pLxIS-containing domains are biochemically flexible regulators of interferons and metabolism.

Signal transduction proteins containing a pLxIS motif induce interferon (IFN) responses central to antiviral immunity. Apart from their established roles in activating the IFN regulator factor (IRF) transcription factors, the existence of additional pathways and functions associated with the pLxIS motif is unknown. Using a synthetic biology-based platform, we identified two orphan pLxIS-containing proteins that stimulate IFN responses independent of all known pattern-recognition receptor pathways. We further uncovered a diversity of pLxIS signaling mechanisms, where the pLxIS motif represents one component of a multi-motif signaling entity, which has variable functions in activating IRF3, the TRAF6 ubiquitin ligase, IκB kinases, mitogen-activated protein kinases, and metabolic activities. The most diverse pLxIS signaling mechanisms were associated with the highest antiviral activities in human cells. The flexibility of domains that regulate IFN signaling may explain their prevalence in nature.

Inhibiting Inflammation with Myeloid Cell-Specific Nanobiologics Promotes Organ Transplant Acceptance.

Inducing graft acceptance without chronic immunosuppression remains an elusive goal in organ transplantation. Using an experimental transplantation mouse model, we demonstrate that local macrophage activation through dectin-1 and toll-like receptor 4 (TLR4) drives trained immunity-associated cytokine production during allograft rejection. We conducted nanoimmunotherapeutic studies and found that a short-term mTOR-specific high-density lipoprotein (HDL) nanobiologic treatment (mTORi-HDL) averted macrophage aerobic glycolysis and the epigenetic modifications underlying inflammatory cytokine production. The resulting regulatory macrophages prevented alloreactive CD8+ T cell-mediated immunity and promoted tolerogenic CD4+ regulatory T (Treg) cell expansion. To enhance therapeutic efficacy, we complemented the mTORi-HDL treatment with a CD40-TRAF6-specific nanobiologic (TRAF6i-HDL) that inhibits co-stimulation. This synergistic nanoimmunotherapy resulted in indefinite allograft survival. Together, we show that HDL-based nanoimmunotherapy can be employed to control macrophage function in vivo. Our strategy, focused on preventing inflammatory innate immune responses, provides a framework for developing targeted therapies that promote immunological tolerance.

A TLR4/TRAF6-dependent signaling pathway mediates NCoR coactivator complex formation for inflammatory gene activation.

The nuclear receptor corepressor (NCoR) forms a complex with histone deacetylase 3 (HDAC3) that mediates repressive functions of unliganded nuclear receptors and other transcriptional repressors by deacetylation of histone substrates. Recent studies provide evidence that NCoR/HDAC3 complexes can also exert coactivator functions in brown adipocytes by deacetylating and activating PPARγ coactivator 1α (PGC1α) and that signaling via receptor activator of nuclear factor kappa-B (RANK) promotes the formation of a stable NCoR/HDAC3/PGC1ß complex that coactivates nuclear factor kappa-B (NFκB)- and activator protein 1 (AP-1)-dependent genes required for osteoclast differentiation. Here, we demonstrate that activation of Toll-like receptor (TLR) 4, but not TLR3, the interleukin 4 (IL4) receptor nor the Type I interferon receptor, also promotes assembly of an NCoR/HDAC3/PGC1ß coactivator complex. Receptor-specific utilization of TNF receptor-associated factor 6 (TRAF6) and downstream activation of extracellular signal-regulated kinase 1 (ERK1) and TANK-binding kinase 1 (TBK1) accounts for the common ability of RANK and TLR4 to drive assembly of an NCoR/HDAC3/PGC1ß complex in macrophages. ERK1, the p65 component of NFκB, and the p300 histone acetyltransferase (HAT) are also components of the induced complex and are associated with local histone acetylation and transcriptional activation of TLR4-dependent enhancers and promoters. These observations identify a TLR4/TRAF6-dependent signaling pathway that converts NCoR from a corepressor of nuclear receptors to a coactivator of NFκB and AP-1 that may be relevant to functions of NCoR in other developmental and homeostatic processes.

Non-canonical Activation of the DNA Sensing Adaptor STING by ATM and IFI16 Mediates NF-κB Signaling after Nuclear DNA Damage.

DNA damage can be sensed as a danger-associated molecular pattern by the innate immune system. Here we find that keratinocytes and other human cells mount an innate immune response within hours of etoposide-induced DNA damage, which involves the DNA sensing adaptor STING but is independent of the cytosolic DNA receptor cGAS. This non-canonical activation of STING is mediated by the DNA binding protein IFI16, together with the DNA damage response factors ATM and PARP-1, resulting in the assembly of an alternative STING signaling complex that includes the tumor suppressor p53 and the E3 ubiquitin ligase TRAF6. TRAF6 catalyzes the formation of K63-linked ubiquitin chains on STING, leading to the activation of the transcription factor NF-κB and the induction of an alternative STING-dependent gene expression program. We propose that STING acts as a signaling hub that coordinates a transcriptional response depending on its mode of activation.

Dual-specificity kinase DYRK3 phosphorylates p62 at the Thr-269 residue and promotes melanoma progression.

Melanoma is a type of skin cancer that originates in melanin-producing melanocytes. It is considered a multifactorial disease caused by both genetic and environmental factors, such as UV radiation. Dual-specificity tyrosine-phosphorylation-regulated kinase (DYRK) phosphorylates many substrates involved in signaling pathways, cell survival, cell cycle control, differentiation, and neuronal development. However, little is known about the cellular function of DYRK3, one of the five members of the DYRK family. Interestingly, it was observed that the expression of DYRK3, as well as p62 (a multifunctional signaling protein), is highly enhanced in most melanoma cell lines. This study aimed to investigate whether DYRK3 interacts with p62, and how this affects melanoma progression, particularly in melanoma cell lines. We found that DYRK3 directly phosphorylates p62 at the Ser-207 and Thr-269 residue. Phosphorylation at Thr-269 of p62 by DYRK3 increased the interaction of p62 with tumor necrosis factor receptor-associated factor 6 (TRAF6), an already known activator of mammalian target of rapamycin complex 1 (mTORC1) in the mTOR-involved signaling pathways. Moreover, the phosphorylation of p62 at Thr-269 promoted the activation of mTORC1. We also found that DYRK3-mediated phosphorylation of p62 at Thr-269 enhanced the growth of melanoma cell lines and melanoma progression. Conversely, DYRK3 knockdown or blockade of p62-T269 phosphorylation inhibited melanoma growth, colony formation, and cell migration. In conclusion, we demonstrated that DYRK3 phosphorylates p62, positively modulating the p62-TRAF6-mTORC1 pathway in melanoma cells. This finding suggests that DYRK3 suppression may be a novel therapy for preventing melanoma progression by regulating the mTORC1 pathway.

14-3-3ζ suppresses RANKL signaling by destabilizing TRAF6.

Macrophages are essential regulators of inflammation and bone loss. Receptor activator of nuclear factor-κß ligand (RANKL), a pro-inflammatory cytokine, is responsible for macrophage differentiation to osteoclasts and bone loss. We recently showed that 14-3-3ζ-knockout (YwhazKO) rats exhibit increased bone loss in the inflammatory arthritis model. 14-3-3ζ is a cytosolic adaptor protein that actively participates in many signaling transductions. However, the role of 14-3-3ζ in RANKL signaling or bone remodeling is unknown. We investigated how 14-3-3ζ affects osteoclast activity by evaluating its role in RANKL signaling. We utilized 14-3-3ζ-deficient primary bone marrow-derived macrophages obtained from wildtype and YwhazKO animals and RAW264.7 cells generated using CRISPR-Cas9. Our results showed that 14-3-3ζ-deficient macrophages, upon RANKL stimulation, have bigger and stronger tartrate-resistant acid phosphatase-positive multinucleated cells and increased bone resorption activity. The presence of 14-3-3ζ suppressed RANKL-induced MAPK and AKT phosphorylation, transcription factors (NFATC1 and p65) nuclear translocation, and subsequently, gene induction (Rank, Acp5, and Ctsk). Mechanistically, 14-3-3ζ interacts with TRAF6, an essential component of the RANKL receptor complex. Upon RANKL stimulation, 14-3-3ζ-TRAF6 interaction was increased, while RANK-TRAF6 interaction was decreased. Importantly, 14-3-3ζ supported TRAF6 ubiquitination and degradation by the proteasomal pathway, thus dampening the downstream RANKL signaling. Together, we show that 14-3-3ζ regulates TRAF6 levels to suppress inflammatory RANKL signaling and osteoclast activity. To the best of our knowledge, this is the first report on 14-3-3ζ regulation of RANKL signaling and osteoclast activation.

Dysregulation of innate immune signaling in animal models of spinal muscular atrophy.

BACKGROUND: Spinal muscular atrophy (SMA) is a devastating neuromuscular disease caused by hypomorphic loss of function in the survival motor neuron (SMN) protein. SMA presents across a broad spectrum of disease severity. Unfortunately, genetic models of intermediate SMA have been difficult to generate in vertebrates and are thus unable to address key aspects of disease etiology. To address these issues, we developed a Drosophila model system that recapitulates the full range of SMA severity, allowing studies of pre-onset biology as well as late-stage disease processes. RESULTS: Here, we carried out transcriptomic and proteomic profiling of mild and intermediate Drosophila models of SMA to elucidate molecules and pathways that contribute to the disease. Using this approach, we elaborated a role for the SMN complex in the regulation of innate immune signaling. We find that mutation or tissue-specific depletion of SMN induces hyperactivation of the immune deficiency (IMD) and Toll pathways, leading to overexpression of antimicrobial peptides (AMPs) and ectopic formation of melanotic masses in the absence of an external challenge. Furthermore, the knockdown of downstream targets of these signaling pathways reduced melanotic mass formation caused by SMN loss. Importantly, we identify SMN as a negative regulator of a ubiquitylation complex that includes Traf6, Bendless, and Diap2 and plays a pivotal role in several signaling networks. CONCLUSIONS: In alignment with recent research on other neurodegenerative diseases, these findings suggest that hyperactivation of innate immunity contributes to SMA pathology. This work not only provides compelling evidence that hyperactive innate immune signaling is a primary effect of SMN depletion, but it also suggests that the SMN complex plays a regulatory role in this process in vivo. In summary, immune dysfunction in SMA is a consequence of reduced SMN levels and is driven by cellular and molecular mechanisms that are conserved between insects and mammals.

Exosome-delivered miR-410-3p reverses epithelial-mesenchymal transition, migration and invasion of trophoblasts in spontaneous abortion.

Current studies have indicated that insufficient trophoblast epithelial-mesenchymal transition (EMT), migration and invasion are crucial for spontaneous abortion (SA) occurrence and development. Exosomal miRNAs play significant roles in embryonic development and cellular communication. Hereon, we explored the roles of serum exosomes derived from SA patients on trophoblast EMT, migration and invasion. Exosomes were isolated from normal control (NC) patients with abortion for unplanned pregnancy and SA patients, then characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blotting. Exosomal miRNA profiles were identified by miRNA sequencing. The effects of serum exosomes on trophoblast migration and invasion were detected by scratch wound healing and transwell assays, and other potential mechanisms were revealed by quantitative real-time PCR (RT-PCR), western blotting and dual-luciferase reporter assay. Finally, animal experiments were used to explore the effects of exosomal miR-410-3p on embryo absorption in mice. The serum exosomes from SA patients inhibited trophoblast EMT and reduced their migration and invasion ability in vitro. The miRNA sequencing showed that miR-410-3p was upregulated in SA serum exosomes. The functional experiments showed that SA serum exosomes restrained trophoblast EMT, migration and invasion by releasing miR-410-3p. Mechanistically, SA serum exosomal miR-410-3p inhibited trophoblast cell EMT, migration and invasion by targeting TNF receptor-associated factor 6 (TRAF6) at the post-transcriptional level. Besides, SA serum exosomal miR-410-3p inhibited the p38 MAPK signalling pathway by targeting TRAF6 in trophoblasts. Moreover, milk exosomes loaded with miR-410-3p mimic reached the maternal-fetal interface and aggravated embryo absorption in female mice. Clinically, miR-410-3p and TRAF6 expression were abnormal and negatively correlated in the placental villi of SA patients. Our findings indicated that exosome-derived miR-410-3p plays an important role between SA serum and trophoblasts in intercellular communication, suggesting a novel mechanism by which serum exosomal miRNA regulates trophoblasts in SA patients.

c-Src regulates δ-secretase activation and truncated Tau production by phosphorylating the E3 ligase Traf6.

The accumulation of abnormal Tau protein is a common feature of various neurodegenerative diseases. Truncated Tau, resulting from cleavage by asparaginyl endopeptidase (AEP, δ-secretase), promotes its own phosphorylation and aggregation. Our study focused on understanding the regulatory mechanisms of AEP activation and its interaction with other proteins. We discovered that c-Src plays a critical role in mediating the activation and polyubiquitination of AEP in response to epidermal growth factor stimulation. In addition, we investigated the involvement of tumor necrosis factor receptor-associated factor 6 (Traf6), an E3 ligase, in the regulation of AEP levels and its interaction with c-Src. Knockdown of Traf6 effectively inhibited c-Src-induced AEP activation. To gain further insights into the molecular mechanisms, we employed mass spectrometry to identify the specific tyrosine residues of Traf6 that are phosphorylated by c-Src. By mutating these phosphorylation sites to phenylalanine, we disrupted Traf6-mediated polyubiquitination and subsequently observed the inactivation of AEP. This finding suggests that the phosphorylation of Traf6 by c-Src is crucial for AEP activation. Pharmacological inhibition of c-Src reduced the phosphorylation of Traf6 and inhibited AEP activation in neurons derived from human-induced pluripotent stem cells. Conditional knockout of Traf6 in neurons prevented c-Src-induced AEP activation and subsequent Tau truncation in vivo. Moreover, phosphorylation of Traf6 is highly correlated with AEP activation, Tau368 and pathological Tau (AT8) in Alzheimer's disease brain. Overall, our study elucidates the role of c-Src in regulating AEP-cleaved Tau through phosphorylating Traf6. Targeting the c-Src-Traf6 pathway may hold potential for the treatment of Alzheimer's disease and other tauopathies.

Phosphorylation of USP20 on Ser334 by IRAK1 promotes IL-1ß-evoked signaling in vascular smooth muscle cells and vascular inflammation.

Reversible lysine-63 (K63) polyubiquitination regulates proinflammatory signaling in vascular smooth muscle cells (SMCs) and plays an integral role in atherosclerosis. Ubiquitin-specific peptidase 20 (USP20) reduces NFκB activation triggered by proinflammatory stimuli, and USP20 activity attenuates atherosclerosis in mice. The association of USP20 with its substrates triggers deubiquitinase activity; this association is regulated by phosphorylation of USP20 on Ser334 (mouse) or Ser333 (human). USP20 Ser333 phosphorylation was greater in SMCs of atherosclerotic segments of human arteries as compared with nonatherosclerotic segments. To determine whether USP20 Ser334 phosphorylation regulates proinflammatory signaling, we created USP20-S334A mice using CRISPR/Cas9-mediated gene editing. USP20-S334A mice developed ∼50% less neointimal hyperplasia than congenic WT mice after carotid endothelial denudation. WT carotid SMCs showed substantial phosphorylation of USP20 Ser334, and WT carotids demonstrated greater NFκB activation, VCAM-1 expression, and SMC proliferation than USP20-S334A carotids. Concordantly, USP20-S334A primary SMCs in vitro proliferated and migrated less than WT SMCs in response to IL-1ß. An active site ubiquitin probe bound to USP20-S334A and USP20-WT equivalently, but USP20-S334A associated more avidly with TRAF6 than USP20-WT. IL-1ß induced less K63-linked polyubiquitination of TRAF6 and less downstream NFκB activity in USP20-S334A than in WT SMCs. Using in vitro phosphorylation with purified IRAK1 and siRNA-mediated gene silencing of IRAK1 in SMCs, we identified IRAK1 as a novel kinase for IL-1ß-induced USP20 Ser334 phosphorylation. Our findings reveal novel mechanisms regulating IL-1ß-induced proinflammatory signaling: by phosphorylating USP20 Ser334, IRAK1 diminishes the association of USP20 with TRAF6 and thus augments NFκB activation, SMC inflammation, and neointimal hyperplasia.

CLG promotes mTOR/ULK1 pathway-mediated autophagy to inhibit OS development by inhibiting TRAF6-mediated FLT3 ubiquitination.

Corilagin (CLG) has antitumor activities in certain human malignant cancers. Herein, the effects and mechanisms of CLG on osteosarcoma (OS) were investigated. OS cell viability and proliferation were detected by MTT and colony formation assay. Cell cycle and apoptosis were examined using flow cytometry. The interaction between TRAF6 and FLT3 was investigated using a co-immunoprecipitation assay. Results demonstrated that CLG treatment inhibited OS cell viability and proliferation but promoted OS cell autophagy and apoptosis in a concentration-dependent manner. Mechanically, CLG inhibited TRAF6-mediated FLT3 ubiquitination degradation. TRAF6 overexpression abolished the effects of CLG on OS cell proliferation, autophagy, and apoptosis. Finally, CLG administration inhibited OS tumor growth in mice by inducing autophagy-dependent apoptosis. Taken together, CLG inhibited OS progression by facilitating mTOR/ULK1 pathway-mediated autophagy through inhibiting TRAF6-mediated FLT3 ubiquitination, which indicated that CLG was a promising candidate for the treatment of OS.

Targeting PYK2 with heterobifunctional T6BP helps mitigate MASLD and MASH-HCC progression.

BACKGROUND & AIMS: The mechanisms underlying the regulation of hepatocyte non-receptor tyrosine kinases in metabolic dysfunction-associated steatohepatitis (MASH) remain largely unclear. METHODS: Hepatocyte-specific overexpression or deletion and anti-protein tyrosine kinase 2 beta (PYK2) or anti-TRAF6-binding protein (T6BP) crosslinking were utilised to study fatty liver protection by T6BP. P-PTC, a peptide-proteolysis targeting chimaera, degrades PYK2 to block MASH progression. RESULTS: Since PYK2 activation is promoter signalling in steatohepatitis development, we find that T6BP is a novel and critical suppressor of PYK2 that reduces hepatic lipid accumulation, pro-inflammatory factor release, and pro-fibrosis production by ubiquitin ligase CBL to degrade PYK2. Mechanistic evidence suggests that T6BP directly targets PYK2 and prevents its N-terminal FERM domain-triggered dimerization, disrupting downstream PYK2-JNK signalling hyperactivation. Additionally, T6BP favourably recruits CBL, a particular E3 ubiquitin ligase targeting PYK2, to form a complex and degrade PYK2. T6BP (F1), a core fragment of T6BP, directly blocks N-terminal FERM domain-associated dimerization of PYK2, followed by T6BP-recruiting CBL-mediated PYK2 degradation in a typical T6BP-dependent manner when the tiny fragment is specifically expressed using thyroxine binding globulin (TBG)-ground vectors. This inhibits the progression of MASH, metabolic dysfunction-associated steatotic liver disease (MASLD)-related HCC (MASH-HCC), and metabolic syndrome in dietary rodent models. First-ever peptide-proteolysis targeting chimaera (P-PTC) based on the core segment of T6BP as a ligand for targeted recruitment of CBL targeting metabolic disorders like MASH has been devised and validated in animal models. CONCLUSIONS: Our study revealed a previously unknown mechanism: identification of T6BP as a key eliminator of fatty liver strongly contributes to the development of promising therapeutic targets, and the discovery of crucial fragments of T6BP-based pharmacon that interrupt PYK2 dimerization are novel and viable treatments for fatty liver and its advanced symptoms and complications. IMPACT AND IMPLICATIONS: Excessive high-energy diet ingestion is critical in driving steatohepatitis via regulation of hepatocyte non-receptor tyrosine kinases. The mechanisms under lying the regulation of hepatocyte PYK2 in metabolic dysfunction-associated steatohepatitis (MASH) remain largely unclear. Here, we found that T6BP as a critical fatty liver eliminator has a significant impact on the development of promising therapeutic targets. Additionally, vital T6BP-based pharmacon fragments that impede PYK2 dimerization have been found, offering new and effective treatments for advanced fatty liver symptoms and complications.

Toxoplasma GRA15 limits parasite growth in IFNγ-activated fibroblasts through TRAF ubiquitin ligases.

The protozoan parasite Toxoplasma gondii lives inside a vacuole in the host cytosol where it is protected from host cytoplasmic innate immune responses. However, IFNγ-dependent cell-autonomous immunity can destroy the vacuole and the parasite inside. Toxoplasma strain differences in susceptibility to human IFNγ exist, but the Toxoplasma effector(s) that determine these differences are unknown. We show that in human primary fibroblasts, the polymorphic Toxoplasma-secreted effector GRA15 mediates the recruitment of ubiquitin ligases, including TRAF2 and TRAF6, to the vacuole membrane, which enhances recruitment of ubiquitin receptors (p62/NDP52) and ubiquitin-like molecules (LC3B, GABARAP). This ultimately leads to lysosomal degradation of the vacuole. In murine fibroblasts, GRA15-mediated TRAF6 recruitment mediates the recruitment of immunity-related GTPases and destruction of the vacuole. Thus, we have identified how the Toxoplasma effector GRA15 affects cell-autonomous immunity in human and murine cells.

Mechanism of miR-7 mediating TLR4/TRAF6/NF-κB inflammatory pathway in colorectal cancer.

This study is aimed at investigating the roles of Toll-like receptor 4 (TLR4) and microRNA-7 (miR-7) in colorectal cancer (CRC) development and progression. We assessed TLR4 and miR-7 expression in CRC cells and tissues using reverse transcription-quantitative polymerase chain reaction. The relationship between miR-7 and TLR4 was analyzed through dual luciferase reporter assays. MTT, wound healing, and cell invasion assays were conducted to examine the effects of TLR4 and miR-7 on CRC cell proliferation, migration, and invasion. Western blotting was used to explore the involvement of the TRAF6/NF-κB signaling pathway. miR-7 was underexpressed in CRC, while TLR4 levels were increased. miR-7 negatively regulated TLR4 expression and its knockdown enhanced CRC cell proliferation, migration, and invasion. TLR4 knockdown had the opposite effects. The TRAF6/NF-κB pathway was linked to TLR4's role in tumor progression. miR-7 might inhibit TRAF6/NF-κB target a signaling pathway of TLR4 and promote CRC occurrence. miR-7 may therefore be used as a sensitive biomarker in CRC patients.

Epimedin A inhibits the PI3K/AKT/NF-κB signalling axis and osteoclast differentiation by negatively regulating TRAF6 expression.

BACKGROUND: Epimedin A (EA) has been shown to suppress extensive osteoclastogenesis and bone resorption, but the effects of EA remain incompletely understood. The aim of our study was to investigate the effects of EA on osteoclastogenesis and bone resorption to explore the corresponding signalling pathways. METHODS: Rats were randomly assigned to the sham operation or ovariectomy group, and alendronate was used for the positive control group. The therapeutic effect of EA on osteoporosis was systematically analysed by measuring bone mineral density and bone biomechanical properties. In vitro, RAW264.7 cells were treated with receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) to induce osteoclast differentiation. Cell viability assays, tartrate-resistant acid phosphatase (TRAP) staining, and immunofluorescence were used to elucidate the effects of EA on osteoclastogenesis. In addition, the expression of bone differentiation-related proteins or genes was evaluated using Western blot analysis or quantitative polymerase chain reaction (PCR), respectively. RESULTS: After 3 months of oral EA intervention, ovariectomized rats exhibited increased bone density, relative bone volume, trabecular thickness, and trabecular number, as well as reduced trabecular separation. EA dose-dependently normalized bone density and trabecular microarchitecture in the ovariectomized rats. Additionally, EA inhibited the expression of TRAP and NFATc1 in the ovariectomized rats. Moreover, the in vitro results indicated that EA inhibits osteoclast differentiation by suppressing the TRAF6/PI3K/AKT/NF-κB pathway. Further studies revealed that the effect on osteoclast differentiation, which was originally inhibited by EA, was reversed when the TRAF6 gene was overexpressed. CONCLUSIONS: The findings indicated that EA can negatively regulate osteoclastogenesis by inhibiting the TRAF6/PI3K/AKT/NF-κB axis and that ameliorating ovariectomy-induced osteoporosis in rats with EA may be a promising potential therapeutic strategy for the treatment of osteoporosis.

Regulation mechanisms of CARMA1-Bcl10-MALT1 complex assembly inferred from the analysis of TRAF6-deficient cells.

The CARMA1-Bcl10-MALT1 (CBM) signalosome is a crucial module of NF-κB activation in B cell receptor (BCR) signaling. Biophysical studies have shown that the E3 ubiquitin ligase TRAF6 cooperatively modifies the CBM signalosome; however, the specific details regarding how TRAF6 is involved in BCR signal-induced CBM formation remain unclear. In this study, we aimed to reveal the influences of TRAF6 on CBM formation and TAK1 and IKK activities using DT40 B cells which lack all the exons of TRAF6. In TRAF6-null cells we found: (i) attenuation of TAK1 activity and abolishment of IKK activity and (ii) sustained binding of CARMA1 to Bcl10. To account for the molecular mechanism causing these dynamics, we performed a mathematical model analysis. The mathematical model analysis showed that the regulation of IKK activation by TRAF6 can reproduce TAK1 and IKK activities in TRAF6 null cells, and that the TRAF6 related signal-dependent inhibitor suppresses CARMA1 binding to Bcl10 in wild-type cells. These results suggest that TRAF6 contributes to the positive regulation of IKK activation via TAK1, alongside the negative signal-dependent regulation of CARMA1 binding to Bcl10.

Role of inflammatory signaling pathways involving the CD40-CD40L-TRAF cascade in diabetes and hypertension-insights from animal and human studies.

CD40L-CD40-TRAF signaling plays a role in atherosclerosis progression and affects the pathogenesis of coronary heart disease (CHD). We tested the hypothesis that CD40L-CD40-TRAF signaling is a potential therapeutic target in hyperlipidemia, diabetes, and hypertension. In mouse models of hyperlipidemia plus diabetes (db/db mice) or hypertension (1 mg/kg/d angiotensin-II for 7 days), TRAF6 inhibitor treatment (2.5 mg/kg/d for 7 or 14 days) normalized markers of oxidative stress and inflammation. As diabetes and hypertension are important comorbidities aggravating CHD, we explored whether the CD40L-CD40-TRAF signaling cascade and their associated inflammatory pathways are expressed in CHD patients suffering from comorbidities. Therefore, we analyzed vascular bypass material (aorta or internal mammary artery) and plasma from patients with CHD with diabetes and/or hypertension. Our Olink targeted plasma proteomic analysis using the IMMUNO-ONCOLOGY panel revealed a pattern of step-wise increase for 13/92 markers of low-grade inflammation with significant changes. CD40L or CD40 significantly correlated with 38 or 56 other inflammatory targets. In addition, specific gene clusters that correlate with the comorbidities were identified in isolated aortic mRNA of CHD patients through RNA-sequencing. These signaling clusters comprised CD40L-CD40-TRAF, immune system, hemostasis, muscle contraction, metabolism of lipids, developmental biology, and apoptosis. Finally, immunological analysis revealed key markers correlated with comorbidities in CHD patients, such as CD40L, NOX2, CD68, and 3-nitrotyrosine. These data indicate that comorbidities increase inflammatory pathways in CHD, and targeting these pathways will be beneficial in reducing cardiovascular events in CHD patients with comorbidities.

TGFßR1 inhibition drives hepatocellular carcinoma proliferation through induction of toll-like-receptor signalling.

Transforming growth factor (TGF)-ß and toll-like receptors (TLRs) have been shown to independently modulate the proliferation of hepatocellular carcinoma (HCC). Since a direct cross-talk between these two signalling pathways in HCC has not been clearly described before, we aimed here to explore the possibility of such interaction. A human HCC tissue array (n = 20 vs. four control samples), human HCC samples (n = 10) and steatohepatitis-driven murine HCC samples (control, NASH and HCC; n = 6/group) were immunostained for TGFßR1, pSMAD2, TRAF6, IRAK1 and PCNA. The results were confirmed by immunoblotting. Effects of constant activation of the SMAD pathway by constitutive expression of ALK5 or knockdown of mediators of TLR signalling, IRAK1 and MyD88, on HCC proliferation, were investigated in the HCC cell line (HUH-7) after treatment with TGFß1 cytokine or TGFßR1 kinase inhibitor (LY2157299) using PCNA and MTS assay. TGFßR1 expression is decreased in human and murine HCC and associated with downregulated pSMAD2, but increased IRAK1, TRAF6 and PCNA staining. TGFßR1 kinase inhibition abolished the cytostatic effects of TGFß1 and led to the induction of IRAK1, pIRAK1 and elevated mRNA levels of TLR-9. Overexpression of ALK5 and knockdown of MyD88 or IRAK1 augmented the cytostatic effects of TGFß1 on HUH-7. In another epithelial HCC cell line, that is, HepG2, TGFßR1 kinase inhibitor similarly elevated cellular proliferation. There is a balance between the canonical SMAD-driven tumour-suppressing arm and the non-canonical tumour-promoting arm of TGFß signalling. Disruption of this balance, by inhibition of the canonical pathway, induces HCC proliferation through TLR signalling.

The protective mechanism of sevoflurane in pulmonary arterial hypertension via downregulation of TRAF6.

Pulmonary arterial hypertension (PAH) is an obstructive vasculopathy that, if not promptly treated, culminates in right heart failure. Therefore, pre-clinical studies are needed to support and optimize therapeutic approaches of PAH. Here, we explore a prospective function of sevoflurane in experimental PAH through regulating TRAF6. Monocrotaline (MCT)-induced PAH rats were subjected to sevoflurane inhalation and intratracheal instillation of lentivirus overexpressing TRAF6. Platelet-derived growth factor (PDGF)-treated pulmonary artery smooth muscle cells (PASMCs) were exposed to sevoflurane and genetically manipulated for TRAF6 overexpression. It was found that MCT and PDGF challenge upregulated the levels of TRAF6 in rat lung tissues and PASMCs, but sevoflurane treatment led to reduced TRAF6 expression. Sevoflurane inhalation in MCT-induced rats resulted in alleviative pulmonary vascular remodeling, mitigated right ventricular dysfunction and hypertrophy, improved mitochondrial function and dynamics, and inactivation of NF-κB pathway. In vitro studies confirmed that exposure to sevoflurane repressed PDGF-induced proliferation, migration, and phenotype switching of PASMCs, and suppressed mitochondrial dysfunction and NF-κB activation in PDGF-stimulated PASMCs. The beneficial impact of sevoflurane on pathological changes of lung and cell phenotype of PASMCs were reversed by overexpression of TRAF6. In summary, our study suggested the protective properties of sevoflurane in targeting PAH by downregulating TRAF6 expression, providing a novel avenue for the management of PAH.

TRAF6-mediated ubiquitination of AKT in the nucleus is a critical event underlying the desensitization of G protein-coupled receptors.

BACKGROUND: Desensitization of G protein-coupled receptors (GPCRs) refers to the attenuation of receptor responsiveness by prolonged or intermittent exposure to agonists. The binding of ß-arrestin to the cytoplasmic cavity of the phosphorylated receptor, which competes with the G protein, has been widely accepted as an extensive model for explaining GPCRs desensitization. However, studies on various GPCRs, including dopamine D2-like receptors (D2R, D3R, D4R), have suggested the existence of other desensitization mechanisms. The present study employed D2R/D3R variants with different desensitization properties and utilized loss-of-function approaches to uncover the mechanisms underlying GPCRs homologous desensitization, focusing on the signaling cascade that regulates the ubiquitination of AKT. RESULTS: AKT undergoes K8/14 ubiquitination by TRAF6, which occurs in the nucleus and promotes its membrane recruitment, phosphorylation and activation under receptor desensitization conditions. The nuclear entry of TRAF6 relies on the presence of the importin complex. Src regulates the nuclear entry of TRAF6 by mediating the interaction between TRAF6 and importin ß1. Ubiquitinated AKT translocates to the plasma membrane where it associates with Mdm2 to phosphorylate it at the S166 and S186 residues. Thereafter, phosphorylated Mdm2 is recruited to the nucleus, resulting in the deubiquitination of ß-Arr2. The deubiquitinated ß-Arr2 then forms a complex with Gßγ, which serves as a biomarker for GPCRs desensitization. Like in D3R, ubiquitination of AKT is also involved in the desensitization of ß2 adrenoceptors. CONCLUSION: Our study proposed that the property of a receptor that causes a change in the subcellular localization of TRAF6 from the cytoplasm to the nucleus to mediate AKT ubiquitination could initiate the desensitization of GPCRs.