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The Role of Ubiquitin in NF-κB Regulatory Pathways
Abstract
Nuclear factor kappa enhancer binding protein (NF-kappaB) regulates diverse biological processes including immunity, inflammation, and apoptosis. A vast array of cellular stimuli converges on NF-kappaB, and ubiquitination plays an essential role in the coordination of these signals to regulate NF-kappaB activity. At least three steps in NF-kappaB activation directly involve ubiquitination: proteasomal degradation of inhibitor of NF-kappaB (IkappaB), processing of NF-kappaB precursors, and activation of the transforming growth factor (TGF)-beta-activated kinase (TAK1) and IkappaB kinase (IKK) complexes. In this review, we discuss recent advances in the identification and characterization of ubiquitination and deubiquitination machinery that regulate NF-kappaB. Particular emphasis is given to proteasome-independent functions of ubiquitin, specifically its role in the activation of protein kinase complexes and in coordination of cell survival and apoptosis signals downstream of tumor necrosis factor alpha (TNFalpha).
... Emerging evidence suggests that K63-linked ubiquitination also plays a pivotal role in the regulation of pathways that have been implicated in inflammatory response and cancer development, such as NF-κB (Skaug et al., 2009). The canonical NF-κB activation pathway is mediated through IKK, an upstream kinase responsible for IκB phosphorylation and subsequent degradation (Figure 2). ...
... The canonical NF-κB activation pathway is mediated through IKK, an upstream kinase responsible for IκB phosphorylation and subsequent degradation (Figure 2). IKK is consisted of IKKα kinase, IKKβ kinase, and IKKγ regulatory subunit (also known as NEMO; Chen, 2005;Bhoj and Chen, 2009;Skaug et al., 2009), which phosphorylates IκB and promotes SCF-βTrCP-mediated K48-linked ubiquitination and subsequent degradation by proteasome. Degradation of IκB results in NF-κB nuclear translocation and activation. ...
... Degradation of IκB results in NF-κB nuclear translocation and activation. In the non-canonical pathway, NIK-mediated-phosphorylation of IKKα results in its activation and subsequent phosphorylation of p100, which recruits a K48 E3 ligase to targetp100 for partial proteasomal degradation (Martinez-Forero et al., 2009;Skaug et al., 2009). A stop signal within the p100 sequence will terminate the proteasomal degradation process and the p52 transcription factor is then released and forms heterodimer NF-κB2 transcription factor with REL-B, which translocates to the nucleus and regulates gene expression. ...
... This, in turn, prevents the activation of NF-kB, which would otherwise produce a variety of different proinflammatory mediators via a cascade effect. In addition, taraxerol has been shown to suppress the activity of acetylcholinesterase (AChE) in a dose-dependent way [49,50]. Taraxerol is a bioactive metabolite found in certain higher plants. ...
... Specifically, heart rate and systolic blood pressure (SBP) were recorded using a rat tail-cuff plethysmography technique and an attached pressure gauge. This procedure was consistent with previous descriptions in the literature and was intended to provide a thorough assessment of cardiovascular function in animal models used for research purposes [50]. ...
Myocardial infarction (MI) continues to be an important issue in healthcare systems worldwide, leading to high rates of morbidity and mortality. Despite ongoing efforts towards the development of preventive measures and treatments, addressing the challenges posed by MI remains difficult both in developed and developing countries. However, researchers recently investigated the potential cardioprotective effects of taraxerol utilizing an isoproterenol (ISO)-induced cardiotoxicity model among Sprague Dawley rats. Specifically, subcutaneous tissue injections consisting of 5.25 mg/kg or 8.5 mg/kg ISO were administered over two consecutive days as stimuli to induce cardiac injury. To investigate the possibility of preventing damage caused by ISO-induced cardiotoxicity by taraxerol treatment, five groups were formed: a normal control group (1% Tween 80), an ISO control group, an amlodipine group administered 5 mg/kg/day, and various doses of taraxerol. The study results showed that treatment significantly reduced cardiac marker enzymes. Additionally, pretreatment with taraxerol increased myocardial activity in SOD and GPx, leading to significant reductions in serum CK-MB levels along with MDA, TNF-α, and IL-6. Further histopathological analysis supported these observations, as treated animals had less cellular infiltration compared to untreated ones. These multifaceted findings suggest that oral administration of taraxerol could potentially protect hearts from ISO-caused damage by increasing endogenous antioxidant concentrations while decreasing pro-inflammatory cytokines.
... ATRA also suppresses NF-κB activity and associated cytokine discharge through its inhibitory activity on the ubiquitin proteasome system (UPS) [212,237,238]. In the inflammatory tissue environment, with reduced amount or lack of ATRA, the UPS activation through TRAF and cytokine signaling is not properly inhibited and as a result, UPS-mediated NF-κB activity increases in the adaptive immune cells leading to the development of naive T cells into Th17 cells [41,212,234,237,[239][240][241]. ...
... ATRA also suppresses NF-κB activity and associated cytokine discharge through its inhibitory activity on the ubiquitin proteasome system (UPS) [212,237,238]. In the inflammatory tissue environment, with reduced amount or lack of ATRA, the UPS activation through TRAF and cytokine signaling is not properly inhibited and as a result, UPS-mediated NF-κB activity increases in the adaptive immune cells leading to the development of naive T cells into Th17 cells [41,212,234,237,[239][240][241]. Low ATRA levels are initially responsible for diverting the immune response towards inflammation [234]. ...
... ATRA also suppresses NF-κB activity and associated cytokine discharge through its inhibitory activity on the ubiquitin proteasome system (UPS) [212,237,238]. In the inflammatory tissue environment, with reduced amount or lack of ATRA, the UPS activation through TRAF and cytokine signaling is not properly inhibited and as a result, UPS-mediated NF-κB activity increases in the adaptive immune cells leading to the development of naive T cells into Th17 cells [41,212,234,237,[239][240][241]. ...
... ATRA also suppresses NF-κB activity and associated cytokine discharge through its inhibitory activity on the ubiquitin proteasome system (UPS) [212,237,238]. In the inflammatory tissue environment, with reduced amount or lack of ATRA, the UPS activation through TRAF and cytokine signaling is not properly inhibited and as a result, UPS-mediated NF-κB activity increases in the adaptive immune cells leading to the development of naive T cells into Th17 cells [41,212,234,237,[239][240][241]. Low ATRA levels are initially responsible for diverting the immune response towards inflammation [234]. ...
The SARS-CoV-2 virus has caused a worldwide COVID-19 pandemic. In less than a year and a half, more than 200 million people have been infected and more than four million have died. Despite some improvement in the treatment strategies, no definitive treatment protocol has been developed. The pathogenesis of the disease has not been clearly elucidated yet. A clear understanding of its pathogenesis will help develop effective vaccines and drugs. The immunopathogenesis of COVID-19 is characteristic with acute respiratory distress syndrome and multiorgan involvement with impaired Type I interferon response and hyperinflammation. The destructive systemic effects of COVID-19 cannot be explained simply by the viral tropism through the ACE2 and TMPRSS2 receptors. In addition, the recently identified mutations cannot fully explain the defect in all cases of Type I interferon synthesis. We hypothesize that retinol depletion and resulting impaired retinoid signaling play a central role in the COVID-19 pathogenesis that is characteristic for dysregulated immune system, defect in Type I interferon synthesis, severe inflammatory process, and destructive systemic multiorgan involvement. Viral RNA recognition mechanism through RIG-I receptors can quickly consume a large amount of the body's retinoid reserve, which causes the retinol levels to fall below the normal serum levels. This causes retinoid insufficiency and impaired retinoid signaling, which leads to interruption in Type I interferon synthesis and an excessive inflammation. Therefore, reconstitution of the retinoid signaling may prove to be a valid strategy for management of COVID-19 as well for some other chronic, degenerative, inflammatory, and autoimmune diseases.
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Keywords
Vitamin AVitamin DRetinolRetinoic acidRetinoid signalingCOVID-19Type-I IFNRIG-ICYP450NF-κBRORγtFoxP3Th17TregInflammationAutoimmunity
Abbreviations
Aktserine/threonine specific protein kinase BATRAAll-trans retinoic acidBALFbronchoalveolar lavage fluidcisRAcis isomers of retinoic acidc-Myccellular Myc proto-oncogeneCDCCenters for Disease Control and PreventionCOVID-19coronavirus disease 2019CRBP-Icytoplasmic retinol binding protein 1CRABP-1cytoplasmic retinoic acid binding protein 1CXCL9C-X-C motif chemokine ligand 9CYP26cytochrome P450 family 26CYP27cytochrome P450 family 27CYP450cytochrome P450 superfamily of enzymesErkextracellular-signal-regulated kinaseFoxP3Forkhead box P3 transcription factorGluR1glutamate receptor 1IFNinterferonILinterleukin cytokinesIL2RAinterleukin 2 receptor-alpha subunitIRFinterferon-regulatory factorsJakJanus kinasesLynLyn protein kinase, a member of Src family tyrosine kinasesMAPKmitogen-activated protein kinaseMDA5melanoma differentiation-associated protein 5NCoAnuclear receptor co-activatorsNCoRnuclear receptor co-repressorsNF-κBnuclear factor kappa light chain enhancer of activated B cellsPI3KPhosphatidylinositol-3-KinasePPARperoxisome proliferator-activated receptorPRRpattern recognition receptorsRAretinoic acidRARretinoic acid receptorsRAREretinoic acid response elements (regulatory DNA sequences bound by retinoic acid receptors)RBPretinol binding proteinRALDHretinaldehyde dehydrogenase enzymeRDHretinol dehydrogenase enzymesRIG-Iretinoic acid-inducible gene-IRLRRIG-I-like receptorRORγtRetinoic acid-related orphan receptor gT transcription factorRXRretinoid X receptorsSARS-CoV-2severe acute respiratory syndrome coronavirus 2sST2soluble suppression of tumorigenicity 2STATsignal transducer and activator of transcriptionsTNFRSF1Asoluble TNF receptor superfamily member 1ASTRA6signaling receptor and transporter of retinolTGF-βtransforming growth factor-betaTh17T helper 17 cellsTLRToll-like receptorsTNFTumor necrosis factorTRAFTNF receptor (TNF-R)-associated factorTregregulatory T cellsType-I IFNType I interferon (IFN-α and IFN-β)VADvitamin A deficiencyVitAvitamin AVitDvitamin DWHOWorld Health Organization
... ATRA also suppresses NF-κB activity and associated cytokine discharge through its inhibitory activity on the ubiquitin proteasome system (UPS) [212,237,238]. In the inflammatory tissue environment, with reduced amount or lack of ATRA, the UPS activation through TRAF and cytokine signaling is not properly inhibited and as a result, UPS-mediated NF-κB activity increases in the adaptive immune cells leading to the development of naive T cells into Th17 cells [41,212,234,237,[239][240][241]. ...
... ATRA also suppresses NF-κB activity and associated cytokine discharge through its inhibitory activity on the ubiquitin proteasome system (UPS) [212,237,238]. In the inflammatory tissue environment, with reduced amount or lack of ATRA, the UPS activation through TRAF and cytokine signaling is not properly inhibited and as a result, UPS-mediated NF-κB activity increases in the adaptive immune cells leading to the development of naive T cells into Th17 cells [41,212,234,237,[239][240][241]. Low ATRA levels are initially responsible for diverting the immune response towards inflammation [234]. ...
The SARS-CoV-2 virus has caused a worldwide COVID-19 pandemic. In less than a year and a half, more than 200 million people have been infected and more than four million have died. Despite some improvement in the treatment strategies, no definitive treatment protocol has been developed. The pathogenesis of the disease has not been clearly elucidated yet. A clear understanding of its pathogenesis will help develop effective vaccines and drugs. The immunopathogenesis of COVID-19 is characteristic with acute respiratory distress syndrome and multiorgan involvement with impaired Type I interferon response and hyperinflammation. The destructive systemic effects of COVID-19 cannot be explained simply by the viral tropism through the ACE2 and TMPRSS2 receptors. In addition, the recently identified mutations cannot fully explain the defect in all cases of Type I interferon synthesis. We hypothesize that retinol depletion and resulting impaired retinoid signaling play a central role in the COVID-19 pathogenesis that is characteristic for dysregulated immune system, defect in Type I interferon synthesis, severe inflammatory process, and destructive systemic multiorgan involvement. Viral RNA recognition mechanism through RIG-I receptors can quickly consume a large amount of the body's retinoid reserve, which causes the retinol levels to fall below the normal serum levels. This causes retinoid insufficiency and impaired retinoid signaling, which leads to interruption in Type I interferon synthesis and an excessive inflammation. Therefore, reconstitution of the retinoid signaling may prove to be a valid strategy for management of COVID-19 as well for some other chronic, degenerative, inflammatory, and autoimmune diseases.
... 333polyubiquitylation, and then TAK1 undergoes auto-phosphorylation at Ser192 and Thr184/187 334 to achieve full activation(Skaug, Jiang et al., 2009, Sorrentino, Thakur et al., 2008. ...
TAK1, a serine/threonine protein kinase, has been identified as a key regulator in a wide variety of cellular processes. However, its function and involved mechanism in cancer metastasis are still not well understood. Here, we found that knockdown of TAK1 promoted esophageal squamous cancer cell (ESCC) migration and invasion, whereas overexpression of TAK1 resulted in an opposite outcome. Moreover, these in vitro findings could be recapitulated in vivo in a xenograft metastasis mouse model. Mechanistically, co-immunoprecipitation combined with mass spectrometry demonstrated that TAK1 interacted with phospholipase C epsilon 1 (PLCE1), and phosphorylated PLCE1 at serine 1060 (S1060). Functional studies revealed that phosphorylation at S1060 in PLCE1 resulted in decreased enzyme activity, leading to a repression on PIP2 hydrolysis. As a result, the degradation products of PIP2 including DAG and inositol IP3 were reduced, which thereby suppressed signal transduction in the axis of PKC/GSK-3beta/beta-Catenin. Consequently, cancer metastasis related genes were impeded by TAK1. Overall, our data indicate that TAK1 plays a negative role in ESCC metastasis, which depends on TAK1 induced phosphorylation of PLCE1 at S1060.
... Given that the interaction between RIPK1 and IKKs occurs in the proximal TNFR1 complex-I, including RIPK1 and adaptor molecules such as TRADD and TRAF2 [33,34], we subsequently examined whether eupatolide affects the recruitment of polyubiquitinated RIPK1 to the TNFR1 complex. As expected, treatment of MEFs with TNF led to the transient recruitment of polyubiquitinated RIPK1 to TNFR1, and this enhanced RIPK1 recruitment upon TNF treatment was completely abolished in eupatolide-pretreated MEFs (Fig. 2C, top panel). ...
Ubiquitination of RIPK1 plays an essential role in the recruitment of the IKK complex, an upstream component of pro-survival NF-κB. It also limits TNF-induced programmed cell death by inhibiting the spatial transition from TNFR1-associated complex-I to RIPK1-dependent death-inducing complex-II or necrosome. Thus, the targeted disruption of RIPK1 ubiquitination, which induces RIPK1-dependent cell death, has proven to be a useful strategy for improving the therapeutic efficacy of TNF. In this study, we found that eupatolide, isolated from Liriodendron tulipifera, is a potent activator of the cytotoxic potential of RIPK1 by disrupting the ubiquitination of RIPK1 upon TNFR1 ligation. Analysis of events upstream of NF-κB signaling revealed that eupatolide inhibited IKKβ-mediated NF-κB activation while having no effect on IKKα-mediated non-canonical NF-κB activation. Pretreatment with eupatolide drastically interfered with RIPK1 recruitment to the TNFR1 complex-I by disrupting RIPK1 ubiquitination. Moreover, eupatolide was sufficient to upregulate the activation of RIPK1, facilitating the TNF-mediated dual modes of apoptosis and necroptosis. Thus, we propose a novel mechanism by which eupatolide activates the cytotoxic potential of RIPK1 at the TNFR1 level and provides a promising anti-cancer therapeutic approach to overcome TNF resistance.
... In the present study, we confirmed that miR-125a-5p could downregulate downstream molecular TRAF6. TRAF6 is crucial in the induction of inflammatory response, 59,60 and is also significantly associated with proinflammatory cytokine production and neutrophil infiltration in the myocardium after ischemic/reperfusion injury. 61,62 TRAF6-mediated signals play a crucial role in the immune system by significantly impacting the development, stability, and stimulation of innate immune cells including macrophages, 63 particularly regulating macrophage phenotypic switching. ...
Background
Exosomes derived from bone marrow mesenchymal stem cells (MSC-exo) have been considered as a promising cell-free therapeutic strategy for ischemic heart disease. Cardioprotective drug pretreatment could be an effective approach to improve the efficacy of MSC-exo. Nicorandil has long been used in clinical practice for cardioprotection. This study aimed to investigate whether the effects of exosomes derived from nicorandil pretreated MSC (MSCNIC-exo) could be enhanced in facilitating cardiac repair after acute myocardial infarction (AMI).
Methods
MSCNIC-exo and MSC-exo were collected and injected into the border zone of infarcted hearts 30 minutes after coronary ligation in rats. Macrophage polarization was detected 3 days post-infarction, cardiac function as well as histological pathology were measured on the 28th day after AMI. Macrophages were separated from the bone marrow of rats for in vitro model. Exosomal miRNA sequencing was conducted to identify differentially expressed miRNAs between MSCNIC-exo and MSC-exo. MiRNA mimics and inhibitors were transfected to MSCs or macrophages to explore the specific mechanism.
Results
Compared to MSC-exo, MSCNIC-exo showed superior therapeutic effects on cardiac functional and structural recovery after AMI and markedly elevated the ratio of CD68⁺ CD206⁺/ CD68⁺cells in infarcted hearts 3 days post-infarction. The notable ability of MSCNIC-exo to promote macrophage M2 polarization was also confirmed in vitro. Exosomal miRNA sequencing and both in vivo and in vitro experiments identified and verified that miR-125a-5p was an effector of the roles of MSCNIC-exo in vivo and in vitro. Furthermore, we found miR-125a-5p promoted macrophage M2 polarization by inhibiting TRAF6/IRF5 signaling pathway.
Conclusion
This study suggested that MSCNIC-exo could markedly facilitate cardiac repair post-infarction by promoting macrophage M2 polarization by upregulating miR-125a-5p targeting TRAF6/IRF5 signaling pathway, which has great potential for clinical translation.
... 12,13 Notably, ubiquitin conjugation/deconjugation in NF-kB signaling has been demonstrated to be frequently deregulated in numerous human cancer types. 5,14 By coordinately regulating repertoires of target genes, microRNAs (miRNAs) have been reported to potentially modulate multiple steps of cancer development, progression, and metastasis. [15][16][17][18][19][20][21] A growing body of studies has showed that miRNAs play crucial roles in sustaining NF-kB activity by ubiquitination-mediated mechanism. ...
The prime issue derived from prostate cancer (PCa) is its high prevalence to metastasize to bone. MicroRNA-204-5p (miR-204-5p) has been reported to be involved in the development and metastasis in a variety of cancers. However, the clinical significance and biological functions of miR-204-5p in bone metastasis of PCa are still not reported yet. In this study, we find that miR-204-5p expression is reduced in PCa tissues and serum sample with bone metastasis compared with that in PCa tissues and serum sample without bone metastasis, which is associated with advanced clinicopathological characteristics and poor bone metastasis-free survival in PCa patients. Moreover, upregulation of miR-204-5p inhibits the migration and invasion of PCa cells in vitro, and importantly, upregulat-ing miR-204-5p represses bone metastasis of PCa cells in vivo. Our results further demonstrated that miR-204-5p suppresses invasion, migration, and bone metastasis of PCa cells via inac-tivating nuclear factor kB (NF-kB) signaling by simultaneously targeting TRAF1, TAB3, and MAP3K3. In clinical PCa samples , miR-204-5p expression negatively correlates with TRAF1, TAB3, and MAP3K3 expression and NF-kB signaling activity. Therefore, our findings reveal a new mechanism underpinning the bone metastasis of PCa, as well as provide evidence that miR-204-5p might serve as a novel serum biomarker in bone metastasis of PCa. This study identifies a novel functional role of miR-204-5p in bone metastasis of prostate cancer and supports the potential clinical value of miR-204-5p as a serum biomarker in bone metastasis of PCa.
... TAK1 then phosphorylates downstream signaling molecules, including MAP kinases and various transcription factors. [11][12][13][14][15] TAK1 is involved in numerous cellular processes, including the regulation of immune responses, inflammation, cell survival, and differentiation. 15 Dysregulation of TAK1 has been implicated in various diseases, including cancer, autoimmune disorders, and inflammatory diseases. ...
Current treatment options for patients with multiple myeloma (MM) include proteasome inhibitors, anti-CD38 antibodies, and immunomodulatory agents. However, if patients have continued disease progression after administration of these treatments, there are limited options. There is a need for effective targeted therapies of MM. Recent studies have shown that the transforming growth factor-β activated kinase (TAK1) is upregulated and overexpressed in MM. We have discovered that 6-substituted morpholine or piperazine imidazo[1,2-b]pyridazines, with an appropriate aryl substituent at position-3, inhibit TAK1 at nanomolar concentrations. The lead compound, 26, inhibits the enzymatic activity of TAK1 with an IC50 of 55 nM. Under similar conditions, the known TAK1 inhibitor, takinib, inhibits the kinase with an IC50 of 187 nM. Compound 26 and analogs thereof inhibit the growth of multiple myeloma cell lines MPC-11 and H929 with GI50 values as low as 30 nM. These compounds have the potential to be translated into anti-MM therapeutics.
... Inflammatory responses are critical for the initiation of bone repair [7]. When inflammation occurs, inflammatory cells recruit BMSCs, which direct their migration and differentiation, and the bone marrow microenvironment changes considerably [7], promoting TNF-receptor-related factor 6 (TRAF6) hemagglutination and autoubiquitination [8,9], which provide signals that inhibit osteogenesis through mediating various signaling molecules and activating the NF-κB and MAPK signaling pathways [10,11], thus providing a signal to inhibit osteogenesis [12]. Therefore, the identification of antagonistic signs of osteogenic differentiation of MSCs from the bone marrow microenvironment is key to elucidating the molecular mechanisms of inflammatory bone loss [13][14][15][16]. ...
Osteoporosis is a chronic inflammatory disease that severely affects quality of life. Cornus officinalis is a Chinese herbal medicine with various bioactive ingredients, among which morroniside is its signature ingredient. Although anti-bone resorption drugs are the main treatment for bone loss, promoting bone anabolism is more suitable for increasing bone mass. Therefore, identifying changes in bone formation induced by morroniside may be conducive to developing effective intervention methods. In this study, morroniside was found to promote the osteogenic differentiation of bone marrow stem cells (BMSCs) and inhibit inflammation-induced bone loss in an in vivo mouse model of inflammatory bone loss. Morroniside enhanced bone density and bone microstructure, and inhibited the expression of IL6, IL1β, and ALP in serum (p < 0.05). Furthermore, in in vitro experiments, BMSCs exposed to 0–256 μM morroniside did not show cytotoxicity. Morroniside inhibited the expression of IL6 and IL1β and promoted the expression of the osteogenic transcription factors Runx2 and OCN. Furthermore, morroniside promoted osteocalcin and Runx2 expression and inhibited TRAF6-mediated NF-κB and MAPK signaling, as well as osteoblast growth and NF-κB nuclear transposition. Thus, morroniside promoted osteogenic differentiation of BMSCs, slowed the occurrence of the inflammatory response, and inhibited bone loss in mice with inflammatory bone loss.
... In addition, EAAs prevented the increase of TNFα in the Doxo-treated mice, while increasing the expression of NFkB, which seems to suggest a possible antiapoptotic activity. Although RIP1 is known to mediate cell death in response to DNA damage, it is also crucial for cell survival through activation of the NFkB pathway [63]. The anti-apoptotic activity of NFkB may avoid unnecessary and/or redundant cell death in response to apoptotic stimuli such as TNFα activation [64]. ...
Background:
Doxorubicin (Doxo) is a widely prescribed drug against many malignant cancers. Unfortunately, its utility is limited by its toxicity, in particular a progressive induction of congestive heart failure. Doxo acts primarily as a mitochondrial toxin, with consequent increased production of reactive oxygen species (ROS) and attendant oxidative stress, which drives cardiac dysfunction and cell death. A diet containing a special mixture of all essential amino acids (EAAs) has been shown to increase mitochondriogenesis, and reduce oxidative stress both in skeletal muscle and heart. So, we hypothesized that such a diet could play a favorable role in preventing Doxo-induced cardiomyocyte damage.
Methods:
Using transmission electron microscopy, we evaluated cells' morphology and mitochondria parameters in adult mice. In addition, by immunohistochemistry, we evaluated the expression of pro-survival marker Klotho, as well as markers of necroptosis (RIP1/3), inflammation (TNFα, IL1, NFkB), and defense against oxidative stress (SOD1, glutathione peroxidase, citrate synthase).
Results:
Diets with excess essential amino acids (EAAs) increased the expression of Klotho and enhanced anti-oxidative and anti-inflammatory responses, thereby promoting cell survival.
Conclusion:
Our results further extend the current knowledge about the cardioprotective role of EAAs and provide a novel theoretical basis for their preemptive administration to cancer patients undergoing chemotherapy to alleviate the development and severity of Doxo-induced cardiomyopathy.
... Signalling cascades associated with these TFs are activated by a number of extracellular ligands and membrane-bound receptors, most often represented by members of the super-families of Toll-like receptors (TLRs), TNFR, IL-1R, and antigen receptors. Lately, there have been discovered new signalling pathways, which regulate the activity of NFκB and AP1 in response to changes in the redox potential of cells and intracellular stresses, accompanied by DNA damage, ROS/RNS generation, endoplasmic reticulum stress, the action of intracellular pathogenic factors and are mediated by protein families of RIG-I-like receptors (RLRs) and NOD-like receptors (NLRs) [49,50]. It has been shown the NFκB activation by cytokines TNF and IL-1β requires NADPH-dependent ROS production [51,52] and a decrease in the level of reduced glutathione [53]. ...
One of the adverse outcomes of acute inflammatory response is progressing to the chronic stage or transforming into an aggressive process, which can develop rapidly and result in the multiple organ dysfunction syndrome. The leading role in this process is played by the Systemic Inflammatory Response that is accompanied by the production of pro- and anti-inflammatory cytokines, acute phase proteins, and reactive oxygen and nitrogen species.The purpose of this review that highlights both the recent reports and the results of the authors' own research is to encourage scientists to develop new approaches to the differentiated therapy of various SIR manifestations (low- and high-grade systemic inflammatory response phenotypes) by modulating redox-sensitive transcription factors with polyphenols and to evaluate the saturation of the pharmaceutical market with appropriate dosage forms tailored for targeted delivery of these compounds.Redox-sensitive transcription factors such as NFκB, STAT3, AP1 and Nrf2 have a leading role in mechanisms of the formation of low- and high-grade systemic inflammatory phenotypes as variants of SIR. These phenotypic variants underlie the pathogenesis of the most dangerous diseases of internal organs, endocrine and nervous systems, surgical pathologies, and post-traumatic disorders.The use of individual chemical compounds of the class of polyphenols, or their combinations can be an effective technology in the therapy of SIR. Administering natural polyphenols in oral dosage forms is very beneficial in the therapy and management of the number of diseases accompanied with low-grade systemic inflammatory phenotype. The therapy of diseases associated with high-grade systemic inflammatory phenotype requires medicinal phenol preparations manufactured for parenteral administration.
... Phosphorylated IκB are degraded through the proteasomal pathway. The ubiquitin-proteasome system plays a crucial role in regulating NF-κB activity (Skaug et al., 2009). ...
Periprosthetic osteolysis (PPO) induced by wear particles is an important cause of aseptic loosening after artificial joint replacement, among which the imbalance of osteogenesis and osteoclastic processes occupies a central position. The cells involved in PPO mainly include osteoclasts (macrophages), osteoblasts, osteocytes, and fibroblasts. RANKL/RANK/OGP axis is a typical way for osteolysis. Autophagy, a mode of regulatory cell death and maintenance of cellular homeostasis, has a dual role in PPO. Although autophagy is activated in various periprosthetic cells and regulates the release of inflammatory cytokines, osteoclast activation, and osteoblast differentiation, its beneficial or detrimental role remains controversy. In particular, differences in the temporal control and intensity of autophagy may have different effects. This article focuses on the role of autophagy in PPO, and expects the regulation of autophagy to become a powerful target for clinical treatment of PPO.
... In terms of the mechanism, on the basis of relevant literatures, TAK1 phosphorylates NF-kB-inducing kinase, the IkB kinase complex is activated, which promotes osteoclastogenesis(Ajibade, Wang et al. 2013). Phosphorylation of IKB-α can promote its ubiquitination degradation(Skaug, Jiang et al. 2009). It was further discovered that NaHS can inhibit the phosphorylation of IKB-α, and thus inhibit the ubiquitination degradation of IKB-α. ...
Background: Although several effective therapies are available for the treatment of postmenopausal osteoporosis (PMO), the most common type of primary osteoporosis (OP). More effective and acceptable drugs to cure postmenopausal osteoporosis were needed. NaHS, the donor of H2S, may be one of the drugs to treat PMO, but its role and mechanism are still unclear.
Methods：Ovariectomized mice and Sham operation mice, BMMs and RAW264.7 cell lines were used to illustrate the in vivo and in vitro effects of NaHS on the osteoclast differentiation. On the other hand, molecular and histological methods were applied to evaluate the osteoclast differentiation and investigate the in vivo and in vitro mechanism.
Results: Phenotypically, NaHS treatment can increase the bone mineral density and bone quality of osteoporosis models induced by ovariectomy (OVX) in mice. Mechanistically, NaHS inhibited the nuclear translocation of p65 by inhibiting the ubiquitination and proteasome degradation of IkB-α.
Conclusions: NaHS protects against OVX-induced bone loss by inhibiting osteoclastic bone resorption. It plays an important role in inhibiting osteoclast differentiation and protecting against bone loss in PMO and it is potential for preventing and treating PMO.
... TRAF3 ubiquitination through K63 provides a platform for assembling and activating the NEMO-TBK1/IKKε complex [58]. Ubiquitination and deubiquitination of TRAF3 are critical to regulating interferon production and resisting viral infection [59][60][61]. ...
Seven tumor necrosis factor receptor- (TNFR-) associated factors (TRAFs) have been found in mammals, which are primarily involved in the signal translation of the TNFR superfamily, the Toll-like receptor (TLR) family, and the retinoic acid-inducible gene I- (RIG-I-) like receptor (RLR) family. TRAF3 is one of the most diverse members of the TRAF family. It can positively regulate type I interferon production while negatively regulating signaling pathways of classical nuclear factor-κB, nonclassical nuclear factor-κB, and mitogen-activated protein kinase (MAPK). This review summarizes the roles of TRAF3 signaling and the related immune receptors (e.g., TLRs) in several preclinical and clinical diseases and focuses on the roles of TRAF3 in immune responses, the regulatory mechanisms, and its role in disease.
... For the activation of TPL2 it is necessary a stimulatory signal. The critical first step is the pattern recognition receptors (PRR) identification of pathogen-associated molecular patterns (PAMP) or damageassociated molecular patterns (DAMP), which through the toll-like receptor (TLR), interleukin (IL) 1 receptor (IL-1-R), T-cell receptor (TCR), tumor necrosis factor receptor (TNFR) and CD40L receptors, will lead to the activation of the IKK complex (IKKα, IKKβ and IKKγ (NEMO)), by TAK1 [11,23,24]. This complex will be in charge of phosphorylating p105 at S927 and S932, creating a binding site for SCF βTrCP ubiquitin E3 ligase complex which links to IκBα. ...
The cytoplasmic protein tumor progression locus 2 (TPL2), also known as cancer Osaka thyroid (Cot), or MAP3K8, is thought to have a significant role in a variety of cancers and illnesses and it is a key component in the activation pathway for the expression of inflammatory mediators. Despite the tight connection between inflammation and TPL2, its function has not been extensively studied in chronic liver disease (CLD), a major cause of morbidity and mortality worldwide. Here, we analyze more in detail the significance of TPL2 in CLD to shed light on the pathological and molecular transduction pattern of TPL2 during the progression of CLD. This might result in important advancements and enable progress in the diagnosis and treatment of CLD.
... Interestingly, the same Ub linkage could result in distinct outcomes. For instance, the K63-linked poly-Ub chain plays a role of a degradation inducer for octamer-binding transcription factor-4 (Oct4) in mouse embryonic stem cells [14][15][16]. The K11-linked poly-Ub chain does not serve as a "death label" for β-catenin but increases its stability in human colorectal cancer cells [17,18]. ...
Ubiquitination is extensively involved in critical signaling pathways through monitoring protein stability, subcellular localization, and activity. Dysregulation of this process results in severe diseases including malignant cancers. To develop drugs targeting ubiquitination-related factors is a hotspot in research to realize better therapy of human diseases. Ubiquitination comprises three successive reactions mediated by Ub-activating enzyme E1, Ub-conjugating enzyme E2, and Ub ligase E3. As expected, multiple ubiquitination enzymes have been highlighted as targets for anticancer drug development due to their dominant effect on tumorigenesis and cancer progression. In this review, we discuss recent progresses in anticancer drug development targeting enzymatic machinery components.
... The NF-κB pathway plays an important role in pathological neuroinflammation [1]. In the activated state, NF-κB regulates the expression of pro-inflammatory genes through IKKβ phosphorylation, IκBα degradation, and subsequent p65 translocation to the nucleus [15][16][17]. The results of western blot (WB) assay showed that in BV2 cells activated by LPS, the expression of p-IKK and p-p65 increased significantly, while the expression of IκBα decreased significantly. ...
The occurrence and development of neurodegenerative diseases is related to a variety of physiological and pathological changes. Neuroinflammation is one of the major factors that induces and aggravates neurodegenerative diseases. The most important manifestation of neuroinflammation is the activation of microglia. Therefore, inhibiting the abnormal activation of microglia is an important way to alleviate the occurrence of neuroinflammatory diseases. In this research, the inhibitory effect of tabersonine (Tab) on neuroinflammation was evaluated by establishing the BV2 neuroinflammation model induced by lipopolysaccharide (LPS). It was found that Tab significantly inhibited the production and expression of nitric oxide (NO), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and reactive oxygen species (ROS) in BV-2 cells stimulated by LPS. In addition, Tab can also inhibit the activation of nuclear factor-κB (NF-κB) induced by LPS, thus regulating inflammatory mediators such as inducible nitric oxide synthase (iNOS). These results indicated that Tab regulated the release of inflammatory mediators such as NO, IL-1β, TNF-α, and IL-6 by inhibiting NF-κB signaling pathway, and exerting its anti-neuroinflammatory effect. This is the first report regarding the inhibition on LPS-induced neuroinflammation in BV2 microglia cells of Tab, which indicated the drug development potential of Tab for the treatment of neurodegenerative diseases.
... The results showed that the pH240R-induced NEMO degradation was rescued by chloroquine or BafA1 but not by MG132 (Fig. 5H), indicating that pH240R downregulates NEMO levels via autophagy-mediated lysosomal degradation. Ik Ba is a regulator of NF-k B activation and binds tightly to NF-k B dimers (39). Phosphorylation by the IKK complex triggers Ik Ba ubiquitination and degradation. ...
African swine fever (ASF), a lethal hemorrhagic disease, is caused by African swine fever virus (ASFV). There are no commercially available vaccines or antivirals for the disease.
... Previous studies have shown that the stimulation of inflammation causes TNF receptor-associated factor 6 (TRAF6), a critical protein of the classical NF-κB signaling pathway, to activate ubiquitinase and synthesize a K63-Ub chain which recruits and binds TAK1-TABs complexes and IκB kinase (IKK) α/β/γ complexes. Later the phosphorylation of IκB ultimately releases the transcription factor, p50/RELA(p65), into the nuclei to initiate downstream signal transduction [11,12]. The abnormal activation of the NF-κB signaling pathway is an important precipitating factor for inflammation-cancer transformation and for the occurrence and development of CRC [13,14]. ...
The abnormal activation of the nuclear factor-kappa B (NF-κB) signaling pathway is an important precipitating factor for the inception and development of colorectal cancer (CRC), one of the most common tumors worldwide. As a pro-apoptotic transcription factor, monocyte chemotactic protein-induced protein 1 (MCPIP1) has been closely associated with many tumor types. In the present study, the expression of MCPIP1 was firstly discovered reduced in CRC tissues and correlated with poor patient prognosis. The decreased expression was caused by promoter hypermethylation. Overexpressed MCPIP1 was found to inhibit the proliferative and migratory abilities of CRC cells, whereas knockdown of MCPIP1 produced the opposite result. The subsequent investigation demonstrated that MCPIP1 exerted its “anti-cancer” effect by suppression of the NF-κB signaling pathway through negative regulation of K63-linked ubiquitylation of TNF receptor associated factor 6 (TRAF6). Therefore, our results indicate a prognostic marker for CRC and a theoretical basis for MCPIP1 as a treatment.
... It is well established that NF-κB is involved in the regulation of multiple pro-inflammatory mechanisms. Activation of NF-κB is necessary and sufficient for the transcriptional activation of IL-1β, IL-6, IL-17, MCP-1, and TNF-α receptor activator of NF-κB ligand (RANKL), and cyclooxygenase 2 (COX-2), all of which are required for the initiation, amplification, and maintenance of chronic inflammations, including that seen in RA (Ghosh and Hayden 2008;Skaug et al. 2009;Sun 2012;Napetschnig and Wu 2013). Especially, TNF-α is critical in the pathogenesis of RA. ...
Rheumatoid arthritis (RA) is a great concern across the globe. It is characterized as an autoimmune disease, where the body's immune system mistakenly attacks bone joints causing them to erode which leads to deformity in structure. Thus, the present study was conducted to elucidate the effect of Lidang Tang (LDT) decoction, a traditional Chinese medicine against rheumatoid arthritis. The rheumatoid arthritis was induced in Sprague-Dawley albino rats by intradermal injection of bovine collagen II type at the tail. Results suggested that LDT reduces paw swelling and arthritic scores in rats. The protective effect of LDT against RA was further substantiated by histopathological analysis of synovial tissue of rats, where LDT improves the architecture of the tissues as compared to CIA rats. It also reduces oxidative stress and inflammation in CIA rats as compared to the disease model group. The serum level of anti-collagen II-specific immunoglobulins (IgG1 and IgG2a) was reduced significantly in LDT treated group. In a western blot analysis, LDT treated group showed a significant reduction in the expression of NF-ĸB and COX-2 as compared to CIA rats. Collectively, our study for the first time demonstrated the protective effect of Lidang Tang decoction against rheumatoid arthritis.
... This is key to mediate protein ubiquitination as well as oligomerization and association of TRAF members with their upstream receptors, adaptors, and downstream effector proteins. Through the lysine (K)-48 and K63linked polyubiquitin chain, TRAF members control multiple signaling pathways that in turn are important for the induction of genes associated with innate immunity, inflammation, and cell survival (34,35). Recent studies demonstrated that some TRAF proteins, especially TRAF2 and TRAF3, could function as negative regulators in NF-κB signaling pathways, in which their E3 activity is key for their regulatory roles (35)(36)(37). ...
SGK1 is a serine/threonine kinase that plays important roles in the cellular stress response. While SGK1 has been reported to restrain inflammatory immune responses, the molecular mechanism involved remains elusive, especially in the oral bacteria-induced inflammatory milieu. In this study, we found that SGK1 curtails Porphyromonas gingivalis-induced inflammatory responses through maintaining levels of TNF receptor-associated factor (TRAF) 3, and thereby suppressing NF-κB signaling. We show pharmacological inhibition of SGK1 significantly enhances the production of pro-inflammatory cytokines including TNFα, IL-6, IL-1β, and IL-8 in P. gingivalis-stimulated innate immune cells. The results were confirmed with siRNA and LysM-Cre-mediated SGK1 knockout mice. Moreover, SGK1 deletion robustly increased NF-κB activity and c-Jun expression, but failed to alter activation of MAPK signaling pathways. Further mechanistic data revealed that SGK1 deletion elevates phosphorylation of TRAF2, which leads to TRAF3 degradation in a proteasome-dependent manner. Importantly, we show siRNA-mediated traf3 silencing or overexpression of c-Jun mimics the effect of SGK1 inhibition on P. gingivalis-induced inflammatory cytokines and NF-κB activation. Additionally, using a P. gingivalis infection-induced periodontal bone loss model, we found inhibition of SGK1 modulates expression of TRAF3 and c-Jun, aggravates inflammatory responses in gingival tissues, and exacerbates alveolar bone loss. Altogether, we demonstrated for the first time that SGK1 acts as a rheostat to limit P. gingivalis-induced inflammatory immune responses and mapped out a novel SGK1-TRAF2/3-c-Jun/NF-κB signaling axis. These findings provide novel insights into the anti-inflammatory molecular mechanisms of SGK1 and suggest novel interventional targets relevant to inflammatory diseases that could extend beyond the oral cavity.
... A pathway could be activated if inhibitor proteins get ubiquitinated and degraded, or it could stop the transduction by targeting active enzymes for degradation, after the signal disappears. K63-linked chains instead usually modulate the function of proteins or serve as platform for subsequent signalling steps, instead of mediating degradation (Skaug et al. 2009;Zhang et al. 2017). ...
Lysosomal-dependent self-degradative (autophagic) mechanisms are essential for the maintenance of normal homeostasis in all eukaryotic cells. Several types of such self-degradative and recycling pathways have been identified, based on how the cellular self material can incorporate into the lysosomal lumen. Ubiquitination, a well-known and frequently occurred posttranslational modification has essential role in all cell biological processes, thus in autophagy too. The second most common type of polyubiquitin chain is the K63-linked polyubiquitin, which strongly connects to some self-degradative mechanisms in the cells. In this review, we discuss the role of this type of polyubiquitin pattern in numerous autophagic processes.
... TRAF6 is an ubiquitin E3 ligase that catalyzes lysine-63 (K63) polyubiquitination in target proteins such as IκB kinase or as a self-polyubiquitination of TRAF6, which in turn forms a recognition signal for the recruitment of proteins such as TAB2/3 and TAK1, leading to NF-κB activation. TRAF6 can also be modified by K48-linked ubiquitination that targets proteasome degradation [53][54][55]. Therefore, future analysis should investigate if RANKL stimulation routes TRAF6 to degradation by increasing TRAF6-K48 ubiquitination in macrophages and blocking the inflammatory process. ...
High serum levels of osteoprotegerin (OPG) are found in patients with obesity, type 2 diabetes, sepsis, or septic shock and are associated with a high mortality rate in stroke. The primary known function of OPG is to bind to the receptor activator of NF-κB ligand (RANKL), and by doing so, it inhibits the binding between RANKL and its receptor (RANK). TLR4 signaling in macrophages involves TRAF6 recruitment and contributes to low-grade chronic inflammation in adipose tissue. LPS is a classical activator of the TLR4 pathway and induces the expression of inflammatory cytokines in macrophages. We have previously observed that in the presence of RANKL, there is no LPS-induced activation of TLR4 in macrophages. In this study, we investigated the crosstalk between RANK and TLR4 pathways in macrophages stimulated with both RANKL and LPS to unveil the role of OPG in inflammatory processes. We found that RANKL inhibits TLR4 activation by binding to RANK, promoting the binding between TRAF6 and RANK, lowering TLR4 activation and the expression of proinflammatory mediators. Furthermore, high OPG levels aggravate inflammation by inhibiting RANKL. Our findings elect RANKL as a candidate for drug development as a way to mitigate the impact of obesity-induced inflammation in patients.
... Among them, IRAK1 is a key signal transducer [25], which can be recruited by myeloid differentiation marker 88 (MyD88) and leads to the phosphorylation of IRAK1 and IRAK4 [45]. Phosphorylation of IRAK1 and IRAK4 facilitates oligomerization and auto-ubiquitination of TRAF6 [46], which is followed by the activation of IKK, and ultimately leads to the activation of NF-κB [47]. A few studies have reported that lncRNAs can influence the modification of these signaling molecules and affect the activation of the NF-κB pathway. ...
Escherichia coli is the most common gram-negative pathogenic bacterium causing meningitis. It penetrates the blood–brain barrier (BBB) and activates nuclear factor kappa B (NF-κB) signaling, which are vital events leading to the development of meningitis. Long non-coding RNAs (lncRNAs) have been implicated in regulating neuroinflammatory signaling, and our previous study showed that E. coli can induce differential expression of lncRNAs, including lncC11orf54-1, in human brain microvascular endothelial cells (hBMECs). The hBMECs constitute the structural and functional basis for the BBB, however, it is unclear whether lncRNAs are involved in the regulation of inflammatory responses of hBMECs during meningitic E. coli infection. In this study, we characterized an abundantly expressed lncRNA, lncC11orf54-1, which was degraded by translocated coilin to produce mgU2-19 and mgU2-30 in hBMECs during E. coli infection. Functionally, lncC11orf54-1-originated non-coding RNA mgU2-30 interacted with interleukin-1 receptor-associated kinase 1 (IRAK1) to induce its oligomerization and autophosphorylation, thus promoting the activation of NF-κB signaling and facilitating the production of pro-inflammatory cytokines. In summary, our study uncovers the involvement of lncC11orf54-1 in IRAK1–NF-κB signaling, and it functions as a positive regulator of inflammatory responses in meningitic E. coli -induced neuroinflammation, which may be a valuable therapeutic and diagnostic target for bacterial meningitis.
... Deubiquitination is a process that counteracts ubiquitination by detaching Ub moieties or Ub-like conjugates from target proteins by DUBs, thereby emerging as a key regulator of ubiquitin-mediated signaling pathways by maintaining a balance of ubiquitin dynamics [130]. The human genome encodes more than 100 DUBs that have been categorized into seven families: Ubiquitin C-terminal hydrolases (UCHs), ubiquitin-specific proteases (USPs), ovarian tumor proteases (OTUs), Jab1/MPN domain-associated metalloisopeptidases (JAMM), Josephin or Machado-Joseph disease domain proteases (MJDs), motif interacting with Ub-containing novel DUB family (MINDY), and Zn-finger and UFSP domain proteins (ZUFSPs) [130][131][132][133]. Deubiquitination is a highly coordinated process that has implications in various cellular functions, such as cell-cycle regulation, DNA repair [22], proteasome-and lysosome-dependent protein degradation [134], kinase activation [135,136], microbial pathogenesis [137,138], and gene expression. Owing to the role of DUBs as key regulators of many ubiquitin-mediated processes, their localization, abundance, and catalytic activity are strongly controlled by a variety of mechanisms. ...
Reactive oxygen species (ROS) act as a double-edged sword in cancer, where low levels of ROS are beneficial but excessive accumulation leads to cancer progression. Elevated levels of ROS in cancer are counteracted by the antioxidant defense system. An imbalance between ROS generation and the antioxidant system alters gene expression and cellular signaling, leading to cancer progression or death. Post-translational modifications, such as ubiquitination, phosphorylation, and SUMOylation, play a critical role in the maintenance of ROS homeostasis by controlling ROS production and clearance. Recent evidence suggests that deubiquitinating enzymes (DUBs)-mediated ubiquitin removal from substrates is regulated by ROS. ROS-mediated oxidation of the catalytic cysteine (Cys) of DUBs, leading to their reversible inactivation, has emerged as a key mechanism regulating DUB-controlled cellular events. A better understanding of the mechanism by which DUBs are susceptible to ROS and exploring the ways to utilize ROS to pharmacologically modulate DUB-mediated signaling pathways might provide new insight for anticancer therapeutics. This review assesses the recent findings regarding ROS-mediated signaling in cancers, emphasizes DUB regulation by oxidation, highlights the relevant recent findings, and proposes directions of future research based on the ROS-induced modifications of DUB activity.
... TRAF6 is the major transducer of TLR signaling, and the activated TRAF6 then synthesizes lysine-63-linked polyubiquitin chains on itself and the protein kinase complex including transforming growth factor b (TGF-b)-activated kinase 1 (TAK1) (also called mitogen-activated protein kinase kinase kinase 7 [MAP3K7]), TAK1-binding protein 1 (TAB1) (also called MAP3K7P1), and TAB2 (also called MAP3K7P2) (4)(5)(6). Then, activated TAK1 phosphorylates the Ik B kinase (IKK) complex, which results in the activation of activated protein 1 (AP-1) and NF-k B transcription factors (7,8). ...
Upon recognition of the pathogen components by PRR (pattern recognition receptors), then the cells could be activated to produce inflammatory cytokines and type I interferons. The inflammation is tightly modulated by the host to prevent inappropriate inflammatory responses. MicroRNAs (miRNAs) are non-coding and small RNAs that can inhibit gene expression and participate in various biological functions, including maintaining a balanced immune response in the host. To maintain the balance of the immune response, these pathways are closely regulated by the host to prevent inappropriate reactions of the cells. However, in low vertebrates, the miRNA-mediated inflammatory response regulatory networks remain largely unknown. Here, we report that two miRNAs, miR-20-1 and miR-101a are identified as negative regulators in teleost inflammatory responses. Initially, we find that both miR-20-1 and miR-101a dramatically increased after lipopolysaccharide (LPS) stimulation and Vibrio harveyi infection. Upregulated miR-20-1 and miR-101a inhibit LPS-induced inflammatory cytokines production by targeting TNF receptor-associated factor 6 (TRAF6), thus avoiding excessive inflammation. Moreover, miR-20-1 and miR-101a regulate the inflammatory responses through the TRAF6-mediated nuclear factor kappa (NF-κB) signaling pathways. Collectively, these data indicate that miR-20-1 and miR-101a act as negative regulators through regulating the TRAF6-mediated NF-κB signaling pathway, and participate in the host antibacterial immune responses, which will provide new insight into the intricate networks of the host-pathogen interaction in the lower vertebrates.
... [15] TAK1 is a MAP3K protein and has been defined as a critical upstream molecule of MAPKs. [16] Thus, to further investigate whether breviscapine protects against NASH by regulating the MAPK signaling pathway, western blotting was used to analyze the activation of MAPK signaling in vitro and in vivo. The phosphorylation of TAK1, c-Jun N-terminal kinase (JNK), and p38 was significantly suppressed by breviscapine treatment after PA, HFD, or HFHC diet treatment ( Figure 7G). ...
Background & aims:
Nonalcoholic fatty liver disease (NAFLD) is a key component of metabolic syndrome ranging from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH) and is now becoming the leading cause of cirrhosis and hepatocellular carcinoma worldwide. However, due to the complex and unclear pathophysiological mechanism, there are no specific approved agents for treating NASH. Breviscapine, a natural flavonoid prescription drug isolated from the traditional Chinese herb Erigeron breviscapus, exhibits a wide range of pharmacological properties, including effects on metabolism. However, the anti-NASH efficacy and mechanisms of breviscapine have not yet been characterized.
Approach & results:
We evaluated the effects of breviscapine on the development of hepatic steatosis, inflammation and fibrosis in vivo and in vitro under metabolic stress. Breviscapine treatment significantly reduced lipid accumulation, inflammatory cell infiltration, liver injury and fibrosis in mice fed a high-fat diet (HFD), a high-fat/high-cholesterol (HFHC) diet, or a methionine- and choline-deficient (MCD) diet. In addition, breviscapine attenuated lipid accumulation, inflammation and lipotoxicity in hepatocytes undergoing metabolic stress. RNA sequencing and multiomics analyses further indicated that the key mechanism linking the anti-NASH effects of breviscapine was inhibition of TAK1 phosphorylation and the subsequent MAPK signaling cascade. Treatment with the TAK1 inhibitor 5Z-7-oxozeaenol abrogated breviscapine-mediated hepatoprotection under metabolic stress. Molecular docking illustrated that breviscapine directly bound to TAK1.
Conclusion:
Breviscapine prevents metabolic stress-induced NASH progression through direct inhibition of TAK1 signaling. Breviscapine might be a novel therapeutic candidate for the treatment of NASH.
... K63-linked polyubiquitination of TRAF6 is essential for NF-kB signaling (30). Such self-linked polyubiquitin chains serve as a scaffold to assemble the downstream signaling complex consisting of TAB2, NEMO, TAK1, and IKKs, which subsequently leads to IkB degradation and NF-kB activation (31). K48-linked polyubiquitination of TRAF6, however, might lead to protein degradation and hence negatively regulate the NF-kB pathway (32). ...
The apextrin C-terminal (ApeC) domain is a class of newly discovered protein domains with an origin dating back to prokaryotes. ApeC-containing proteins (ACPs) have been found in various marine and aquatic invertebrates, but their functions and the underlying mechanisms are largely unknown. Early studies suggested that amphioxus ACP1 and ACP2 bind to bacterial cell walls and have a role in immunity. Here we identified another two amphioxus ACPs (ACP3 and ACP5), which belong to the same phylogenetic clade with ACP1/2, but show distinct expression patterns and sequence divergence (40-50% sequence identities). Both ACP3 and ACP5 were mainly expressed in the intestine and hepatic cecum, and could be up-regulated after bacterial challenge. Both prokaryotic-expressed recombinant ACP3 and ACP5 could bind with several species of bacteria and yeasts, showing agglutinating activity but no microbicidal activity. ELISA assays suggested that their ApeC domains could interact with peptidoglycan (PGN), but not with lipoteichoic acid (LTA), lipopolysaccharides (LPS) and zymosan A. Furthermore, they can only bind to Lys-type PGN from Staphylococcus aureus, but not to DAP-type PGN from Bacillus subtilis and not to moieties of PGN such as MDPs, NAMs and NAGs. This recognition spectrum is different from that of ACP1/2. We also found that when expressed in mammalian cells, ACP3 could interact with TRAF6 via a conserved non-ApeC region, which inhibited the ubiquitination of TRAF6 and hence suppressed downstream NF-κB activation. This work helped define a novel subfamily of ACPs, which have conserved structures, and have related yet diversified molecular functions. Its members have dual roles, with ApeC as a lectin and a conserved unknown region as a signal transduction regulator. These findings expand our understanding of the ACP functions and may guide future research on the role of ACPs in different animal clades.
... The essential roles ubiquitylation plays in the activation of NF-κB pathways has been discussed in detail elsewhere [64,65]. The focus here will be the reversal of ubiquitylation by deubiquitylating enzymes (DUBs). ...
Cells must adapt to changes in their environment to maintain cell, tissue and organismal integrity in the face of mechanical, chemical or microbiological stress. Nuclear factor-κB (NF-κB) is one of the most important transcription factors that controls inducible gene expression as cells attempt to restore homeostasis. It plays critical roles in the immune system, from acute inflammation to the development of secondary lymphoid organs, and also has roles in cell survival, proliferation and differentiation. Given its role in such critical processes, NF-κB signalling must be subject to strict spatiotemporal control to ensure measured and context-specific cellular responses. Indeed, deregulation of NF-κB signalling can result in debilitating and even lethal inflammation and also underpins some forms of cancer. In this review, we describe the homeostatic feedback mechanisms that limit and ‘re-set’ inducible activation of NF-κB. We first describe the key components of the signalling pathways leading to activation of NF-κB, including the prominent role of protein phosphorylation and protein ubiquitylation, before briefly introducing the key features of feedback control mechanisms. We then describe the array of negative feedback loops targeting different components of the NF-κB signalling cascade including controls at the receptor level, post-receptor signalosome complexes, direct regulation of the critical ‘inhibitor of κB kinases’ (IKKs) and inhibitory feedforward regulation of NF-κB-dependent transcriptional responses. We also review post-transcriptional feedback controls affecting RNA stability and translation. Finally, we describe the deregulation of these feedback controls in human disease and consider how feedback may be a challenge to the efficacy of inhibitors.
... After TNF-α binds to its receptor, TNFR1, two protein complexes can subsequently be formed. The first is called the TNFR1 signaling complex (TNF-RSC, also known as complex-I), which is related to NF-κB's transcriptional activity and cell survival; the second complex is called complex-II and is related to apoptosis [2]. Generally, the signal pathway induced by complex-I can inhibit the apoptotic signal pathway of complex-II. ...
Monocyte chemoattractant protein-1-induced protein 1 (MCPIP1) is rapidly produced under proinflammatory stimuli, thereby feeding back to downregulate excessive inflammation. In this study, we used the stable, inducible expressions of wild-type (WT) MCPIP1 and an MCPIP1-D141N mutant in T-REx-293 cells by means of a tetracycline on (Tet-on) system. We found that WT MCPIP1 but not MCPIP1-D141N mutant expression dramatically increased apoptosis, caspase-3, -7, -8, and -9 activation, and c-Jun N-terminal kinase (JNK) phosphorylation in TNF-α-treated cells. The pan-caspase inhibitor, z-VAD-fmk, and the caspase-1 inhibitor, z-YVAD-fmk, but not the JNK inhibitor, SP600125, significantly reversed apoptosis and caspase activation in TNF-α/MCPIP1-treated cells. Surprisingly, MCPIP1 itself was also cleaved, and the cleavage was suppressed by treatment with the pan-caspase inhibitor and caspase-1 inhibitor. Moreover, MCPIP1 was found to contain a caspase-1/-4 consensus recognition sequence located in residues 234~238. As expected, the WT MCPIP1 but not the MCPIP1-D141N mutant suppressed NF-κB activation, as evidenced by inhibition of IκB kinase (IKK) phosphorylation and IκB degradation using Western blotting, IKK activity using in vitro kinase activity, and NF-κB translocation to nuclei using an immunofluorescence assay. Interestingly, MCPIP1 also significantly inhibited importin α3 and importin α4 expressions, which are major nuclear transporter receptors for NF-κB. Inhibition of NF-κB activation further downregulated expression of the caspase-8 inhibitor, cFLIP. In summary, the results suggest that MCPIP1 could enhance the TNF-α-induced apoptotic pathway through decreasing NF-κB activation and cFLIP expression.
... In addition to TRAF6, a recent study found that the activation of TIR-domain-containing adapter-inducing interferon-β (TRIF)-dependent signaling induced-by Pellino1-mediated ubiquitination of RIP1 also contributed to the upregulation of NF-κB (Sato et al. 2003;Chang et al. 2009). Moreover, Pellino1 deficiency may interfere with the ubiquitination of RIP1 and TRAF6 which are needed for TLR3/4-mediated pro-inflammatory gene induction, thereby increasing the resistance of mice to LPS-induced lethality (Chang et al. 2009;Skaug et al. 2009). Collectively, the importance of Pellino1 in LPS-induced inflammatory signaling makes it reasonable to assume that suppressing Pel-lino1 do be especially effective in attenuating LPS-induced myocarditis. ...
Pellino1 has been shown to regulate proinflammatory genes by activating the nuclear factor kappa B (NF-κB) and Toll-like receptor (TLR) signaling pathways, which are important in the pathological development of lipopolysaccharide (LPS)-induced myocarditis. However, it is still unknown whether silencing Pellino1 (si-Pellino1) has a therapeutic effect on this disease. Here, we showed that silencing Pellino1 can be a potential protective strategy for abnormal myocardial energy metabolism in LPS-induced myocarditis. We used liquid chromatography electrospray–ionization tandem mass spectrometry (LC–MS/MS) to analyze samples from si-Pellino1 neonatal rat cardiac myocytes (NRCMs) treated with LPS or left untreated. After normalization of the data, metabolite interaction analysis of matched KEGG pathway associations following si-Pellino1 treatment was applied, accompanied by interaction analysis of gene and metabolite associations after this treatment. Moreover, we used western blot (WB) and polymerase chain reaction (PCR) analyses to determine the expression of genes involved in regulating cardiac energy and energy metabolism in different groups. LC–MS-based metabolic profiling analysis demonstrated that si-Pellino1 treatment could alleviate or even reverse LPS-induced cellular damage by altering cardiomyocytes energy metabolism accompanied by changes in key genes (Cs, Cpt2, and Acadm) and metabolites (3-oxoocotanoyl-CoA, hydroxypyruvic acid, lauroyl-CoA, and NADPH) in NRCMs. Overall, our study unveiled the promising cardioprotective effect of silencing Pellino1 in LPS-induced myocarditis through fuel and energy metabolic regulation, which can also serve as biomarkers for this disease.
Targeting lipopolysaccharide (LPS)/toll-like receptor 4 signaling in mononuclear phagocytes has been explored for the treatment of inflammation and inflammation-related disorders. However, only a few key targets have been translated into clinical applications. Flavonoids, a class of ubiquitous plant secondary metabolites, possess a privileged scaffold which serves as a valuable template for designing pharmacologically active compounds directed against diseases with inflammatory components. This perspective provides a general overview of the diversity of flavonoids and their multifaceted mechanisms that interfere with LPS-induced signaling in monocytes and macrophages. Focus is placed on flavonoids targeting MD-2, IκB kinases, c-Jun N-terminal kinases, extracellular signal-regulated kinase, p38 MAPK and PI3K/Akt or modulating LPS-related gene expression.
The COVID-19 disease, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), emerged in late 2019 and rapidly spread worldwide, becoming a pandemic that infected millions of people and caused significant deaths. COVID-19 continues to be a major threat, and there is a need to deepen our understanding of the virus and its mechanisms of infection. To study the cellular responses to SARS-CoV-2 infection, we performed an RNA sequencing of infected vs. uninfected Calu-3 cells. Total RNA was extracted from infected (0.5 MOI) and control Calu-3 cells and converted to cDNA. Sequencing was performed, and the obtained reads were quality-analyzed and pre-processed. Differential expression was assessed with the EdgeR package, and functional enrichment was performed in EnrichR for Gene Ontology, KEGG pathways, and WikiPathways. A total of 1040 differentially expressed genes were found in infected vs. uninfected Calu-3 cells, of which 695 were up-regulated and 345 were down-regulated. Functional enrichment analyses revealed the predominant up-regulation of genes related to innate immune response, response to virus, inflammation, cell proliferation, and apoptosis. These transcriptional changes following SARS-CoV-2 infection may reflect a cellular response to the infection and help to elucidate COVID-19 pathogenesis, in addition to revealing potential biomarkers and drug targets.
Posttranslational modifications are crucial in determining the functions of proteins in the cell. Modification of the NLRP3 inflammasome by the ubiquitin system has recently emerged as a new level of regulation of the inflammasome complex. Here we describe a method to detect poly-ubiquitination of NRLP3 using two different approaches: (i) detection with a ubiquitin antibody or (ii) using TUBEs (Tandem Ubiquitin Binding entities). This approach can be used to detect ubiquitination of other NLRs or other proteins.Key wordsImmunoprecipitationUbiquitinationInflammasomeNLRP3TUBE
Tumor-associated macrophages (TAMs) are key components of tumor immune microenvironment and play a dual role in promoting tumor growth and anti-tumor immunity. Therefore, regulating TAMs has become a promising method in cancer immunotherapy. NF- κB pathway is the key regulatory pathway of TAMs. Targeting this pathway has shown the potential to improve tumor immune microenvironment. At present, there are still some controversies and the idea of combined therapy in this field. This article reviews the progress in the field of immunotherapy in improving tumor immune microenvironment by exploring the mechanism of regulating TAMs (including promoting M1 polarization, inhibiting M2 polarization and regulating TAMs infiltration).
The specificity of CRISPR-Cas9 in response to particular pathological stimuli remains largely unexplored. Hence, we designed an inflammation-inducible CRISPR-Cas9 system by grafting a sequence that binds with NF-κB to the CRISPR-Cas9 framework, termed NBS-CRISPR. The genetic scissor function of this developed genome-editing tool is activated on encountering an inflammatory attack and is inactivated or minimized in non-inflammation conditions. Furthermore, we employed this platform to reverse inflammatory conditions by targeting the MyD88 gene, a crucial player in the NF-κB signaling pathway, and achieved impressive therapeutic effects. Finally, during inflammation, P65 (RELA) can translocate to the nucleus from the cytoplasm. Herein, to avoid Cas9 leaky DNA cleavage activity i, we constructed an NBS-P65-CRISPR system expressing the Cas9-p65 fusion protein. Our inflammation inducible Cas9-mediated genome editing strategy provides new perspectives and avenues for pathological gene interrogation.
Research on obesity- and diabetes mellitus (DM)-related carcinogenesis has expanded exponentially since these two diseases were recognized as important risk factors for cancers. The growing interest in this area is prominently actuated by the increasing obesity and DM prevalence, which is partially responsible for the slight but constant increase in pancreatic cancer (PC) occurrence. PC is a highly lethal malignancy characterized by its insidious symptoms, delayed diagnosis, and devastating prognosis. The intricate process of obesity and DM promoting pancreatic carcinogenesis involves their local impact on the pancreas and concurrent whole-body systemic changes that are suitable for cancer initiation. The main mechanisms involved in this process include the excessive accumulation of various nutrients and metabolites promoting carcinogenesis directly while also aggravating mutagenic and carcinogenic metabolic disorders by affecting multiple pathways. Detrimental alterations in gastrointestinal and sex hormone levels and microbiome dysfunction further compromise immunometabolic regulation and contribute to the establishment of an immunosuppressive tumor microenvironment (TME) for carcinogenesis, which can be exacerbated by several crucial pathophysiological processes and TME components, such as autophagy, endoplasmic reticulum stress, oxidative stress, epithelial-mesenchymal transition, and exosome secretion. This review provides a comprehensive and critical analysis of the immunometabolic mechanisms of obesity- and DM-related pancreatic carcinogenesis and dissects how metabolic disorders impair anticancer immunity and influence pathophysiological processes to favor cancer initiation.
Optineurin/OPTN polymorphism, M98K is associated with normal tension glaucoma in certain populations, and genetic evidence shows its interaction with tumour necrosis factor–alpha (TNFα) polymorphism in causing glaucoma. Endoplasmic reticulum (ER) stress is also associated with glaucoma. We hypothesized that M98K‐OPTN may sensitize retinal ganglion cells to various types of stress. To test this hypothesis, stable clones of a retinal cell line, 661W, expressing either wild‐type (WT)‐OPTN or M98K‐OPTN were generated and examined for their survival under various stress conditions. Compared with WT‐OPTN expressing cells, M98K‐OPTN expressing cells showed significantly lower cell survival and higher activation of caspase‐3 and caspase‐8 upon treatment with tunicamycin (an inducer of ER stress) or TNFα. Levels of ER stress sensors IRE1α, PERK and ATF6 were significantly higher in M98K‐OPTN expressing cells. Tunicamycin treatment resulted in significantly higher induction of ER stress marker CHOP and several other ER stress response genes regulated by IRE1α‐XBP1, PERK‐ATF4 and ATF6 pathways, in M98K‐OPTN expressing cells. Splicing of XBP1 and ATF6 activation was higher in tunicamycin‐treated M98K‐OPTN expressing cells. Increased levels of PERK and IRE1α proteins in M98K‐OPTN expressing cells were dependent on autophagy. Overall, our results show that M98K‐OPTN sensitizes retinal cells to TNFα and ER stress‐induced cell death. We also show that M98K‐OPTN alters ER stress response signalling, which possibly enhances the sensitivity of retinal cells to ER stress. Our results provide support to the hypothesis that M98K‐OPTN may cooperate with other genetic or environmental factors to cause retinal ganglion cell death associated with glaucoma.
Persistent hyperglycemia increases inflammation response, promoting the development of myocardial fibrosis. Based on our previous research that exercise and metformin alone or their combination intervention could attenuate myocardial fibrosis in db/db mice, this study aimed to further explore the underlying mechanisms by which these interventions attenuate myocardial fibrosis in early diabetic cardiomyopathy. Forty BKS db/db mice were randomly divided into four groups. Diabetic db/db mice without intervention were in the C group. Aerobic exercise (7–12 m/min, 30–40 min/day, 5 days/week) was performed in the E group. Metformin (300 mg·kg⁻¹·day⁻¹) was administered in the M group. Exercise combined with metformin was performed in the EM group. Ten wild-type mice were in the WT group. All interventions were administered for 8 weeks. Results showed that the expression levels of α-SMA, Collagen I, and Collagen III were increased in 16-week-old db/db mice, which were reversed by exercise and metformin alone or their combination intervention. All interventions attenuated the level of TGF-β1/Smad2/3 pathway–related proteins and reduced the expression of inflammatory signaling pathway–regulated proteins TNF-α, p-IκBα/IκBα, and p-NF-κB p65/NF-κB p65 in db/db mice. Furthermore, metformin intervention inhibited HNF4α expression via AMPK activation, whereas exercise intervention increased the expression of IL-6 instead of activating AMPK. In conclusion, exercise and metformin alone or their combination intervention inhibited the TGF-β1/Smad pathway to attenuate myocardial fibrosis by reducing NF-κB–mediated inflammatory response. The anti-fibrotic effects were regulated by metformin-activated AMPK or exercise-induced elevation of IL-6, whereas their combination intervention showed no synergistic effects.
The type III secretion system (T3SS) effector EseN is encoded on the Edwardsiella ictaluri chromosome and is homologous to a family of T3SS effector proteins with phosphothreonine lyase activity. Previously we demonstrated that E. ictaluri invasion activates extracellular signal-regulated kinases 1 and 2 (ERK1/2) early in the infection, which are subsequently inactivated by EseN. Comparative transcriptomic analysis showed a total of 753 significant differentially expressed genes in head-kidney-derived macrophages (HKDM) infected with an EseN mutant (∆EseN) compared to HKDM infected with wild-type (WT) strains. This data strongly indicates classical activation of macrophages (the M1 phenotype) in response to E. ictaluri infection and a significant role for EseN in the manipulation of this process. Our data also indicates that E. ictaluri EseN is involved in the modulation of pathways involved in the immune response to infection and expression of several transcription factors, including NF-κβ (c-rel and relB), creb3L4, socs6 and foxo3a. Regulation of transcription factors leads to regulation of proinflammatory interleukins (IL-8, IL-12a, IL-15, IL-6) and cyclooxygenase-2 (COX-2) expression. Inhibition of COX-2 mRNA by WT E. ictaluri leads to decreased production of prostaglandin E2 (PGE2), which is the product of COX-2 activity. Collectively, our results indicate that E. ictaluri EseN is an important player in the modulation of host immune responses to E.ictaluri infection.
To aid in the prioritization of deubiquitinases (DUBs) as anticancer targets, we developed an approach combining activity-based protein profiling (ABPP) with mass spectrometry in both non-small cell lung cancer (NSCLC) tumor tissues and cell lines along with analysis of available RNA interference and CRISPR screens. We identified 67 DUBs in NSCLC tissues, 17 of which were overexpressed in adenocarcinoma or squamous cell histologies and 12 of which scored as affecting lung cancer cell viability in RNAi or CRISPR screens. We used the CSN5 inhibitor, which targets COPS5/CSN5, as a tool to understand the biological significance of one of these 12 DUBs, COPS6, in lung cancer. Our study provides a powerful resource to interrogate the role of DUB signaling biology and nominates druggable targets for the treatment of lung cancer subtypes.
Renal ischemia-reperfusion (I/R) injury is a limiting factor for the success of renal grafts and is deemed greatly responsible for the mortality. A novel series of Ketamine inspired compounds were synthesized and subjected to NF-ĸB transcriptional inhibitory activity in LPS-stimulated RAW264.7 cells, where entire set of compounds showed mild to significant NF-ĸB transcriptional inhibitory activity (IC50 6.53 - 67.52 µM). Compound 6d showed highest inhibitory activity among the tested series (IC50 2.62 µM), and found more potent as compared to Ketamine as standard. The effect of compound 6d was further quantified in I/R injury in Wistar rats, where it dose-dependently improves kidney function of rats with significant amelioration of kidney injury as suggested by histopathological examination of renal tissues. It further showed reduction in the generation of pro-inflammatory cytokines and improves the anti-oxidant status of experimental rats. Compound 6d inhibited apoptosis and increases the expression of Bcl2 and decreases Bax, and cleaved caspase-3 level. It further reduces TLR-4 and NF-κB expression in renal cells of rats, with increases in IκB-α level in western blot analysis as compared to I/R group. In summary, our current study showed the development of a novel class of Ketamine-inspired derivatives against renal ischemia/reperfusion injury.
Epidemiological and clinical studies have shown that exposure to particulate matter (PM) is associated with an increased incidence of lung cancer and metastasis. However, the underlying mechanism remains unclear. Here, we demonstrated the central role of PM-induced neutrophil recruitment in promoting lung cancer metastasis. We found that reactive oxygen species (ROS)-mediated alveolar epithelial macroautophagy/autophagy was essential for initiating neutrophil chemotaxis and pre-metastatic niche formation in the lungs in response to PM exposure. During PM-induced autophagy, the E3 ubiquitin ligase TRIM37 was degraded and protected TRAF6 from proteasomal degradation in lung epithelial cells, which promoted the NFKB-dependent production of chemokines to recruit neutrophils. Importantly, ROS blockade, autophagy inhibition or TRAF6 knockdown abolished PM-induced neutrophil recruitment and lung metastasis enhancement. Our study indicates that host lung epithelial cells and neutrophils coordinate to promote cancer metastasis to the lungs in response to PM exposure and provides ideal therapeutic targets for metastatic progression.
Abbreviations: ACTA2/α-SMA: actin alpha 2, smooth muscle, aorta; ATII: alveolar type II; Cho-Traf6 siRNA: 5ʹ-cholesterol-Traf6 siRNA; EMT: epithelial-mesenchymal transition; HBE: human bronchial epithelial; HCQ: hydroxychloroquine; MAPK: mitogen-activated protein kinase; NAC: N-acetyl-L-cysteine; NFKB: nuclear factor of kappa light polypeptide gene enhancer in B cells; NS: normal saline; PM: particulate matter; ROS: reactive oxygen species; TRAF6: TNF receptor-associated factor 6; TRIM37: tripartite motif-containing 37.
In tumor necrosis factor (TNF) signaling, IκB kinase (IKK) complex-mediated activation of NF-κB is a well-known protective mechanism against cell death via transcriptional induction of pro-survival genes occurring as a late checkpoint. However, recent belief holds that IKK functions as an early cell death checkpoint to suppress the death-inducing signaling complex by regulating receptor interacting protein kinase1 (RIPK1) phosphorylation. In this study, we propose that two major gernaylated 7-hydroxy coumarins, 6-geranyl-7-hydroxycoumarin (ostruthin) and 8-geranyl-7-hydroxycoumarin (8-geranylumbelliferone, 8-GU) isolated from Paramignya timera, facilitate RIPK1-dependent dual modes of apoptosis and necroptosis by targeting IKKβ upon TNF receptor1 (TNFR1) ligation. Analysis of events upstream of NF-κB revealed that 8-GU and ostruthin drastically inhibited TNF-induced IKK phosphorylation, while having no effect on TAK1 phosphorylation and TNFR1 complex-I formation. Interestingly, 8-GU did not affect the cell death induced by Fas ligand or TNF‐related apoptosis-inducing ligand or that induced by DNA-damaging agents, indicating that 8-GU sensitizes TNF-induced cell death exclusively. Moreover, 8-GU accelerated TNF-driven necroptosis by up-regulating necrosome formation in FADD deficient cancer cells harboring RIPK3. Thus, the present study provides new insights into the molecular mechanism underlying geranylated 7-hydroxy coumarin-mediated control of the RIPK1-dependent early cell death checkpoint and suggests that 8-GU is a potential anti-cancer therapeutic via an alternative apoptosis-independent strategy to overcome TNF resistance.
p65 is one of the important subunits of the inflammation-related transcription factor NF-κB. In the present study, we cloned and identified the p65 from Megalobrama amblycephala (Mnp65) by homologous cloning and RACE technique. The full-length Mnp65 cDNA consisted of 2331 bp, and included one open reading frame encoding a 604-amino acid putative protein. The protein sequence included a DNA binding motif, a well conserved N-terminal Rel-homology domain (RHD), and a C-terminal IG-like plexins transcription (IPT). Mnp65 was closely related with the other p65 proteins of Cypriniformes and clearly distinct from that of Perciformes and Salmoniformes in terms of sequence homology. Mnp65 homodimer may interact with IκBα in the IPT domain based on the predicted 3D structure of IκBα/Mnp65 complex. Mnp65 was ubiquitously expressed in M. amblycephala tissues, and the highest levels were detected in muscle and liver. Intragastric infection with Aeromonas hydrophila caused respiratory burst and cytokine storm from 8 h to 48 h, showing significantly higher level of respiratory burst activities and significantly high cytokines levels, such as TNF-α, IL-1β, IL-6, IL-8 etc., compared to 0 h. In addition, the bacterial challenge downregulated the IkBα, and upregulated Mnp65 and TNF-α in the liver. IkBα-Mnp65 was regulated by the negative feedback of cytokine storm, to increase IkBα and decrease Mnp65. Then cytokine storm was relieved at 96 h. Finally, severe intestinal inflammation was observed from 24 to 48 h after infection, characterized by extensive villous necrosis, epithelial hyperplasia and lymphocyte infiltration, all of which were relieved at 96 h. Taken together, Mnp65 plays a crucial role in the physiological response of teleost fish to bacterial infection.
Background
: Nephrotic syndrome (NS) is a common nephropathy with a complex and diverse aetiology. Both Imperatae rhizoma and Hedyotis diffusa Willd. are herbs that are widely used as medicine and functional food. In traditional Chinese medicine theory, they are used as an herbal pair (HP) to treat inflammation-related diseases in the clinic, especially disorders of the kidney.
Purpose
: This study aimed to investigate the anti-inflammatory and hypolipidaemic effects of HP in an NS rat model and provide scientific data for its clinical application.
Methods
: An NS model was established by two-dose injection of Sprague-Dawley rats with adriamycin. Seven groups, including the sham, model, HP treatment (0.25, 0.5 and 1.0 g/kg/d), prednisone (positive control, 5 mg/kg/d), and atorvastatin (positive control, 4 mg/kg/d) groups, were tested. The biochemical indexes of renal function and inflammatory cytokines were determined by ELISA kits and/or qPCR assays, and the crucial protein involved in the signalling pathway were subsequently tested by qPCR and/or Western blotting. Based on specific compounds identified by LC-Q-TOF-MS, network pharmacological study was carried out.
Results
: The levels of BUN, Scr, Upro, UA, Alb, TC, TG, and LDL-C were significantly elevated in model rats. HP treatment for four weeks improved the renal function and the dyslipidaemia by decreasing the levels of all parameters, except BUN and Scr. HP treatment (0.5 and 1.0 g/kg/d) upregulated the expression of PPARγ, CYP7b1, and LDLR in the liver, while it down-regulated PCSK9, showing a regulatory effect on lipid metabolism disorder. The levels of TNF-α and IL-1β in the plasma and the mRNA expression of TNF-α, IL-1β, MCP-1, and TGF-β1 in the kidney were decreased in HP groups, revealing its anti-inflammatory effect in NS rats. The HP exerted an alleviation effect on the inflammatory response through the NF-κB pathway by inhibiting the mRNA and protein expression of p50 and p65. There were 34 compounds identified or tentatively characterized in HP. In the network pharmacological study, PPARG(PPARγ), PCSK9, RELA(p65), and NF-κB1(p50) were the top 20 targets for HP, supporting the animal experimental results.
Conclusion
: HP exhibited protective effects on NS rats. These effects might be closely related to the inhibition of NF-κB and PCSK9-LDLR and activation of the PPARγ-CYP7B1 signalling pathways.
Transcription is an elaborate process that is required to establish and maintain the identity of the more than two hundred cell types of a metazoan organism. Strict regulation of gene expression is therefore vital for tissue formation and homeostasis. An accumulating body of work found that ubiquitylation of histones, transcription factors, or RNA polymerase II is crucial for ensuring that transcription occurs at the right time and place during development. Here, we will review principles of ubiquitin-dependent control of gene expression and discuss how breakdown of these regulatory circuits leads to a wide array of human diseases.
Processing of the p105 NF-κB precursor to yield the p50 active subunit is a unique and rare case in which the ubiquitin system
is involved in limited processing rather than in complete destruction of its target. The mechanisms involved in this process
are largely unknown, although a glycine repeat in the middle of p105 has been identified as a processing stop signal. IκB
kinase (IKK)β-mediated phosphorylation at the C-terminal domain with subsequent recruitment of the SCFβ-TrCP ubiquitin ligase leads to accelerated processing and degradation of the precursor, yet the roles that the kinase and ligase
play in each of these two processes have not been elucidated. Here we demonstrate that IKKβ has two distinct functions: (i)
stimulation of degradation and (ii) stimulation of processing. IKKβ-induced degradation is dependent on SCFβ-TrCP, which acts through multiple lysine residues in the IκBγ domain. In contrast, IKKβ-induced processing of p105 is β-transduction
repeat-containing protein (β-TrCP) independent, as it is not affected by expression of a dominant-negative β-TrCP or following
its silencing by small inhibitory RNA. Furthermore, removal of all 30 lysine residues from IκBγ results in complete inhibition
of IKK-dependent degradation but has no effect on IKK-dependent processing. Yet processing still requires the activity of
the ubiquitin system, as it is inhibited by dominant-negative UbcH5a. We suggest that IKKβ mediates its two distinct effects
by affecting, directly and indirectly, two different E3s.
NF-κB/Rel transcription factors have recently emerged as crucial regulators of cell survival. Activation of NF-κB antagonizes programmed cell death (PCD) induced by tumor necrosis factor-receptors (TNF-Rs) and several other triggers. This prosurvival activity of NF-κB participates in a wide range of biological processes, including immunity, lymphopoiesis and development. It is also crucial for pathogenesis of various cancers, chronic inflammation and certain hereditary disorders. This participation of NF-κB in survival signaling often involves an antagonism of PCD triggered by TNF-R-family receptors, and is mediated through a suppression of the formation of reactive oxygen species (ROS) and a control of sustained activation of the Jun-N-terminal kinase (JNK) cascade. Effectors of this antagonistic activity of NF-κB on this ROS/JNK pathway have been recently identified. Indeed, further delineating the mechanisms by which NF-κB promotes cell survival might hold the key to developing new highly effective therapies for treatment of widespread human diseases.
NF-κB transcription factors mediate the effects of pro-inflammatory cytokines such as tumour necrosis factor-α and interleukin-1β1. Failure to downregulate NF-κB transcriptional activity results in chronic inflammation and cell death, as observed in A20-deficient mice2. A20 is a potent inhibitor of NF-κB signalling, but its mechanism of action is unknown2. Here we show that A20 downregulates NF-κB signalling through the cooperative activity of its two ubiquitin-editing domains. The amino-terminal domain of A20, which is a de-ubiquitinating (DUB) enzyme of the OTU (ovarian tumour) family3, removes lysine-63 (K63)-linked ubiquitin chains from receptor interacting protein (RIP), an essential mediator of the proximal TNF receptor 1 (TNFR1) signalling complex4, 5. The carboxy-terminal domain of A20, composed of seven C2/C2 zinc fingers6, then functions as a ubiquitin ligase by polyubiquitinating RIP with K48-linked ubiquitin chains, thereby targeting RIP for proteasomal degradation. Here we define a novel ubiquitin ligase domain and identify two sequential mechanisms by which A20 downregulates NF-κB signalling. We also provide an example of a protein containing separate ubiquitin ligase and DUB domains, both of which participate in mediating a distinct regulatory effect.
Ubiquitin-binding domains (UBDs) are a collection of modular protein domains that non-covalently bind to ubiquitin. These recently discovered motifs interpret and transmit information conferred by protein ubiquitylation to control various cellular events. Detailed molecular structures are known for a number of UBDs, but to understand their mechanism of action, we also need to know how binding specificity is determined, how ubiquitin binding is regulated, and the function of UBDs in the context of full-length proteins. Such knowledge will be key to our understanding of how ubiquitin regulates cellular proteins and processes.
Familial cylindromatosis is an autosomal dominant genetic predisposition to multiple tumours of the skin appendages. The susceptibility gene (CYLD) has previously been localized to chromosome 16q and has the genetic attributes of a tumour-suppressor gene (recessive oncogene). Here we have identified CYLD by detecting germline mutations in 21 cylindromatosis families and somatic mutations in 1 sporadic and 5 familial cylindromas. All mutations predict truncation or absence of the encoded protein. CYLD encodes three cytoskeletal-associated-protein-glycine-conserved (CAP-GLY) domains, which are found in proteins that coordinate the attachment of organelles to microtubules. CYLD also has sequence homology to the catalytic domain of ubiquitin carboxy-terminal hydrolases (UCH).
Ubc13 is a ubiquitin-conjugating enzyme responsible for noncanonical ubiquitination of TNF receptor-associated factor (TRAF)-family adapter proteins involved in Toll-like receptor and TNF-family cytokine receptor signaling, which are regulators of innate immunity. Gene ablation was used to study the function of Ubc13 in mice. Whereas homozygous ubc13 gene disruption resulted in embryonic lethality, heterozygous ubc13 +/− mice appeared normal, without alterations in immune cell populations. Haploinsufficient ubc13 +/− mice were resistant to lipopolysaccharide-induced lethality, and demonstrated reduced in vivo ubiquitination of TRAF6. Macrophages and splenocytes isolated from ubc13 +/− mice exhibited reduced lipopolysaccharide-inducible cytokine secretion and impaired activation of TRAF-dependent signal transduction pathways (NF-κB, JNK, and p38 MAPK). These findings document a critical role for Ubc13 in inflammatory responses and suggest that agents reducing Ubc13 activity could have therapeutic utility.
• inflammation
• Toll-like receptor
• sepsis
• innate immunity
Recent studies suggest that nuclear factor kappaB-inducing kinase (NIK) is suppressed through constitutive proteasome-mediated degradation regulated by TRAF2, TRAF3 and cIAP1 or cIAP2. Here we demonstrated that the degradation of NIK occurs upon assembly of a regulatory complex through TRAF3 recruitment of NIK and TRAF2 recruitment of cIAP1 and cIAP2. In contrast to TRAF2 and TRAF3, cIAP1 and cIAP2 seem to play redundant roles in the degradation of NIK, as inhibition of both cIAPs was required for noncanonical NF-kappaB activation and increased survival and proliferation of primary B lymphocytes. Furthermore, the lethality of TRAF3 deficiency in mice could be rescued by a single NIK gene, highlighting the importance of tightly regulated NIK.
The adaptor and signaling proteins TRAF2, TRAF3, cIAP1 and cIAP2 may inhibit alternative nuclear factor-kappaB (NF-kappaB) signaling in resting cells by targeting NF-kappaB-inducing kinase (NIK) for ubiquitin-dependent degradation, thus preventing processing of the NF-kappaB2 precursor protein p100 to release p52. However, the respective functions of TRAF2 and TRAF3 in NIK degradation and activation of alternative NF-kappaB signaling have remained elusive. We now show that CD40 or BAFF receptor activation result in TRAF3 degradation in a cIAP1-cIAP2- and TRAF2-dependent way owing to enhanced cIAP1, cIAP2 TRAF3-directed ubiquitin ligase activity. Receptor-induced activation of cIAP1 and cIAP2 correlated with their K63-linked ubiquitination by TRAF2. Degradation of TRAF3 prevented association of NIK with the cIAP1-cIAP2-TRAF2 ubiquitin ligase complex, which resulted in NIK stabilization and NF-kappaB2-p100 processing. Constitutive activation of this pathway causes perinatal lethality and lymphoid defects.
TAK1 kinase is an indispensable intermediate in several cytokine signaling pathways including tumor necrosis factor, interleukin-1, and transforming growth factor-beta signaling pathways. TAK1 also participates in stress-activated intracellular signaling pathways such as osmotic stress signaling pathway. TAK1-binding protein 1 (TAB1) is constitutively associated with TAK1 through its C-terminal region. Although TAB1 is known to augment TAK1 catalytic activity when it is overexpressed, the role of TAB1 under physiological conditions has not yet been identified. In this study, we determined the role of TAB1 in TAK1 signaling by analyzing TAB1-deficient mouse embryonic fibroblasts (MEFs). Tumor necrosis factor- and interleukin-1-induced activation of TAK1 was entirely normal in Tab1-deficient MEFs and could activate both mitogen-activated protein kinases and NF-kappaB. In contrast, we found that osmotic stress-induced activation of TAK1 was largely impaired in Tab1-deficient MEFs. Furthermore, we showed that the C-terminal 68 amino acids of TAB1 were sufficient to mediate osmotic stress-induced TAK1 activation. Finally, we attempted to determine the mechanism by which TAB1 activates TAK1. We found that TAK1 is spontaneously activated when the concentration is increased and that it is totally dependent on TAB1. Cell shrinkage under the osmotic stress condition increases the concentration of TAB1-TAK1 and may oligomerize and activate TAK1 in a TAB1-dependent manner. These results demonstrate that TAB1 mediates TAK1 activation only in a subset of TAK1 pathways that are mediated through spontaneous oligomerization of TAB1-TAK1.
A20 was first identified as a tumor necrosis factor (TNF) primary response transcript encoding a 790-amino acid protein with a unique zinc finger motif. Recently, A20 was shown to protect cells from TNF-induced cytotoxicity in a variety of cell lines. Nuclear run-on studies previously established that TNF activates A20 at the transcriptional level. To further characterize the mechanism by which TNF activates the A20 gene, we have cloned the A20 5'-flanking sequences and identified TNF-responsive elements within the promoter. The transcription initiation site was mapped by both primer extension and S1 nuclease protection experiments to a position 4.2 kilobases (kb) upstream of the initiator methionine; the first and second exon were separated by a 3.9-kb intron. Sequences upstream of the transcription start site were 76% GC-rich and contained six Sp1 binding sites and a TATA-like sequence at -29 but lacked a consensus CCAAT site. Transfection of Jurkat T-cells with an array of A20 promoter CAT constructs showed that two kappa B elements residing at -54 and -66 were required for induction by TNF. Supporting this notion, DNA electrophoretic mobility shift assays using nuclear extracts from unstimulated and TNF-stimulated Jurkat cells demonstrated kappa B-specific binding of a TNF-activated factor to an end-labeled probe containing the two A20 kappa B sequences. Finally, evidence obtained from cotransfection experiments showed that A20 negatively regulated its own expression.
Non-agouti-lethal 18H (a18H) mice are dark agouti with black pinna hairs. What makes these mice unique is that they develop a spectrum of immunological diseases not seen in other agouti mutant mice. On the JU/Ct background, a18H mice develop an inflammatory disease of the large intestine. On the C57BL/6J background, they develop a fatal disease characterized by pulmonary chronic interstitial inflammation and alveolar proteinosis, inflammation of the glandular stomach and skin resulting in scarring due to constant itching, and hyperplasia of lymphoid cells, haematopoietic cells and the forestomach epithelium. Previous studies suggested that the a18H mutation results from a paracentric inversion that affects two loci: agouti and another, as yet unidentified locus designated itchy (the provisional gene symbol is Itch), that is responsible for the immunological phenotype of a18H mice. Here we confirm that a18H results from an inversion and show that Itch encodes a novel E3 ubiquitin protein ligase, a protein involved in ubiquitin-mediated protein degradation. Our results indicate that ubiquitin-dependent proteolysis is an important mediator of the immune response in vivo and provide evidence for Itch's role in inflammation and the regulation of epithelial and haematopoietic cell growth.
Bone resorption and remodeling is an intricately controlled, physiological process that requires the function of osteoclasts. The processes governing both the differentiation and activation of osteoclasts involve signals induced by osteoprotegerin ligand (OPGL), a member of tumor necrosis factor (TNF) superfamily, and its cognate receptor RANK. The molecular mechanisms of the intracellular signal transduction remain to be elucidated. Here we report that mice deficient in TNF receptor-associated factor 6 (TRAF6) are osteopetrotic with defects in bone remodeling and tooth eruption due to impaired osteoclast function. Using in vitro assays, we demonstrate that TRAF6 is crucial not only in IL-1 and CD40 signaling but also, surprisingly, in LPS signaling. Furthermore, like TRAF2 and TRAF3, TRAF6 is essential for perinatal and postnatal survival. These findings establish unexpectedly diverse and critical roles for TRAF6 in perinatal and postnatal survival, bone metabolism, LPS, and cytokine signaling.
To determine why proteasome inhibitors prevent thymocyte death, we examined whether proteasomes degrade anti-apoptotic molecules in cells induced to undergo apoptosis. The c-IAP1 and XIAP inhibitors of apoptosis were selectively lost in glucocorticoid- or etoposide-treated thymocytes in a proteasome-dependent manner before death. IAPs catalyzed their own ubiquitination in vitro, an activity requiring the RING domain. Overexpressed wild-type c-IAP1, but not a RING domain mutant, was spontaneously ubiquitinated and degraded, and stably expressed XIAP lacking the RING domain was relatively resistant to apoptosis-induced degradation and, correspondingly, more effective at preventing apoptosis than wild-type XIAP. Autoubiquitination and degradation of IAPs may be a key event in the apoptotic program.
We report the identification of a TRAF-interacting protein, T6BP, that specifically associates with TRAF6. This interaction occurs between the coiled-coil region of T6BP and the N-terminal ring finger and zinc finger domains of TRAF6. IL-1, but not tumor necrosis factor, induces TRAF6-T6BP complex formation in a ligand-dependent manner. Formation of the TRAF6-T6BP complex depends on the presence of the IL-1 receptor-associated kinase (IRAK). After IL-1 stimulation, TRAF6 can exist in two separate complexes, TRAF6-IRAK or TRAF6-T6BP, but IRAK is not present in TRAF6-T6BP complexes. T6BP does not seem to play a direct role in activation of IkappaB kinases or Jun N-terminal kinase.
The molecular basis of X-linked recessive anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) has remained elusive. Here we report hypomorphic mutations in the gene IKBKG in 12 males with EDA-ID from 8 kindreds, and 2 patients with a related and hitherto unrecognized syndrome of EDA-ID with osteopetrosis and lymphoedema (OL-EDA-ID). Mutations in the coding region of IKBKG are associated with EDA-ID, and stop codon mutations, with OL-EDA-ID. IKBKG encodes NEMO, the regulatory subunit of the IKK (IkappaB kinase) complex, which is essential for NF-kappaB signaling. Germline loss-of-function mutations in IKBKG are lethal in male fetuses. We show that IKBKG mutations causing OL-EDA-ID and EDA-ID impair but do not abolish NF-kappaB signaling. We also show that the ectodysplasin receptor, DL, triggers NF-kappaB through the NEMO protein, indicating that EDA results from impaired NF-kappaB signaling. Finally, we show that abnormal immunity in OL-EDA-ID patients results from impaired cell responses to lipopolysaccharide, interleukin (IL)-1beta, IL-18, TNFalpha and CD154. We thus report for the first time that impaired but not abolished NF-kappaB signaling in humans results in two related syndromes that associate specific developmental and immunological defects.
Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) were identified as signal transducers for the TNF receptor
superfamily. However, the exact roles of TRAF2 and TRAF5 in TNF-induced NF-κB activation still remain controversial. To address
this issue, we generated TRAF2 and TRAF5 double knockout (DKO) mice. TNF- but not interleukin-1-induced nuclear translocation
of NF-κB was severely impaired in murine embryonic fibroblasts (MEFs) derived from DKO mice. Moreover, DKO MEFs were more
susceptible to TNF-induced cytotoxicity than TRAF2 knockout MEFs. Collectively, these results indicate that both TRAF2 and
TRAF5 are involved in TNF-induced NF-κB activation and protection from cell death.
Signal-induced activation of the transcription factor NF-κB requires specific phosphorylation of the inhibitor IκBα and its subsequent proteolytic degradation. Phosphorylation of serine residues 32 and 36 targets IκBα to the ubiquitin (Ub)–proteasome pathway. Here we report the identification of a large, multisubunit kinase (molecular mass ∼700 kDa) that phosphorylates IκBα at S32 and S36. Remarkably, the activity of this kinase requires the Ub-activating enzyme (E1), a specific Ub carrier protein (E2) of the Ubc4/Ubc5 family, and Ub. We also show that a ubiquitination event in the kinase complex is a prerequisite for specific phosphorylation of IκBα. Thus, ubiquitination serves a novel regulatory function that does not involve proteolysis.
Processing of the nfκb2 gene product p100 to generate p52 is an important step in NF-κB regulation. We show that this step is negatively regulated by a processing-inhibitory domain (PID) within p100 and positively regulated by the NF-κB-inducing kinase (NIK). While the PID suppresses the constitutive processing of p100, NIK induces p100 processing by stimulating site-specific phosphorylation and ubiquitination of this precursor protein. Further, a natural mutation of the gene encoding NIK in alymphoplasia (aly) mice cripples the function of NIK in p100 processing, causing a severe defect in p52 production. These data suggest that NIK is a specific kinase regulating p100 processing and explain why the aly and nfκb2 knockout mice exhibit similar immune deficiencies.
TRAF6 is a signal transducer that activates IκB kinase (IKK) and Jun amino-terminal kinase (JNK) in response to pro-inflammatory mediators such as interleukin-1 (IL-1) and lipopolysaccharides (LPS)1-4. IKK activation by TRAF6 requires two intermediary factors, TRAF6-regulated IKK activator 1 (TRIKA1) and TRIKA2 (ref. 5). TRIKA1 is a dimeric ubiquitin-conjugating enzyme complex composed of Ubc13 and Uev1A (or the functionally equivalent Mms2). This Ubc complex, together with TRAF6, catalyses the formation of a Lys 63 (K63)-linked polyubiquitin chain that mediates IKK activation through a unique proteasome-independent mechanism. Here we report the purification and identification of TRIKA2, which is composed of TAK1, TAB1 and TAB2, a protein kinase complex previously implicated in IKK activation through an unknown mechanism. We find that the TAK1 kinase complex phosphorylates and activates IKK in a manner that depends on TRAF6 and Ubc13-Uev1A. Moreover, the activity of TAK1 to phosphorylate MKK6, which activates the JNK-p38 kinase pathway, is directly regulated by K63-linked polyubiquitination. We also provide evidence that TRAF6 is conjugated by the K63 polyubiquitin chains. These results indicate that ubiquitination has an important regulatory role in stress response pathways, including those of IKK and INK.
Stimulation of the Drosophila immune response activates NF-κB and JNK signaling pathways. For example, infection by Gram-negative bacteria induces the
Imd signaling pathway, leading to the activation of the NF-κB-like transcription factor Relish and the expression of a battery
of genes encoding antimicrobial peptides. Bacterial infection also activates the JNK pathway, but the role of this pathway
in the immune response has not yet been established. Genetic experiments suggest that the Drosophila homolog of the mammalian MAPK kinase kinase, TAK1 (transforming growth factor β-activated kinase 1), activates both the JNK
and NF-κB pathways following immune stimulation. In this report, we demonstrate that Drosophila TAK1 functions as both the Drosophila IκB kinase-activating kinase and the JNK kinase-activating kinase. However, we found that JNK signaling is not required for
antimicrobial peptide gene expression but is required for the activation of other immune inducible genes, including Punch, sulfated, and malvolio. Thus, JNK signaling appears to play an important role in the cellular immune response and the stress response.
BackgroundTRAF6, a member of the tumour necrosis factor receptor-associated factor family, was first identified as a transducer of CD40 and interleukin-1 receptor (IL-1R) signals based on the interaction of TRAF6 with the cytoplasmic tail of CD40 and with the IL-1R associated kinase in vitro. However, the functions of TRAF6 in vivo remain unidentified.ResultsWe show that TRAF6−/− mice exhibit severe osteopetrosis and are defective in osteoclast formation. In vitro culture experiments revealed that osteoclast precursor cells derived from TRAF6−/− mice are unable to differentiate to functional osteoclasts in response to osteoclast differentiation factor (ODF). In bone marrow of TRAF6−/− mice, the number of sIgM+B220+ immature B cells is markedly reduced while the ratio of proB to preB cells is not affected. In contrast, development of thymocytes is not affected. Furthermore, TRAF6−/− mice are defective in lymph node organogenesis and IL-1 signalling in thymocytes.Conclusions
The results identify TRAF6 as an essential component of ODF signalling pathway, and also show that TRAF6 plays pivotal roles in immune and inflammatory systems in vivo.
The transcription factor NF-kappaB has served as a standard for inducible transcription factors for more than 20 years. The numerous stimuli that activate NF-kappaB, and the large number of genes regulated by NF-kappaB, ensure that this transcription factor is still the subject of intense research. Here, we attempt to synthesize some of the basic principles that have emerged from studies of NF-kappaB, and we aim to generate a more unified view of NF-kappaB regulation.
The proinflammatory cytokine tumor necrosis factor (TNF) alpha signals both cell survival and death. The biological outcome of TNFalpha treatment is determined by the balance between NF-kappaB and Jun kinase (JNK) signaling; NF-kappaB promotes survival, whereas JNK enhances cell death. Critically, identity of a JNK substrate that promotes TNFalpha-induced apoptosis has been outstanding. Here we show that TNFalpha-mediated JNK activation accelerates turnover of the NF-kappaB-induced antiapoptotic protein c-FLIP, an inhibitor of caspase-8. This is not due to direct c-FLIP phosphorylation but depends on JNK-mediated phosphorylation and activation of the E3 ubiquitin ligase Itch, which specifically ubiquitinates c-FLIP and induces its proteasomal degradation. JNK1 or Itch deficiency or treatment with a JNK inhibitor renders mice resistant in three distinct models of TNFalpha-induced acute liver failure, and cells from these mice do not display inducible c-FLIP(L) ubiquitination and degradation. Thus, JNK antagonizes NF-kappaB during TNFalpha signaling by promoting the proteasomal elimination of c-FLIP(L).
To examine the role of the TNF-R superfamily signaling protein TRAF2 in mature B cell development and NF-kappaB activation, conditionally TRAF2-deficient mice were produced. B cells lacking TRAF2 expression in these mice possessed a selective survival advantage, accumulated in the lymph nodes and splenic marginal zone, were larger in size, and expressed increased levels of CD21/35. These TRAF2-deficient B cells could not proliferate or activate the canonical NF-kappaB pathway in response to CD40 ligation. By contrast, noncanonical NF-kappaB activation was constitutively hyperactive, with TRAF2-deficient B cells exhibiting close to maximal processing of NF-kappaB2 from p100 to p52 and high levels of constitutive p52 and RelB DNA binding activity. These findings establish TRAF2 as a multifunctional regulator of NF-kappaB activation that mediates activation of the canonical pathway but acts as a negative regulator of the noncanonical pathway. This dual functionality explains the contrasting roles of TRAF2 in B cell maturation and activation.
The NFkappaB1 gene encodes two functionally distinct proteins termed p50 and p105. p50 corresponds to the N terminus of p105 and with p65 (RelA) forms the prototypical NF-kappaB transcription factor complex. In contrast, p105 functions as a Rel-specific inhibitor (IKB) and has been proposed to be the precursor of p50. Our studies now demonstrate that p50 is generated by a unique cotranslational processing event involving the 26S proteasome, whereas cotranslational folding of sequences near the C terminus of p50 abrogates proteasome processing and leads to p105 production. These results indicate that p105 is not the precursor of p50 and reveal a novel mechanism of gene regulation that ensures the balanced production and independent function of the p50 and p105 proteins.
Lindsey Criswell and colleagues report an association between three independent variants near TNFAIP3 and systemic lupus erythematosus (SLE). In a related study, Patrick Gaffney and colleagues report results of a genome-wide association study for SLE, also identifying variants in the TNFAIP3 region on 6q23 that are strongly associated with the disease. The same region on 6q23 has recently been associated with rheumatoid arthritis, but only a subset of risk alleles in this region seem to be common to both diseases. The TNFAIP3 (tumor necrosis factor alpha–induced protein 3) gene encodes a ubiquitin editing enzyme, A20, that restricts NF-κB–dependent signaling and prevents inflammation. We show that three independent SNPs in the TNFAIP3 region (rs13192841, rs2230926 and rs6922466) are associated with systemic lupus erythematosus (SLE) among individuals of European ancestry. These findings provide critical links between A20 and the etiology of SLE.
To characterize proteins that bind to the immunoglobulin (Ig) heavy chain and the kappa light chain enhancers, an electrophoretic mobility shift assay with end-labeled DNA fragments was used. Three binding proteins have been found. One is NF-A, a factor found in all tested cell types that binds to the octamer sequence found upstream of all Ig variable region gene segments and to the same octamer in the heavy chain enhancer. The second, also ubiquitous, protein binds to a sequence in both the heavy chain and the kappa enhancers that was previously shown to be protected from methylation in vivo. Other closely related sites do not compete for this binding, implying a restriction enzyme-like binding specificity. The third protein binds to a sequence in the kappa enhancer (and to an identical sequence in the SV40 enhancer) and is restricted in its occurrence to B cells.
We demonstrate an essential role for the proteasome complex in two proteolytic processes required for activation of the transcription factor NF-kappa B. The p105 precursor of the p50 subunit of NF-kappa B is processed in vitro by an ATP-dependent process that requires proteasomes and ubiquitin conjugation. The C-terminal region of p105 is rapidly degraded, leaving the N-terminal p50 domain. p105 processing can be blocked in intact cells with inhibitors of the proteasome or in yeast with proteasome mutants. These inhibitors also block the activation of NF-kappa B and the rapid degradation of I kappa B alpha induced by tumor necrosis factor alpha. Thus, the ubiquitin-proteasome pathway functions not only in the complete degradation of polypeptides, but also in the regulated processing of precursors into active proteins.
The 75 kDa tumor necrosis factor receptor (TNFR2) transduces extracellular signals via receptor-associated cytoplasmic proteins. Two of these signal transducers, TRAF1 and TRAF2, were isolated and characterized previously. We report here the biochemical purification and subsequent molecular cloning of two novel TNFR2-associated proteins, designated c-IAP1 and c-IAP2, that are closely related mammalian members of the inhibitor of apoptosis protein (IAP) family originally identified in baculoviruses. The viral and cellular IAPs contain N-terminal baculovirus IAP repeat (BIR) motifs and a C-terminal RING finger. The c-IAPs do not directly contact TNFR2, but rather associate with TRAF1 and TRAF2 through their N-terminal BIR motif-comprising domain. The recruitment of c-IAP1 or c-IAP2 to the TNFR2 signaling complex requires a TRAF2-TRAF1 heterocomplex.
Tumor necrosis factor (TNF) can induce apoptosis and activate NF-kappa B through signaling cascades emanating from TNF receptor 1 (TNFR1). TRADD is a TNFR1-associated signal transducer that is involved in activating both pathways. Here we show that TRADD directly interacts with TRAF2 and FADD, signal transducers that activate NF-kappa B and induce apoptosis, respectively. A TRAF2 mutant lacking its N-terminal RING finger domain is a dominant-negative inhibitor of TNF-mediated NF-kappa B activation, but does not affect TNF-induced apoptosis. Conversely, a FADD mutant lacking its N-terminal 79 amino acids is a dominant-negative inhibitor of TNF-induced apoptosis, but does not inhibit NF-kappa B activation. Thus, these two TNFR1-TRADD signaling cascades appear to bifurcate at TRADD.
Transcription factor NF-kappaB is generally considered to be a heterodimer with two subunits, p50 and p65. The p50 subunit
has been suggested to be generated from its precursor, p105, via the ubiquitin-proteasome pathway. During processing, the
C-terminal portion of p105 is rapidly degraded whereas the N-terminal portion (p50) is left intact. We report here that a
23-amino-acid, glycine-rich region (GRR) in p105 functions as a processing signal for the generation of p50. A GRR-dependent
endoproteolytic cleavage downstream of the GRR releases p50 from p105, and this cleavage does not require any specific downstream
sequences. p50 can be generated from chimeric precursor p105N-GRR-IkappaBalpha, while the C-terminal portion (IkappaBalpha)
can also be recovered, suggesting that p105 processing includes two steps: a GRR-dependent endoproteolytic cleavage and the
subsequent degradation of the C-terminal portion. We have also demonstrated that the GRR can direct a similar processing event
when it is inserted into a protein unrelated to the NF-kappaB family and that it is therefore an independent signal for processing.
Many cytokines signal through different cell-surface receptors to activate the transcription factor NF-kappaB. Members of the TRAF protein family have been implicated in the activation of NF-kappaB by the tumour-necrosis factor (TNF)-receptor superfamily. Here we report the identification of a new TRAF family member, designated TRAF6. When overexpressed in human 293 cells, TRAF6 activates NF-kappaB. A dominant-negative mutant of TRAF6 inhibits NF-kappaB activation signalled by interleukin-1 (IL-1) but not by TNF. IL-1 treatment of 293 cells induces the association of TRAF6 with IRAK, a serine/threonine kinase that is rapidly recruited to the IL-1 receptor after IL-1 induction. These findings indicate that TRAF proteins may function as signal transducers for distinct receptor families and that TRAF6 participates in IL-1 signalling.
TRAF2 is an intracellular signal-transducing protein recruited to the TNFR1 and TNFR2 receptors following TNF stimulation. To investigate the physiological role of TRAF2, we generated TRAF2-deficient mice. traf2-/- mice appeared normal at birth but became progressively runted and died prematurely. Atrophy of the thymus and spleen and depletion of B cell precursors also were observed. Thymocytes and other hematopoietic progenitors were highly sensitive to TNF-induced cell death and serum TNF levels were elevated in these TRAF2-deficient animals. Examination of traf2-/- cells revealed a severe reduction in TNF-mediated JNK/SAPK activation but a mild effect on NF-kappaB activation. These results suggest that TRAF2-independent pathways of NF-kappaB activation exist and that TRAF2 is required for an NF-kappaB-independent signal that protects against TNF-induced apoptosis.
TRAF2 is believed to mediate the activation of NF-kappaB and JNK induced by the tumor necrosis factor receptor (TNFR) superfamily, which elicits pleiotropic responses in lymphocytes. We have investigated the physiological roles of TRAF2 in these processes by expressing a lymphocyte-specific dominant negative form of TRAF2, thereby blocking this protein's effector function. We find that the TNFR superfamily signals require TRAF2 for activation of JNK but not NF-kappaB. In addition, we show that TRAF2 induces NF-kappaB-independent antiapoptotic pathways during TNF-induced apoptosis. Inhibition of TRAF2 leads to splenomegaly, lymphadenopathy, and an increased number of B cells. These findings indicate that TRAF2 is involved in the regulation of lymphocyte function and growth in vivo.
NF-kappaB, a ubiquitous, inducible transcription factor involved in immune, inflammatory, stress and developmental processes, is retained in a latent form in the cytoplasm of non-stimulated cells by inhibitory molecules, IkappaBs. Its activation is a paradigm for a signal-transduction cascade that integrates an inducible kinase and the ubiquitin-proteasome system to eliminate inhibitory regulators. Here we isolate the pIkappaBalpha-ubiquitin ligase (pIkappaBalpha-E3) that attaches ubiquitin, a small protein which marks other proteins for degradation by the proteasome system, to the phosphorylated NF-kappaB inhibitor pIkappaBalpha. Taking advantage of its high affinity to pIkappaBalpha, we isolate this ligase from HeLa cells by single-step immunoaffinity purification. Using nanoelectrospray mass spectrometry, we identify the specific component of the ligase that recognizes the pIkappaBalpha degradation motif as an F-box/WD-domain protein belonging to a recently distinguished family of beta-TrCP/Slimb proteins. This component, which we denote E3RSIkappaB (pIkappaBalpha-E3 receptor subunit), binds specifically to pIkappaBalpha and promotes its in vitro ubiquitination in the presence of two other ubiquitin-system enzymes, E1 and UBC5C, one of many known E2 enzymes. An F-box-deletion mutant of E3RS(IkappaB), which tightly binds pIkappaBalpha but does not support its ubiquitination, acts in vivo as a dominant-negative molecule, inhibiting the degradation of pIkappaBalpha and consequently NF-kappaB activation. E3RS(IkappaB) represents a family of receptor proteins that are core components of a class of ubiquitin ligases. When these receptor components recognize their specific ligand, which is a conserved, phosphorylation-based sequence motif, they target regulatory proteins containing this motif for proteasomal degradation.
Signal-induced phosphorylation of IkappaBalpha targets this inhibitor of NF-kappaB for ubiquitination and subsequent degradation, thus allowing NF-kappaB to enter the nucleus to turn on its target genes. We report here the identification of an IkappaB-ubiquitin (Ub) ligase complex containing the F-box/WD40-repeat protein, beta-TrCP, a vertebrate homolog of Drosophila Slimb. beta-TrCP binds to IkappaBalpha only when the latter is specifically phosphorylated by an IkappaB kinase complex. Moreover, immunopurified beta-TrCP ubiquitinates phosphorylated IkappaBalpha at specific lysines in the presence of Ub-activating (E1) and -conjugating (Ubch5) enzymes. A beta-TrCP mutant lacking the F-box inhibits the signal-induced degradation of IkappaBalpha and subsequent activation of NF-kappaB-dependent transcription. Furthermore, Drosophila embryos deficient in slimb fail to activate twist and snail, two genes known to be regulated by the NF-kappaB homolog, Dorsal. These biochemical and genetic data strongly suggest that Slimb/beta-TrCP is the specificity determinant for the signal-induced ubiquitination of IkappaBalpha.
Ubiquitin-mediated proteolysis has a central role in controlling the intracellular levels of several important regulatory molecules such as cyclins, CKIs, p53, and IkappaBalpha. Many diverse proinflammatory signals lead to the specific phosphorylation and subsequent ubiquitin-mediated destruction of the NF-kappaB inhibitor protein IkappaBalpha. Substrate specificity in ubiquitination reactions is, in large part, mediated by the specific association of the E3-ubiquitin ligases with their substrates. One class of E3 ligases is defined by the recently described SCF complexes, the archetype of which was first described in budding yeast and contains Skp1, Cdc53, and the F-box protein Cdc4. These complexes recognize their substrates through modular F-box proteins in a phosphorylation-dependent manner. Here we describe a biochemical dissection of a novel mammalian SCF complex, SCFbeta-TRCP, that specifically recognizes a 19-amino-acid destruction motif in IkappaBalpha (residues 21-41) in a phosphorylation-dependent manner. This SCF complex also recognizes a conserved destruction motif in beta-catenin, a protein with levels also regulated by phosphorylation-dependent ubiquitination. Endogenous IkappaBalpha-ubiquitin ligase activity cofractionates with SCFbeta-TRCP. Furthermore, recombinant SCFbeta-TRCP assembled in mammalian cells contains phospho-IkappaBalpha-specific ubiquitin ligase activity. Our results suggest that an SCFbeta-TRCP complex functions in multiple transcriptional programs by activating the NF-kappaB pathway and inhibiting the beta-catenin pathway.
Interleukin-1 (IL-1) and tumor necrosis factor (TNF-alpha) stimulate transcription factors AP-1 and NF-kappaB through activation of the MAP kinases JNK and p38 and the IkappaB kinase (IKK), respectively. The TNF-alpha and IL-1 signals are transduced through TRAF2 and TRAF6, respectively. Overexpressed TRAF2 or TRAF6 activate JNK, p38, or IKK in the absence of extracellular stimulation. By replacing the carboxy-terminal TRAF domain of TRAF2 and TRAF6 with repeats of the immunophilin FKBP12, we demonstrate that their effector domains are composed of their amino-terminal Zn and RING fingers. Oligomerization of the TRAF2 effector domain results in specific binding to MEKK1, a protein kinase capable of JNK, p38, and IKK activation, and induction of TNF-alpha and IL-1 responsive genes. TNF-alpha also enhances the binding of native TRAF2 to MEKK1 and stimulates the kinase activity of the latter. Thus, TNF-alpha and IL-1 signaling is based on oligomerization of TRAF2 and TRAF6 leading to activation of effector kinases.
Biochemical coupling of transcription factor NF-kappaB to antigen and co-stimulatory receptors is required for the temporal control of T-cell proliferation. In contrast to its transitory activation during normal growth-signal transduction, NF-kappaB is constitutively deployed in T-cells transformed by the type 1 human T-cell leukemia virus (HTLV-1). This viral/host interaction is mediated by the HTLV-1-encoded Tax protein, which has potent oncogenic properties. As reviewed here, Tax activates NF-kappaB primarily via a pathway leading to the chronic phosphorylation and degradation of IkappaBalpha, a cytoplasmic inhibitor of NF-kappaB. To access this pathway, Tax associates stably with a cytokine-inducible IkappaB kinase (IKK), which contains both catalytic (IKKalpha and IKKbeta) and noncatalytic (IKKgamma) subunits. Unlike their transiently induced counterparts in cytokine-treated cells, Tax-associated forms of IKKalpha and IKKbeta are persistently activated in HTLV-1-infected T cells. Acquisition of the deregulated IKK phenotype is contingent on the presence of IKKgamma, which functions as a molecular adaptor in the assembly of pathologic Tax/IkappaB kinase complexes. These findings highlight a key mechanistic role for IKK in the Tax/NF-kappaB signaling axis and define new intracellular targets for the therapeutic control of HTLV-1-associated disease.
The adapter protein RIP plays a crucial role in NF-kappaB activation by TNF. Here we show that triggering of the p55 TNF receptor induces binding of RIP to NEMO (IKKgamma), a component of the I-kappa-B-kinase (IKK) "signalosome" complex, as well as recruitment of RIP to the receptor together with the three major signalosome components, NEMO, IKK1 and IKK2, and some kind of covalent modification of the recruited RIP molecules. It also induces binding of NEMO to the signaling inhibitor A20, and recruitment of A20 to the receptor. Enforced expression of NEMO in cells revealed that NEMO can both promote and block NF-kappaB activation and dramatically augments the phosphorylation of c-Jun. The findings suggest that the signaling activities of the IKK signalosome are regulated through binding of NEMO to RIP and A20 within the p55 TNF receptor complex.
We report here the identification of a novel protein, Smac, which promotes caspase activation in the cytochrome c/Apaf-1/caspase-9 pathway. Smac promotes caspase-9 activation by binding to inhibitor of apoptosis proteins, IAPs, and removing their inhibitory activity. Smac is normally a mitochondrial protein but is released into the cytosol when cells undergo apoptosis. Mitochondrial import and cleavage of its signal peptide are required for Smac to gain its apoptotic activity. Overexpression of Smac increases cells' sensitivity to apoptotic stimuli. Smac is the second mitochondrial protein, along with cytochrome c, that promotes apoptosis by activating caspases.
A20 is a cytoplasmic zinc finger protein that inhibits nuclear factor kappaB (NF-kappaB) activity and tumor necrosis factor (TNF)-mediated programmed cell death (PCD). TNF dramatically increases A20 messenger RNA expression in all tissues. Mice deficient for A20 develop severe inflammation and cachexia, are hypersensitive to both lipopolysaccharide and TNF, and die prematurely. A20-deficient cells fail to terminate TNF-induced NF-kappaB responses. These cells are also more susceptible than control cells to undergo TNF-mediated PCD. Thus, A20 is critical for limiting inflammation by terminating TNF-induced NF-kappaB responses in vivo.
TRAF6 is a signal transducer in the NF-kappaB pathway that activates IkappaB kinase (IKK) in response to proinflammatory cytokines. We have purified a heterodimeric protein complex that links TRAF6 to IKK activation. Peptide mass fingerprinting analysis reveals that this complex is composed of the ubiquitin conjugating enzyme Ubc13 and the Ubc-like protein Uev1A. We find that TRAF6, a RING domain protein, functions together with Ubc13/Uev1A to catalyze the synthesis of unique polyubiquitin chains linked through lysine-63 (K63) of ubiquitin. Blockade of this polyubiquitin chain synthesis, but not inhibition of the proteasome, prevents the activation of IKK by TRAF6. These results unveil a new regulatory function for ubiquitin, in which IKK is activated through the assembly of K63-linked polyubiquitin chains.
Processing of the nf(kappa)b2 gene product p100 to generate p52 is an important step in NF-kappaB regulation. We show that this step is negatively regulated by a processing-inhibitory domain (PID) within p100 and positively regulated by the NF-kappaB-inducing kinase (NIK). While the PID suppresses the constitutive processing of p100, NIK induces p100 processing by stimulating site-specific phosphorylation and ubiquitination of this precursor protein. Further, a natural mutation of the gene encoding NIK in alymphoplasia (aly) mice cripples the function of NIK in p100 processing, causing a severe defect in p52 production. These data suggest that NIK is a specific kinase regulating p100 processing and explain why the aly and nf(kappa)b2 knockout mice exhibit similar immune deficiencies.
Signals emanating from the receptor for interleukin-1 (IL-1), lipopolysaccharide (LPS) or osteoclast differentiation factor/receptor activator of NF kappa B ligand (ODF/RANKL) stimulate transcription factors AP-1 through mitogen-activated protein kinase (MAPK) activation and NF kappa B through I kappa B kinase (IKK) activation. These kinases are thought to be activated by tumor necrosis factor receptor-associated factor 6 (TRAF6). However, molecular mechanisms by which TRAF6 activates various downstream kinases remain to be elucidated. We identified functional domains of TRAF6 under physiological conditions established by appropriate expression of TRAF6 mutants in TRAF6-deficient cells. In IL-1 and LPS signaling pathways, the RING finger and first zinc finger domains are not required for NF kappa B activation but are required for full activation of MAPK. However, IL-1 and LPS signals utilize distinct regions within the zinc finger domains of TRAF6 to activate NF kappa B. Furthermore, the RING finger domain is not required for differentiation of splenocytes to multinuclear osteoclasts, but is essential for osteoclast maturation. Thus, TRAF6 plays essential roles in both the differentiation and maturation of osteoclasts by activating various kinases via its multiple domains.
In most cases, target proteins of the ubiquitin system are completely degraded. In several exceptions, such as the first step in the activation of the transcriptional regulator NF-kappaB, the substrate, the precursor protein p105, is processed in a limited manner to yield the active subunit p50. p50 is derived from the N-terminal domain of p105, whereas the C-terminal domain is degraded. The mechanisms involved in this unique process have remained elusive. We have shown that a Gly-rich region (GRR) at the C-terminal domain of p50 is one important processing signal and that it interferes with processing of the ubiquitinated precursor by the 26S proteasome. Also, amino acid residues 441-454 are important for processing under non-stimulated conditions. Lys 441 and 442 serve as ubiquitination targets, whereas residues 446-454 may serve as a ligase recognition motif. Following IkappaB kinase (IKK)-mediated phosphorylation, the C-terminal domain of p105, residues 918-934, recruits the SCF(beta-TrCP) ubiquitin ligase, and ubiquitination by this complex leads to accelerated processing. The two sites appear to be recognized under different physiological conditions by two different ligases, targeting two distinct recognition motifs. We have shown that ubiquitin conjugation and processing of a series of precursors of p105 that lack the C-terminal IKK phosphorylation/TrCP binding domain, is progressively inhibited with increasing number of ankyrin repeats. Inhibition is due to docking of active NF-kappaB subunits to the ankyrin repeat domain in the C-terminal half of p105 (IkappaBgamma). Inhibition is alleviated by phosphorylation of the C-terminal domain that leads to ubiquitin-mediated degradation of the ankyrin repeat domain and release of the anchored subunits. We propose a model that may explain the requirement for two sites: a) a basal site that may be involved in co-translational processing prior to the synthesis of the ankyrin repeat domain; and b) a signal-induced site that is involved in processing/degradation of the complete molecule following cell activation, with rapid release of stored, transcriptionally active subunits.
The conjugation of ubiquitin to other cellular proteins regulates a broad range of eukaryotic cell functions. The high efficiency and exquisite selectivity of ubiquitination reactions reflect the properties of enzymes known as ubiquitin-protein ligases or E3s. An E3 recognizes its substrates based on the presence of a specific ubiquitination signal, and catalyzes the formation of an isopeptide bond between a substrate (or ubiquitin) lysine residue and the C terminus of ubiquitin. Although a great deal is known about the molecular basis of E3 specificity, much less is known about molecular mechanisms of catalysis by E3s. Recent findings reveal that all known E3s utilize one of just two catalytic domains--a HECT domain or a RING finger--and crystal structures have provided the first detailed views of an active site of each type. The new findings shed light on many aspects of E3 structure, function, and mechanism, but also emphasize that key features of E3 catalysis remain to be elucidated.
In mammals, TAK1, a MAPKKK kinase, is implicated in multiple signaling processes, including the regulation of NF-kappaB activity via the IL1-R/TLR pathways. TAK1 function has largely been studied in cultured cells, and its in vivo function is not fully understood. We have isolated null mutations in the Drosophila dTAK1 gene that encodes dTAK1, a homolog of TAK1. dTAK1 mutant flies are viable and fertile, but they do not produce antibacterial peptides and are highly susceptible to Gram-negative bacterial infection. This phenotype is similar to the phenotypes generated by mutations in components of the Drosophila Imd pathway. Our genetic studies also indicate that dTAK1 functions downstream of the Imd protein and upstream of the IKK complex in the Imd pathway that controls the Rel/NF-kappaB like transactivator Relish. In addition, our epistatic analysis places the caspase, Dredd, downstream of the IKK complex, which supports the idea that Relish is processed and activated by a caspase activity. Our genetic demonstration of dTAK1's role in the regulation of Drosophila antimicrobial peptide gene expression suggests an evolutionary conserved role for TAK1 in the activation of Rel/NF-kappaB-mediated host defense reactions.