Publications by authors named "Parameswaran Ramakrishnan"

23 Publications

  • Page 1 of 1

Negative Regulation of FOXP3 Expression by c-Rel O-GlcNAcylation.

Glycobiology 2021 Jan 12. Epub 2021 Jan 12.

Department of Pathology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106.

O-GlcNAcylation is a reversible posttranslational protein modification that regulates fundamental cellular processes including immune responses and autoimmunity. Previously, we showed that hyperglycemia increases O-GlcNAcylation of the transcription factor, Nuclear Factor kappaB (NF-κB) c-Rel at serine residue 350 and enhances the transcription of the c-Rel-dependent proautoimmune cytokines interleukin-2 (IL-2), interferon gamma (IFNG), and granulocyte macrophage colony stimulating factor (GM-CSF) in T cells. c-Rel also plays a critical role in the transcriptional regulation of forkhead box P3 (FOXP3), the master transcription factor that governs development and function of Treg cells. Here we show that the regulatory effect of c-Rel O-GlcNAcylation is gene-dependent, and in contrast to its role in enhancing the expression of proautoimmune cytokines, it suppresses the expression of FOXP3. Hyperglycemia-induced O-GlcNAcylation-dependent suppression of FOXP3 expression was found in vivo in two mouse models of autoimmune diabetes; streptozotocin (STZ)-induced diabetes and spontaneous diabetes in non-obese diabetic (NOD) mice. Mechanistically, we show that both hyperglycemia-induced and chemically enhanced cellular O-GlcNAcylation decreases c-Rel binding at the FOXP3 promoter and negatively regulates FOXP3 expression. Mutation of the O-GlcNAcylation site in c-Rel, (serine 350 to alanine), augments T cell receptor-induced FOXP3 expression and resists the O-GlcNAcylation-dependent repression of FOXP3 expression. This study reveals c-Rel S350 O-GlcNAcylation as a novel molecular mechanism inversely regulating immunosuppressive FOXP3 expression and proautoimmune gene expression in autoimmune diabetes with potential therapeutic implications.
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http://dx.doi.org/10.1093/glycob/cwab001DOI Listing
January 2021

Molecular dynamics simulations and functional studies reveal that hBD-2 binds SARS-CoV-2 spike RBD and blocks viral entry into ACE2 expressing cells.

bioRxiv 2021 Jan 7. Epub 2021 Jan 7.

New approaches to complement vaccination are needed to combat the spread of SARS-CoV-2 and stop COVID-19 related deaths and long-term medical complications. Human beta defensin 2 (hBD-2) is a naturally occurring epithelial cell derived host defense peptide that has antiviral properties. Our comprehensive studies demonstrate that hBD-2 binds the site on the CoV-2-RBD that docks with the ACE2 receptor. Biophysical and biochemical assays confirm that hBD-2 indeed binds to the CoV-2-receptor binding domain (RBD) (K ∼ 300 nM), preventing it from binding to ACE2 expressing cells. Importantly, hBD-2 shows specificity by blocking CoV-2/spike pseudoviral infection, but not VSV-G mediated infection, of ACE2 expressing human cells with an IC of 2.4± 0.1 μM. These promising findings offer opportunities to develop hBD-2 and/or its derivatives and mimetics to safely and effectively use as novel agents to prevent SARS-CoV-2 infection.
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http://dx.doi.org/10.1101/2021.01.07.425621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7805467PMC
January 2021

A tale of two proteins: PACT and PKR and their roles in inflammation.

FEBS J 2021 Jan 2. Epub 2021 Jan 2.

Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, OH, USA.

Inflammation is a pathological hallmark associated with bacterial and viral infections, autoimmune diseases, genetic disorders, obesity and diabetes, as well as environmental stresses including physical and chemical trauma. Among numerous proteins regulating proinflammatory signaling, very few such as Protein kinase R (PKR), have been shown to play an all-pervading role in inflammation induced by varied stimuli. PKR was initially characterized as an interferon-inducible gene activated by viral double-stranded RNA with a role in protein translation inhibition. However, it has become increasingly clear that PKR is involved in multiple pathways that promote inflammation in response to stress activation, both dependent on and independent of its cellular protein activator of PKR (PACT). In this review, we discuss the signaling pathways that contribute to the initiation of inflammation, including Toll-like receptor, interferon, and RIG-I-like receptor signaling, as well as inflammasome activation. We go on to discuss the specific roles that PKR and PACT play in such proinflammatory signaling, as well as in metabolic syndrome- and environmental stress-induced inflammation.
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http://dx.doi.org/10.1111/febs.15691DOI Listing
January 2021

PACT-mediated PKR activation acts as a hyperosmotic stress intensity sensor weakening osmoadaptation and enhancing inflammation.

Elife 2020 03 16;9. Epub 2020 Mar 16.

Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, United States.

The inability of cells to adapt to increased environmental tonicity can lead to inflammatory gene expression and pathogenesis. The Rel family of transcription factors TonEBP and NF-κB p65 play critical roles in the switch from osmoadaptive homeostasis to inflammation, respectively. Here we identified PACT-mediated PKR kinase activation as a marker of the termination of adaptation and initiation of inflammation in embryonic fibroblasts. We found that high stress-induced PACT-PKR activation inhibits the interaction between NF-κB c-Rel and TonEBP essential for the increased expression of TonEBP-dependent osmoprotective genes. This resulted in enhanced formation of TonEBP/NF-κB p65 complexes and enhanced proinflammatory gene expression. These data demonstrate a novel role of c-Rel in the adaptive response to hyperosmotic stress, which is inhibited via a PACT/PKR-dependent dimer redistribution of the Rel family transcription factors. Our results suggest that inhibiting PACT-PKR signaling may prove a novel target for alleviating stress-induced inflammatory diseases.
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http://dx.doi.org/10.7554/eLife.52241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145421PMC
March 2020

Regulation of B-cell function by NF-kappaB c-Rel in health and disease.

Cell Mol Life Sci 2020 Sep 4;77(17):3325-3340. Epub 2020 Mar 4.

Department of Pathology, School of Medicine, Case Western Reserve University, University Hospitals Cleveland Medical Center, 6526, Wolstein Research Building, 2103 Cornell Road, Cleveland, OH, 44106, USA.

B cells mediate humoral immune response and contribute to the regulation of cellular immune response. Members of the Nuclear Factor kappaB (NF-κB) family of transcription factors play a major role in regulating B-cell functions. NF-κB subunit c-Rel is predominantly expressed in lymphocytes, and in B cells, it is required for survival, proliferation, and antibody production. Dysregulation of c-Rel expression and activation alters B-cell homeostasis and is associated with B-cell lymphomas and autoimmune pathologies. Based on its essential roles, c-Rel may serve as a potential prognostic and therapeutic target. This review summarizes the current understanding of the multifaceted role of c-Rel in B cells and B-cell diseases.
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http://dx.doi.org/10.1007/s00018-020-03488-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7725409PMC
September 2020

NF-κB c-Rel Dictates the Inflammatory Threshold by Acting as a Transcriptional Repressor.

iScience 2020 Mar 1;23(3):100876. Epub 2020 Feb 1.

Department of Pathology, School of Medicine, Case Western Reserve University and University Hospitals Cleveland Medical Center, 6526, Wolstein Research Building, 2103 Cornell Road, Cleveland, OH 44106, USA; Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA; The Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA. Electronic address:

NF-κB/Rel family of transcription factors plays a central role in initiation and resolution of inflammatory responses. Here, we identified a function of the NF-κB subunit c-Rel as a transcriptional repressor of inflammatory genes. Genetic deletion of c-Rel substantially potentiates the expression of several TNF-α-induced RelA-dependent mediators of inflammation. v-Rel, the viral homologue of c-Rel, but not RelB, also possesses this repressive function. Mechanistically, we found that c-Rel selectively binds to the co-repressor HDAC1 and competitively binds to the DNA mediating HDAC1 recruitment to the promoters of inflammatory genes. A specific point mutation at tyrosine in c-Rel's DNA-binding domain, for which a missense single nucleotide variation (Y25H) exists in humans, completely abrogated its ability to bind DNA and repress TNF-α-induced, RelA-mediated transcription. Our findings reveal that the transactivator NF-κB subunit c-Rel also plays a role as a transcriptional repressor in the maintenance of inflammatory homeostasis.
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http://dx.doi.org/10.1016/j.isci.2020.100876DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031323PMC
March 2020

Differential regulation of basal expression of inflammatory genes by NF-κB family subunits.

Cell Mol Immunol 2019 08 29;16(8):720-723. Epub 2019 May 29.

Department of Pathology, School of Medicine, Case Western Reserve University, Cleveland, OH, 44106, USA.

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http://dx.doi.org/10.1038/s41423-019-0242-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6804592PMC
August 2019

Hexosamine Biosynthetic Pathway Inhibition Leads to AML Cell Differentiation and Cell Death.

Mol Cancer Ther 2018 10 6;17(10):2226-2237. Epub 2018 Aug 6.

Division of Hematology/Oncology, Department of Medicine, Case Western Reserve University, 2103 Cornell Road, Cleveland, Ohio.

Treatment for acute myeloid leukemia (AML) has remained unchanged for past 40 years. Targeting cell metabolism is a promising avenue for future cancer therapy. We found that enzymes involved in metabolic hexosamine biosynthetic pathway (HBP) are increased in patients with AML. Inhibiting GFAT (the rate-limiting enzyme of HBP) induced differentiation and apoptosis in AML cells, sparing normal cells. UDP-GlcNAc, the end product of HBP, is the substrate for O-GlcNAcylation, a posttranslational modification. O-GlcNAc transferase (OGT) is the enzyme which transfers GlcNAc from UDP-GlcNAc to target proteins. Inhibition of O-GlcNAcylation, using OGT inhibitors as well as genetic knockdown of OGT, also led to cell differentiation and apoptosis of AML cells. Finally, HBP inhibition reduced the tumor growth in a subcutaneous AML xenograft model and tumor cells showed signs of differentiation A circulating AML xenograft model also showed clearance of tumor load in bone marrow, spleen, and blood, after HBP inhibition, with no signs of general toxicity. This study reveals an important role of HBP/O-GlcNAcylation in keeping AML cells in an undifferentiated state and sheds light into a new area of potential AML therapy by HBP/O-GlcNAc inhibition. .
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http://dx.doi.org/10.1158/1535-7163.MCT-18-0426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6168390PMC
October 2018

Too sweet to resist: Control of immune cell function by O-GlcNAcylation.

Cell Immunol 2018 11 2;333:85-92. Epub 2018 Jun 2.

Department of Pathology, School of Medicine, Case Western Reserve University, 6526, Wolstein Research Building, 2103 Cornell Road, Cleveland, OH 44106, USA. Electronic address:

O-linked β-N-acetyl glucosamine modification (O-GlcNAcylation) is a dynamic, reversible posttranslational modification of cytoplasmic and nuclear proteins. O-GlcNAcylation depends on nutrient availability and the hexosamine biosynthetic pathway (HBP), which produces the donor substrate UDP-GlcNAc. O-GlcNAcylation is mediated by a single enzyme, O-GlcNAc transferase (OGT), which adds GlcNAc and another enzyme, O-GlcNAcase (OGA), which removes O-GlcNAc from proteins. O-GlcNAcylation controls vital cellular processes including transcription, translation, the cell cycle, metabolism, and cellular stress. Aberrant O-GlcNAcylation has been implicated in various pathologies including Alzheimer's disease, diabetes, obesity, and cancer. Growing evidences indicate that O-GlcNAcylation plays crucial roles in regulating immunity and inflammatory responses, especially under hyperglycemic conditions. This review will highlight the emerging functions of O-GlcNAcylation in mammalian immunity under physiological and various pathological conditions.
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http://dx.doi.org/10.1016/j.cellimm.2018.05.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6275141PMC
November 2018

Protein Kinase R Mediates the Inflammatory Response Induced by Hyperosmotic Stress.

Mol Cell Biol 2017 02 1;37(4). Epub 2017 Feb 1.

Department of Genetics, Case Western Reserve University, Cleveland, Ohio, USA

High extracellular osmolarity results in a switch from an adaptive to an inflammatory gene expression program. We show that hyperosmotic stress activates the protein kinase R (PKR) independently of its RNA-binding domain. In turn, PKR stimulates nuclear accumulation of nuclear factor κB (NF-κB) p65 species phosphorylated at serine-536, which is paralleled by the induction of a subset of inflammatory NF-κB p65-responsive genes, including inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), and IL-1β. The PKR-mediated hyperinduction of iNOS decreases cell survival in mouse embryonic fibroblasts via mechanisms involving nitric oxide (NO) synthesis and posttranslational modification of proteins. Moreover, we demonstrate that the PKR inhibitor C16 ameliorates both iNOS amplification and disease-induced phenotypic breakdown of the intestinal epithelial barrier caused by an increase in extracellular osmolarity induced by dextran sodium sulfate (DSS) in vivo Collectively, these findings indicate that PKR activation is an essential part of the molecular switch from adaptation to inflammation in response to hyperosmotic stress.
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http://dx.doi.org/10.1128/MCB.00521-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5288580PMC
February 2017

Sam68 is a regulator of Toll-like receptor signaling.

Cell Mol Immunol 2017 01 4;14(1):107-117. Epub 2016 Jul 4.

Department of Pathology, School of Medicine, Case Western Reserve University and University Hospitals Case Medical Center, Cleveland, OH 44106, USA.

Recognition of pathogens by Toll-like receptors (TLR) activate multiple signaling cascades and expression of genes tailored to mount a primary immune response, inflammation, cell survival and apoptosis. Although TLR-induced activation of pathways, such as nuclear factor kappaB (NF-κB) and mitogen-activated protein kinases (MAPK), has been well studied, molecular entities controlling quantitative regulation of these pathways during an immune response remain poorly defined. We identified Sam68 as a novel regulator of TLR-induced NF-κB and MAPK activation. We found that TLR2 and TLR3 are totally dependent, whereas TLR4 is only partially dependent on Sam68 to induce the activation of NF-κB c-Rel. Absence of Sam68 greatly decreased TLR2- and TLR3-induced NF-κB p65 activation, whereas TLR4-induced p65 activation in a Sam68-independent manner. In contrast, Sam68 appeared to be a negative regulator of MAPK pathways because absence of Sam68 enhanced TLR2-induced activation of extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK). Interestingly, TLR2- and TLR3-induced gene expression showed a differential requirement of Sam68. Absence of Sam68 impaired TLR2-induced gene expression, suggesting that Sam68 has a critical role in myeloid differentiation primary response gene 88-dependent TLR2 signaling. TLR3-induced gene expression that utilize Toll/Interleukin-1 receptor-domain-containing adapter-inducing beta interferon pathway, depend only partially on Sam68. Our findings suggest that Sam68 may function as an immune rheostat that balances the activation of NF-κB p65 and c-Rel, as well as MAPK, revealing a potential novel target to manipulate TLR signaling.
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http://dx.doi.org/10.1038/cmi.2016.32DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5214940PMC
January 2017

Deficiency of Nuclear Factor-κB c-Rel Accelerates the Development of Autoimmune Diabetes in NOD Mice.

Diabetes 2016 08 23;65(8):2367-79. Epub 2016 May 23.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA

The nuclear factor-κB protein c-Rel plays a critical role in controlling autoimmunity. c-Rel-deficient mice are resistant to streptozotocin-induced diabetes, a drug-induced model of autoimmune diabetes. We generated c-Rel-deficient NOD mice to examine the role of c-Rel in the development of spontaneous autoimmune diabetes. We found that both CD4(+) and CD8(+) T cells from c-Rel-deficient NOD mice showed significantly decreased T-cell receptor-induced IL-2, IFN-γ, and GM-CSF expression. Despite compromised T-cell function, c-Rel deficiency dramatically accelerated insulitis and hyperglycemia in NOD mice along with a substantial reduction in T-regulatory (Treg) cell numbers. Supplementation of isogenic c-Rel-competent Treg cells from prediabetic NOD mice reversed the accelerated diabetes development in c-Rel-deficient NOD mice. The results suggest that c-Rel-dependent Treg cell function is critical in suppressing early-onset autoimmune diabetogenesis in NOD mice. This study provides a novel natural system to study autoimmune diabetes pathogenesis and reveals a previously unknown c-Rel-dependent mechanistic difference between chemically induced and spontaneous diabetogenesis. The study also reveals a unique protective role of c-Rel in autoimmune diabetes, which is distinct from other T-cell-dependent autoimmune diseases such as arthritis and experimental autoimmune encephalomyelitis, where c-Rel promotes autoimmunity.
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http://dx.doi.org/10.2337/db15-1607DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4955991PMC
August 2016

Repression of GSK3 restores NK cell cytotoxicity in AML patients.

Nat Commun 2016 Apr 4;7:11154. Epub 2016 Apr 4.

Department of Pathology, Case Western Reserve University, Cleveland, Ohio 44106, USA.

Natural killer cells from acute myeloid leukaemia patients (AML-NK) show a dramatic impairment in cytotoxic activity. The exact reasons for this dysfunction are not fully understood. Here we show that the glycogen synthase kinase beta (GSK3β) expression is elevated in AML-NK cells. Interestingly, GSK3 overexpression in normal NK cells impairs their ability to kill AML cells, while genetic or pharmacological GSK3 inactivation enhances their cytotoxic activity. Mechanistic studies reveal that the increased cytotoxic activity correlates with an increase in AML-NK cell conjugates. GSK3 inhibition promotes the conjugate formation by upregulating LFA expression on NK cells and by inducing ICAM-1 expression on AML cells. The latter is mediated by increased NF-κB activation in response to TNF-α production by NK cells. Finally, GSK3-inhibited NK cells show significant efficacy in human AML mouse models. Overall, our work provides mechanistic insights into the AML-NK dysfunction and a potential NK cell therapy strategy.
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http://dx.doi.org/10.1038/ncomms11154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4822012PMC
April 2016

Dual mechanisms by which miR-125b represses IRF4 to induce myeloid and B-cell leukemias.

Blood 2014 Aug 8;124(9):1502-12. Epub 2014 Jul 8.

Department of Biology, California Institute of Technology, Pasadena, CA;

The oncomir microRNA-125b (miR-125b) is upregulated in a variety of human neoplastic blood disorders and constitutive upregulation of miR-125b in mice can promote myeloid and B-cell leukemia. We found that miR-125b promotes myeloid and B-cell neoplasm by inducing tumorigenesis in hematopoietic progenitor cells. Our study demonstrates that miR-125b induces myeloid leukemia by enhancing myeloid progenitor output from stem cells as well as inducing immortality, self-renewal, and tumorigenesis in myeloid progenitors. Through functional and genetic analyses, we demonstrated that miR-125b induces myeloid and B-cell leukemia by inhibiting interferon regulatory factor 4 (IRF4) but through distinct mechanisms; it induces myeloid leukemia through repressing IRF4 at the messenger RNA (mRNA) level without altering the genomic DNA and induces B-cell leukemia via genetic deletion of the gene encoding IRF4.
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http://dx.doi.org/10.1182/blood-2014-02-553842DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4148772PMC
August 2014

Activation of the transcriptional function of the NF-κB protein c-Rel by O-GlcNAc glycosylation.

Sci Signal 2013 Aug 27;6(290):ra75. Epub 2013 Aug 27.

Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.

The transcription factor nuclear factor κB (NF-κB) rapidly reprograms gene expression in response to various stimuli, and its activity is regulated by several posttranslational modifications, including phosphorylation, methylation, and acetylation. The addition of O-linked β-N-acetylglucosamine (a process known as O-GlcNAcylation) is an abundant posttranslational modification that is enhanced in conditions such as hyperglycemia and cellular stress. We report that the NF-κB subunit c-Rel is modified and activated by O-GlcNAcylation. We identified serine 350 as the site of O-GlcNAcylation, which was required for the DNA binding and transactivation functions of c-Rel. Blocking the O-GlcNAcylation of this residue abrogated c-Rel-mediated expression of the cytokine-encoding genes IL2, IFNG, and CSF2 in response to T cell receptor (TCR) activation, whereas increasing the extent of O-GlcNAcylation of cellular proteins enhanced the expression of these genes. TCR- or tumor necrosis factor (TNF)-induced expression of other NF-κB target genes, such as NFKBIA (which encodes IκBα) and TNFAIP3 (which encodes A20), occurred independently of the O-GlcNAcylation of c-Rel. Our findings suggest a stimulus-specific role for hyperglycemia-induced O-GlcNAcylation of c-Rel in promoting T cell-mediated autoimmunity in conditions such as type 1 diabetes by enhancing the production of T helper cell cytokines.
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http://dx.doi.org/10.1126/scisignal.2004097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4066889PMC
August 2013

miR-146a controls the resolution of T cell responses in mice.

J Exp Med 2012 Aug 13;209(9):1655-70. Epub 2012 Aug 13.

Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.

T cell responses in mammals must be tightly regulated to both provide effective immune protection and avoid inflammation-induced pathology. NF-κB activation is a key signaling event induced by T cell receptor (TCR) stimulation. Dysregulation of NF-κB is associated with T cell-mediated inflammatory diseases and malignancies, highlighting the importance of negative feedback control of TCR-induced NF-κB activity. In this study we show that in mice, T cells lacking miR-146a are hyperactive in both acute antigenic responses and chronic inflammatory autoimmune responses. TCR-driven NF-κB activation up-regulates the expression of miR-146a, which in turn down-regulates NF-κB activity, at least partly through repressing the NF-κB signaling transducers TRAF6 and IRAK1. Thus, our results identify miR-146a as an important new member of the negative feedback loop that controls TCR signaling to NF-κB. Our findings also add microRNA to the list of regulators that control the resolution of T cell responses.
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http://dx.doi.org/10.1084/jem.20112218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3428948PMC
August 2012

Modulation of NF-κB-dependent gene transcription using programmable DNA minor groove binders.

Proc Natl Acad Sci U S A 2012 Jan 27;109(4):1023-8. Epub 2011 Dec 27.

Division of Chemistry and Chemical Engineering and Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.

Nuclear factor κB (NF-κB) is a transcription factor that regulates various aspects of immune response, cell death, and differentiation as well as cancer. In this study we introduce the Py-Im polyamide 1 that binds preferentially to the sequences 5'-WGGWWW-3' and 5'GGGWWW-3'. The compound is capable of binding to κB sites and reducing the expression of various NF-κB-driven genes including IL6 and IL8 by qRT-PCR. Chromatin immunoprecipitation experiments demonstrate a reduction of p65 occupancy within the proximal promoters of those genes. Genome-wide expression analysis by RNA-seq compares the DNA-binding polyamide with the well-characterized NF-κB inhibitor PS1145, identifies overlaps and differences in affected gene groups, and shows that both affect comparable numbers of TNF-α-inducible genes. Inhibition of NF-κB DNA binding via direct displacement of the transcription factor is a potential alternative to the existing antagonists.
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http://dx.doi.org/10.1073/pnas.1118506109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3268328PMC
January 2012

Sam68 is required for both NF-κB activation and apoptosis signaling by the TNF receptor.

Mol Cell 2011 Jul 27;43(2):167-79. Epub 2011 May 27.

Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.

The RNA-binding protein Sam68 is implicated in various cellular processes including RNA metabolism, apoptosis, and signal transduction. Here we identify a role of Sam68 in TNF-induced NF-κB activation and apoptosis. We found that Sam68 is recruited to the TNF receptor, and its deficiency dramatically reduces RIP recruitment and ubiquitylation. It also impairs cIAP1 recruitment and maintenance of recruited TRAF2 at the TNF receptor. In its absence, activation of the TAK1-IKK kinase complex is defective, greatly reducing signal transduction. Sam68 is also found as a part of the TNF-induced cytoplasmic caspase-8-FADD complex. RIP is not recruited to this complex in Sam68 knockout cells, and caspase activation is virtually absent. These findings delineate previously unknown functions for Sam68 in the TNF signaling pathway, where it acts as a signaling adaptor both in the membrane-associated complex I and in the cytoplasmic complex II, regulating both NF-κB activation and apoptosis.
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http://dx.doi.org/10.1016/j.molcel.2011.05.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3142289PMC
July 2011

NF-kappaB-inducing kinase is involved in the activation of the CD28 responsive element through phosphorylation of c-Rel and regulation of its transactivating activity.

J Immunol 2006 Apr;176(8):4666-74

Centro de Biología Molecular, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Madrid, Spain.

Previous evidence suggested that NF-kappaB-inducing kinase (NIK) might regulate IL-2 synthesis. However, the molecular mechanism is not understood. In this study, we show that NIK is involved in CD3 plus CD28 activation of IL-2 transcription. Splenic T cells from aly/aly mice (that have a defective NIK protein) have a severe impairment in IL-2 and GM-CSF but not TNF secretion in response to CD3/CD28. This effect takes place at the transcriptional level as overexpression of alyNIK inhibits IL-2 promoter transcription. NIK activates the CD28 responsive element (CD28RE) of the IL-2 promoter and strongly synergizes with c-Rel in this activity. We found that NIK interacts with the N-terminal domain of c-Rel, mapping this interaction to aa 771-947 of NIK. Moreover, NIK phosphorylates the c-Rel C-terminal transactivation domain (TAD) and induces Gal4-c-Rel-transactivating activity. Anti-CD28 activated Gal4-c-Rel transactivation activity, and this effect was inhibited by a NIK-defective mutant. Deletion studies mapped the region of c-Rel responsive to NIK in aa 456-540. Mutation of several serines, including Ser471, in the TAD of c-Rel abrogated the NIK-enhancing activity of its transactivating activity. Interestingly, a Jurkat mutant cell line that expresses one of the mutations of c-Rel (Ser471Asn) has a severe defect in IL-2 and CD28RE-dependent transcription in response to CD3/CD28 or to NIK. Our results support that NIK may be controlling CD28RE-dependent transcription and T cell activation by modulating c-Rel phosphorylation of the TAD. This leads to more efficient transactivation of genes which are dependent on CD28RE sites where c-Rel binds such as the IL-2 promoter.
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http://dx.doi.org/10.4049/jimmunol.176.8.4666DOI Listing
April 2006

Hsp90 recognizes a common surface on client kinases.

J Biol Chem 2006 May 21;281(20):14361-9. Epub 2006 Mar 21.

Department of Biological Regulation, Weizmann Institute of Science, Rehovot 97100, Israel.

Hsp90 is a highly abundant chaperone whose clientele includes hundreds of cellular proteins, many of which are central players in key signal transduction pathways and the majority of which are protein kinases. In light of the variety of Hsp90 clientele, the mechanism of selectivity of the chaperone toward its client proteins is a major open question. Focusing on human kinases, we have demonstrated that the chaperone recognizes a common surface in the amino-terminal lobe of kinases from diverse families, including two newly identified clients, NFkappaB-inducing kinase and death-associated protein kinase, and the oncoprotein HER2/ErbB-2. Surface electrostatics determine the interaction with the Hsp90 chaperone complex such that introduction of a negative charge within this region disrupts recognition. Compiling information on the Hsp90 dependence of 105 protein kinases, including 16 kinases whose relationship to Hsp90 is first examined in this study, reveals that surface features, rather than a contiguous amino acid sequence, define the capacity of the Hsp90 chaperone machine to recognize client kinases. Analyzing Hsp90 regulation of two major signaling cascades, the mitogen-activated protein kinase and phosphatidylinositol 3-kinase, leads us to propose that the selectivity of the chaperone to specific kinases is functional, namely that Hsp90 controls kinases that function as hubs integrating multiple inputs. These lessons bear significance to pharmacological attempts to target the chaperone in human pathologies, such as cancer.
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http://dx.doi.org/10.1074/jbc.M512613200DOI Listing
May 2006

TNF receptor (TNFR)-associated factor (TRAF) 3 serves as an inhibitor of TRAF2/5-mediated activation of the noncanonical NF-kappaB pathway by TRAF-binding TNFRs.

Proc Natl Acad Sci U S A 2005 Feb 11;102(8):2874-9. Epub 2005 Feb 11.

Institut für Immunologie der Universität München, Goethestrasse 31, 80366 Munich, Germany.

TNF family members and their receptors contribute to increased gene expression for inflammatory processes and intracellular cascades leading to programmed cell death, both via activation of NF-kappaB. TNF receptor (TNFR)-associated factors (TRAFs) are cytoplasmic adaptor proteins binding to various receptors of the TNFR family. In an attempt to delineate the role of individual TRAFs, we compared NF-kappaB activation by CD40(wt) and CD40 mutants with different TRAF recruitment patterns. Recognized only recently, NF-kappaB signaling occurs at least via two different pathways. Each pathway results in nuclear translocation of two different Reldimers, the canonical p50/RelA and the noncanonical p52/RelB. Here, we show that via TRAF6, CD40 mediates only the activation of the canonical NF-kappaB pathway. Via TRAF2/5, CD40 activates both the canonical and the noncanonical NF-kappaB pathways. We observed that TRAF3 specifically blocked the NF-kappaB activation via TRAF2/5. This inhibitory effect of TRAF3 depends on the presence of an intact zinc finger domain. Paradoxically, suppression of TRAF2/5-mediated NF-kappaB activation by TRAF3 resulted in enhanced transcriptional activity of TRAF6-mediated canonical NF-kappaB emanating from CD40. We also observed that 12 TNFR family members (p75TNFR, LTbetaR, RANK, HVEM, CD40, CD30, CD27, 4-1BB, GITR, BCMA, OX40, and TACI) are each capable of activating the alternative NF-kappaB pathway and conclude that TRAF3 serves as a negative regulator of this pathway for all tested receptors.
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http://dx.doi.org/10.1073/pnas.0500187102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC549490PMC
February 2005

Receptor-specific signaling for both the alternative and the canonical NF-kappaB activation pathways by NF-kappaB-inducing kinase.

Immunity 2004 Oct;21(4):477-89

Department of Biological Chemistry, The Weizmann Institute of Science, 76100 Rehovot, Israel.

The NF-kappaB-inducing kinase (NIK) induces proteolytic processing of NF-kappaB2/p100 and, hence, the generation of NF-kappaB dimers such as p52:RelB but was suggested not to signal for the processing of IkappaB. Here, we show that although the induction of IkappaB degradation in lymphocytes by TNF is independent of NIK, its induction by CD70, CD40 ligand, and BLyS/BAFF, which all also induce NF-kappaB2/p100 processing, does depend on NIK function. Both CD70 and TNF induce recruitment of the IKK kinase complex to their receptors. In the case of CD70, but not TNF, this process is associated with NIK recruitment and is followed by prolonged receptor association of just IKK1 and NIK. Recruitment of the IKK complex to CD27, but not that of NIK, depends on NIK kinase function. Our findings indicate that NIK participates in a unique set of proximal signaling events initiated by specific inducers, which activate both canonical and noncanonical NF-kappaB dimers.
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http://dx.doi.org/10.1016/j.immuni.2004.08.009DOI Listing
October 2004

Caspase-8 serves both apoptotic and nonapoptotic roles.

J Immunol 2004 Sep;173(5):2976-84

Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel.

Knockout of caspase-8, a cysteine protease that participates in the signaling for cell death by receptors of the TNF/nerve growth factor family, is lethal to mice in utero. To explore tissue-specific roles of this enzyme, we established its conditional knockout using the Cre/loxP recombination system. Consistent with its role in cell death induction, deletion of caspase-8 in hepatocytes protected them from Fas-induced caspase activation and death. However, application of the conditional knockout approach to investigate the cause of death of caspase-8 knockout embryos revealed that this enzyme also serves cellular functions that are nonapoptotic. Its deletion in endothelial cells resulted in degeneration of the yolk sac vasculature and embryonal death due to circulatory failure. Caspase-8 deletion in bone-marrow cells resulted in arrest of hemopoietic progenitor functioning, and in cells of the myelomonocytic lineage, its deletion led to arrest of differentiation into macrophages and to cell death. Thus, besides participating in cell death induction by receptors of the TNF/nerve growth factor family, caspase-8, apparently independently of these receptors, also mediates nonapoptotic and perhaps even antiapoptotic activities.
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http://dx.doi.org/10.4049/jimmunol.173.5.2976DOI Listing
September 2004