Mutation of the extracellular domain of tumour necrosis factor receptor 1 causes reduced NF-kappaB activation due to decreased surface expression

Tumour necrosis factor receptor-associated periodic syndrome (TRAPS) results from point mutations in the extracellular domain of TNF receptor 1 (TNFRSF1A), but the effects of the mutations are controversial. This study shows that reduced NF-kappaB signalling is a feature of four TRAPS mutations. Reduced signalling correlates with reduced surface expression, measured by flow cytometry and microscopy. This suggests that correct formation of the extracellular domain of TNFRSF1A is important for localisation and receptor function. Importantly, our data provides a mechanism for the reduced TNFRSF1 signalling observed in a patient cell line.     

Agonist antibody activates death receptor 6 downstream signaling involving TRADD recruitment

Death receptor 6 (DR6) is a member of the death domain-containing receptors that belong to the TNFR superfamily. To date, the ligand for DR6 is still not clearly defined. Here, we developed a functional agonist monoclonal antibody (DQM3) against DR6, which bound to the first cysteine-rich domain. Importantly, DR6 signaling could be clearly activated by DQM3, which was dependent on its intracellular death domain. In addition, we demonstrated that the association between DR6 and TRADD was enhanced upon DQM3 stimulation and TRADD was involved in DR6-induced signaling activation. Taken together, our findings provide new insight into a novel mechanism by which DR6 induces downstream signaling in response to an agonist antibody.     

The crystal structure of death receptor 6 (DR6): a potential receptor of the amyloid precursor protein (APP)

Death receptors belong to the tumor necrosis factor receptor (TNFR) super family and are intimately involved in the signal transduction during apoptosis, stress response and cellular survival. Here we present the crystal structure of recombinantly expressed death receptor six (DR6), one family member that was recently shown to bind to the amyloid precursor protein (APP) and hence to be probably involved in the development of Alzheimer's disease. The extracellular cysteine rich region of DR6, the typical ligand binding region of all TNFRs, was refined to 2.2 Å resolution and shows that its four constituting cysteine rich domains (CRDs) are arranged in a rod-like overall structure, which presents DR6-specific surface patches responsible for the exclusive recognition of its ligand(s). Based on the structural data, the general ligand binding modes of TNFRs and molecular modeling experiments we were able to elucidate structural features of the potential DR6-APP signaling complex.     

Early embryonic lethality caused by targeted disruption of the TRAF-interacting protein (TRIP) gene

Tumor necrosis factor receptor (TNFR)-associated factors (TRAFs) are key adaptor molecules in the TNFR-signaling complexes that promote a wide variety of signaling cascades including cell proliferation, activation, differentiation, and apoptosis. TRAF-interacting protein (TRIP) is required for the inhibitory regulation of TNF-induced NF-κB signaling via the TNFR/TRAF-signaling complexes in vitro. TRIP also directly interacts with the familial cylindromatosis tumor suppressor gene (CYLD) and negatively regulates NF-κB activation in vitro. However, although there appears to be a relationship between TRIP, the TRAFs and also CYLD as modulators of NF-κB signaling in vitro, the functional role of TRIP in vivo is still unclear. To identify the role of TRIP in vivo, we have generated TRIP-deficient mice. Homozygous mouse embryos were found to die shortly after implantation due to proliferation defects and excessive cell death. These results indicate that TRIP is an essential factor during early mouse embryonic development in vivo.     

Structure-Function Relationship of Tumor Necrosis Factor (TNF) and Its Receptor Interaction Based on 3D Structural Analysis of a Fully Active TNFR1-Selective TNF Mutant

Tumor necrosis factor (TNF) is an important cytokine that suppresses carcinogenesis and excludes infectious pathogens to maintain homeostasis. TNF activates its two receptors [TNF receptor (TNFR) 1 and TNFR2], but the contribution of each receptor to various host defense functions and immunologic surveillance is not yet clear. Here, we used phage display techniques to generate receptor-selective TNF mutants that activate only one TNFR. These TNF mutants will be useful in the functional analysis of TNFR.Six amino acids in the receptor binding interface (near TNF residues 30, 80, and 140) were randomly mutated by polymerase chain reaction. Two phage libraries comprising over 5 million TNF mutants were constructed. By selecting the mutants without affinity for TNFR1 or TNFR2, we successfully isolated 4 TNFR2-selective candidates and 16 TNFR1-selective candidates, respectively. The TNFR1-selective candidates were highly mutated near residue 30, whereas TNFR2-selective candidates were highly mutated near residue 140, although both had conserved sequences near residues 140 and 30, respectively. This finding suggested that the phage display technique was suitable for identifying important regions for the TNF interaction with TNFR1 and TNFR2. Purified clone R1-6, a TNFR1-selective candidate, remained fully bioactive and had full affinity for TNFR1 without activating TNFR2, indicating the usefulness of the R1-6 TNF mutant in analyzing TNFR1 receptor function.To further elucidate the receptor selectivity of R1-6, we examined the structure of R1-6 by X-ray crystallography. The results suggested that R31A and R32G mutations strongly influenced electrostatic interaction with TNFR2, and that L29K mutation contributed to the binding of R1-6 to TNFR1. This phage display technique can be used to efficiently construct functional mutants for analysis of the TNF structure-function relationship, which might facilitate in silico drug design based on receptor selectivity.     

Comparative Biochemical and Functional Analysis of Viral and Human Secreted Tumor Necrosis Factor (TNF) Decoy Receptors

The blockade of tumor necrosis factor (TNF) by etanercept, a soluble version of the human TNF receptor 2 (hTNFR2), is a well established strategy to inhibit adverse TNF-mediated inflammatory responses in the clinic. A similar strategy is employed by poxviruses, encoding four viral TNF decoy receptor homologues (vTNFRs) named cytokine response modifier B (CrmB), CrmC, CrmD, and CrmE. These vTNFRs are differentially expressed by poxviral species, suggesting distinct immunomodulatory properties. Whereas the human variola virus and mouse ectromelia virus encode one vTNFR, the broad host range cowpox virus encodes all vTNFRs. We report the first comprehensive study of the functional and binding properties of these four vTNFRs, providing an explanation for their expression profile among different poxviruses. In addition, the vTNFRs activities were compared with the hTNFR2 used in the clinic. Interestingly, CrmB from variola virus, the causative agent of smallpox, is the most potent TNFR of those tested here including hTNFR2. Furthermore, we demonstrate a new immunomodulatory activity of vTNFRs, showing that CrmB and CrmD also inhibit the activity of lymphotoxin β. Similarly, we report for the first time that the hTNFR2 blocks the biological activity of lymphotoxin β. The characterization of vTNFRs optimized during virus-host evolution to modulate the host immune response provides relevant information about their potential role in pathogenesis and may be used to improve anti-inflammatory therapies based on soluble decoy TNFRs.     

Binding Studies of TNF Receptor Superfamily (TNFRSF) Receptors on Intact Cells

Ligands of the tumor necrosis factor superfamily (TNFSF) interact with members of the TNF receptor superfamily (TNFRSF). TNFSF ligand-TNFRSF receptor interactions have been intensively evaluated by many groups. The affinities of TNFSF ligand-TNFRSF receptor interactions are highly dependent on the oligomerization state of the receptor, and cellular factors (e.g. actin cytoskeleton and lipid rafts) influence the assembly of ligand-receptor complexes, too. Binding studies on TNFSF ligand-TNFRSF receptor interactions were typically performed using cell-free assays with recombinant fusion proteins that contain varying numbers of TNFRSF ectodomains. It is therefore not surprising that affinities determined for an individual TNFSF ligand-TNFRSF interaction differ sometimes by several orders of magnitude and often do not reflect the ligand activity observed in cellular assays. To overcome the intrinsic limitations of cell-free binding studies and usage of recombinant receptor domains, we performed comprehensive binding studies with Gaussia princeps luciferase TNFSF ligand fusion proteins for cell-bound TNFRSF members on intact cells at 37 °C. The affinities of the TNFSF ligand G. princeps luciferase-fusion proteins ranged between 0.01 and 19 nm and offer the currently most comprehensive and best suited panel of affinities for in silico studies of ligand-receptor systems of the TNF family.     

Structure of CrmE, a virus-encoded tumour necrosis factor receptor

Vaccinia virus (VACV), the smallpox vaccine, encodes many proteins that subvert the host immune response. One of these, cytokine response modifier E (CrmE), is secreted by infected cells and protects these cells from apoptotic challenge by tumour necrosis factor alpha (TNFalpha). We have expressed recombinant CrmE from VACV strain Lister in Escherichia coli, shown that the purified protein is monomeric in solution and competent to bind TNFalpha, and solved the structure to 2.0 A resolution. This is the first structure of a virus-encoded tumour necrosis factor receptor (TNFR). CrmE shares significant sequence similarity with mammalian type 2 TNF receptors (TNFSFR1B, p75; TNFR type 2). The structure confirms that CrmE adopts the canonical TNFR fold but only one of the two "ligand-binding" loops of TNFRSF1A is conserved in CrmE, suggesting a mechanism for the higher affinity of poxvirus TNFRs for TNFalpha over lymphotoxin-alpha. The roles of dimerisation and pre-ligand-assembly domains (PLADs) in poxvirus and mammalian TNFR activity are discussed.     

Molecular basis for herpesvirus entry mediator recognition by the human immune inhibitory receptor CD160 and its relationship to the cosignaling molecules BTLA and LIGHT

CD160 was recently identified as a T cell coinhibitory molecule that interacts with the herpesvirus entry mediator (HVEM) on antigen-presenting cells to deliver a potent inhibitory signal to CD4⁺ T cells. HVEM also binds to the coinhibitory receptor BTLA (B- and T-lymphocyte attenuator) and the costimulatory receptor LIGHT (which is homologous to lymphotoxins, exhibits inducible expression, and competes with the herpes simplex virus glycoprotein D for HVEM, a receptor expressed by T lymphocytes, or TNFSF14), thus regulating the CD160/BTLA/LIGHT/HVEM signaling pathway. To date, the detailed properties of the formation of these complexes, especially HVEM binding to the newly identified receptor CD160, and the relationship of CD160 with BTLA and LIGHT are still unclear. We performed N-terminal sequencing and a mass spectrometric analysis, which revealed that the extracellular domain of CD160 exists primarily in the monomeric form. The surface plasmon resonance analysis revealed that CD160 binds directly to the cysteine-rich domain 1-3 of HVEM with a similar affinity to, but slower dissociation rate than, that of BTLA. Notably, CD160 competed with BTLA for binding to HVEM; in contrast, LIGHT did not affect HVEM binding to either CD160 or BTLA. The results of a mutagenesis study of HVEM also suggest that the CD160 binding region on HVEM was slightly different from, but overlapped with, the BTLA binding site. Interestingly, an anti-CD160 antibody exhibiting antiangiogenic properties blocked CD160/HVEM binding. These results provide insight into the molecular architecture of the CD160/BTLA/LIGHT/HVEM signaling complex that regulates immune function.     

Progranulin directly binds to the CRD2 and CRD3 of TNFR extracellular domains

We previously reported that PGRN directly bound to TNF receptors (TNFR) in vitro and in chondrocytes (Tang, et al., Science, 2011). Here we report that PGRN also associated with TNFR in splenocytes, and inhibited the binding of TNFα to immune cells. Proper folding of PGRN is essential for its binding to TNFR, as DTT treatment abolished its binding to TNFR. In contrast, the binding of PGRN to Sortilin was enhanced by DTT. Protein interaction assays with mutants of the TNFR extracellular domain demonstrated that CRD2 and CRD3 of TNFR are important for the interaction with PGRN, similar to the binding to TNFα. Taken together, these findings provide the molecular basis underlying PGRN/TNFR interaction and PGRN-mediated anti-inflammatory activity in various autoimmune diseases and conditions.     

Activation of the proapoptotic death receptor DR5 by oligomeric peptide and antibody agonists

The cell-extrinsic apoptotic pathway triggers programmed cell death in response to certain ligands that bind to cell-surface death receptors. Apoptosis is essential for normal development and homeostasis in metazoans, and furthermore, selective activation of the cell-extrinsic pathway in tumor cells holds considerable promise for cancer therapy. We used phage display to identify peptides and synthetic antibodies that specifically bind to the human proapoptotic death receptor DR5. Despite great differences in overall size and structure, the DR5-binding peptides and antibodies shared a tripeptide motif, which was conserved within a disulfide-constrained loop of the peptides and the third complementarity determining region of the antibody heavy chains. The X-ray crystal structure of an antibody in complex with DR5 revealed that the tripeptide motif is buried at the core of the interface, confirming its central role in antigen recognition. We found that certain peptides and antibodies exhibited potent proapoptotic activity against DR5-expressing SK-MES-1 lung carcinoma cells. These phage-derived ligands may be useful for elucidating DR5 activation at the molecular level and for creating synthetic agonists of proapoptotic death receptors.     

TNFR1 and the TNFα axis as a targetable mediator of liver injury from stereotactic body radiation therapy

IntroductionRadiation therapy for the management of intrahepatic malignancies can adversely affect liver function. Liver damage has been associated with increased levels of inflammatory cytokines, including tumor necrosis factor alpha (TNFα). We hypothesized that an inflammatory state, characterized by increased soluble TNFα receptor (sTNFR1), mediates sensitivity of the liver to radiation.Materials/MethodsPlasma samples collected during 3 trials of liver radiation for liver malignancies were assayed for sTNFR1 level via enzyme-linked immunosorbent assay (ELISA). Univariate and multivariate logistic regression and longitudinal models were used to characterize associations between liver toxicity (defined as a ≥2-point increase in Child-Pugh [CP] score within 6 months of radiation treatment) and sTNFR1 levels, ALBI score, biocorrected mean liver dose (MLD), age, and baseline laboratory values.ResultsSamples from 78 patients given liver stereotactic body radiation therapy [SBRT] (92%) or hypofractionated radiation were examined. There was a significant association between liver toxicity and sTNFR1 levels, and higher values were associated with increased toxicity over a range of mean liver doses. When ALBI score and biocorrected dose were included in the model with sTNFR1, baseline ALBI score and change in ALBI (ΔALBI) were significantly associated with toxicity, but sTNFR1 was not. Baseline aminotransferase levels also predicted toxicity but not independently of ALBI score.ConclusionsElevated plasma sTNFR1 levels are associated with liver injury after liver radiation, suggesting that elevated inflammatory cytokine activity is a predictor of radiation-induced liver dysfunction. Future studies should determine whether administration of agents that decrease inflammation prior to treatment is warranted.     

Tumor necrosis factor receptor-1-induced neuronal death by TRADD contributes to the pathogenesis of Japanese encephalitis

While a number of studies have documented the neurotropism of Japanese encephalitis virus (JEV), little is known regarding the molecular mechanism of neuronal death following viral infection. The tumor necrosis factor receptor (TNFR)-associated death domain (TRADD) has been suggested to be the crucial signal adaptor that mediates all intracellular responses from TNFR-1. Using mouse (Neuro2a) and human (SK-N-SH) neuroblastoma cell lines, we have shown that the altered expression of TNFR-1 and TRADD following JEV infection regulates the downstream apoptotic cascades. Activation of TRADD led to mitochondria-mediated neuronal apoptosis. As TRADD-knockout animals or deficient cell lines are unavailable, it has been difficult to definitively address the physiological role of TRADD in diseases pathology following JEV infection. We circumvented this problem by silencing TRADD expression with small-interfering RNA (siRNA) and have found that TRADD is required for TNFR-1-initiated neuronal apoptosis following in vitro infection with JEV. Interestingly, siRNA against TRADD also decreased the viral load in Neuro2a cells. Furthermore, siRNA against TRADD increased the survival of JEV-infected mice by altering the expression of pro apoptotic versus antiapoptotic molecules. These studies show that the engagement of TNFR-1 and TRADD following JEV infection plays a crucial role in neuronal apoptosis.     

Leptin and leptin receptor: Analysis of a structure to function relationship in interaction and evolution from humans to fish

Leptin is a circulating protein which regulates dietary intake through binding the leptin receptor. Numerous labs have used known structures and mutagenesis to study this binding process in common animal models (human, mouse and rat). Understanding this binding process in other vertebrate species will allow for a better understanding of leptin and leptin receptor function. The binding site between leptin and leptin receptor is highly conserved in mammals as confirmed through sequence alignments mapped onto structures of both leptin and leptin receptor. More variation in this interaction is found in lizard and frog sequences. Using our models, we show that the avian leptin sequences have far less variation in the binding site than does the leptin receptor. This analysis further suggests that avian leptins are artifactual. In fish, gene duplication events have led to the expression of multiple leptin proteins. These multiple leptin proteins have variation in the regions interacting with leptin receptor. In zebrafish and the Japanese rice fish, we propose that leptin A has a higher binding energy than does B. Differing binding energies are evidence of either divergent functions, different binding confirmations, or other protein partners of leptin B.     

Functional role of the Ca-activated Cl channel DOG1/TMEM16A in gastrointestinal stromal tumor cells

DOG1, a Ca²⁺-activated Cl⁻ channel (CaCC), was identified in 2004 to be robustly expressed in gastrointestinal stromal tumors (GIST). It was rapidly included as a tumor marker in routine diagnostics, but the functional role remained unknown. CaCCs are important regulators of normal physiological functions, but also implicated in tumorigenesis, cancer progression, metastasis, cell migration, apoptosis, proliferation and viability in several malignancies. We therefore investigated whether DOG1 plays a role in the three latter in GIST by utilizing in vitro cell model systems. Confocal microscopy identified different subcellular localizations of DOG1 in imatinib-sensitive and imatinib-resistant cells. Electrophysiological studies confirmed that DOG1-specific pharmacological agents possess potent activating and inhibiting properties. Proliferation assays showed small effects up to 72 h, and flow cytometric analysis of adherent cells with 7-AAD/Annexin V detected no pharmacological effects on viable GIST cells. However, inhibition of DOG1 conveyed pro-apoptotic effects among early apoptotic imatinib-resistant cells. In conclusion, DOG1 generates Cl⁻ currents in GIST that can be regulated pharmacologically, with small effects on cell viability and proliferation in vitro. Inhibition of DOG1 might act pro-apoptotic on some early apoptotic GIST cell populations. Further studies are warranted to fully illuminate the function of DOG1 and its potential as therapeutic target.     

Molecular Characterization and Evolution of Self-Incompatibility Genes in Arabidopsis thaliana: The Case of the Sc Haplotype

The switch from an outcrossing mode of mating enforced by self-incompatibility to self-fertility in the Arabidopsis thaliana lineage was associated with mutations that inactivated one or both of the two genes that comprise the self-incompatibility (SI) specificity-determining S-locus haplotype, the S-locus receptor kinase (SRK) and the S-locus cysteine-rich (SCR) genes, as well as unlinked modifier loci required for SI. All analyzed A. thaliana S-locus haplotypes belong to the SA, SB, or SC haplotypic groups. Of these three, the SC haplotype is the least well characterized. Its SRKC gene can encode a complete open-reading frame, although no functional data are available, while its SCRC sequences have not been isolated. As a result, it is not known what mutations were associated with inactivation of this haplotype. Here, we report on our analysis of the Lz-0 accession and the characterization of its highly rearranged SC haplotype. We describe the isolation of its SCRC gene as well as the subsequent isolation of SCRC sequences from other SC-containing accessions and from the A. lyrata S36 haplotype, which is the functional equivalent of the A. thaliana SC haplotype. By performing transformation experiments using chimeric SRK and SCR genes constructed with SC- and S36-derived sequences, we show that the SRKC and SCRC genes of Lz-0 and at least a few other SC-containing accessions are nonfunctional, despite SCRC encoding a functional full-length protein. We identify the probable mutations that caused the inactivation of these genes and discuss our results in the context of mechanisms of S-locus inactivation in A. thaliana.     

A Review of Prostanoid Receptors: Expression,Characterization, Regulation,and Mechanism of Action

Prostaglandin signaling controls a wide range of biological processes from blood pressure homeostasis to inflammation and resolution thereof to the perception of pain to cell survival. Disruption of normal prostanoid signaling is implicated in numerous disease states. Prostaglandin signaling is facilitated by G-protein-coupled, prostanoid-specific receptors and the array of associated G-proteins. This review focuses on the expression, characterization, regulation, and mechanism of action of prostanoid receptors with particular emphasis on human isoforms.     

Inhibition of Na,K-ATPase-suppressive activity of translationally controlled tumor protein by sorting nexin 6

Translationally controlled tumor protein (TCTP) has both extra- and intracellular functions. Our group recently reported that TCTP interacts with Na,K-ATPase and suppresses its activity. Our studies led to the identification of sorting nexin 6 (SNX6) which binds with TCTP as a potential negative regulator of TCTP. SNX6 does not interact directly with any cytoplasmic domains of Na,K-ATPase. However, when overexpressed, it restores the Na,K-ATPase activity suppressed by TCTP. This was confirmed by measurements of purified plasma membrane Na,K-ATPase activity after incubation with recombinant TCTP and SNX6. SNX6 alone has no effect on Na,K-ATPase activity, but activates Na,K-ATPase via inhibition of TCTP. Inhibition of endogenous TCTP by the overexpression of SNX6 or knockdown of TCTP expression by siTCTP increased Na,K-ATPase activity above the basal level. The interaction between SNX6 and TCTP thus appears to regulate Na,K-ATPase activity.