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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/IL1β: 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.

Mitochondria support multiple cell functions, but an accumulation of dysfunctional or excessive mitochondria is detrimental to cells. We previously demonstrated that a defect in the autophagic removal of mitochondria, termed mitophagy, leads to the acceleration of apoptosis induced by herpesvirus productive infection. However, the exact molecular mechanisms underlying activation of mitophagy and regulation of apoptosis remain poorly understood despite the identification of various mitophagy-associated proteins. Here, we report that the mitochondrial translation elongation factor Tu, a mitophagy-associated protein encoded by the TUFM gene, locates in part on the outer membrane of mitochondria (OMM) where it acts as an inhibitor of altered mitochondria-induced apoptosis through its autophagic function. Inducible depletion of TUFM potentiated caspase-8-mediated apoptosis in virus-infected cells with accumulation of altered mitochondria. In addition, TUFM depletion promoted caspase-8 activation induced by treatment with TNF-related apoptosis-inducing ligand in cancer cells, potentially via dysregulation of mitochondrial dynamics and mitophagy. Importantly, we revealed the existence of and structural requirements for autophagy-competent TUFM on the OMM; the GxxxG motif within the N-terminal mitochondrial targeting sequences of TUFM was required for self-dimerization and mitophagy. Furthermore, we found that autophagy-competent TUFM was subject to ubiquitin-proteasome-mediated degradation but stabilized upon mitophagy or autophagy activation. Moreover, overexpression of autophagy-competent TUFM could inhibit caspase-8 activation. These studies extend our knowledge of mitophagy regulation of apoptosis and could provide a novel strategic basis for targeted therapy of cancer and viral diseases.

TAX1BP1 is a selective autophagy receptor which inhibits NF-κB and RIG-I-like receptor (RLR) signaling to prevent excessive inflammation and maintain homeostasis. Selective autophagy receptors such as p62/SQSTM1 and OPTN are phosphorylated by the noncanonical IκB 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 the noncanonical IκB kinases TBK1 and IKKi phosphorylate TAX1BP1 to regulate its basal turnover, whereas the canonical IκB kinase IKKα and the core autophagy factor ATG9 play essential roles in RNA virus-mediated TAX1BP1 autophagosomal degradation. TAX1BP1 phosphorylation by canonical and noncanonical IκB kinases promotes its localization to lysosomes resulting in its degradation. Furthermore, TAX1BP1 plays a critical role in the clearance of MAVS aggregates, and phosphorylation of TAX1BP1 augments its MAVS aggrephagy function. Together, our data support a model whereby IκB kinases license TAX1BP1 selective autophagy function to inhibit MAVS and RLR signaling.Author SummaryThe RIG-I-like receptor (RLR) pathway induces type I interferon (IFN) and proinflammatory cytokines in response to RNA virus infection. MAVS is a mitochondrial adaptor protein in the RLR pathway that forms prion-like aggregates upon activation; however, how MAVS aggregates are cleared to restore homeostasis is unclear. Autophagy is a lysosomal degradation pathway important for the clearance of potentially cytotoxic protein aggregates that could induce inflammation and/or cell death. TAX1BP1 is a selective autophagy receptor that inhibits RLR signaling, but the precise mechanisms remain unknown. Here, we found that TAX1BP1 is a substrate for multi-site phosphorylation by canonical and noncanonical IκB kinases which triggered its lysosomal localization and degradation. We also found that TAX1BP1 was critical for the clearance of MAVS aggregates in a phosphorylation-dependent manner. Overall, our data suggest that phosphorylation serves a key regulatory function for TAX1BP1 to inhibit RLR signaling.

// Mai-Tram Vo 1 , Seong Hee Choi 1 , Ji-Heon Lee 2 , Chung Hwan Hong 1 , Jong Soo Kim 2 , Unn Hwa Lee 1 , Hyung-Min Chung 2 , Byung Ju Lee 1 , Jeong Woo Park 1 and Wha Ja Cho 1 1 Department of Biological Sciences, University of Ulsan, Ulsan, 680-749, Korea 2 Department of Stem Cell Biology, School of Medicine, Konkuk University, Gwangjin-Gu, Seoul, 143-701, Korea Correspondence to: Jeong Woo Park, email: jwpark@ulsan.ac.kr Wha Ja Cho, email: wjcho26@ulsan.ac.kr Keywords: tristetraprolin, mitochondrial dynamics, α-Synuclein Received: December 14, 2016 Accepted: March 19, 2017 Published: March 30, 2017 ABSTRACT Mitochondrial dynamics play critical roles in maintaining mitochondrial functions. Here, we report a novel mechanism for regulation of mitochondrial dynamics mediated by tristetraprolin (TTP), an AU-rich element (ARE)-binding protein. Overexpression of TTP resulted in elongated mitochondria, down-regulation of mitochondrial oxidative phosphorylation, reduced membrane potential, cytochrome c release, and increased apoptotic cell death in cancer cells. TTP overexpression inhibited the expression of α-Synuclein (α-Syn). TTP bound to the ARE within the mRNA 3'-untranslated regions (3′-UTRs) of α-Syn and enhanced the decay of α-Syn mRNA. Overexpression of α-Syn without the 3′-UTR restored TTP-induced defects in mitochondrial morphology, mitochondrial oxidative phosphorylation, membrane potential, and apoptotic cell death. Taken together, our data demonstrate that TTP acts as a regulator of mitochondrial dynamics through enhancing degradation of α-Syn mRNA in cancer cells. This finding will increase understanding of the molecular basis of mitochondrial dynamics.

Malignant metastatic melanoma (MM) is the most lethal of all skin cancers, but detailed mechanisms for regulation of melanoma metastasis are not fully understood. Here, we demonstrated that developmentally regulated GTP-binding protein 2 (DRG2) is required for the growth of primary tumors and for metastasis. DRG2 expression was significantly increased in MM compared with primary melanoma (PM) and dysplastic nevi. A correlation between DRG2 expression and poor disease-specific survival in melanoma patients was also identified. Furthermore, inhibition of DRG2 suppressed the binding of Hypoxia-inducible factor 1α to the VEGF-A promoter region, expression of vascular endothelial growth factor (VEGF)-A, and formation of endothelial cell tubes. In experimental mice, DRG2 depletion inhibited the growth of PM and lung metastases and increased survival. These results identify DRG2 as a critical regulator of VEGF-A expression and of growth of PMs and lung metastases.

Viral control of mitochondrial quality and content has emerged as an important mechanism for counteracting the host response to virus infection. Despite the knowledge of this crucial function of some viruses, little is known about how herpesviruses regulate mitochondrial homeostasis during infection. Human herpesvirus 8 (HHV-8) is an oncogenic virus causally related to AIDS-associated malignancies. Here, we found that HHV-8-encoded viral interferon regulatory factor 1 (vIRF-1) promotes mitochondrial clearance by activating mitophagy to support virus replication. Genetic interference with vIRF-1 expression or targeting to the mitochondria inhibits HHV-8 replication-induced mitophagy and leads to an accumulation of mitochondria. Moreover, vIRF-1 binds directly to a mitophagy receptor, NIX, on the mitochondria and activates NIX-mediated mitophagy to promote mitochondrial clearance. Genetic and pharmacological interruption of vIRF-1/NIX-activated mitophagy inhibits HHV-8 productive replication. In short, our findings uncover an essential role of vIRF-1 in mitophagy activation for successful lytic replication of HHV-8.

Interleukin 23 (IL-23) is an inflammatory cytokine that plays an important role in tumor promotion. Expression of IL-23 is increased in cancer cells and correlates with tumor progression. However, the mechanisms regulating IL-23 expression in cancer cells are still unclear. Here we report that tristetraprolin (TTP), an AU-rich element (ARE)-binding protein, inhibits IL-23 production in CT26 mouse colon cancer cells. Overexpression of TTP decreased the stability of IL-23 mRNA and the expression level of IL-23 in CT26 cells. Conversely, inhibition of TTP by siRNA increased IL-23 production. TTP destabilized a luciferase mRNA reporter containing the IL-23 mRNA 3’UTR, which contains five AREs. Analyses of deletion and point mutants of the IL-23 mRNA 3’UTR demonstrated that the ARE cluster between the third and fifth AREs was responsible for TTP-mediated destabilization of IL-23 mRNA. A RNA electrophoretic mobility shift assay confirmed that TTP binds to this ARE cluster. Taken together, these results demonstrate that TTP acts as a negative regulator of IL-23 gene expression in mouse colon cancer cells and suggest its potential application as a novel therapeutic target to control IL-23-mediated tumor promotion.

The proviral integration site for Moloney murine leukemia virus 1 (Pim-1) is an oncogenic serine/threonine kinase that is up-regulated in several human cancers, facilitates cell cycle progression, and suppresses apoptosis. Previously, it has been reported that the Pim-1 3′-UTR plays important roles in the regulation of Pim-1 mRNA stability. However, the mechanisms explaining how Pim-1 mRNA stability is determined by its 3′-UTR are not well known. Here, we demonstrate that tristetraprolin (TTP) plays a critical role in the regulation of Pim-1 mRNA stability. Our results show that the level of Pim-1 expression is inversely correlated with TTP expression in human cancer cells. Pim-1 mRNA contains two AU-rich elements (ARE1 and ARE2) in the 3′-UTR. TTP bound to ARE2 and enhanced the decay of Pim-1 mRNA. Overexpression of TTP decreased Pim-1 expression and p21 and p27 phosphorylation and inhibited cell growth. Overexpression of Pim-1 cDNA without the 3′-UTR attenuated the inhibitory effects of TTP on p21 phosphorylation and cell growth. In addition, inhibition of p21 by siRNA attenuated the inhibitory effect of TTP on cell growth. Our results suggest that TTP post-transcriptionally down-regulates Pim-1 expression and that the overexpression of TTP may contribute to tumor suppression in part by down-regulating Pim-1 expression.

Tristetraprolin (TTP) is a AU-rich element (ARE) binding protein and exhibits suppressive effects on cell growth through down-regulation of ARE-containing oncogenes. The let-7 microRNA has emerged as a significant factor in tumor suppression. Both TTP and let-7 are often repressed in human cancers, thereby promoting oncogenesis by derepressing their target genes. In this work, an unexpected link between TTP and let-7 has been found in human cancer cells. TTP promotes an increase in expression of mature let-7, which leads to the inhibition of let-7 target gene CDC34 expression and suppresses cell growth. This event is associated with TTP-mediated inhibition of Lin28, which has emerged as a negative modulator of let-7. Lin28 mRNA contains ARE within its 3′-UTR and TTP enhances the decay of Lin28 mRNA through binding to its 3′-UTR. This suggests that the TTP-mediated down-regulation of Lin28 plays a key role in let-7 miRNA biogenesis in cancer cells.

Tristetraprolin (TTP) is an AU-rich element-binding protein that regulates mRNA stability. We previously showed that TTP acts as a negative regulator of VEGF gene expression in colon cancer cells. The p38 MAPK pathway is known to suppress the TTP activity. However, until now the signaling pathway to enhance TTP function is not well known. Here, we show that casein kinase 2 (CK2) enhances the TTP function in the regulation of the VEGF expression in colon cancer cells. CK2 increased TTP protein levels and enhanced VEGF mRNA decaying activity of TTP. TTP was not a direct target of CK2. Instead, CK2 increased the phosphorylation of MKP-1, which led to a decrease in the phosphorylation of p38 MAPK. Inhibition of MKP-1 by siRNA attenuated the increase in TTP function and the decrease of p38 phosphorylation induced by CK2α overexpression. TGF-β1 increased the expressions of CK2 and TTP and the TTP function. The siRNA against CK2α or TTP reversed TGF-β1-induced increases in the expression of CK2 and TTP and the TTP function. Our data suggest that CK2 enhances the protein level and activity of TTP via the modulation of the MKP-1-p38 MAPK signaling pathway and that TGF-β1 enhances the activity of CK2.

LATS2 is a tumor suppressor gene implicated in the control of cell growth and the cell cycle. Here, we investigated the post-transcriptional regulation of LATS2 expression by tristetraprolin (TTP). Our results show that the expression level of LATS2 is inversely correlated with TTP expression in human cancer cell lines. Overexpression of TTP reduced the expression level of LATS2. Conversely, treatment with small interfering RNA against TTP increased the expression level of LATS2 through stabilization of LATS2 mRNA and suppressed the proliferation of A549 human lung cancer cells. LATS2 mRNA contains AU-rich elements (AREs) within the 3′-untranslated region, and TTP destabilized a luciferase mRNA containing LATS2 ARE. In addition, RNA electrophoretic mobility shift assay revealed that TTP directly bound to the ARE of LATS2 mRNA. These results establish LATS2 mRNA as a physiological target of TTP and suggest the possibility that TTP controls cell growth through regulation of LATS2 mRNA stability.

Epithelial-Mesenchymal Transition (EMT) enhances tumor cell migration. However, detail mechanisms for the regulation of EMT are not well known. Tristetraprolin (TTP) is an AU-rich element (ARE) binding protein that can promote the decay of the transcripts. This study demonstrates that TTP plays a critical role in EMT process. Overexpression of TTP in ovarian cancer cells decreased the expression of mesenchymal markers (N-cadherin and Vimentin), increased the expression of epithelial marker (E-cadherin), and inhibited cell migration and invasion activities. On the contrary, inhibition of TTP by siRNA decreased the expression of E-cadherin, increased the expression of N-cadherin and Vimentin, and promoted cell migration and invasion. The cDNA microarray analysis revealed that Twist1 was significantly down-regulated in TTP-overexpressed cells. Twist1 mRNA contains ARE within its 3′-UTR and TTP enhances the decay of Twist1 mRNA through binding to its 3′-UTR. In addition, TTP directly bound to the ARE of Twist1 mRNA by EMSA assay. Ectopic expression of Twist1 gene without 3′UTR blocked the inhibitory of TTP on the EMT. We also observed enhanced levels of Twist1 and reduced TTP levels in human ovarian adenocarcinoma. Collectively, our data indicate that TTP conducts anti-metastatic activity by down-regulating Twist1, presenting a novel mechanism of TTP-mediated regulation of tumor metastasis. Citation Format: Wha Ja Cho, Nal Ae Yoon, Mai-Tram Vo, Young Joo Min, Jeong Woo Park. Tristetraprolin inhibits Twist1-induced cancer cell migration. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1126. doi:10.1158/1538-7445.AM2014-1126

An excess of interleukin 17 (IL-17) may contribute to chronic inflammatory disorders, but mechanisms that regulate IL-17 in immune cells are unclear. Here we report that tristetraprolin (TTP) inhibits IL-17 production in human T cell lines. Overexpression of TTP decreased the expression of IL-17. Conversely, TTP inhibition by siRNA increased IL-17 production. IL-17 mRNA contains eight AREs within its 3′UTR. TTP bound directly to the IL-17 mRNA 3′UTR at a location between the fourth and seventh AREs and enhanced decay of IL-17 transcripts. These results suggest that TTP could control IL-17-mediated inflammation.