12 results on '"Harhaj, Edward William"'
Search Results
2. The E3/E4 ubiquitin conjugation factor UBE4B interacts with and ubiquitinates the HTLV-1 Tax oncoprotein to promote NF-κB activation.
- Author
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Mohanty, Suchitra, Han, Teng, Choi, Young Bong, Lavorgna, Alfonso, Zhang, Jiawen, and Harhaj, Edward William
- Subjects
UBIQUITINATION ,HTLV-I ,SYNTAXINS ,ADULT T-cell leukemia ,UBIQUITIN ,CELL communication - Abstract
Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), and the neurological disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 Tax protein persistently activates the NF-κB pathway to enhance the proliferation and survival of HTLV-1 infected T cells. Lysine 63 (K63)-linked polyubiquitination of Tax provides an important regulatory mechanism that promotes Tax-mediated interaction with the IKK complex and activation of NF-κB; however, the host proteins regulating Tax ubiquitination are largely unknown. To identify new Tax interacting proteins that may regulate its ubiquitination we conducted a yeast two-hybrid screen using Tax as bait. This screen yielded the E3/E4 ubiquitin conjugation factor UBE4B as a novel binding partner for Tax. Here, we confirmed the interaction between Tax and UBE4B in mammalian cells by co-immunoprecipitation assays and demonstrated colocalization by proximity ligation assay and confocal microscopy. Overexpression of UBE4B specifically enhanced Tax-induced NF-κB activation, whereas knockdown of UBE4B impaired Tax-induced NF-κB activation and the induction of NF-κB target genes in T cells and ATLL cell lines. Furthermore, depletion of UBE4B with shRNA resulted in apoptotic cell death and diminished the proliferation of ATLL cell lines. Finally, overexpression of UBE4B enhanced Tax polyubiquitination, and knockdown or CRISPR/Cas9-mediated knockout of UBE4B attenuated both K48- and K63-linked polyubiquitination of Tax. Collectively, these results implicate UBE4B in HTLV-1 Tax polyubiquitination and downstream NF-κB activation. Author summary: Infection with the retrovirus HTLV-1 leads to the development of either CD4+CD25+ leukemia/lymphoma (ATLL) or a demyelinating neuroinflammatory disease (HAM/TSP) in a subset of infected individuals. The HTLV-1 Tax protein is a regulatory protein which regulates viral gene expression and persistently activates cellular signaling pathways such as NF-κB to drive the clonal expansion and longevity of HTLV-1 infected CD4+ T cells. Polyubiquitination of Tax is a key mechanism of NF-κB activation by assembling and activating IκB kinase (IKK) signaling complexes; however, the host factors regulating Tax ubiquitination have remained elusive. Here, we have identified the E3/E4 ubiquitin conjugation factor UBE4B as a novel Tax binding protein that promotes both K48- and K63-linked polyubiquitination of Tax. Knockdown or knockout of UBE4B impairs Tax-induced NF-κB activation and triggers apoptosis of HTLV-1-transformed cells. Therefore, UBE4B is an integral host factor that supports HTLV-1 Tax polyubiquitination, NF-κB activation and cell survival. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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- View/download PDF
3. NF‐κB signaling mechanisms in HTLV‐1‐induced adult T‐cell leukemia/lymphoma.
- Author
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Harhaj, Edward William and Giam, Chou‐Zen
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T cells , *LEUKEMIA , *HEMATOLOGIC malignancies , *CELL proliferation , *GENE expression - Abstract
The human T‐cell leukemia virus type 1 (HTLV‐1) is a complex deltaretrovirus linked to adult T‐cell leukemia/lymphoma (ATLL), a fatal CD4 + malignancy in 3–5% of infected individuals. The HTLV‐1 Tax regulatory protein plays indispensable roles in regulating viral gene expression and activating cellular signaling pathways that drive the proliferation and clonal expansion of T cells bearing HTLV‐1 proviral integrations. Tax is a potent activator of NF‐κB, a key signaling pathway that is essential for the survival and proliferation of HTLV‐1‐infected T cells. However, constitutive NF‐κB activation by Tax also triggers a senescence response, suggesting the possibility that only T cells capable of overcoming NF‐κB‐induced senescence can selectively undergo clonal expansion after HTLV‐1 infection. Tax expression is often silenced in the majority of ATLL due to genetic alterations in the tax gene or DNA hypermethylation of the 5′‐LTR. Despite the loss of Tax, NF‐κB activation remains persistently activated in ATLL due to somatic mutations in genes in the T/B‐cell receptor (T/BCR) and NF‐κB signaling pathways. In this review, we focus on the key events driving Tax‐dependent and ‐independent mechanisms of NF‐κB activation during the multistep process leading to ATLL. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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4. Peroxisomes support human herpesvirus 8 latency by stabilizing the viral oncogenic protein vFLIP via the MAVS-TRAF complex.
- Author
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Choi, Young Bong, Choi, Yeeun, and Harhaj, Edward William
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PEROXISOMES ,HERPESVIRUSES ,ONCOGENIC proteins ,GENE expression ,CYTOLOGY ,IMMUNOSTAINING - Abstract
Human herpesvirus 8 (HHV-8) is causally related to human malignancies. HHV-8 latent viral FLICE-inhibitory protein (vFLIP) is a viral oncoprotein that is linked to pathogenesis, but how its expression is regulated is largely unknown. In an attempt to understand the role of the mitochondrial antiviral signaling (MAVS) adaptor in HHV-8 infection, we discovered that vFLIP expression was post-translationally up-regulated by the MAVS signaling complex on peroxisomes. Furthermore, we demonstrated that vFLIP could be targeted to the peroxisomes, where it was oncogenically active, in a PEX19-dependent manner. Targeted disruption of vFLIP and MAVS interaction resulted in a decrease in vFLIP expression and selectively promoted death of latently HHV-8-infected cells, providing therapeutic potential for treating HHV-8 diseases. Collectively, our experimental results suggest novel involvement of peroxisomes and MAVS in the stabilization of vFLIP and thereby in the establishment or maintenance of HHV-8 latency and associated pathogenesis. [ABSTRACT FROM AUTHOR]
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- 2018
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5. TAX1BP1 Restrains Virus-Induced Apoptosis by Facilitating Itch-Mediated Degradation of the Mitochondrial Adaptor MAVS.
- Author
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Young Bong Choi, Shembade, Noula, Parvatiyar, Kislay, Balachandran, Siddharth, and Harhaj, Edward William
- Subjects
VIRUS-induced enzymes ,APOPTOSIS ,RNA virus infections ,NATURAL immunity ,VIRAL replication - Abstract
The host response to RNA virus infection consists of an intrinsic innate immune response and the induction of apoptosis as mechanisms to restrict viral replication. The mitochondrial adaptor molecule MAVS plays critical roles in coordinating both virus-induced type I interferon production and apoptosis; however, the regulation of MAVS-mediated apoptosis is poorly understood. Here, we show that the adaptor protein TAX1BP1 functions as a negative regulator of virus-induced apoptosis. TAX1BP1-deficient cells are highly sensitive to apoptosis in response to infection with the RNA viruses vesicular stomatitis virus and Sendai virus and to transfection with poly(I·C). TAX1BP1 undergoes degradation during RNA virus infection, and loss of TAX1BP1 is associated with apoptotic cell death. TAX1BP1 deficiency augments virus-induced activation of proapoptotic c-Jun N-terminal kinase (JNK) signaling. Virus infection promotes the mitochondrial localization of TAX1BP1 and concomitant interaction with the mitochondrial adaptor MAVS. TAX1BP1 recruits the E3 ligase Itch to MAVS to trigger its ubiquitination and degradation, and loss of TAX1BP1 or Itch results in increased MAVS protein expression. Together, these results indicate that TAX1BP1 functions as an adaptor molecule for Itch to target MAVS during RNA virus infection and thus restrict virus-induced apoptosis. [ABSTRACT FROM AUTHOR]
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- 2017
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6. Regulation of HTLV-1 Tax Stability, Cellular Trafficking and NF-κB Activation by the Ubiquitin-Proteasome Pathway.
- Author
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Lavorgna, Alfonso and Harhaj, Edward William
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HTLV-I , *UBIQUITIN , *NF-kappa B , *ADULT T-cell leukemia , *RETROVIRUSES , *DNA damage , *PROTEASOMES - Abstract
Human T-cell leukemia virus type 1 (HTLV-1) is a complex retrovirus that infects CD4+ T cells and causes adult T-cell leukemia/lymphoma (ATLL) in 3%-5% of infected individuals after a long latent period. HTLV-1 Tax is a trans-activating protein that regulates viral gene expression and also modulates cellular signaling pathways to enhance T-cell proliferation and cell survival. The Tax oncoprotein promotes T-cell transformation, in part via constitutive activation of the NF-κB transcription factor; however, the underlying mechanisms remain unknown. Ubiquitination is a type of post-translational modification that occurs in a three-step enzymatic cascade mediated by E1, E2 and E3 enzymes and regulates protein stability as well as signal transduction, protein trafficking and the DNA damage response. Emerging studies indicate that Tax hijacks the ubiquitin machinery to activate ubiquitin-dependent kinases and downstream NF-κB signaling. Tax interacts with the E2 conjugating enzyme Ubc13 and is conjugated on C-terminal lysine residues with lysine 63-linked polyubiquitin chains. Tax K63-linked polyubiquitination may serve as a platform for signaling complexes since this modification is critical for interactions with NEMO and IKK. In addition to NF-κB signaling, mono- and polyubiquitination of Tax also regulate its subcellular trafficking and stability. Here, we review recent advances in the diverse roles of ubiquitin in Tax function and how Tax usurps the ubiquitin-proteasome pathway to promote oncogenesis. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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7. HTLV-1 Tax Stabilizes MCL-1 via TRAF6-Dependent K63-Linked Polyubiquitination to Promote Cell Survival and Transformation.
- Author
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Choi, Young Bong and Harhaj, Edward William
- Subjects
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HTLV , *SYNTAXINS , *UBIQUITIN , *T cells , *CELL transformation - Abstract
The human T-cell leukemia virus type 1 (HTLV-1) Tax protein hijacks the host ubiquitin machinery to activate IκB kinases (IKKs) and NF-κB and promote cell survival; however, the key ubiquitinated factors downstream of Tax involved in cell transformation are unknown. Using mass spectrometry, we undertook an unbiased proteome-wide quantitative survey of cellular proteins modified by ubiquitin in the presence of Tax or a Tax mutant impaired in IKK activation. Tax induced the ubiquitination of 22 cellular proteins, including the anti-apoptotic BCL-2 family member MCL-1, in an IKK-dependent manner. Tax was found to promote the nondegradative lysine 63 (K63)-linked polyubiquitination of MCL-1 that was dependent on the E3 ubiquitin ligase TRAF6 and the IKK complex. Tax interacted with and activated TRAF6, and triggered its mitochondrial localization, where it conjugated four carboxyl-terminal lysine residues of MCL-1 with K63-linked polyubiquitin chains, which stabilized and protected MCL-1 from genotoxic stress-induced degradation. TRAF6 and MCL-1 played essential roles in the survival of HTLV-1 transformed cells and the immortalization of primary T cells by HTLV-1. Therefore, K63-linked polyubiquitination represents a novel regulatory mechanism controlling MCL-1 stability that has been usurped by a viral oncogene to precipitate cell survival and transformation. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
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8. A Critical Role for IL-17RB Signaling in HTLV-1 Tax-Induced NF-κB Activation and T-Cell Transformation.
- Author
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Lavorgna, Alfonso, Matsuoka, Masao, and Harhaj, Edward William
- Subjects
ADULT T-cell leukemia ,HTLV-I ,NF-kappa B ,T cells ,PARAPARESIS - Abstract
Human T-cell leukemia virus type 1 (HTLV-1) infection is linked to the development of adult T-cell leukemia (ATL) and the neuroinflammatory disease HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 Tax protein functions as a potent viral oncogene that constitutively activates the NF-κB transcription factor to transform T cells; however, the underlying mechanisms remain obscure. Here, using next-generation RNA sequencing we identified the IL-25 receptor subunit IL-17RB as an aberrantly overexpressed gene in HTLV-1 immortalized T cells. Tax induced the expression of IL-17RB in an IκB kinase (IKK) and NF-κB-dependent manner. Remarkably, Tax activation of the canonical NF-κB pathway in T cells was critically dependent on IL-17RB expression. IL-17RB and IL-25 were required for HTLV-1-induced immortalization of primary T cells, and the constitutive NF-κB activation and survival of HTLV-1 transformed T cells. IL-9 was identified as an important downstream target gene of the IL-17RB pathway that drives the proliferation of HTLV-1 transformed cells. Furthermore, IL-17RB was overexpressed in leukemic cells from a subset of ATL patients and also regulated NF-κB activation in some, but not all, Tax-negative ATL cell lines. Together, our results support a model whereby Tax instigates an IL-17RB-NF-κB feed-forward autocrine loop that is obligatory for HTLV-1 leukemogenesis. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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9. HSP90 Protects the Human T-Cell Leukemia Virus Type 1 (HTLV-1) Tax Oncoprotein from Proteasomal Degradation To Support NF-κB Activation and HTLV-1 Replication.
- Author
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Gao, Linlin and Harhaj, Edward William
- Subjects
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HTLV , *STATHMIN , *PROTEASOMES , *HEAT shock proteins , *VIRAL replication - Abstract
Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/ tropical spastic paraparesis (HAM/TSP). The HTLV-1 genome encodes the Tax protein that plays essential regulatory roles in HTLV-1 replication and oncogenic transformation of T lymphocytes. Despite intensive study of Tax, how Tax interfaces with host signaling pathways to regulate virus replication and drive T-cell proliferation and immortalization remains poorly understood. To gain new insight into the mechanisms of Tax function and regulation, we used tandem affinity purification and mass spectrometry to identify novel cellular Tax-interacting proteins. This screen identified heat shock protein 90 (HSP90) as a new binding partner of Tax. The interaction between HSP90 and Tax was validated by coimmunoprecipitation assays, and colocalization between the two proteins was observed by confocal microscopy. Treatment of HTLV-1-transformed cells with the HSP90 inhibitor 17-DMAG elicited proteasomal degradation of Tax in the nuclear matrix with concomitant inhibition of NF-κB and HTLV-1 long terminal repeat (LTR) activation. Knockdown of HSP90 by lentiviral shRNAs similarly provoked a loss of Tax protein in HTLV-1-transformed cells. Finally, treatment of HTLV-1-transformed cell lines with 17-DMAG suppressed HTLV-1 replication and promoted apoptotic cell death. Taken together, our results reveal that Tax is a novel HSP90 client protein and HSP90 inhibitors may exert therapeutic benefits for ATL and HAM/TSP patients. [ABSTRACT FROM AUTHOR]
- Published
- 2013
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10. Oncogenic mutations in IKKβ function through global changes induced by K63-linked ubiquitination and result in autocrine stimulation
- Author
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April N. Meyer, Alexandre Rosa Campos, Katelyn N. Nelson, Asma Siari, Daniel J. Donoghue, Juyeon Ko, Guillermo Cardenas, Thomas Whisenant, Leandro H. Gallo, and Harhaj, Edward William
- Subjects
Proteomics ,0301 basic medicine ,MAPK/ERK pathway ,Cell signaling ,Receptor complex ,Kinase Inhibitors ,lcsh:Medicine ,Signal transduction ,Pathology and Laboratory Medicine ,Biochemistry ,Mice ,0302 clinical medicine ,Models ,Medicine and Health Sciences ,Cytokine Receptor gp130 ,Enzyme assays ,Colorimetric assays ,Protein Interaction Maps ,Post-Translational Modification ,Phosphorylation ,Enzyme Inhibitors ,STAT3 ,lcsh:Science ,Bioassays and physiological analysis ,Immune Response ,Cancer ,MTT assay ,Multidisciplinary ,biology ,Chemistry ,Kinase ,Hematology ,I-kappa B Kinase ,Ubiquitin ligase ,Cell biology ,STAT signaling ,Autocrine Communication ,030220 oncology & carcinogenesis ,Signal Transduction ,Research Article ,STAT3 Transcription Factor ,General Science & Technology ,Immunoblotting ,Immunology ,Molecular Probe Techniques ,Research and Analysis Methods ,Models, Biological ,03 medical and health sciences ,Rare Diseases ,Signs and Symptoms ,Diagnostic Medicine ,Genetics ,Animals ,Humans ,Molecular Biology Techniques ,Autocrine signalling ,Molecular Biology ,Cell Proliferation ,Janus Kinases ,Oncogenic Signaling ,Inflammation ,Lysine ,lcsh:R ,Ubiquitination ,Biology and Life Sciences ,Proteins ,Oncogenes ,Biological ,HEK293 Cells ,030104 developmental biology ,Biochemical analysis ,Mutation ,Enzymology ,biology.protein ,STAT protein ,Mutant Proteins ,lcsh:Q - Abstract
Mutations at position K171 in the kinase activation loop of Inhibitor of κB kinase beta (IKKβ) occur in multiple myeloma, spleen marginal zone lymphoma and mantle cell lymphoma. Previously, we demonstrated that these result in constitutive kinase activation and stimulate Signal Transducer and Activator of Transcription 3 (STAT3). This work also identified K147 as a site of K63-linked regulatory ubiquitination required for activation of signaling pathways. We now present a more detailed analysis of ubiquitination sites together with a comprehensive examination of the signaling pathways activated by IKKβ K171E mutants. Downstream activation of STAT3 is dependent upon the activity of: UBE2N, the E2 ubiquitin ligase involved in K63-linked ubiquitination; TAK1 (MAP3K7), or TGFβ Activated Kinase, which forms a complex required for NFκB activation; JAK kinases, involved proximally in the phosphorylation of STAT transcription factors in response to inflammatory cytokines; and gp130, or IL-6 Receptor Subunit Beta which, upon binding IL-6 or other specific cytokines, undergoes homodimerization leading to activation of associated JAKs, resulting in STAT activation. We further demonstrate, using an IL-6-responsive cell line, that IKKβ K171E mutants stimulate the release of IL-6 activity into conditioned media. These results show that IKKβ K171E mutants trigger an autocrine loop in which IL-6 is secreted and binds to the IL-6 receptor complex gp130, resulting in JAK activation. Lastly, by examining the differential abundance of proteins associated with K63-only-ubiquitinated IKKβ K171E, proteomic analysis demonstrates the global activation of proliferative responses. As cancers harboring K171-mutated IKKβ are likely to also exhibit activated STAT3 and p44/42 MAPK (Erk1/2), this suggests the possibility of using MAPK (Erk1/2) and JAK inhibitors, or specific ubiquitination inhibitors. K63-linked ubiquitination occurs in other kinases at sites homologous to K147 in IKKβ, including K578 in BRAF V600E, which serves as an oncogenic driver in melanoma and other cancers.
- Published
- 2018
11. Deletion of FADD in Macrophages and Granulocytes Results in RIP3- and MyD88-Dependent Systemic Inflammation
- Author
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Suruchi N. Schock, Tina H. He, Astar Winoto, Yuefang Sun, Jennifer A. Young, and Harhaj, Edward William
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Male ,Fas-Associated Death Domain Protein ,T-Lymphocytes ,lcsh:Medicine ,Apoptosis ,Systemic inflammation ,Inbred C57BL ,Transgenic ,Mice ,0302 clinical medicine ,2.1 Biological and endogenous factors ,FADD ,Aetiology ,lcsh:Science ,Mice, Knockout ,0303 health sciences ,Multidisciplinary ,biology ,Blotting ,3. Good health ,Cell biology ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Receptor-Interacting Protein Serine-Threonine Kinases ,Female ,medicine.symptom ,Western ,Research Article ,Signal Transduction ,General Science & Technology ,Necroptosis ,T cell ,Knockout ,1.1 Normal biological development and functioning ,Blotting, Western ,Inflammation ,Enzyme-Linked Immunosorbent Assay ,Mice, Transgenic ,03 medical and health sciences ,Necrosis ,Immune system ,Underpinning research ,medicine ,Animals ,030304 developmental biology ,Innate immune system ,Integrases ,Inflammatory and immune system ,Macrophages ,lcsh:R ,Dendritic cell ,Dendritic Cells ,Mice, Inbred C57BL ,Myeloid Differentiation Factor 88 ,biology.protein ,lcsh:Q ,Granulocytes - Abstract
Myeloid cells, which include monocytes, macrophages, and granulocytes, are important innate immune cells, but the mechanism and downstream effect of their cell death on the immune system is not completely clear. Necroptosis is an alternate form of cell death that can be triggered when death receptor-mediated apoptosis is blocked, for example, in stimulated Fas-associated Death Domain (FADD) deficient cells. We report here that mice deficient for FADD in myeloid cells (mFADD-/-) exhibit systemic inflammation with elevated inflammatory cytokines and increased levels of myeloid and B cell populations while their dendritic and T cell numbers are normal. These phenotypes were abolished when RIP3 deficiency was introduced, suggesting that systemic inflammation is caused by RIP3-dependent necroptotic and/or inflammatory activity. We further found that loss of MyD88 can rescue the systemic inflammation observed in these mice. These phenotypes are surprisingly similar to that of dendritic cell (DC)-specific FADD deficient mice with the exception that DC numbers are normal in mFADD-/- mice. Together these data support the notion that innate immune cells are constantly being stimulated through the MyD88-dependent pathway and aberrations in their cell death machinery can result in systemic effects on the immune system.
- Published
- 2015
12. TAX1BP1 Restrains Virus-Induced Apoptosis by Facilitating Itch-Mediated Degradation of the Mitochondrial Adaptor MAVS.
- Author
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Choi YB, Shembade N, Parvatiyar K, Balachandran S, and Harhaj EW
- Subjects
- Animals, Apoptosis, HEK293 Cells, HeLa Cells, Humans, Mice, Sendai virus pathogenicity, Ubiquitination, Vesiculovirus pathogenicity, Adaptor Proteins, Signal Transducing metabolism, Intracellular Signaling Peptides and Proteins metabolism, Mitochondria metabolism, Neoplasm Proteins metabolism, RNA Viruses pathogenicity, Repressor Proteins metabolism, Ubiquitin-Protein Ligases metabolism
- Abstract
The host response to RNA virus infection consists of an intrinsic innate immune response and the induction of apoptosis as mechanisms to restrict viral replication. The mitochondrial adaptor molecule MAVS plays critical roles in coordinating both virus-induced type I interferon production and apoptosis; however, the regulation of MAVS-mediated apoptosis is poorly understood. Here, we show that the adaptor protein TAX1BP1 functions as a negative regulator of virus-induced apoptosis. TAX1BP1-deficient cells are highly sensitive to apoptosis in response to infection with the RNA viruses vesicular stomatitis virus and Sendai virus and to transfection with poly(I·C). TAX1BP1 undergoes degradation during RNA virus infection, and loss of TAX1BP1 is associated with apoptotic cell death. TAX1BP1 deficiency augments virus-induced activation of proapoptotic c-Jun N-terminal kinase (JNK) signaling. Virus infection promotes the mitochondrial localization of TAX1BP1 and concomitant interaction with the mitochondrial adaptor MAVS. TAX1BP1 recruits the E3 ligase Itch to MAVS to trigger its ubiquitination and degradation, and loss of TAX1BP1 or Itch results in increased MAVS protein expression. Together, these results indicate that TAX1BP1 functions as an adaptor molecule for Itch to target MAVS during RNA virus infection and thus restrict virus-induced apoptosis., (Copyright © 2016 American Society for Microbiology.)
- Published
- 2016
- Full Text
- View/download PDF
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