July 2018

Triad3a induces the degradation of early necrosome to limit RipK1-dependent cytokine production and necroptosis.

Cell Death Dis 2018 05 22;9(6):592. Epub 2018 May 22.

Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Canada.

Understanding the molecular signaling in programmed cell death is vital to a practical understanding of inflammation and immune cell function. Here we identify a previously unrecognized mechanism that functions to downregulate the necrosome, a central signaling complex involved in inflammation and necroptosis. We show that RipK1 associates with RipK3 in an early necrosome, independent of RipK3 phosphorylation and MLKL-induced necroptotic death. We find that formation of the early necrosome activates K48-ubiquitin-dependent proteasomal degradation of RipK1, Caspase-8, and other necrosomal proteins. Our results reveal that the E3-ubiquitin ligase Triad3a promotes this negative feedback loop independently of typical RipK1 ubiquitin editing enzymes, cIAPs, A20, or CYLD. Finally, we show that Triad3a-dependent necrosomal degradation limits necroptosis and production of inflammatory cytokines. These results reveal a new mechanism of shutting off necrosome signaling and may pave the way to new strategies for therapeutic manipulation of inflammatory responses.
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http://dx.doi.org/10.1038/s41419-018-0672-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5964080PMC
May 2018

Isolation of Camelid Single-Domain Antibodies Against Native Proteins Using Recombinant Multivalent Peptide Ligands.

Methods Mol Biol 2015 ;1348:167-89

Human Health Therapeutics Portfolio, National Research Council Canada, 100 Sussex Drive, Ottawa, ON, Canada, K1A 0R6.

Generation of antibodies against desired epitopes on folded proteins may be hampered by various characteristics of the target protein, including antigenic and immunogenic dominance of irrelevant epitopes and/or steric occlusion of the desired epitope. In such cases, peptides encompassing linear epitopes of the native protein represent attractive alternative reagents for immunization and screening. Peptide antigens are typically prepared by fusing or conjugating the peptide of interest to a carrier protein. The utility of such antigens depends on many factors including the peptide's amino acid sequence, display valency, display format (synthetic conjugate vs. recombinant fusion) and characteristics of the carrier. Here we provide detailed protocols for: (1) preparation of DNA constructs encoding peptides fused to verotoxin (VT) multimerization domain; (2) expression, purification, and characterization of the multivalent peptide-VT ligands; (3) concurrent panning of a non-immune phage-displayed camelid VHH library against the peptide-VT ligands and native protein; and (4) identification of VHHs enriched via panning using next-generation sequencing techniques. These methods are simple, rapid and can be easily adapted to yield custom peptide-VT ligands that appear to maintain the antigenic structures of the peptide. However, we caution that peptide sequences should be chosen with great care, taking into account structural, immunological, and biophysical information on the protein of interest.
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http://dx.doi.org/10.1007/978-1-4939-2999-3_16DOI Listing
June 2016

Type-I interferon signaling through ISGF3 complex is required for sustained Rip3 activation and necroptosis in macrophages.

Proc Natl Acad Sci U S A 2014 Aug 21;111(31):E3206-13. Epub 2014 Jul 21.

Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada K1N 6N5;

Myeloid cells play a critical role in perpetuating inflammation during various chronic diseases. Recently the death of macrophages through programmed necrosis (necroptosis) has emerged as an important mechanism in inflammation and pathology. We evaluated the mechanisms that lead to the induction of necrotic cell death in macrophages. Our results indicate that type I IFN (IFN-I) signaling is a predominant mechanism of necroptosis, because macrophages deficient in IFN-α receptor type I (IFNAR1) are highly resistant to necroptosis after stimulation with LPS, polyinosinic-polycytidylic acid, TNF-α, or IFN-β in the presence of caspase inhibitors. IFN-I-induced necroptosis occurred through both mechanisms dependent on and independent of Toll/IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF) and led to persistent phosphorylation of receptor-interacting protein 3 (Rip3) kinase, which resulted in potent necroptosis. Although various IFN-regulatory factors (IRFs) facilitated the induction of necroptosis in response to IFN-β, IRF-9-STAT1- or -STAT2-deficient macrophages were highly resistant to necroptosis. Our results indicate that IFN-β-induced necroptosis of macrophages proceeds through tonic IFN-stimulated gene factor 3 (ISGF3) signaling, which leads to persistent expression of STAT1, STAT2, and IRF9. Induction of IFNAR1/Rip3-dependent necroptosis also resulted in potent inflammatory pathology in vivo. These results reveal how IFN-I mediates acute inflammation through macrophage necroptosis.
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http://dx.doi.org/10.1073/pnas.1407068111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4128105PMC
August 2014
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