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.

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.

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.

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.