ANXA2 (annexin A2) is crucial to ATG7-mediated autophagy, leading to tumor aggressiveness in triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) is associated with a poor prognosis and metastatic growth. TNBC cells frequently undergo macroautophagy/autophagy, contributing to tumor progression and chemotherapeutic resistance. ANXA2 (annexin A2), a potential therapeutic target for TNBC, has been reported to stimulate autophagy. In this study, we investigated the role of ANXA2 in autophagic processes in TNBC cells. TNBC patients exhibited high levels of ANXA2, which correlated with poor outcomes. ANXA2 increased LC3B-II levels following bafilomycin A1 treatment and enhanced autophagic flux in TNBC cells. Notably, ANXA2 upregulated the phosphorylation of HSF1 (heat shock transcription factor 1), resulting in the transcriptional activation of ATG7 (autophagy related 7). The mechanistic target of rapamycin kinase complex 2 (MTORC2) played an important role in ANXA2-mediated ATG7 transcription by HSF1. MTORC2 did not affect the mRNA level of ANXA2, but it was involved in the protein stability of ANXA2. HSPA (heat shock protein family A (Hsp70)) was a potential interacting protein with ANXA2, which may protect ANXA2 from lysosomal proteolysis. ANXA2 knockdown significantly increased sensitivity to doxorubicin, the first-line chemotherapeutic regimen for TNBC treatment, suggesting that the inhibition of autophagy by ANXA2 knockdown may overcome doxorubicin resistance. In a TNBC xenograft mouse model, we demonstrated that ANXA2 knockdown combined with doxorubicin administration significantly inhibited tumor growth compared to doxorubicin treatment alone, offering a promising avenue to enhance the effectiveness of chemotherapy. In summary, our study elucidated the molecular mechanism by which ANXA2 modulates autophagy, suggesting a potential therapeutic approach for TNBC treatment.Abbreviation: ATG: autophagy related; ChIP: chromatin-immunoprecipitation; HBSS: Hanks' balanced salt solution; HSF1: heat shock transcription factor 1; MTOR: mechanistic target of rapamycin kinase; TNBC: triple-negative breast cancer; TFEB: transcription factor EB; TFE3: transcription factor binding to IGHM enhancer 3.

HCMV-encoded viral protein US12 promotes autophagy by inducing autophagy flux.

The human cytomegalovirus (HCMV)-encoded US12 gene family is a group of ten predicted seven-transmembrane domain proteins that are structurally similar to G-protein-coupled receptors or transmembrane Bax inhibitor-1 motif-containing proteins; however, the roles of US12 family proteins in virus-host interactions remain to be discovered. Here, we suggest a new function of the US12 protein in regulating cellular autophagy. US12 is predominantly located to the lysosome and interacts with the lysosomal membrane protein 2 (LAMP2). A liquid chromatography-mass spectrometry (MS)/MS-based targeted proteomics analysis shows that US12 is tightly correlated with autophagy. US12 induces autophagy via upregulating ULK1 phosphorylation and subsequent LC3-II conversion, thereby accelerating autophagic flux. Moreover, HeLa cells overexpressing US12 displays intense LC3-specific staining and autolysosome formation even under nutrient-sufficient conditions. Furthermore, the physical interaction of p62/SQSTM1 with US12 is involved in the resistance to the degradation of p62/SQSTM1 by autophagy, despite the induction of both autolysosome formation and autophagic flux. Although the effect of US12 expression in HCMV infection on autophagy remains undetermined, these findings provide new insights into the viral drivers of host autophagy during HCMV evolution and pathogenesis.

TRIM40 is a pathogenic driver of inflammatory bowel disease subverting intestinal barrier integrity.

The cortical actin cytoskeleton plays a critical role in maintaining intestinal epithelial integrity, and the loss of this architecture leads to chronic inflammation, as seen in inflammatory bowel disease (IBD). However, the exact mechanisms underlying aberrant actin remodeling in pathological states remain largely unknown. Here, we show that a subset of patients with IBD exhibits substantially higher levels of tripartite motif-containing protein 40 (TRIM40), a gene that is hardly detectable in healthy individuals. TRIM40 is an E3 ligase that directly targets Rho-associated coiled-coil-containing protein kinase 1 (ROCK1), an essential kinase involved in promoting cell-cell junctions, markedly decreasing the phosphorylation of key signaling factors critical for cortical actin formation and stabilization. This causes failure of the epithelial barrier function, thereby promoting a long-lived inflammatory response. A mutant TRIM40 lacking the RING, B-box, or C-terminal domains has impaired ability to accelerate ROCK1 degradation-driven cortical actin disruption. Accordingly, Trim40-deficient male mice are highly resistant to dextran sulfate sodium (DSS)-induced colitis. Our findings highlight that aberrant upregulation of TRIM40, which is epigenetically silenced under healthy conditions, drives IBD by subverting cortical actin formation and exacerbating epithelial barrier dysfunction.

The nucleolus is the site for inflammatory RNA decay during infection.

Inflammatory cytokines are key signaling molecules that can promote an immune response, thus their RNA turnover must be tightly controlled during infection. Most studies investigate the RNA decay pathways in the cytosol or nucleoplasm but never focused on the nucleolus. Although this organelle has well-studied roles in ribosome biogenesis and cellular stress sensing, the mechanism of RNA decay within the nucleolus is not completely understood. Here, we report that the nucleolus is an essential site of inflammatory pre-mRNA instability during infection. RNA-sequencing analysis reveals that not only do inflammatory genes have higher intronic read densities compared with non-inflammatory genes, but their pre-mRNAs are highly enriched in nucleoli during infection. Notably, nucleolin (NCL) acts as a guide factor for recruiting cytosine or uracil (C/U)-rich sequence-containing inflammatory pre-mRNAs and the Rrp6-exosome complex to the nucleolus through a physical interaction, thereby enabling targeted RNA delivery to Rrp6-exosomes and subsequent degradation. Consequently, Ncl depletion causes aberrant hyperinflammation, resulting in a severe lethality in response to LPS. Importantly, the dynamics of NCL post-translational modifications determine its functional activity in phases of LPS. This process represents a nucleolus-dependent pathway for maintaining inflammatory gene expression integrity and immunological homeostasis during infection.

Prostaglandin E2 receptor PTGER4-expressing macrophages promote intestinal epithelial barrier regeneration upon inflammation.

OBJECTIVE: Dysfunctional resolution of intestinal inflammation and altered mucosal healing are essential features in the pathogenesis of inflammatory bowel disease (IBD). Intestinal macrophages are vital in the process of inflammation resolution, but the mechanisms underlying their mucosal healing capacity remain elusive. DESIGN: We investigated the role of the prostaglandin E2 (PGE2) receptor PTGER4 on the differentiation of intestinal macrophages in patients with IBD and mouse models of intestinal inflammation. We studied mucosal healing and intestinal epithelial barrier regeneration in Csf1r-iCre Ptger4fl/fl mice during dextran sulfate sodium (DSS)-induced colitis. The effect of PTGER4+ macrophage secreted molecules was investigated on epithelial organoid differentiation. RESULTS: Here, we describe a subset of PTGER4-expressing intestinal macrophages with mucosal healing properties both in humans and mice. Csf1r-iCre Ptger4fl/fl mice showed defective mucosal healing and epithelial barrier regeneration in a model of DSS colitis. Mechanistically, an increased mucosal level of PGE2 triggers chemokine (C-X-C motif) ligand 1 (CXCL1) secretion in monocyte-derived PTGER4+ macrophages via mitogen-activated protein kinases (MAPKs). CXCL1 drives epithelial cell differentiation and proliferation from regenerating crypts during colitis. Specific therapeutic targeting of macrophages with liposomes loaded with an MAPK agonist augmented the production of CXCL1 in vivo in conditional macrophage PTGER4-deficient mice, restoring their defective epithelial regeneration and favouring mucosal healing. CONCLUSION: PTGER4+ intestinal macrophages are essential for supporting the intestinal stem cell niche and regeneration of the injured epithelium. Our results pave the way for the development of a new class of therapeutic targets to promote macrophage healing functions and favour remission in patients with IBD.

O-Linked N-Acetylglucosamine Modification of Mitochondrial Antiviral Signaling Protein Regulates Antiviral Signaling by Modulating Its Activity.

Post-translational modifications, including O-GlcNAcylation, play fundamental roles in modulating cellular events, including transcription, signal transduction, and immune signaling. Several molecular targets of O-GlcNAcylation associated with pathogen-induced innate immune responses have been identified; however, the direct regulatory mechanisms linking O-GlcNAcylation with antiviral RIG-I-like receptor signaling are not fully understood. In this study, we found that cellular levels of O-GlcNAcylation decline in response to infection with Sendai virus. We identified a heavily O-GlcNAcylated serine-rich region between amino acids 249-257 of the mitochondrial antiviral signaling protein (MAVS); modification at this site disrupts MAVS aggregation and prevents MAVS-mediated activation and signaling. O-GlcNAcylation of the serine-rich region of MAVS also suppresses its interaction with TRAF3; this prevents IRF3 activation and production of interferon-ß. Taken together, these results suggest that O-GlcNAcylation of MAVS may be a master regulatory event that promotes host defense against RNA viruses.

CEP41-mediated ciliary tubulin glutamylation drives angiogenesis through AURKA-dependent deciliation.

The endothelial cilium is a microtubule-based organelle responsible for blood flow-induced mechanosensation and signal transduction during angiogenesis. The precise function and mechanisms by which ciliary mechanosensation occurs, however, are poorly understood. Although posttranslational modifications (PTMs) of cytoplasmic tubulin are known to be important in angiogenesis, the specific roles of ciliary tubulin PTMs play remain unclear. Here, we report that loss of centrosomal protein 41 (CEP41) results in vascular impairment in human cell lines and zebrafish, implying a previously unknown pro-angiogenic role for CEP41. We show that proper control of tubulin glutamylation by CEP41 is necessary for cilia disassembly and that is involved in endothelial cell (EC) dynamics such as migration and tubulogenesis. We show that in ECs responding to shear stress or hypoxia, CEP41 activates Aurora kinase A (AURKA) and upregulates expression of VEGFA and VEGFR2 through ciliary tubulin glutamylation, as well as leads to the deciliation. We further show that in hypoxia-induced angiogenesis, CEP41 is responsible for the activation of HIF1α to trigger the AURKA-VEGF pathway. Overall, our results suggest the CEP41-HIF1α-AURKA-VEGF axis as a key molecular mechanism of angiogenesis and demonstrate how important ciliary tubulin glutamylation is in mechanosense-responded EC dynamics.

HCMV-encoded US7 and US8 act as antagonists of innate immunity by distinctively targeting TLR-signaling pathways.

The mechanisms by which many human cytomegalovirus (HCMV)-encoded proteins help the virus to evade immune surveillance remain poorly understood. In particular, it is unknown whether HCMV proteins arrest Toll-like receptor (TLR) signaling pathways required for antiviral defense. Here, we report that US7 and US8 as key suppressors that bind both TLR3 and TLR4, facilitating their destabilization by distinct mechanisms. US7 exploits the ER-associated degradation components Derlin-1 and Sec61, promoting ubiquitination of TLR3 and TLR4. US8 not only disrupts the TLR3-UNC93B1 association but also targets TLR4 to the lysosome, resulting in rapid degradation of the TLR. Accordingly, a mutant HCMV lacking the US7-US16 region has an impaired ability to hinder TLR3 and TLR4 activation, and the impairment is reversed by the introduction of US7 or US8. Our findings reveal an inhibitory effect of HCMV on TLR signaling, which contributes to persistent avoidance of the host antiviral response to achieve viral latency.

CNBP controls tumor cell biology by regulating tumor-promoting gene expression.

Cellular nucleic acid-binding protein (CNBP) is associated with cell proliferation, and its expression is elevated in human tumors, but the molecular mechanisms of CNBP in tumor cell biology have not been fully elucidated. In this study, we report that CNBP is a transcription factor essential for regulating matrix metalloproteinases mmp-2, mmp-14, and transcription factor e2f2 gene expression by binding to their promoter regions via a sequence-specific manner. Importantly, epidermal growth factor stimulation is required to induce CNBP phosphorylation and nuclear transport, thereby promoting the expression of mmp-2, mmp-14, and e2f2 genes. As a consequence, loss of cnbp attenuates the ability of tumor cell growth, invasion, and migration. Conversely, overexpression of cnbp is associated with tumor cell biology. Collectively, our findings reveal CNBP as a key transcriptional regulator of tumor-promoting target genes to control tumor cell biology.

Human cytomegalovirus-encoded US9 targets MAVS and STING signaling to evade type I interferon immune responses.

Human cytomegalovirus (HCMV) has evolved sophisticated immune evasion mechanisms that target both the innate and adaptive immune responses. However, how HCMV encoded proteins are involved in this immune escape is not clear. Here, we show that HCMV glycoprotein US9 inhibits the IFN-ß response by targeting the mitochondrial antiviral-signaling protein (MAVS) and stimulator of interferon genes (STING)-mediated signaling pathways. US9 accumulation in mitochondria attenuates the mitochondrial membrane potential, leading to promotion of MAVS leakage from the mitochondria. Furthermore, US9 disrupts STING oligomerization and STING-TBK1 association through competitive interaction. Intriguingly, US9 blocks interferon regulatory factor 3 (IRF3) nuclear translocation and its cytoplasmic domain is essential for inhibiting IRF3 activation. Mutant HCMV lacking US7-16 is impaired in antagonism of MAVS/STING-mediated IFN-ß expression, an effect that is reversible by the introduction of US9. Our findings indicate that HCMV US9 is an antagonist of IFN signaling to persistently evade host innate antiviral responses.

STRAP positively regulates TLR3-triggered signaling pathway.

Toll-like receptor (TLR) signaling drives the innate immune response by activating nuclear factor-κB (NF-κB) and interferon regulatory factor (IRF). We have previously shown that STRAP interacts with TAK1 and IKKα along with NF-κB subunit p65, leading to the activation of pro-inflammatory cytokines. However, the roles of STRAP in TRIF/TBK1-mediated TLR3 activation and the subsequent type I interferon (IFN) production are not fully elucidated. Here, we demonstrate that STRAP acts as a scaffold protein in TLR3-triggered signaling. STRAP strongly interacts with TBK1 and IRF3, which enhances IFN-ß production. As a consequence, STRAP knockdown reduces the level of both pro-inflammatory cytokine and IFN in TLR3 agonist-stimulated macrophages, whereas its overexpression significantly enhances production of these cytokines. Furthermore, the C-terminus of STRAP is essential for its functional activity in TLR3-mediated IL-6 and IFN-ß production. These data suggest that STRAP is a positive regulator of the TLR3-meditated NF-κB and IRF signaling pathway.

CNBP acts as a key transcriptional regulator of sustained expression of interleukin-6.

The transcription of inflammatory genes is an essential step in host defense activation. Here, we show that cellular nucleic acid-binding protein (CNBP) acts as a transcription regulator that is required for activating the innate immune response. We identified specific CNBP-binding motifs present in the promoter region of sustained inflammatory cytokines, thus, directly inducing the expression of target genes. In particular, lipopolysaccharide (LPS) induced cnbp expression through an NF-κB-dependent manner and a positive autoregulatory mechanism, which enables prolonged il-6 gene expression. This event depends strictly on LPS-induced CNBP nuclear translocation through phosphorylation-mediated dimerization. Consequently, cnbp-depleted zebrafish are highly susceptible to Shigella flexneri infection in vivo. Collectively, these observations identify CNBP as a key transcriptional regulator required for activating and maintaining the immune response.

STRAP Acts as a Scaffolding Protein in Controlling the TLR2/4 Signaling Pathway.

The WD40-repeat protein serine/threonine kinase receptor-associated protein (STRAP) is involved in the regulation of several biological processes, including cell proliferation and apoptosis, in response to various stresses. Here, we show that STRAP is a new scaffold protein that functions in Toll-like receptor (TLR)-mediated immune responses. STRAP specifically binds transforming growth factor ß-activated kinase 1 (TAK1) and IκB kinase alpha (IKKα) along with nuclear factor-κB (NF-κB) subunit p65, leading to enhanced association between TAK1, IKKα, and p65, and subsequent facilitation of p65 phosphorylation and nuclear translocation. Consequently, the depletion of STRAP severely impairs interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and IL-1ß production, whereas its overexpression causes a significant increase in the secretion of these pro-inflammatory cytokines by TLR2 or TLR4 agonist-stimulated macrophages. Notably, STRAP translocates to the nucleus and subsequently binds to NF-κB at later times after lipopolysaccharide (LPS) stimulation, resulting in prolonged IL-6 mRNA production. Moreover, the C-terminal region of STRAP is essential for its functional activity in facilitating IL-6 production. Collectively, these observations suggest that STRAP acts as a scaffold protein that positively contributes to innate host defenses against pathogen infections.

TRIM31 promotes Atg5/Atg7-independent autophagy in intestinal cells.

Autophagy is responsible for the bulk degradation of cytosolic constituents and plays an essential role in the intestinal epithelium by controlling beneficial host-bacterial relationships. Atg5 and Atg7 are thought to be critical for autophagy. However, Atg5- or Atg7-deficient cells still form autophagosomes and autolysosomes, and are capable of removing proteins or bacteria. Here, we report that human TRIM31 (tripartite motif), an intestine-specific protein localized in mitochondria, is essential for promoting lipopolysaccharide-induced Atg5/Atg7-independent autophagy. TRIM31 directly interacts with phosphatidylethanolamine in a palmitoylation-dependent manner, leading to induction of autolysosome formation. Depletion of endogenous TRIM31 significantly increases the number of intestinal epithelial cells containing invasive bacteria. Crohn's disease patients display TRIM31 downregulation. Human cytomegalovirus-infected intestinal cells show a decrease in TRIM31 expression as well as a significant increase in bacterial load, reversible by the introduction of wild-type TRIM31. We provide insight into an alternative autophagy pathway that protects against intestinal pathogenic bacterial infection.

Hyperproduction of IL-6 caused by aberrant TDP-43 overexpression in high-fat diet-induced obese mice.

Inclusion of Tat-activating regulatory DNA-binding protein-43 (TDP-43) due to hyperphosphorylation or hyperubiquitination is a cause of neurodegenerative disease. Cellular TDP-43 expression is tightly controlled through a negative feedback loop involving its mRNA. Recently, we reported that the TDP-43-mediated sub-nuclear body is an essential site of interleukin-6 (IL-6) pre-mRNA processing. Here we show that mice fed on a high-fat diet exhibit increased TDP-43 expression in the liver and adipose tissue with a prominent increase in IL-6. TDP-43 depletion in vivo reduces IL-6 production in the liver. Overexpression or depletion of TDP-43 in pre-adipose and adipose cells causes reciprocal alteration of IL-6 expression and RNA processing. Our findings provide evidence for a link between homeostasis of TDP-43 expression and the risk of developing obesity.

InSAC: A novel sub-nuclear body essential for Interleukin-6 and -10 RNA processing and stability.

Dysregulation of cytokine expression causes inflammatory diseases or chronic infection conditions. We have identified that Tat-activating regulatory DNA-binding protein-43 (TDP-43) is involved in cytokine RNA processing in order to promote an optimal immune response. The interaction of TDP-43 with spliceosomal components from the Cajal body leads to the formation of a novel sub-nuclear body called the Interleukin (IL)-6 and IL-10 Splicing Activating Compartment (InSAC). TDP-43 binds to the IL-6 and IL-10 RNAs in a sequence-dependent manner. In cell-based studies, we observed that lipopolysaccharide (LPS) stimulation induces the formation of the InSAC through TDP-43 ubiquitination, thereby influencing the processing and expression levels of IL-6 RNA. Moreover, TDP-43 knockdown in vivo results in a decrease in IL-6 production and its RNA splicing and stability. Thus, these findings demonstrate that the InSAC is linked to the activation and modulation of the immune response.

Identification of a subnuclear body involved in sequence-specific cytokine RNA processing.

Processing of interleukin RNAs must be tightly controlled during the immune response. Here we report that a subnuclear body called the interleukin-6 and -10 splicing activating compartment (InSAC) is a nuclear site of cytokine RNA production and stability. Tat-activating regulatory DNA-binding protein-43 (TDP-43) acts as an InSAC scaffold that selectively associates with IL-6 and IL-10 RNAs in a sequence-specific manner. TDP-43 also recruits key spliceosomal components from Cajal bodies. LPS induces posttranslational modifications of TDP-43; in particular, TDP-43 ubiquitination provides a driving force for InSAC formation. As a consequence, in vivo depletion of TDP-43 leads to a dramatic reduction in the RNA processing and the protein levels of IL-6 in serum. Collectively, our findings highlight the importance of TDP-43-mediated InSAC biogenesis in immune regulation.

Differential control of interleukin-6 mRNA levels by cellular distribution of YB-1.

Cytokine production is essential for innate and adaptive immunity against microbial invaders and must be tightly controlled. Cytokine messenger RNA (mRNA) is in constant flux between the nucleus and the cytoplasm and in transcription, splicing, or decay; such processes must be tightly controlled. Here, we report a novel function of Y-box-binding protein 1 (YB-1) in modulating interleukin-6 (IL-6) mRNA levels in a cell type-specific manner. In lipopolysaccharide (LPS)-stimulated macrophages, YB-1 interacts with IL-6 mRNA and actively transports it to the extracellular space by YB-1-enriched vesicles, resulting in the proper maintenance of intracellular IL-6 mRNA levels. YB-1 secretion occurs in a cell type-specific manner. Whereas macrophages actively secret YB-1, dendritic cells maintain it predominantly in the cytoplasm even in response to LPS. Intracellular YB-1 has the distinct function of regulating IL-6 mRNA stability in dendritic cells. Moreover, because LPS differentially regulates the expression of histone deacetylase 6 (HDAC6) in macrophages and dendritic cells, this stimulus might control YB-1 acetylation differentially in both cell types. Taken together, these results suggest a unique feature of YB-1 in controlling intracellular IL-6 mRNA levels in a cell type-specific manner, thereby leading to functions that are dependent on the extracellular and intracellular distribution of YB-1.

Negative self-regulation of TLR9 signaling by its N-terminal proteolytic cleavage product.

TLR signaling is essential to innate immunity against microbial invaders and must be tightly controlled. We have previously shown that TLR9 undergoes proteolytic cleavage processing by lysosomal proteases to generate two distinct fragments. The C-terminal cleavage product plays a critical role in activating TLR9 signaling; however, the precise role of the N-terminal fragment, which remains in lysosomes, in the TLR9 response is still unclear. In this article, we report that the N-terminal cleavage product negatively regulates TLR9 signaling. Notably, the N-terminal fragment promotes the aspartic protease-mediated degradation of the C-terminal fragment in endolysosomes. Furthermore, the N-terminal TLR9 fragment physically interacts with the C-terminal product, thereby inhibiting the formation of homodimers of the C-terminal fragment; this suggests that the monomeric C-terminal product is more susceptible to attack by aspartic proteases. Together, these results suggest that the N-terminal TLR9 proteolytic cleavage product is a negative self-regulator that prevents excessive TLR9 signaling activity.

An unconventional role for miRNA: let-7 activates Toll-like receptor 7 and causes neurodegeneration.

Activation of innate immune receptors by host-derived factors exacerbates CNS damage, but the identity of these factors remains elusive. We uncovered an unconventional role for the microRNA let-7, a highly abundant regulator of gene expression in the CNS, in which extracellular let-7 activates the RNA-sensing Toll-like receptor (TLR) 7 and induces neurodegeneration through neuronal TLR7. Cerebrospinal fluid (CSF) from individuals with Alzheimer's disease contains increased amounts of let-7b, and extracellular introduction of let-7b into the CSF of wild-type mice by intrathecal injection resulted in neurodegeneration. Mice lacking TLR7 were resistant to this neurodegenerative effect, but this susceptibility to let-7 was restored in neurons transfected with TLR7 by intrauterine electroporation of Tlr7(−/−) fetuses. Our results suggest that microRNAs can function as signaling molecules and identify TLR7 as an essential element in a pathway that contributes to the spread of CNS damage.