Atypical TNF-TNFR superfamily binding interface in the GITR-GITRL complex for T cell activation.

Glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) is a critical regulatory molecule in modulation of T cell immune responses. Here we report the mouse GITR (mGITR) and mGITR ligand (mGITRL) complex structure and find that the binding interface of mGITR and mGITRL is distinct from the typical tumor necrosis factor superfamily (TNFSF)/TNF receptor superfamily (TNFRSF) members. mGITR binds to its ligand with a single domain, whereas the binding interface on mGITRL is located on the side, which is distal from conserved binding sites of TNFSF molecules. Mutational analysis reveals that the binding interface of GITR/GITRL in humans is conserved with that in the mouse. Substitution of key interacting D93-I94-V95 (DIV) in mGITR with the corresponding K93-F94-S95 (KFS) in human GITR enables cross-recognition with human GITRL and cross-activation of receptor signaling. The findings of this study substantially expand our understanding of the interaction of TNFSF/TNFRSF superfamily molecules and can benefit the future design of biologics by targeting GITR/GITRL.

Structures of mouse and human GITR-GITRL complexes reveal unique TNF superfamily interactions.

Glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) and GITR ligand (GITRL) are members of the tumor necrosis superfamily that play a role in immune cell signaling, activation, and survival. GITR is a therapeutic target for directly activating effector CD4 and CD8 T cells, or depleting GITR-expressing regulatory T cells (Tregs), thereby promoting anti-tumor immune responses. GITR activation through its native ligand is important for understanding immune signaling, but GITR structure has not been reported. Here we present structures of human and mouse GITR receptors bound to their cognate ligands. Both species share a receptor-ligand interface and receptor-receptor interface; the unique C-terminal receptor-receptor enables higher order structures on the membrane. Human GITR-GITRL has potential to form a hexameric network of membrane complexes, while murine GITR-GITRL complex forms a linear chain due to dimeric interactions. Mutations at the receptor-receptor interface in human GITR reduce cell signaling with in vitro ligand binding assays and minimize higher order membrane structures when bound by fluorescently labeled ligand in cell imaging experiments.

Generation and Evaluation of Bispecific Anti-TNF Antibodies Based on Single-Chain VHH Domains.

Systemic cytokine inhibition may be an effective therapeutic strategy for several autoimmune diseases. However, recent studies suggest that tissue or cell type-specific targeting of certain cytokines, including TNF, may have distinct advantages and show fewer side effects. Here we describe protocols for generating and testing bispecific cytokine inhibitors using variable domain of single-chain antibodies from Camelidae (VHH) with a focus on cell-specific TNF inhibitors.

TNFPred: identifying tumor necrosis factors using hybrid features based on word embeddings.

BACKGROUND: Cytokines are a class of small proteins that act as chemical messengers and play a significant role in essential cellular processes including immunity regulation, hematopoiesis, and inflammation. As one important family of cytokines, tumor necrosis factors have association with the regulation of a various biological processes such as proliferation and differentiation of cells, apoptosis, lipid metabolism, and coagulation. The implication of these cytokines can also be seen in various diseases such as insulin resistance, autoimmune diseases, and cancer. Considering the interdependence between this kind of cytokine and others, classifying tumor necrosis factors from other cytokines is a challenge for biological scientists. METHODS: In this research, we employed a word embedding technique to create hybrid features which was proved to efficiently identify tumor necrosis factors given cytokine sequences. We segmented each protein sequence into protein words and created corresponding word embedding for each word. Then, word embedding-based vector for each sequence was created and input into machine learning classification models. When extracting feature sets, we not only diversified segmentation sizes of protein sequence but also conducted different combinations among split grams to find the best features which generated the optimal prediction. Furthermore, our methodology follows a well-defined procedure to build a reliable classification tool. RESULTS: With our proposed hybrid features, prediction models obtain more promising performance compared to seven prominent sequenced-based feature kinds. Results from 10 independent runs on the surveyed dataset show that on an average, our optimal models obtain an area under the curve of 0.984 and 0.998 on 5-fold cross-validation and independent test, respectively. CONCLUSIONS: These results show that biologists can use our model to identify tumor necrosis factors from other cytokines efficiently. Moreover, this study proves that natural language processing techniques can be applied reasonably to help biologists solve bioinformatics problems efficiently.

Effects of protein-protein interface disruptors at the ligand of the glucocorticoid-induced tumor necrosis factor receptor-related gene (GITR).

The tumor necrosis factor (TNF) superfamily (TNFSF) includes about thirty structurally related receptors (TNFSFRs) and about twenty protein ligands that bind to one or more of these receptors. Receptors of the tumor necrosis factor (TNF) superfamily (TNFSFRs) are pharmacological targets for treatment of inflammatory and autoimmune diseases. Currently, drugs targeting TNFSFR signaling are biological drugs (monoclonal antibodies, decoy receptors) aimed at binding and sequestering TNFSFR ligands. The glucocorticoid-induced tumor necrosis factor receptor-related gene (GITR) signaling is involved in a series of inflammatory and autoimmune diseases, such as rheumatoid arthritis and Crohn's disease. Our study aimed at repurposing FDA approved small molecules as protein-protein disruptors at the GITR ligand (GITRL) trimer, in order to inhibit the binding of GITRL to its receptor (GITR). A structure based molecular modeling approach was carried out to identify, through high throughput virtual screening, GITRL monomer-monomer disruptors. We used a database of ~8,000 FDA approved drugs, and after virtual screening, we focused on two hit compounds, minocycline and oxytetracycline. These two compounds were tested for their capability to modulate IL-17, IL-21 and RORγT expression in T lymphocytes, isolated from wild-type and GITR knock-out (GITR-/-) mice. Minocycline showed immunomodulatory effects specific to GITR activation and could represent a novel pharmacological tool to treat inflammatory diseases.

HERA-GITRL activates T cells and promotes anti-tumor efficacy independent of FcγR-binding functionality.

BACKGROUND: Glucocorticoid-induced TNFR-related protein (TNFRSF18, GITR, CD357), expressed by T cells, and its ligand (TNFSF18, GITRL), expressed by myeloid populations, provide co-stimulatory signals that boost T cell activity. Due to the important role that GITR plays in regulating immune functions, agonistic stimulation of GITR is a promising therapeutic concept. Multiple strategies to induce GITR signaling have been investigated. The limited clinical efficacy of antibody-based GITR agonists results from structural and functional characteristics of antibodies that are unsuitable for stimulating the well-defined trimeric members of the TNFRSF. METHODS: To overcome limitations of antibody-based TNFRSF agonists, we have developed HERA-GITRL, a fully human hexavalent TNF receptor agonist (HERA) targeting GITR and mimicking the natural signaling concept. HERA-GITRL is composed of a trivalent but single-chain GITRL-receptor-binding-domain (scGITRL-RBD) unit fused to an IgG1 derived silenced Fc-domain serving as dimerization scaffold. A specific mouse surrogate, mmHERA-GITRL, was also generated to examine in vivo activity in respective mouse tumor models. RESULTS: For functional characterization of HERA-GITRL in vitro, human immune cells were isolated from healthy-donor blood and stimulated with anti-CD3 antibody in the presence of HERA-GITRL. Consistently, HERA-GITRL increased the activity of T cells, including proliferation and differentiation, even in the presence of regulatory T cells. In line with these findings, mmHERA-GITRL enhanced antigen-specific clonal expansion of both CD4+ (OT-II) and CD8+ (OT-I) T cells in vivo while having no effect on non-specific T cells. In addition, mmHERA-GITRL showed single-agent anti-tumor activity in two subcutaneous syngeneic colon cancer models (CT26wt and MC38-CEA). Importantly, this activity is independent of its FcγR-binding functionality, as both mmHERA-GITRL with a functional Fc- and a silenced Fc-domain showed similar tumor growth inhibition. Finally, in a direct in vitro comparison to a bivalent clinical benchmark anti-GITR antibody and a trivalent GITRL, only the hexavalent HERA-GITRL showed full biological activity independent of additional crosslinking. CONCLUSION: In this manuscript, we describe the development of HERA-GITRL, a true GITR agonist with a clearly defined mechanism of action. By clustering six receptor chains in a spatially well-defined manner, HERA-GITRL induces potent agonistic activity without being dependent on additional FcγR-mediated crosslinking.

3D Modeling of Tumor Necrosis Factor Receptor and Tumor Necrosis Factor-bound Receptor Systems.

The interactions between the tumor necrosis factor (TNF) and its receptor molecule are responsible for various signaling networks that are central to the functioning of human immune homeostasis. The present work is a computational study of certain structural aspects of this cell-signaling protein, specifically focusing on the molecular level analyses of the TNF receptor (TNF-R), guided by its crystallographic structure. We also examine the possible binding sites of the TNF onto TNF-R, and the associated interactions. The structural and conformational variations in the TNF-R and TNF bound TNF-R systems are examined in this context using molecular dynamics (MD) simulations. The time dependent variations of the dimeric TNF-R structures are compared with, and shown to be steadier than their isolated monomers. This dimeric stability is favored under acidic conditions. The results are used to further illustrate how 3D modeling and computer simulations can aid the structure-based approach to probing a ligand-receptor system.

Construction of Artificial TNF-Binding Proteins Based on the 10th Human Fibronectin Type III Domain Using Bacterial Display.

Construction of antibody mimetics on the base of alternative scaffold proteins is a promising strategy for obtaining new products for medicine and biotechnology. The aim of our work was to optimize the cell display system for the 10th human fibronectin type III domain (10Fn3) scaffold protein based on the AT877 autotransporter from Psychrobacter cryohalolentis K5T and to construct new artificial TNF-binding proteins. We obtained a 10Fn3 gene combinatorial library and screened it using the bacterial display method. After expression of the selected 10Fn3 variants in Escherichia coli cells and analysis of their TNF-binding activity, we identified proteins that display high affinity for TNF and characterized their properties.

Modeling the structural implications of an alternatively spliced Exoc3l2, a paralog of the tunneling nanotube-forming M-Sec.

The exocyst is a molecular tether that retains secretory vesicles at the plasma membrane prior to SNARE-mediated docking and fusion. However, individual exocyst complex components (EXOCs) may also function independently of exocyst assembly. Alternative splice variants of EXOC mRNA and paralogs of EXOC genes have been described and several have been attributed functions that may be independent of the exocyst complex. Here we describe a novel splice variant of murine Exoc3l2, which we term Exoc3l2a. We discuss possible functional implications of the resulting domain excision from this isoform of EXOC3L2 based on structural similarities with its paralog M-Sec (EXOC3L3), which is implicated in tunneling nanotube formation. The identification of this Exoc3l2 splice variant expands the potential for subunit diversity within the exocyst and for alternative functionality of this component independently of the exocyst.

Current Status and Future Prospects of Small-molecule Protein-protein Interaction (PPI) Inhibitors of Tumor Necrosis Factor (TNF) and Receptor Activator of NF-κB Ligand (RANKL).

The overexpression of Tumor Necrosis Factor (TNF) is directly related to the development of several autoimmune diseases, such as rheumatoid and psoriatic arthritis, inflammatory bowel disease, Crohn's disease, refractory asthma, and multiple sclerosis. Receptor Activator of Nuclear Factor Kappa- B Ligand (RANKL) belongs to the TNF family and is the primary mediator of osteoclast-induced bone resorption through interaction with its receptor RANK. The function of RANKL is physiologically inhibited by the action of osteoprotegerin (OPG), which is a decoy receptor that binds to RANKL and prevents the process of osteoclastogenesis. Malfunction among RANK/RANKL/OPG can also result in bone loss diseases, including postmenopausal osteoporosis, rheumatoid arthritis, bone metastasis and multiple myeloma. To disrupt the unwanted functions of TNF and RANKL, current attempts focus on blocking TNF and RANKL binding to their receptors. In this review, we present the research efforts toward the development of low-molecular-weight pharmaceuticals that directly block the detrimental actions of TNF and RANKL.

[Research progress of complement-C1q/tumor necrosis factor-related protein 3].

Complement-C1q/tumor necrosis factor-related protein 3 (CTRP3) is an adipokine that primarily identified in 2003 and is an important member of CTRP family. CTRP3 is expressed in various tissues and cell types, and is highly conserved among different species. Multiple novel functions of CTRP3 have been reported recently as the further research on this protein develops. CTRP3 not only affects the proliferation of chondrocytes and the pathogenesis of osteoarthritis, but also regulates multiple physiological and pathological processes including the secretion of testosterone and adipokines, glucose and lipid metabolism, mitochondrial biogenesis, inflammatory response, cell apoptosis, angiogenesis, vascular calcification and ventricular remodeling. The present review mainly focuses on the research progresses on CTRP3, including its discovery, gene and protein structure, expression regulation, and biological functions. The progresses summarized may provide new clues for the further investigation of CTRP3.

C1q/TNF-Related Protein 3 (CTRP3) Function and Regulation.

As the largest endocrine organ, adipose tissue secretes many bioactive molecules that circulate in blood, collectively termed adipokines. Efforts to identify such metabolic regulators have led to the discovery of a family of secreted proteins, designated as C1q tumor necrosis factor (TNF)-related proteins (CTRPs). The CTRP proteins, adiponectin, TNF-alpha, as well as other proteins with the distinct C1q domain are collectively grouped together as the C1q/TNF superfamily. Reflecting profound biological potency, the initial characterization of these adipose tissue-derived CTRP factors finds wide-ranging effects upon metabolism, inflammation, and survival-signaling in multiple tissue types. CTRP3 (also known as CORS26, cartducin, or cartonectin) is a unique member of this adipokine family. In this review we provide a comprehensive overview of the research concerning the expression, regulation, and physiological function of CTRP3. © 2017 American Physiological Society. Compr Physiol 7:863-878, 2017.

Molecular identification and functional characterization of a tumor necrosis factor (TNF) gene in Crassostrea hongkongensis.

Tumor necrosis factor superfamily (TNFSF) represents a group of multifunctional inflammatory cytokines that have been shown to participate in a variety of pathological and immunological process. However, the functions of these proteins in oyster are still poorly understood. In the present study, an oyster TNF homolog (named ChTNF) was identified from a cDNA library of Crassostrea hongkongensis. The complete cDNA of ChTNF was 2457bp in length containing an open reading frame (ORF) of 1044bp, a 5'-untranslated region (UTR) of 381bp and a 3'-UTR of 1032bp. The deduced ChTNF protein consisted of 347 amino acids with a characteristic transmembrane domain and a typical TNF homology domain (THD). Quantitative real-time PCR analysis revealed that ChTNF was broadly expressed in various oyster tissues and different stages of embryonic development. In addition, transcriptional analysis indicated that ChTNF transcription levels in hemocytes were increased significantly in pathogen challenge groups (Vibrio alginolyticus and Staphylococcus haemolyticus) compared to that in the control. Moreover, in vitro PAMP (lipopolysaccharide and peptidoglycan) treatments showed a stimulatory effect on the expression of ChTNF in the primary cultured hemocytes of C. hongkongensis. Finally, dual-luciferase reporter assays revealed that ChTNF could activate the NF-κB-Luc reporter in a dose-dependent manner in HEK293T cells. Altogether, these findings may provide valuable information regarding oyster TNFs and its role in innate immunity.

TNF superfamily cytokines in the promotion of Th9 differentiation and immunopathology.

The tumor necrosis factor (TNF) receptors and their corresponding cytokine ligands have been implicated in many aspects of the biology of immune functions. TNF receptors have key roles during various stages of T cell homeostasis. Many of them can co-stimulate lymphocyte proliferation and cytokine production. Additionally, several TNF cytokines can regulate T cell differentiation, including promoting Th1, Th2, Th17, and more recently the newly described Th9 subset. Four TNF family cytokines have been identified as regulators for IL-9 production by T cells. OX40L, TL1A, and GITRL can promote Th9 formation but can also divert iTreg into Th9, while 4-1BBL seems to inhibit IL-9 production from iTreg and has not been studied for its ability to promote Th9 generation. Regulation of IL-9 production by TNF family cytokines has repercussions in vivo, including enhancement of anti-tumor immunity and immunopathology in allergic lung and ocular inflammation. Regulating T cell production of IL-9 through blockade or agonism of TNF family cytokine receptors may be a therapeutic strategy for autoimmune and allergic diseases and in tumor.

Comparative genomic analysis of eutherian tumor necrosis factor ligand genes.

The present analysis made attempts to resolve discrepancies in descriptions of eutherian tumor necrosis factor ligand genes implicated in cell signalling pathways, as well as in major physiological and pathological processes. Among 455 potential coding sequences, the eutherian comparative genomic analysis protocol annotated 211 complete coding sequences using public genomic sequence assemblies. The most comprehensive third party data gene data set first described 8 superclusters of eutherian tumor necrosis factor ligand genes, including 18 major gene clusters. The integrated gene annotations, phylogenetic analysis, and protein molecular evolution analysis proposed new classification and nomenclature of eutherian tumor necrosis factor ligand genes, as new framework of future experiments.

Structural insights of homotypic interaction domains in the ligand-receptor signal transduction of tumor necrosis factor (TNF).

Several members of tumor necrosis factor receptor (TNFR) superfamily that these members activate caspase-8 from death-inducing signaling complex (DISC) in TNF ligand-receptor signal transduction have been identified. In the extrinsic pathway, apoptotic signal transduction is induced in death domain (DD) superfamily; it consists of a hexahelical bundle that contains 80 amino acids. The DD superfamily includes about 100 members that belong to four subfamilies: death domain (DD), caspase recruitment domain (CARD), pyrin domain (PYD), and death effector domain (DED). This superfamily contains key building blocks: with these blocks, multimeric complexes are formed through homotypic interactions. Furthermore, each DD-binding event occurs exclusively. The DD superfamily regulates the balance between death and survival of cells. In this study, the structures, functions, and unique features of DD superfamily members are compared with their complexes. By elucidating structural insights of DD superfamily members, we investigate the interaction mechanisms of DD domains; these domains are involved in TNF ligand-receptor signaling. These DD superfamily members play a pivotal role in the development of more specific treatments of cancer. [BMB Reports 2016; 49(3): 159-166].

Swim training and the genetic expression of adipokines in monosodium glutamate-treated obese rats

Objective The aim of this study was to evaluate the genetic expression of adipokines in the adipocytes of monosodium glutamate (MSG)-treated obese rats submitted to physical activity.Materials and methods Obesity was induced by neonatal MSG administration. Exercised rats (MSG and control) were subjected to swim training for 30 min for 10 weeks, whereas their respective controls remained sedentary. Total RNA was obtained from sections of the mesenteric adipose tissue of the rats. mRNA levels of adiponectin (Adipoq), tumor necrosis factor alpha (Tnf), peroxisome proliferator-activated receptor alpha (Ppara), and peroxisome proliferator-activated receptor gamma (Pparg) adipokines were quantified by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR).Results In the exercise-trained control group, the expression of Adipoq increased compared to the sedentary control, which was not observed in the MSG-obese rats. Increased levels of Tnf in MSG-obese rats were not reversed by the swim training. The expression of Ppara was higher in sedentary MSG-obese rats compared to the sedentary control. Swimming increased this adipokine expression in the exercise-trained control rats compared to the sedentary ones. mRNA levels of Pparg were higher in the sedentary MSG-rats compared to the sedentary control; however, the exercise did not influenced its expression in the groups analyzed.Conclusions In conclusion, regular physical activity was not capable to correct the expression of proinflammatory adipokines in MSG-obese rat adipocytes.

TNF superfamily protein-protein interactions: feasibility of small- molecule modulation.

The tumor necrosis factor (TNF) superfamily (TNFSF) contains about thirty structurally related receptors (TNFSFRs) and about twenty protein ligands that bind to one or more of these receptors. Almost all of these cell surface protein-protein interactions (PPIs) represent high-value therapeutic targets for inflammatory or immune modulation in autoimmune diseases, transplant recipients, or cancers, and there are several biologics including antibodies and fusion proteins targeting them that are in various phases of clinical development. Small-molecule inhibitors or activators could represent possible alternatives if the difficulties related to the targeting of protein-protein interactions by small molecules can be addressed. Compounds proving the feasibility of such approaches have been identified through different drug discovery approaches for a number of these TNFSFR-TNFSF type PPIs including CD40-CD40L, BAFFR-BAFF, TRAIL-DR5, and OX40-OX40L. Corresponding structural, signaling, and medicinal chemistry aspects are briefly reviewed here. While none of these small-molecule modulators identified so far seems promising enough to be pursued for clinical development, they provide proof-of-principle evidence that these interactions are susceptible to small-molecule modulation and can serve as starting points toward the identification of more potent and selective candidates.

The immunomodulation of a novel tumor necrosis factor (CgTNF-1) in oyster Crassostrea gigas.

Tumor necrosis factor (TNF) is one of the most important cytokines involved in many processes in both vertebrate and invertebrate. In the present study, a new tumor necrosis factor with a typical TNF domain was identified in oyster Crassostrea gigas (designated CgTNF-1). CgTNF-1 shared low sequence identity and similarity with the TNF superfamily members from other vertebrate and invertebrate. After LPS stimulation, the mRNA expression of CgTNF-1 in haemocytes increased significantly and peaked at 12h (1.39±0.12, P<0.05) post treatment, and the expression of CgTNF-1 protein in haemolymph also increased obviously during 6-12h. When the oyster haemocytes were incubated with rCgTNF-1, its apoptosis and phagocytosis rate were both effectively induced and peaked at 12h post the treatment of rCgTNF-1 with the concentration of 100ngmL(-1) (23.3±3%, P<0.01), 50ngmL(-1) (5.3±0.6%, P<0.05) and 10ngmL(-1) (6.7±1.2%, P<0.05), respectively. After the co-stimulation of LPS and rCgTNF-1, the apoptosis and phagocytosis rate of oyster haemocytes, and the activities of PO and lysozyme in the haemolymph all increased significantly, and reached the peak at 12h (apoptosis rate 26.7±1.5%, P<0.01), 12h (phagocytosis rate 8.3±0.6%, P<0.01), 6h (PO 1.11±0.01Umg prot(-1), P<0.01) and 12h (lysozyme 168.9±8.3Umg prot(-1), P<0.05), respectively, which were significantly higher than that in the LPS group. Furthermore, the anti-bacteria activity in the LPS+TNF group was significantly higher than that in the LPS group during 6-12h. All the results collectively indicated that CgTNF-1 was involved in the oyster immunity and played a crucial role in the modulation of immune response including apoptosis and phagocytosis of haemocytes, and regulation of anti-bacterial activity as well as the activation of immune relevant enzymes.

Design of vaccine adjuvants incorporating TNF superfamily ligands and TNF superfamily molecular mimics.

TNF superfamily ligands play a critical role in the regulation of adaptive immune responses, including the costimulation of dendritic cells, T cells, and B cells. This costimulation could potentially be exploited for the development of prophylactic vaccines and immunotherapy. Despite this, there have been only a limited number of reports on the use of this family of molecules as gene-based adjuvants to enhance DNA and/or viral vector vaccines. In addition, the molecule latent membrane protein 1 (LMP1), a viral mimic of the TNF superfamily receptor CD40, provides an alternative approach for the design of novel molecular adjuvants. Here, we discuss advances in the development of recombinant TNF superfamily ligands as adjuvants for HIV vaccines and as cancer immunotherapy, including the use of LMP1 and LMP1-CD40 chimeric fusion proteins to mimic constitutive CD40 signaling.