Phosphorylation of the selective autophagy receptor TAX1BP1 by TBK1 and IKBKE/IKKi promotes ATG8-family protein-dependent clearance of MAVS aggregates.

TAX1BP1 is a selective macroautophagy/autophagy receptor that inhibits NFKB and RIGI-like receptor (RLR) signaling to prevent excessive inflammation and maintain homeostasis. Selective autophagy receptors such as SQSTM1/p62 and OPTN are phosphorylated by the kinase TBK1 to stimulate their selective autophagy function. However, it is unknown if TAX1BP1 is regulated by TBK1 or other kinases under basal conditions or during RNA virus infection. Here, we found that TBK1 and IKBKE/IKKi function redundantly to phosphorylate TAX1BP1 and regulate its autophagic turnover through canonical macroautophagy. TAX1BP1 phosphorylation promotes its localization to lysosomes, resulting in its degradation. Additionally, we found that during vesicular stomatitis virus infection, TAX1BP1 is targeted to lysosomes in an ATG8-family protein-independent manner. Furthermore, TAX1BP1 plays a critical role in the clearance of MAVS aggregates, and phosphorylation of TAX1BP1 controls its MAVS aggrephagy function. Together, our data support a model whereby TBK1 and IKBKE license TAX1BP1-selective autophagy function to inhibit MAVS and RLR signaling.Abbreviations: ATG: autophagy related; BafA1: bafilomycin A1; CALCOCO2: calcium binding and coiled-coil domain 2; GFP: green fluorescent protein; IFA: indirect immunofluorescence assay; IFN: interferon; IκB: inhibitor of nuclear factor kappa B; IKK: IκB kinase; IRF: interferon regulatory factor; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; MEF: mouse embryonic fibroblast; MOI: multiplicity of infection; IKBKG/NEMO: inhibitor of nuclear factor kappa B kinase regulatory subunit gamma; NFKB: nuclear factor kappa B; OPTN: optineurin; Poly(I:C): polyinosinic-polycytidylic acid; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RIGI: RNA sensor RIG-I; RLR: RIGI-like receptor; SDD-AGE: semi-denaturing detergent-agarose gel electrophoresis; SeV: Sendai virus; SLR: SQSTM1-like receptor; SQSTM1: sequestosome 1; TAX1BP1: Tax1 binding protein 1; TBK1: TANK binding kinase 1; TNF: tumor necrosis factor; TRAF: TNF receptor associated factor; VSV: vesicular stomatitis virus; ZnF: zinc finger.

The tyrosine kinase KDR is essential for the survival of HTLV-1-infected T cells by stabilizing the Tax oncoprotein.

Human T-cell leukemia virus type 1 (HTLV-1) infection is linked to the development of adult T-cell leukemia/lymphoma (ATLL) and the neuroinflammatory disease, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 Tax oncoprotein regulates viral gene expression and persistently activates NF-κB to maintain the viability of HTLV-1-infected T cells. Here, we utilize a kinome-wide shRNA screen to identify the tyrosine kinase KDR as an essential survival factor of HTLV-1-transformed cells. Inhibition of KDR specifically induces apoptosis of Tax expressing HTLV-1-transformed cell lines and CD4 + T cells from HAM/TSP patients. Furthermore, inhibition of KDR triggers the autophagic degradation of Tax resulting in impaired NF-κB activation and diminished viral transmission in co-culture assays. Tax induces the expression of KDR, forms a complex with KDR, and is phosphorylated by KDR. These findings suggest that Tax stability is dependent on KDR activity which could be exploited as a strategy to target Tax in HTLV-1-associated diseases.

The aryl hydrocarbon receptor-interacting protein in cancer and immunity: Beyond a chaperone protein for the dioxin receptor.

The aryl hydrocarbon receptor (AhR)-interacting protein (AIP) is a ubiquitously expressed, immunophilin-like protein best known for its role as a co-chaperone in the AhR-AIP-Hsp90 cytoplasmic complex. In addition to regulating AhR and the xenobiotic response, AIP has been linked to various aspects of cancer and immunity that will be the focus of this review article. Loss-of-function AIP mutations are associated with pituitary adenomas, suggesting that AIP acts as a tumor suppressor in the pituitary gland. However, the tumor suppressor mechanisms of AIP remain unclear, and AIP can exert oncogenic functions in other tissues. While global deletion of AIP in mice yields embryonically lethal cardiac malformations, heterozygote, and tissue-specific conditional AIP knockout mice have revealed various physiological roles of AIP. Emerging studies have established the regulatory roles of AIP in both innate and adaptive immunity. AIP interacts with and inhibits the nuclear translocation of the transcription factor IRF7 to inhibit type I interferon production. AIP also interacts with the CARMA1-BCL10-MALT1 complex in T cells to enhance IKK/NF-κB signaling and T cell activation. Taken together, AIP has diverse functions that vary considerably depending on the client protein, the tissue, and the species.

Phosphorylation of aryl hydrocarbon receptor interacting protein by TBK1 negatively regulates IRF7 and the type I interferon response.

The innate antiviral response to RNA viruses is initiated by sensing of viral RNAs by RIG-I-like receptors and elicits type I interferon (IFN) production, which stimulates the expression of IFN-stimulated genes that orchestrate the antiviral response to prevent systemic infection. Negative regulation of type I IFN and its master regulator, transcription factor IRF7, is essential to maintain immune homeostasis. We previously demonstrated that AIP (aryl hydrocarbon receptor interacting protein) functions as a negative regulator of the innate antiviral immune response by binding to and sequestering IRF7 in the cytoplasm, thereby preventing IRF7 transcriptional activation and type I IFN production. However, it remains unknown how AIP inhibition of IRF7 is regulated. We show here that the kinase TBK1 phosphorylates AIP and Thr40 serves as the primary target for TBK1 phosphorylation. AIP Thr40 plays critical roles in regulating AIP stability and mediating its interaction with IRF7. The AIP phosphomimetic T40E exhibited increased proteasomal degradation and enhanced interaction with IRF7 compared with wildtype AIP. AIP T40E also blocked IRF7 nuclear translocation, which resulted in reduced type I IFN production and increased viral replication. In sharp contrast, AIP phosphonull mutant T40A had impaired IRF7 binding, and stable expression of AIP T40A in AIP-deficient mouse embryonic fibroblasts elicited a heightened type I IFN response and diminished RNA virus replication. Taken together, these results demonstrate that TBK1-mediated phosphorylation of AIP at Thr40 functions as a molecular switch that enables AIP to interact with and inhibit IRF7, thus preventing overactivation of type I IFN genes by IRF7.

Mechanisms of Innate Immune Sensing of HTLV-1 and Viral Immune Evasion.

Human T lymphotropic virus-1 (HTLV-1) was the first identified oncoretrovirus, which infects and establishes a persistent infection in approximately 10-20 million people worldwide. Although only ~5% of infected individuals develop pathologies such as adult T-cell leukemia/lymphoma (ATLL) or a neuroinflammatory disorder termed HTLV-1-asssociated myelopathy/tropical spastic paraparesis (HAM/TSP), asymptomatic carriers are more susceptible to opportunistic infections. Furthermore, ATLL patients are severely immunosuppressed and prone to other malignancies and other infections. The HTLV-1 replication cycle provides ligands, mainly nucleic acids (RNA, RNA/DNA intermediates, ssDNA intermediates, and dsDNA), that are sensed by different pattern recognition receptors (PRRs) to trigger immune responses. However, the mechanisms of innate immune detection and immune responses to HTLV-1 infection are not well understood. In this review, we highlight the functional roles of different immune sensors in recognizing HTLV-1 infection in multiple cell types and the antiviral roles of host restriction factors in limiting persistent infection of HTLV-1. We also provide a comprehensive overview of intricate strategies employed by HTLV-1 to subvert the host innate immune response that may contribute to the development of HTLV-1-associated diseases. A more detailed understanding of HTLV-1-host pathogen interactions may inform novel strategies for HTLV-1 antivirals, vaccines, and treatments for ATLL or HAM/TSP.

Multifaceted roles of TAX1BP1 in autophagy.

TAX1BP1 is a selective macroautophagy/autophagy receptor that plays a central role in host defense to pathogens and in regulating the innate immune system. TAX1BP1 facilitates the xenophagic clearance of pathogenic bacteria such as Salmonella typhimurium and Mycobacterium tuberculosis and regulates TLR3 (toll-like receptor 3)-TLR4 and DDX58/RIG-I-like receptor (RLR) signaling by targeting TICAM1 and MAVS for autophagic degradation respectively. In addition to these canonical autophagy receptor functions, TAX1BP1 can also exert multiple accessory functions that influence the biogenesis and maturation of autophagosomes. In this review, we will discuss and integrate recent findings related to the autophagy function of TAX1BP1 and highlight outstanding questions regarding its functions in autophagy and regulation of innate immunity and host defense.Abbreviations: ATG: autophagy related; CALCOCO: calcium binding and coiled-coil domain; CC: coiled-coil; CHUK/IKKα: conserved helix-loop-helix ubiquitous kinase; CLIR: noncanonical LC3-interacting region; GABARAP: gamma-aminobutyric acid receptor associated protein; HTLV-1: human T-lymphotropic virus 1; IFN: interferon; IL1B/IL1ß: interleukin 1 beta; LIR: LC3-interacting region; LPS: lipopolysaccharide; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAPK/JNK: mitogen-activated protein kinase; mATG8: mammalian Atg8 homolog; MAVS: mitochondrial antiviral signaling protein; MEF: mouse embryonic fibroblast; MTB: Mycobacterium tuberculosis; MYD88: myeloid differentiation primary response gene 88; NBR1: NBR1, autophagy cargo receptor; NFKB/NF-κB: nuclear factor of kappa light polypeptide gene enhancer in B cells; OPTN: optineurin; Poly(I:C): polyinosinic:polycytidylic acid; PTM: post-translational modification; RB1CC1: RB1-inducible coiled-coil 1; RIPK: receptor (TNFRSF)-interacting serine-threonine kinase; RLR: DDX58/RIG-I-like receptor; RSV: respiratory syncytia virus; SKICH: SKIP carboxyl homology; SLR: SQSTM1 like receptor; SQSTM1: sequestosome 1; TAX1BP1: Tax1 (human T cell leukemia virus type I) binding protein 1; TBK1: TANK-binding kinase 1; TICAM1: toll-like receptor adaptor molecule 1; TLR: toll-like receptor; TNF: tumor necrosis factor; TNFAIP3: TNF alpha induced protein 3; TNFR: tumor necrosis factor receptor; TOM1: target of myb1 trafficking protein; TRAF: TNF receptor-associated factor; TRIM32: tripartite motif-containing 32; UBD: ubiquitin binding domain; ZF: zinc finger.

Regulation of human T-cell leukemia virus type 1 antisense promoter by myocyte enhancer factor-2C in the context of adult T-cell leukemia and lymphoma.

Adult T-cell leukemia and lymphoma (ATLL) is an intractable T-cell neoplasia caused by a retrovirus, namely human T-cell leukemia virus type 1 (HTLV-1). Patients suffering from ATLL present a poor prognosis and have a dearth of treatment options. In contrast to the sporadic expression of viral transactivator protein Tax present at the 5' promoter region long terminal repeats (LTR), HTLV-1 bZIP gene (HBZ) is encoded by 3'LTR (the antisense promoter) and maintains its constant expression in ATLL cells and patients. The antisense promoter is associated with selective retroviral gene expression and has been an understudied phenomenon. Herein, we delineate the activity of transcription factor MEF (myocyte enhancer factor)-2 family members, which were found to be enriched at the 3'LTR and play an important role in the pathogenesis of ATLL. Of the four MEF isoforms (A to D), MEF-2A and 2C were highly overexpressed in a wide array of ATLL cell lines and in acute ATLL patients. The activity of MEF-2 isoforms were determined by knockdown experiments that led to decreased cell proliferation and regulated cell cycle progression. High enrichment of MEF-2C was observed at the 3'LTR along with cofactors Menin and JunD resulting in binding of MEF-2C to HBZ at this region. Chemical inhibition of MEF-2 proteins resulted in the cytotoxicity of ATLL cells in vitro and reduction of proviral load in a humanized mouse model. Taken together, this study provides a novel mechanism of 3'LTR regulation and establishes MEF-2 signaling a potential target for therapeutic intervention for ATLL.

The E3/E4 ubiquitin conjugation factor UBE4B interacts with and ubiquitinates the HTLV-1 Tax oncoprotein to promote NF-κB activation.

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.

Lymphotropic Viruses: Chronic Inflammation and Induction of Cancers.

Inflammation induced by transcription factors, including Signal Transducers and Activators of Transcription (STATs) and NF-κB, in response to microbial pathogenic infections and ligand dependent receptors stimulation are critical for controlling infections. However, uncontrolled inflammation induced by these transcription factors could lead to immune dysfunction, persistent infection, inflammatory related diseases and the development of cancers. Although the induction of innate immunity and inflammation in response to viral infection is important to control virus replication, its effects can be modulated by lymphotropic viruses including human T-cell leukemia virus type 1 (HTLV-1), Κaposi's sarcoma herpesvirus (KSHV), and Epstein Barr virus (EBV) during de novo infection as well as latent infection. These lymphotropic viruses persistently activate JAK-STAT and NF-κB pathways. Long-term STAT and NF-κB activation by these viruses leads to the induction of chronic inflammation, which can support the persistence of these viruses and promote virus-mediated cancers. Here, we review how HTLV-1, KSHV and EBV hijack the function of host cell surface molecules (CSMs), which are involved in the regulation of chronic inflammation, innate and adaptive immune responses, cell death and the restoration of tissue homeostasis. Thus, better understanding of CSMs-mediated chronic activation of STATs and NF-κB pathways in lymphotropic virus-infected cells may pave the way for therapeutic intervention in malignancies caused by lymphotropic viruses.

Mechanisms of Oncogenesis by HTLV-1 Tax.

The human T-cell lymphotropic virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), a neoplasm of CD4+CD25+ T cells that occurs in 2-5% of infected individuals after decades of asymptomatic latent infection. Multiple HTLV-1-encoded regulatory proteins, including Tax and HTLV-1 basic leucine zipper factor (HBZ), play key roles in viral persistence and latency. The HTLV-1 Tax oncoprotein interacts with a plethora of host cellular proteins to regulate viral gene expression and also promote the aberrant activation of signaling pathways such as NF-κB to drive clonal proliferation and survival of T cells bearing the HTLV-1 provirus. Tax undergoes various post-translational modifications such as phosphorylation and ubiquitination that regulate its function and subcellular localization. Tax shuttles in different subcellular compartments for the activation of anti-apoptotic genes and deregulates the cell cycle with the induction of DNA damage for the accumulation of genomic instability that can result in cellular immortalization and malignant transformation. However, Tax is highly immunogenic and therefore HTLV-1 has evolved numerous strategies to tightly regulate Tax expression while maintaining the pool of anti-apoptotic genes through HBZ. In this review, we summarize the key findings on the oncogenic mechanisms used by Tax that set the stage for the development of ATLL, and the strategies used by HTLV-1 to tightly regulate Tax expression for immune evasion and viral persistence.

MEF-2 isoforms' (A-D) roles in development and tumorigenesis.

Myocyte enhancer factor (MEF)-2 plays a critical role in proliferation, differentiation, and development of various cell types in a tissue specific manner. Four isoforms of MEF-2 (A-D) differentially participate in controlling the cell fate during the developmental phases of cardiac, muscle, vascular, immune and skeletal systems. Through their associations with various cellular factors MEF-2 isoforms can trigger alterations in complex protein networks and modulate various stages of cellular differentiation, proliferation, survival and apoptosis. The role of the MEF-2 family of transcription factors in the development has been investigated in various cell types, and the evolving alterations in this family of transcription factors have resulted in a diverse and wide spectrum of disease phenotypes, ranging from cancer to infection. This review provides a comprehensive account on MEF-2 isoforms (A-D) from their respective localization, signaling, role in development and tumorigenesis as well as their association with histone deacetylases (HDACs), which can be exploited for therapeutic intervention.

NF-κB signaling mechanisms in HTLV-1-induced adult T-cell leukemia/lymphoma.

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.

Peroxisomes support human herpesvirus 8 latency by stabilizing the viral oncogenic protein vFLIP via the MAVS-TRAF complex.

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.

Regnase-1, a rapid response ribonuclease regulating inflammation and stress responses.

RNA-binding proteins (RBPs) are central players in post-transcriptional regulation and immune homeostasis. The ribonuclease and RBP Regnase-1 exerts critical roles in both immune cells and non-immune cells. Its expression is rapidly induced under diverse conditions including microbial infections, treatment with inflammatory cytokines and chemical or mechanical stimulation. Regnase-1 activation is transient and is subject to negative feedback mechanisms including proteasome-mediated degradation or mucosa-associated lymphoid tissue 1 (MALT1) mediated cleavage. The major function of Regnase-1 is promoting mRNA decay via its ribonuclease activity by specifically targeting a subset of genes in different cell types. In monocytes, Regnase-1 downregulates IL-6 and IL-12B mRNAs, thus mitigating inflammation, whereas in T cells, it restricts T-cell activation by targeting c-Rel, Ox40 and Il-2 transcripts. In cancer cells, Regnase-1 promotes apoptosis by inhibiting anti-apoptotic genes including Bcl2L1, Bcl2A1, RelB and Bcl3. Together with up-frameshift protein-1 (UPF1), Regnase-1 specifically cleaves mRNAs that are active during translation by recognizing a stem-loop (SL) structure within the 3'UTRs of these genes in endoplasmic reticulum-bound ribosomes. Through this mechanism, Regnase-1 rapidly shapes mRNA profiles and associated protein expression, restricts inflammation and maintains immune homeostasis. Dysregulation of Regnase-1 has been described in a multitude of pathological states including autoimmune diseases, cancer and cardiovascular diseases. Here, we provide a comprehensive update on the function, regulation and molecular mechanisms of Regnase-1, and we propose that Regnase-1 may function as a master rapid response gene for cellular adaption triggered by microenvironmental changes.

TAX1BP1 Restrains Virus-Induced Apoptosis by Facilitating Itch-Mediated Degradation of the Mitochondrial Adaptor MAVS.

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.

Monoubiquitination of survival motor neuron regulates its cellular localization and Cajal body integrity.

Low levels of the survival motor neuron (SMN) protein cause spinal muscular atrophy, the leading genetic disorder for infant mortality. SMN is ubiquitously expressed in various cell types and localizes in both the cytoplasm and the nucleus, where it concentrates in two subnuclear structures termed Cajal body (CB) and gems. In addition, SMN can also be detected in the nucleolus of neurons. Mechanisms that control SMN sorting in the cell remain largely unknown. Here, we report that the ubiquitin (Ub) ligase Itch directly interacts with and monoubiquitinates SMN. Monoubiquitination of SMN has a mild effect on promoting proteasomal degradation of SMN. We generated two SMN mutants, SMN(K0), in which all lysines are mutated to arginines and thereby abolishing SMN ubiquitination, and Ub-SMN(K0), in which a single Ub moiety is fused at the N-terminus of SMN(K0) and thereby mimicking SMN monoubiquitination. Immunostaining assays showed that SMN(K0) mainly localizes in the nucleus, whereas Ub-SMN(K0) localizes in both the cytoplasm and the nucleolus in neuronal SH-SY5Y cells. Interestingly, canonical CB foci and coilin/small nuclear ribonucleoprotein (snRNP) co-localization are significantly impaired in SH-SY5Y cells stably expressing SMN(K0) or Ub-SMN(K0). Thus, our studies discover that Itch monoubiquitinates SMN and monoubiquitination of SMN plays an important role in regulating its cellular localization. Moreover, mislocalization of SMN disrupts CB integrity and likely impairs snRNP maturation.

Aryl Hydrocarbon Receptor Interacting Protein Targets IRF7 to Suppress Antiviral Signaling and the Induction of Type I Interferon.

The transcription factor IRF7 (interferon regulatory factor 7) is a key regulator of type I interferon and plays essential roles in restricting virus infection and spread. IRF7 activation is tightly regulated to prevent excessive inflammation and autoimmunity; however, how IRF7 is suppressed by negative regulators remains poorly understood. Here, we have identified AIP (aryl hydrocarbon receptor interacting protein) as a new binding partner of IRF7. The interaction between AIP and IRF7 is enhanced upon virus infection, and AIP potently inhibits IRF7-induced type I IFN (IFNα/ß) production. Overexpression of AIP blocks virus-induced activation of IFN, whereas knockdown of AIP by siRNA potentiates virally activated IFN production. Consistently, AIP-deficient murine embryonic fibroblasts are highly resistant to virus infection because of increased production of IFNα/ß. AIP inhibits IRF7 function by antagonizing the nuclear localization of IRF7. Together, our study identifies AIP as a novel inhibitor of IRF7 and a negative regulator of innate antiviral signaling.

Myocyte enhancer factor (MEF)-2 plays essential roles in T-cell transformation associated with HTLV-1 infection by stabilizing complex between Tax and CREB.

BACKGROUND: The exact molecular mechanisms regarding HTLV-1 Tax-mediated viral gene expression and CD4 T-cell transformation have yet to be fully delineated. Herein, utilizing virus-infected primary CD4+ T cells and the virus-producing cell line, MT-2, we describe the involvement and regulation of Myocyte enhancer factor-2 (specifically MEF-2A) during the course of HTLV-1 infection and associated disease syndrome. RESULTS: Inhibition of MEF-2 expression by shRNA and its activity by HDAC9 led to reduced viral replication and T-cell transformation in correlation with a heightened expression of MEF-2 in ATL patients. Mechanistically, MEF-2 was recruited to the viral promoter (LTR, long terminal repeat) in the context of chromatin, and constituted Tax/CREB transcriptional complex via direct binding to the HTLV-1 LTR. Furthermore, an increase in MEF-2 expression was observed upon infection in an extent similar to CREB (known Tax-interacting transcription factor), and HATs (p300, CBP, and p/CAF). Confocal imaging confirmed MEF-2 co-localization with Tax and these proteins were also shown to interact by co-immunoprecipitation. MEF-2 stabilization of Tax/CREB complex was confirmed by a novel promoter-binding assay that highlighted the involvement of NFAT (nuclear factor of activated T cells) in this process via Tax-mediated activation of calcineurin (a calcium-dependent serine-threonine phosphatase). MEF-2-integrated signaling pathways (PI3K/Akt, NF-κB, MAPK, JAK/STAT, and TGF-ß) were also activated during HTLV-1 infection of primary CD4+ T cells, possibly regulating MEF-2 activity. CONCLUSIONS: We demonstrate the involvement of MEF-2 in Tax-mediated LTR activation, viral replication, and T-cell transformation in correlation with its heightened expression in ATL patients through direct binding to DNA within the HTLV-1 LTR.

Elucidating dynamic protein-protein interactions and ubiquitination in NF-κB signaling pathways.

The Nuclear factor-kappaB (NF-κB) family of transcription factors plays critical roles in inflammatory responses and host defense; however, uncontrolled NF-κB activation can be deleterious by promoting autoimmune diseases and cancers. Lysine K63 (K63)-linked polyubiquitination has emerged as an important regulatory mechanism in NF-κB signaling by regulating dynamic protein-protein interactions that trigger NF-κB signaling. RIP1 and TRAF6 serve as key substrates of K63-linked polyubiquitin chains in tumor necrosis factor receptor (TNFR) and interleukin-1 receptor (IL-1R) pathways respectively as a mechanism to recruit TAK1 and IKK kinases by associated ubiquitin-binding adaptor molecules. Activation of IKKß by TAK1 induces IκBα phosphorylation, degradation, and downstream NF-κB activation. The ubiquitin-editing enzyme A20 maintains transient NF-κB activation by opposing the K63-linked polyubiquitination of RIP1 and TRAF6. A20 inducibly interacts with the adaptor molecule TAX1BP1 and the E3 ligases Itch and RNF11 to form an A20 ubiquitin-editing enzyme complex. Notably, loss-of-function somatic mutations or polymorphisms in human A20 are associated with B-cell lymphomas or a variety of autoimmune diseases as a result of dysregulated NF-κB activation. In this chapter, we summarize the protocols routinely used in our laboratories to examine ubiquitination and NF-κB signaling.