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UXT plays dual opposing roles on SARM-induced apoptosis
FEBS Letters
Abstract
Apoptosis is a vital defense mechanism for the clearance of infected cells. Ubiquitously expressed transcript (UXT), which exists in two isoforms (V1 and V2), interact with both apoptotic and cellular proteins. By yeast two-hybrid analysis, we found that UXT interacts with SARM (sterile and HEAT armadillo motif-containing protein). Since SARM is a TLR adaptor which induces intrinsic apoptosis following immune activation, we were prompted to query whether UXT and SARM might co-regulate apoptosis. We found that the UXT isoforms elicit dual opposing regulatory effects on SARM-induced apoptosis; while UXT V1, co-expressed with SARM, caused a reduction in caspase 8 activity, UXT V2 strongly increased caspase 8 activity and enhanced SARM-induced apoptosis by activating the extrinsic pathway and depolarizing the mitochondria.
... In addition, UXT can also inhibit P53 activity through murine double minute X (MDMX) or epigenetic mechanisms, thereby suppressing apoptosis (5,22). However, UXT-V2 can also interact with sterile a and HEAT armadillo motif-containing protein (SARM) in mitochondria to promote apoptosis (23). Second, UXT can promote P62-mediated selective autophagy (24,25). ...
... There is a lack of strong evidence on whether UXT affects mitochondrial repositioning through the cytoskeleton and the causal mechanistic studies of this in relation to cell death. Both UXT-V1 and UXT-V2 have been shown to interact with SARM in monocyte mitochondria, as observed under lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly I:C) simulated infection conditions (23). The interaction of UXT-V2 with SARM in the mitochondria increases SARM-induced exogenous apoptosis by increasing caspase-8 activity and mitochondrial membrane potential depolarization. ...
The ubiquitous expressed transcript (UXT), a member of the prefoldin-like protein family, modulates regulated cell death (RCD) such as apoptosis and autophagy-mediated cell death through nuclear factor-κB (NF-κB), tumor necrosis factor-α (TNF-α), P53, P62, and methylation, and is involved in the regulation of cell metabolism, thereby affecting tumor progression. UXT also maintains immune homeostasis and reduces proteotoxicity in neuro-degenerative diseases through selective autophagy and molecular chaperones. Herein, we review and further elucidate the mechanisms by which UXT affects the regulation of cell death, maintenance of immune homeostasis, and neurodegenerative diseases and discuss the possible UXT involvement in the regulation of ferroptosis and immunogenic cell death, and targeting it to improve cancer treatment outcomes by regulating cell death and immune surveillance.
... The apparent discrepancy in reported results linking UXT to cell transformation and tumorigenesis could be attributed in part to the alternative splicing of UXT pre-mRNA, resulting in two protein isoforms, one (UXT-V1) having an additional 12 residues at its N-terminus encoding a TNF receptorassociated factor (TRAF)-binding motif . Research suggests that UXT-V1 is cytoplasmic and binds TRAF2 (or alternatively, TRAF-binding mitochondrial protein SARM1) (Murata et al. 2013;Sethurathinam et al. 2013), preventing recruitment of FADD and caspase 8 to the TRADD-RIP-TRAF2 complex and protecting cells against TNFα-induced apoptosis (Huang et al. 2011). Conversely, the shorter UXT-V2 isoform is predominantly nuclear and potentiates NF-κB transcriptional activity upon TNFα activation, either directly or through EZH1-SUZ12 , thereby promoting glycolysis and proliferation (Qi et al. 2015). ...
... Recently, UXT deletion in somatic cells of mice was shown to be embryonic lethal (Schafler et al. 2018). Two isoforms of UXT have been identified to have opposing roles in SARM-induced apoptosis (Sethurathinam et al. 2013); however, their roles in other contexts has not been well established. The non-coding antisense RNA UXT-AS1 was shown to regulate levels of each isoform through alternative splicing mechanisms of the UXT transcript (Yin et al. 2017). ...
Cellular stability, assembly and activation of a growing list of macromolecular complexes require the action of HSP90 working in concert with the R2TP/Prefoldin-like (R2TP/PFDL) co-chaperone. RNA polymerase II, snoRNPs and complexes of PI3-kinase-like kinases, a family that includes the ATM, ATR, DNA-PKcs, TRAPP, SMG1 and mTOR proteins, are among the clients of the HSP90-R2TP system. Evidence links the R2TP/PFDL pathway with cancer, most likely because of the essential role in pathways commonly deregulated in cancer. R2TP forms the core of the co-cochaperone and orchestrates the recruitment of HSP90 and clients, whereas prefoldin and additional prefoldin-like proteins, including URI, associate with R2TP, but their function is still unclear. The mechanism by which R2TP/PFLD facilitates assembly and activation of such a variety of macromolecular complexes is poorly understood. Recent efforts in the structural characterization of R2TP have started to provide some mechanistic insights. We summarize recent structural findings, particularly how cryo-electron microscopy (cryo-EM) is contributing to our understanding of the architecture of the R2TP core complex. Structural differences discovered between yeast and human R2TP reveal unanticipated complexities of the metazoan R2TP complex, and opens new and interesting questions about how R2TP/PFLD works.
... The N-terminal domain of SARM directs the molecule to the mitochondria where it induces mitochondrial polarity and clustering, which results in apoptosis of the infected cell. Furthermore, UXT-V2, one of the interacting partners of SARM was reported to enhance SARM-mediated apoptosis [65]. Interestingly, it was not possible to block SARM-induced apoptosis by inhibitors of the classical cell death pathways like necroptosis. ...
... UXT and β-actin are reported to mediate mitochondrial clustering [70,71]. This was recently ascertained by demonstration that SARM interacts with these proteins [65]. More importantly, SARM is known to interact with polymerized microtubule and regulate microtubule stability [36,48]. ...
SARM (Sterile alpha and armadillo motif-containing protein) is the recently identified TIR domain-containing cytosolic protein. Classified as a member of the TLR adaptor family, the multiple locations and functions of SARM (sometimes playing opposing roles), provoke an enigma on its biology. Although originally assumed to be a member of the TLR adaptor family (functioning as a negative regulator of TLR signaling pathway), latest findings indicate that SARM regulates signaling differently from other TLR adaptor proteins. Recent studies have highlighted the significant functional role of SARM in mediating apoptosis and antiviral innate immune response. In this review, we provide an update on the evolutionary conservation, spatial distribution, and regulated expression of SARM to highlight its diverse functional roles. The review will summarize findings on the known interacting partners of SARM and provide analogy on how they add new dimensions to the current understanding on the multifaceted roles of SARM in antiviral activities and apoptotic functions. In addition, we provide a future perspective on the roles of SARM in differentiation and development, with substantial emphasis on the molecular insights to its mechanisms of action.
... Whether the SENP1-repressive effect of UXT can explain some of these processes remains to be investigated. But UXT has also been linked to diverse processes beyond transcriptional regulation, such as mitochondrial aggregation, centrosome function and apoptosis (60,96,97). ...
SUMOylation is a post-translational modification frequently found on nuclear proteins, including transcription factors (TFs) and coactivators. By controlling the activity of several TFs, SUMOylation may have far-reaching effects. MYB is an example of a developmental TF being subject to SUMO-mediated regulation, through both SUMO-conjugation and SUMO-binding. How SUMO affects MYB target genes is unknown. Here, we explored the global effect of reduced SUMOylation of MYB on its downstream gene programs. RNA-Seq in K562 cells after MYB knock-down and rescue with mutants having an altered SUMO-status revealed a number of differentially regulated genes as well as distinct gene ontology (GO) term enrichments. Clearly, the SUMO-status of MYB both quantitatively and qualitatively affects its regulome. The transcriptome data further revealed that MYB upregulates the SUMO protease SENP1, a key enzyme that removes SUMO conjugation from SUMOylated proteins. Given this role of SENP1 in the MYB regulome, we expanded the analysis, mapped interaction partners of SENP1 and identified UXT as a novel player affecting the SUMO system by acting as a repressor of SENP1. MYB inhibits the expression of UXT suggesting that MYB is able not only to control a specific gene program directly, but also indirectly by affecting the SUMO landscape through SENP1 and UXT. These findings suggest an autoactivation loop whereby MYB, through enhancing SENP1 and reducing UXT, is itself being activated by a reduced level of repressive SUMOylation. We propose that overexpressed MYB, seen in multiple cancers, may drive this autoactivation loop and contribute to oncogenic activation of MYB.
... UXT-V2 also regulates the androgen receptor signaling pathway through interactions with Androgen Receptor N-terminus [29,30]. In addition, these isoforms have opposing effects in SARM (Sterile and HEAT ARMadillo motif-containing protein)-induced apoptosis, with UXT-V1 promoting a reduction in caspase 8 activity and UXT-V2 increasing caspase 8 activity and enhancing apoptosis by activating the extrinsic pathway through depolarization of mitochondria [31]. UXT-V2 is also implicated in tumorigenesis, being overexpressed in a number of human tumors but not in matching normal tissues. ...
Background
Ubiquitously eXpressed Transcript isoform 2 (UXTV2) is a prefoldin-like protein involved in NF-κB signaling, apoptosis, and the androgen and estrogen response. UXT-V2 is a cofactor in the NF-κB transcriptional enhanceosome, and its knockdown inhibits TNF-α -induced NF-κB activation. Fbxo7 is an F-box protein that interacts with SKP1, Cullin1 and RBX1 proteins to form an SCF(Fbxo7) E3 ubiquitin ligase complex. Fbxo7 negatively regulates NF-κB signaling through TRAF2 and cIAP1 ubiquitination.
Methods
We combine co-immunoprecipitation, ubiquitination in vitro and in vivo, cycloheximide chase assay, ubiquitin chain restriction analysis and microscopy to investigate interaction between Fbxo7 and overexpressed UXT-V2-HA.
Results
The Ubl domain of Fbxo7 contributes to interaction with UXTV2. This substrate is polyubiquitinated by SCF(Fbxo7) with K48 and K63 ubiquitin chain linkages in vitro and in vivo. This post-translational modification decreases UXT-V2 stability and promotes its proteasomal degradation. We further show that UXTV1, an alternatively spliced isoform of UXT, containing 12 additional amino acids at the N-terminus as compared to UXTV2, also interacts with and is ubiquitinated by Fbxo7. Moreover, FBXO7 knockdown promotes UXT-V2 accumulation, and the overexpression of Fbxo7-ΔF-box protects UXT-V2 from proteasomal degradation and enhances the responsiveness of NF-κB reporter. We find that UXT-V2 colocalizes with Fbxo7 in the cell nucleus.
Conclusions
Together, our study reveals that SCF(Fbxo7) mediates the proteasomal degradation of UXT-V2 causing the inhibition of the NF-κB signaling pathway.
General significance
Discovering new substrates of E3 ubiquitin-ligase SCF(Fbxo7) contributes to understand its function in different diseases such as cancer and Parkinson.
... On the one hand, UXT1 is associated with TNF-induced apoptosis [31], and on the other hand, UXT2 can interact with transcription factors, such as the AR (androgen receptor) and NF-κB (nuclear factor κB), and regulates the transcription factor-responsive genes as a transcriptional cofactor [32,33]. Moreover, a recent study showed that UXT1 and UXT2 elicited dual opposing regulatory effects on SARM-induced apoptosis [34]. As shown in the present study, UXT1 regulated cell apoptosis and UXT2 regulated cell proliferation in CRC cells, indicating that UXT1 and UXT2 regulated different cell functions and had the opposite effect on the progression of CRC. ...
Increasing evidence indicates that long non-coding RNAs (lncRNAs) can act as crucial regulators of tumor progression. In the present study, UXT-AS1 was found to be significantly upregulated in colorectal cancer (CRC) and high expression levels of UXT-AS1 were significantly associated with poor prognosis in CRC patients. In addition, upregulation of UXT-AS1 resulted in inhibition of cell apoptosis and the promotion of cell proliferation. Moreover, by regulating the alternative splicing of UXT, upregulation of UXT-AS1 decreased the UXT1 transcript which promoted cell apoptosis and increased the UXT2 transcript which promoted cell proliferation. Thus, aberrant high expression of UXT-AS1 can promote CRC progression by changing the alternative splicing of UXT from the UXT1 transcript to the UXT2 transcript. In conclusion, our findings suggest that the regulation of CRC progression is by UXT-AS1-induced alternative splicing of UXT, and the expression level of UXT-AS1 may be a potential prognostic biomarker and therapy target in CRC patients.
... UXT has also been reported to form an integral component of the NF-B enhanceosome, which is essential for the nuclear function of this transcription factor [Sun et al., 2007]. In the cytoplasm, UXT can associate with the mitochondria-associated leucine-rich pentatricopeptide repeat motif-containing (LRPPRC) protein and cause mitochondrial aggregation and depolarization in a concentration-dependent manner which leads to apoptosis [Moss et al., 2007;Sethurathinam et al., 2013]. Interestingly, UXT has also been identified as a novel component of centrosomal processes associated with - ...
The lysyl oxidase proenzyme propeptide region (LOX-PP) is a tumor suppressor protein whose mechanism of action is not completely understood. Here, the Ubiquitously eXpressed Transcript (UXT) was identified in a yeast two-hybrid assay with LOX-PP as bait and confirmed as a novel LOX-PP associating protein. UXT, a prefoldin-like protein, is ubiquitous in human and mouse. Since UXT modulates androgen receptor transcriptional activity in prostate cancer, we studied its role in breast cancer. Breast tumors and derived cell lines overexpressed UXT. UXT was able to associate with the estrogen receptor alpha (ER) and decrease its transcriptional activity and target gene expression. Conversely, UXT knockdown increased ER element-dependent transcriptional activity. Ectopic LOX-PP relocalized UXT to the cytoplasm and decreased its stability. UXT ubiquitination and depletion in the presence of LOX-PP was rescued by a proteasomal inhibitor. In summary, proteasome-mediated turnover of UXT upon interaction with LOX-PP releases repression of ER transcriptional activity. This article is protected by copyright. All rights reserved.
... Several recent studies suggested that MAVS mediates virus-induced cell apoptosis and that this function is independent of the interferon pathway. MAVS, as well as UXT-V1/V2, have been reported to promote the clearance of infected cells [13]. MAVS overexpression causes cell apoptosis and several viral proteins, such as hepatitis C virus NS3/4A and the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) nonstructural protein (NSP15), are inhibitors of the MAVS-induced apoptosis [14]. ...
Viral infection induces innate immunity and apoptosis. Apoptosis is an effective means to sacrifice virus-infected host cells and therefore restrict the spread of pathogens. However, the underlying mechanisms of this process are still poorly understood. Here, we show that the mitochondrial antiviral signaling protein (MAVS/VISA/Cardif/IPS-1) is critical for SeV (Sendai virus)-induced apoptosis. MAVS specifically activates c-Jun N-terminal kinase 2 (JNK2) but not other MAP kinases. Jnk2-/- cells, but not Jnk1-/- cells, are unable to initiate virus-induced apoptosis and SeV further fails to trigger apoptosis in MAPK kinase 7 (MKK7) knockout (Mkk7-/-) cells. Mechanistically, MAVS recruits MKK7 onto mitochondria via its 3D domain, which subsequently phosphorylates JNK2 and thus activates the apoptosis pathway. Consistently, Jnk2-/- mice, but not Jnk1-/- mice, display marked inflammatory injury in lung and liver after viral challenge. Collectively, we have identified a novel signaling pathway, involving MAVS-MKK7-JNK2, which mediates virus-induced apoptosis and highlights the indispensable role of mitochondrial outer membrane in host defenses.
Cyclic ADP‐ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) are two structural distinct messengers for mobilizing, respectively, the endoplasmic (ER) and endo‐lysosomal Ca 2+ stores. They were discovered soon after inositol trisphosphate (IP 3 ) but were nucleotides totally unrelated to IP 3 . As both were hitherto unknown molecules, no information about their enzymatic synthesis had ever been described. The first enzyme shown to be able to cyclize NAD and produce cADPR was the Aplysia ADP‐ribosyl cyclase. Sequence comparison identified CD38 as a homolog, which had since been established as the main enzyme for metabolizing cADPR in mammalian cells. Both enzymes have now been shown to be multifunctional, capable of producing not only cADPR but NAADP as well. Their catalytic mechanisms have now been fully elucidated using crystallography and mutagenesis. In contrast, the endogenous biogenesis and regulation of cADPR and NAADP in cells are much less understood. Two recent breakthroughs promise to advance our understanding. The first discovery was the natural existence of two topological forms of CD38. The type II form is dominant on cell surface with its catalytic domain facing outside, while the topologically opposite type III form is expressed mostly in the ER. With its catalytic domain facing the cytosol, type III CD38 is efficient in producing cellular cADPR. The natural existence of type III CD38 has been demonstrated and investigations have now begun to uncover the regulatory mechanisms of its activity, folding, and degradation. Recent studies have also shown that the surface Type II CD38 contributes minimally to cellular cADPR but may be responsible to produce NAADP after it is internalized by endocytosis into the acidic endo‐lysosomes. The second surprising finding is SARM1, a regulator of axonal degeneration and a protein without any sequence similarity with CD38 but can catalyze the same multi‐reactions to produce cADPR and NAADP. It is an autoregulated enzyme and its cADPR‐producing activity is activated by nicotinamide mononucleotide. Another metabolite of NAD, ADP‐ribose, that is also produced by CD38 and SARM1, has signaling activity in gating the Ca 2+ ‐permeable TRPM2 channel. Whether it acts as a Ca 2+ ‐messenger under physiological conditions, however, remained to be established. With these advances, the messenger functions of these metabolites derived from NAD(P) have entered a new territory.
SARM is the fifth and most conserved member of the Toll/Il-1 Receptor (TIR) adaptor family. However, unlike the other TIR adaptors, MyD88, Mal, TRIF and TRAM, SARM does not participate in transducing signals downstream of TLRs. By contrast SARM inhibits TLR signalling by interacting with the adaptors TRIF and MyD88. In addition, SARM also has positive roles in innate immunity by activating specific transcriptional programs following immune challenge. SARM has a pivotal role in activating different forms of cell death following cellular stress and viral infection. Many of these functions of mammalian SARM are also reflected in SARM orthologues in lower organisms such as C. elegans and Drosophila. SARM expression is particularly enriched in neurons of the CNS and SARM has a critical role in neuronal death and in axon degeneration. Recent fascinating molecular insights have been revealed as to the molecular mechanism of SARM mediated axon degeneration. SARM has been shown to deplete NAD+ by possessing intrinsic NADase activity in the TIR domain of the protein. This activity can be activated experimentally by forced dimerization of the TIR domain. It is thought that this activity of SARM is normally switched off by the axo-protective activities of NMNAT2 which maintain low levels of the NAD+ precursor NMN. Therefore, there is now great excitement in the field of SARM research as targeting this enzymatic activity of SARM may lead to the development of new therapies for neurodegenerative diseases such as multiple sclerosis and motor neuron disease.
The PAQosome (Particle for Arrangement of Quaternary structure) is a large multisubunit chaperone complex that is essential for the assembly and stabilization of other macromolecular complexes. It also interacts with several chaperones including Hsp90, Hsp70, and CCT. The PAQosome is comprised of the R2TP complex, the URI1 prefoldin complex (also known as the non-canonical prefoldin-like complex), the RNA polymerase subunit RPB5, and the WD40 repeat protein WDR92. The R2TP complex is conserved among eukaryotes and has been comprehensively studied over the last 13 years. The R2TP complex is known for its involvement in the assembly and stabilization of L7Ae ribonucleoproteins, U5 small nuclear ribonucleoprotein, RNA polymerase II, phosphatidylinositol-3-kinase-related proteins (PIKKs), and the tuberous sclerosis complex (TSC1-TSC2). By contrast, the URI1 prefoldin complex has evolved exclusively in higher metazoans. Although the URI1 prefoldin complex was initially reported more than 15 years ago, little is known about its function and its role within the PAQosome. Given that URI1 is overexpressed in many types of cancer, it is surprising that the URI1 prefoldin complex has been overlooked. This chapter provides an update on the recent progress uncovering the physiological roles of each PAQosome subunit and provides an overview of the potential functions of the URI1 prefoldin complex.
The PAQosome, formerly known as the R2TP/PFDL complex, is an eleven-subunit cochaperone complex that assists HSP90 in the assembly of numerous large multisubunit protein complexes involved in essential cellular functions such as protein synthesis, ribosome biogenesis, transcription, splicing, and others. In this review, we discuss possible mechanisms of action and role of phosphorylation in the assembly of client complexes by the PAQosome as well as its potential role in cancer, ciliogenesis and ciliopathies.
Toll-like receptors (TLRs) recognise invading pathogens and initiate an innate immune response by recruiting intracellular adaptor proteins via heterotypic Toll/interleukin-1 receptor (TIR) domain interactions. Of the five TIR domain-containing adaptor proteins identified, Sterile α- and armadillo-motif- containing protein (SARM) is functionally unique; suppressing immune signalling instead of promoting it. Here we demonstrate that the recombinantly expressed and purified SARM TIR domain interacts with both the major human TLR adaptors, MyD88 and TRIF. A single glycine residue located in the BB-loop of the SARM TIR domain, G601, was identified as essential for interaction. A short peptide derived from this domain was also found to interact with MyD88 in vitro. SARM expression in HEK-293 cells was found to significantly suppress lipopolysaccharide (LPS)-mediated upregulation of inflammatory cytokines, IL-8 and TNF-α, an effect lost in the G601A mutant. The same result was observed with cytokine activation initiated by MyD88 expression and stimulation of TLR2 with lipoteichoic acid (LTA), suggesting that SARM is capable of suppressing both TRIF- and MyD88- dependent TLR signalling. Our findings indicate that SARM acts on a broader set of target proteins than previously thought, and that the BB-loop motif is functionally important, giving further insight into the endogenous mechanisms used to suppress inflammation in immune cells.
Protein inhibitor of activated signal transducer and activator of transcription 2 (PIAS2) is a member of the PIAS protein family. This protein family modulates the activity of several transcription factors and acts as an E3 ubiquitin ligase in the sumoylation pathway. To improve understanding of the physiological roles of PIAS2, the current study used a yeast two‑hybrid system to screen mouse stem cell cDNA libraries for proteins that interact with PIAS2. The screening identified an interaction between PIAS2 and ubiquitously expressed transcript (UXT). UXT, also termed androgen receptor trapped clone‑27, is an α‑class prefoldin‑type chaperone that acts as a coregulator for various transcription factors, including nuclear factor‑κB and androgen receptor (AR). A direct interaction between PIAS2 and UXT was confirmed by direct yeast two‑hybrid analysis. In vitro evidence of the association of UXT with PIAS2 was obtained by co‑immunoprecipitation. Colocalization between PIAS2 and UXT was identified in the nucleus and cytoplasm of HEK 293T and human cervical carcinoma HeLa cells. The results of the current study suggested that UXT is a binding protein of PIAS2, and interaction between PIAS2 and UXT may be important for the transcriptional activation of AR.
Autoimmune diseases are immune disorders characterized by T cell hyperactivity and B cell overstimulation leading to overproduction of autoantibodies. Although the pathogenesis of various autoimmune diseases remains to be elucidated, environmental factors have been thought to contribute to the initiation and maintenance of auto-respond inflammation. Toll-like receptors (TLRs) are pattern recognition receptors belonging to innate immunity that recognize and defend invading microorganisms. Besides these exogenous pathogen-associated molecular patterns, TLRs can also bind with damage-associated molecular patterns produced under strike or by tissue damage or cells apoptosis. It is believed that TLRs build a bridge between innate immunity and autoimmunity. There are five adaptors to TLRs including MyD88, TRIF, TIRAP/MAL, TRAM, and SARM. Upon activation, TLRs recruit specific adaptors to initiate the downstream signaling pathways leading to the production of inflammatory cytokines and chemokines. Under certain circumstances, ligation of TLRs drives to aberrant activation and unrestricted inflammatory responses, thereby contributing to the perpetuation of inflammation in autoimmune diseases. In the past, most studies focused on the intracellular TLRs, such as TLR3, TLR7, and TLR9, but recent studies reveal that cell surface TLRs, especially TLR2 and TLR4, also play an essential role in the development of autoimmune diseases and afford multiple therapeutic targets. In this review, we summarized the biological characteristics, signaling mechanisms of TLR2/4, the negative regulators of TLR2/4 pathway, and the pivotal function of TLR2/4 in the pathogenesis of autoimmune diseases including rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, Sjogren's syndrome, psoriasis, multiple sclerosis, and autoimmune diabetes.
The Toll-like receptor (TLR)-mediated NF-κB pathway is tightly controlled because overactivation may result in severe damage to the host, such as in the case of chronic inflammatory diseases and cancer. In mammals, sterile-alpha and armadillo motif-containing protein (SARM) plays an important role in negatively regulating this pathway. While Caenorhabditis elegans SARM is crucial for an efficient immune response against bacterial and fungal infections, it is still unknown whether Drosophila SARM participates in immune responses. Here, Litopenaeus vannamei SARM (LvSARM) was cloned and functionally characterized. LvSARM shared signature domains with and exhibited significant similarities to mammalian SARM. Real-time quantitative PCR analysis indicated that the expression of LvSARM was responsive to Vibrio alginolyticus and white spot syndrome virus (WSSV) infections in the hemocyte, gill, hepatopancreas and intestine. In Drosophila S2 cells, LvSARM was widely distributed in the cytoplasm and could significantly inhibit the promoters of the NF-κB pathway-controlled antimicrobial peptide genes (AMPs). Silencing of LvSARM using dsRNA-mediated RNA interference increased the expression levels of Penaeidins and antilipopolysaccharide factors, which are L.vannamei AMPs, and increased the mortality rate after V. alginolyticus infection. Taken together, our results reveal that LvSARM may be a novel component of the shrimp Toll pathway that negatively regulates shrimp AMPs, particularly Penaeidins and antilipopolysaccharide factors.
Following acute-phase infection, activated T cells are terminated to achieve immune homeostasis, failure of which results in lymphoproliferative and autoimmune diseases. We report that sterile α- and heat armadillo-motif-containing protein (SARM), the most conserved Toll-like receptors adaptor, is proapoptotic during T-cell immune response. SARM expression is significantly reduced in natural killer (NK)/T lymphoma patients compared with healthy individuals, suggesting that decreased SARM supports NK/T-cell proliferation. T cells knocked down of SARM survived and proliferated more significantly compared with wild-type T cells following influenza infection in vivo. During activation of cytotoxic T cells, the SARM level fell before rising, correlating inversely with cell proliferation and subsequent T-cell clearance. SARM knockdown rescued T cells from both activation- and neglect-induced cell deaths. The mitochondria-localized SARM triggers intrinsic apoptosis by generating reactive oxygen species and depolarizing the mitochondrial potential. The proapoptotic function is attributable to the C-terminal sterile alpha motif and Toll/interleukin-1 receptor domains. Mechanistically, SARM mediates intrinsic apoptosis via B cell lymphoma-2 (Bcl-2) family members. SARM suppresses B cell lymphoma-extra large (Bcl-xL) and downregulates extracellular signal-regulated kinase phosphorylation, which are cell survival effectors. Overexpression of Bcl-xL and double knockout of Bcl-2 associated X protein and Bcl-2 homologous antagonist killer substantially reduced SARM-induced apoptosis. Collectively, we have shown how T-cell death following infection is mediated by SARM-induced intrinsic apoptosis, which is crucial for T-cell homeostasis.Cell Death and Differentiation advance online publication, 23 November 2012; doi:10.1038/cdd.2012.144.
Virus infection induces the MAVS-TNFR-associated factor (TRAF) 3 signaling axis on mitochondria. It remains to elucidate the corresponding regulatory processes. In this study, we identify UXT-V1 as a novel TRAF3-binding protein. UXT-V1 is critical for the virus-induced activation of NF-κB and IFN regulatory factor 3. Reduction of UXT-V1 impairs the induction of IFN-β and attenuates the host antiviral responses. The N-terminal TRAF-binding motif of UXT-V1 binds to the C-terminal TRAF domain of TRAF3, thus facilitating the interaction between TRAF3 and MAVS. Notably, TRAF3 and TNFR-associated death domain protein are recruited onto mitochondria upon virus infection. These translocations are blocked when knocking down UXT-V1. Thus, UXT-V1 represents a novel integral component of the MAVS signalosome on mitochondria, mediating the innate antiviral signal transduction.
We investigated C-peptide effects on inflammatory cytokine release and adhesion of monocytes exposed to high glucose and lipopolysaccharide (LPS) in vitro.
Monocytic cells (U-937) were cultured in the presence of 30 mmol/L glucose and stimulated with 0.5 ng/μL LPS in the presence or absence of C-peptide (1 μmol/L) for 24 h to induce inflammatory cytokine secretion. Adhesion of U-937 monocytes to human aortic endothelial cells (HAEC) was also studied in the presence or absence of C-peptide. Concentrations of IL-6, IL-8, macrophage inflammatory protein(MIP)-1α, and MIP-1β in supernatants from LPS-stimulated U-937 monocytes were assessed by Luminex. To gain insights into potential intracellular signaling pathways affected by C-peptide, we investigated nuclear translocation of nuclear factor(NF)-κB p65/p50 subunits by western blot in LPS-treated U-937 cells. The effect of C-peptide on LPS-induced phosphorylation of the cytoplasmic protein IκB-α was also investigated by immunoblotting.
Addition of C-peptide significantly reduced cytokine secretion from LPS-stimulated U-937 monocytes. Adhesion of U-937 cells to HAEC was also significantly reduced by C-peptide. These effects were accompanied by reduced NF-κB p65/p50 nuclear translocation and decreased phosphorylation of IκB-α.
We conclude that, in conditions of hyperglycemia, C-peptide reduces monocytes activation via inhibition of the NF-κB pathway.
Proteins that directly regulate tumor necrosis factor (TNF) signaling have critical roles in determining cell death and survival. Previously we characterized ubiquitously expressed transcript (UXT)-V2 as a novel transcriptional cofactor to regulate nuclear factor-κB in the nucleus. Here we report that another splicing isoform of UXT, UXT-V1, localizes in cytoplasm and regulates TNF-induced apoptosis. UXT-V1 knockdown cells are hypersensitive to TNF-induced apoptosis. We demonstrated that UXT-V1 is a new component of TNF receptor signaling complex. We found that UXT-V1 binds to TNF receptor-associated factor 2 and prevents TNF receptor-associated death domain protein from recruiting Fas-associated protein with death domain. More importantly, UXT-V1 is a short-half-life protein, the degradation of which facilitates the formation of the apoptotic receptor complex II in response to TNF treatment. This study demonstrates that UXT-V1 is a novel regulator of TNF-induced apoptosis and sheds new light on the underlying molecular mechanism of this process.
Sterile alpha and HEAT/Armadillo motif (SARM) is a highly conserved Toll/interleukin-1 receptor (TIR)-containing adaptor protein
that is believed to negatively regulate signaling of the pathogen recognition receptors Toll-like receptor 3 (TLR3) and TLR4.
To test its physiological function in the context of a microbial infection, we generated SARM−/− mice and evaluated the impact of this deficiency on the pathogenesis of West Nile virus (WNV), a neurotropic flavivirus that
requires TLR signaling to restrict infection. Although SARM was preferentially expressed in cells of the central nervous system
(CNS), studies with primary macrophages, neurons, or astrocytes showed no difference in viral growth kinetics. In contrast,
viral replication was increased specifically in the brainstem of SARM−/− mice, and this was associated with enhanced mortality after inoculation with a virulent WNV strain. A deficiency of SARM
was also linked to reduced levels of tumor necrosis factor alpha (TNF-α), decreased microglia activation, and increased neuronal
death in the brainstem after WNV infection. Thus, SARM appears to be unique among the TIR adaptor molecules, since it functions
to restrict viral infection and neuronal injury in a brain region-specific manner, possibly by modulating the activation of
resident CNS inflammatory cells.
The p38 mitogen-activated protein kinase pathway regulates innate immune responses in evolutionarily diverse species. We have previously shown that the Caenorhabditis elegans p38 mitogen-activated protein kinase, PMK-1, functions in an innate immune response pathway that mediates resistance to a variety of microbial pathogens. Here, we show that tir-1, a gene encoding a highly conserved Toll/IL-1 resistance (TIR) domain protein, is also required for C. elegans resistance to microbial pathogens. RNA interference inactivation of tir-1 resulted in enhanced susceptibility to killing by pathogens and correspondingly diminished PMK-1 phosphorylation. Unlike all known TIR-domain adapter proteins, overexpression of the human TIR-1 homologue, SARM, in mammalian cells was not sufficient to induce expression of NF-kappaB or IRF3-dependent reporter genes that are activated by Toll-like receptor signaling. These data reveal the involvement of a previously uncharacterized, evolutionarily conserved TIR domain protein in innate immunity that is functionally distinct from other known TIR domain signaling adapters.
The process of programmed cell death, or apoptosis, is generally characterized by distinct morphological characteristics and energy-dependent biochemical mechanisms. Apoptosis is considered a vital component of various processes including normal cell turnover, proper development and functioning of the immune system, hormone-dependent atrophy, embryonic development and chemical-induced cell death. Inappropriate apoptosis (either too little or too much) is a factor in many human conditions including neurodegenerative diseases, ischemic damage, autoimmune disorders and many types of cancer. The ability to modulate the life or death of a cell is recognized for its immense therapeutic potential. Therefore, research continues to focus on the elucidation and analysis of the cell cycle machinery and signaling pathways that control cell cycle arrest and apoptosis. To that end, the field of apoptosis research has been moving forward at an alarmingly rapid rate. Although many of the key apoptotic proteins have been identified, the molecular mechanisms of action or inaction of these proteins remain to be elucidated. The goal of this review is to provide a general overview of current knowledge on the process of apoptosis including morphology, biochemistry, the role of apoptosis in health and disease, detection methods, as well as a discussion of potential alternative forms of apoptosis.
The innate immune system relies on evolutionally conserved Toll-like receptors (TLRs) to recognize diverse microbial molecular structures. Most TLRs depend on a family of adaptor proteins termed MyD88s to transduce their signals. Critical roles of MyD88-1-4 in host defense were demonstrated by defective immune responses in knockout mice. In contrast, the sites of expression and functions of vertebrate MyD88-5 have remained elusive. We show that MyD88-5 is distinct from other MyD88s in that MyD88-5 is preferentially expressed in neurons, colocalizes in part with mitochondria and JNK3, and regulates neuronal death. We prepared MyD88-5/GFP transgenic mice via a bacterial artificial chromosome to preserve its endogenous expression pattern. MyD88-5/GFP was detected chiefly in the brain, where it associated with punctate structures within neurons and copurified in part with mitochondria. In vitro, MyD88-5 co-immunoprecipitated with JNK3 and recruited JNK3 from cytosol to mitochondria. Hippocampal neurons from MyD88-5-deficient mice were protected from death after deprivation of oxygen and glucose. In contrast, MyD88-5-null macrophages behaved like wild-type cells in their response to microbial products. Thus, MyD88-5 appears unique among MyD88s in functioning to mediate stress-induced neuronal toxicity.
The fifth and the most well-conserved member of the TLR (Toll-like receptor) adaptor, SARM (sterile α- and HEAT/armadillo-motif-containing protein), has been reported to be an important mediator of apoptosis. However, the exact cellular localization of SARM with respect to its role is unclear. In the present study we show that SARM specifically co-localizes with mitochondria. Endogenous SARM is mainly found in the mitochondria. We demonstrate that the N-terminal 27 amino acids (S27) of SARM, which is hydrophobic and polybasic, acts as a mitochondria-targeting signal sequence, associating SARM to the mitochondria. The S27 peptide has an inherent ability to bind to lipids and mitochondria. This sequence effectively translocates the soluble EGFP (enhanced green fluorescence protein) reporter into the mitochondria. Positioning S27 downstream of the EGFP abrogates its mitochondria-targeting ability. Transmission electron microscopy confirms the ability of S27 to import EGFP into the mitochondria. Importantly, by mutagenesis study, we delineated the specificity of the mitochondria-targeting ability to the arginine residue at the 14th position. The R14A SARM mutant also showed reduced apoptotic potential when compared with the wild-type. Taken together, S27, which is a bona fide signal sequence that targets SARM to the mitochondria, explains the pro-apoptotic activity of SARM.
SARM (sterile alpha- and armadillo-motif-containing protein), the fifth identified TIR (Toll-interleukin 1 receptor (IL-1R)) domain-containing adaptors in humans, downregulates NF-kappaB and IRF3 (interferon-regulatory factor 3)-mediated TLR3 and TLR4 signaling. SARM was characterized as a negative regulator of the TRIF (TIR-domain-containing adaptor protein inducing IFN-beta)-dependent pathway via its interaction with TRIF. However, the precise mechanism of action of SARM remains unclear. Here, we demonstrate that SARM inhibits MAPK activation in human embryonic kidney 293 cells, and U937 cells. Both the TRIF- and MyD88-mediated, as well as basal MAPK activity, were repressed, indicating that SARM-mediated inhibition may not be exclusively directed at TRIF or MyD88, but that SARM may also directly inhibit MAPK phosphorylation. The MAPK inhibition effect was verified by RNAi, which increased the basal level of AP-1. Furthermore, LPS challenge upregulated SARM at both the mRNA and protein levels. Finally, we provide evidence to show that truncated SARM changes its subcellular localization, suggesting the importance of the N-terminal and sterile alpha motif domains in the autoregulation of SARM activity.
Incubation of the human U937 histiocytic lymphoma cell line with granulocyte-macrophage colony stimulating factor (GM-CSF) rendered the cells responsive to induction of TNF by LPS. Treatment with IL-6 reduced TNF production in GM-CSF-primed U937 cells. The inhibitory effect was most pronounced (approximately equal to 80%) when IL-6 was added either along with GM-CSF or within the first 3 h of GM-CSF treatment. Both GM-CSF or IL-6 inhibited [3H]TdR uptake in U937 cells, and simultaneous treatment with GM-CSF and IL-6 resulted in an additive inhibitory effect on cell proliferation. However, the inhibition of TNF production could not be explained by the inhibitory effect of IL-6 on cell growth, nor was it due to a reduction in cell viability. An inhibition of TNF production by IL-6 was also demonstrated in cultured human peripheral blood monocytes. Treatment with IL-6 also resulted in a dose-dependent reduction of the 17-kDa TNF band revealed by SDS-PAGE after labeling monocytes with [35S]cysteine and immunoprecipitation with anti-TNF mAb. In addition, treatment with IL-6 resulted in a reduction of monocyte in vitro cytotoxicity for tumor target cells. Finally, in mice sensitized by the administration of Bacillus Calmette-Guérin, the injection of IL-6 significantly reduced the levels of TNF found in the serum upon challenge with LPS. Inasmuch as TNF is known to be an inducer of IL-6, the inhibitory action of IL-6 on TNF production may represent the negative arm of a regulatory circuit. The inhibitory action of IL-6 on TNF production is consistent with a predominantly antiinflammatory role of IL-6 in the intact organism.
Several different disease loci with unknown genetic background map to human Xp11. In a systematic search for novel genes, we identified a novel transcript, UXT (HGMW-approved symbol), close to the ELK1 locus in Xp11.23-p11.22. The gene is composed of seven exons and encodes a protein of 157 amino acids, which is highly conserved in mouse. We showed that UXT is ubiquitously expressed and subject to X-inactivation. No homology to any known genes was found. Database surveys indicate an abundant expression in tumor tissues.
The reliability of an in vitro pyrogen test system based on proinflammatory cytokine release from human monocytic cells was assessed by comparison with a test system based on a human whole blood culture as well as with the conventional rabbit pyrogen test. The human cells used as the pyrogen indicator cells were newly selected by subcloning of a human monocytic cell line, Mono-Mac-6. The selected cells, named MM6-CA8, responded to various pyrogens, including endotoxin, peptidoglycan (PG), Staphylococcus aureus Cowan 1 (SAC), and poly(I x C), with a high sensitivity and produced proinflammatory cytokines, such as interleukin 1 (IL-1), IL-6, and tumor necrosis factor alpha. Among these cytokines, IL-6 was produced most sensitively in response to traces of the pyrogens and detected in the largest quantities in the culture medium. The cytokine-producing responses of MM6-CA8 cells correlated significantly with the responses of cultured human whole blood, which represents an ex vivo culture test system reproducing pyrogen-induced cytokine production in the human body. In terms of cytokine inducibility, the pyrogens were ranked in the order endotoxin > PG > poly (I. C) > SAC in both culture systems, a ranking which almost agreed with the ranking of their pyrogenicity as assessed by the rabbit pyrogen test. These results suggest that the in vitro responsiveness of MM6-CA8 cells to various pyrogens is highly relevant for human pyrogenic reactions. Therefore, the in vitro test system is useful and reliable for detecting the presence of materials that are pyrogenic for humans.
Every cell in a multicellular organism has the potential to die by apoptosis, but tumour cells often have faulty apoptotic pathways. These defects not only increase tumour mass, but also render the tumour resistant to therapy. So, what are the molecular mechanisms of tumour resistance to apoptosis and how can we use this knowledge to resensitize tumour cells to cancer therapy?
Toll-like receptors discriminate between different pathogen-associated molecules and activate signaling cascades that lead to immune responses. The specificity of Toll-like receptor signaling occurs by means of adaptor proteins containing Toll-interleukin 1 receptor (TIR) domains. Activating functions have been assigned to four TIR adaptors: MyD88, Mal, TRIF and TRAM. Here we characterize a fifth TIR adaptor, SARM, as a negative regulator of TRIF-dependent Toll-like receptor signaling. Expression of SARM blocked gene induction 'downstream' of TRIF but not of MyD88. SARM associated with TRIF, and 'knockdown' of endogenous SARM expression by interfering RNA led to enhanced TRIF-dependent cytokine and chemokine induction. Thus, the fifth mammalian TIR adaptor SARM is a negative regulator of Toll-like receptor signaling.
Mitochondria are the bioenergetic and metabolic centers in eukaryotic cells and play a central role in apoptosis. Mitochondrial distribution is controlled by the microtubular cytoskeleton. The perinuclear aggregation of mitochondria is one of the characteristics associated with some types of cell death. Control of mitochondrial aggregation particularly related to cell death events is poorly understood. Previously, we identified ubiquitously expressed transcript (UXT) as a potential component of mitochondrial associated LRPPRC, a multidomain organizer that potentially integrates mitochondria and the microtubular cytoskeleton with chromosome remodeling. Here we show that when overexpressed in mammalian cells, green fluorescent protein-tagged UXT (GFP-UXT) exhibits four types of distribution patterns that are proportional to the protein level, and increase with time. UXT initially was dispersed in the extranuclear cytosol, then appeared in punctate cytosolic dots, then an intense perinuclear aggregation that eventually invaded and disrupted the nucleus. The punctate cytosolic aggregates of GFP-UXT coincided with aggregates of mitochondria and LRPPRC. We conclude that increasing concentrations of UXT contributes to progressive aggregation of mitochondria and cell death potentially through association of UXT with LRPPRC.
Apoptosis: a review of programmed cell death
- S Elmore
Elmore, S. (2007) Apoptosis: a review of programmed cell death. Toxicol.
Pathol. 35, 495-516.
Death and anti-death: tumour resistance to apoptosis
- Igney