TNFSF14-Derived Molecules as a Novel Treatment for Obesity and Type 2 Diabetes.

Obesity is one of the most prevalent metabolic diseases in the Western world and correlates directly with glucose intolerance and insulin resistance, often culminating in Type 2 Diabetes (T2D). Importantly, our team has recently shown that the TNF superfamily (TNFSF) member protein, TNFSF14, has been reported to protect against high fat diet induced obesity and pre-diabetes. We hypothesized that mimics of TNFSF14 may therefore be valuable as anti-diabetic agents. In this study, we use in silico approaches to identify key regions of TNFSF14 responsible for binding to the Herpes virus entry mediator and Lymphotoxin ß receptor. In vitro evaluation of a selection of optimised peptides identified six potentially therapeutic TNFSF14 peptides. We report that these peptides increased insulin and fatty acid oxidation signalling in skeletal muscle cells. We then selected one of these promising peptides to determine the efficacy to promote metabolic benefits in vivo. Importantly, the TNFSF14 peptide 7 reduced high fat diet-induced glucose intolerance, insulin resistance and hyperinsulinemia in a mouse model of obesity. In addition, we highlight that the TNFSF14 peptide 7 resulted in a marked reduction in liver steatosis and a concomitant increase in phospho-AMPK signalling. We conclude that TNFSF14-derived molecules positively regulate glucose homeostasis and lipid metabolism and may therefore open a completely novel therapeutic pathway for treating obesity and T2D.

Innate lymphotoxin receptor mediated signaling promotes HSV-1 associated neuroinflammation and viral replication.

Host anti-viral innate immunity plays important roles in the defense against HSV-1 infection. In this study, we find an unexpected role for innate LT/LIGHT signaling in promoting HSV-1 replication and virus induced inflammation in immunocompromised mice. Using a model of footpad HSV-1 infection in Rag1(-/-) mice, we observed that blocking LT/LIGHT signaling with LTßR-Ig could significantly delay disease progression and extend the survival of infected mice. LTßR-Ig treatment reduced late proinflammatory cytokine release in the serum and nervous tissue, and inhibited chemokine expression and inflammatory cells infiltration in the dorsal root ganglia (DRG). Intriguingly, LTßR-Ig treatment restricted HSV-1 replication in the DRG but not the footpad. These findings demonstrate a critical role for LT/LIGHT signaling in modulating innate inflammation and promoting HSV-1 replication in the nervous system, and suggest a new target for treatment of virus-induced adverse immune response and control of severe HSV-1 infection.

Isolation, molecular cloning, and characterization of a novel porcine lymphotoxin beta receptor gene.

The lymphotoxin beta receptor (LTßR) is a member of the tumor necrosis factor family of receptors (TNFR). It plays a role in regulating lymphoid organogenesis and homeostasis of the immune system. In the present study, the full coding region of a putative LTßR gene of Sus scrofa was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and cloned for the first time (accession Nos. JX457347 and AFU74012). In addition, analysis of the tissue expression profile was carried out via RT-PCR. The full-length coding region of porcine LTßR had 1266 nucleotides (molecular weight, 45.61 kDa; pI, 5.71) and encoded 421 amino acids. Bioinformatic prediction indicates that LTßR belongs to the TNFR superfamily and contains a TNFR domain. The sequence homology analysis revealed that the amino acid sequences of S. scrofa LTßR had 82.9, 82.4, 81.3, 80.5, 78.7, 74.6, and 73.0% identity with those of Equus caballus, Canis lupus, Ailuropoda melanoleuca, Oryctolagus cuniculus, Bos taurus, Mus musculus, and Homo sapiens, respectively. The phylogenetic tree based on the amino acid sequences of LTßR from 8 species revealed that S. scrofa was more closely related to E. caballus, C. lupus, and A. melanoleuca. RT-PCR analysis showed that the porcine LTßR gene was differentially expressed (e.g., high, moderate, low, or nonexistent) in various tissues (e.g., prostate, pituitary, brainstem, and esophagus, respectively). This may be related to differences in the regulation of LTßR in the different tissues.

Redistribution of flexibility in stabilizing antibody fragment mutants follows Le Châtelier's principle.

Le Châtelier's principle is the cornerstone of our understanding of chemical equilibria. When a system at equilibrium undergoes a change in concentration or thermodynamic state (i.e., temperature, pressure, etc.), La Châtelier's principle states that an equilibrium shift will occur to offset the perturbation and a new equilibrium is established. We demonstrate that the effects of stabilizing mutations on the rigidity ⇔ flexibility equilibrium within the native state ensemble manifest themselves through enthalpy-entropy compensation as the protein structure adjusts to restore the global balance between the two. Specifically, we characterize the effects of mutation to single chain fragments of the anti-lymphotoxin-ß receptor antibody using a computational Distance Constraint Model. Statistically significant changes in the distribution of both rigidity and flexibility within the molecular structure is typically observed, where the local perturbations often lead to distal shifts in flexibility and rigidity profiles. Nevertheless, the net gain or loss in flexibility of individual mutants can be skewed. Despite all mutants being exclusively stabilizing in this dataset, increased flexibility is slightly more common than increased rigidity. Mechanistically the redistribution of flexibility is largely controlled by changes in the H-bond network. For example, a stabilizing mutation can induce an increase in rigidity locally due to the formation of new H-bonds, and simultaneously break H-bonds elsewhere leading to increased flexibility distant from the mutation site via Le Châtelier. Increased flexibility within the VH ß4/ß5 loop is a noteworthy illustration of this long-range effect.

Biology and signal transduction pathways of the Lymphotoxin-αß/LTßR system.

This review focuses on the biological functions and signalling pathways activated by Lymphotoxin α (LTα)/Lymphotoxin ß (LTß) and their receptor LTßR. Genetic mouse models shed light on crucial roles for LT/LTßR to build and to maintain the architecture of lymphoid organs and to ensure an adapted immune response against invading pathogens. However, chronic inflammation, autoimmunity, cell death or cancer development are disorders that occur when the LT/LTßR system is twisted. Biological inhibitors, such as antagonist antibodies or decoy receptors, have been developed and used in clinical trials for diseases associated to the LT/LTßR system. Recent progress in the understanding of cellular trafficking and NF-κB signalling pathways downstream of LTα/LTß may bring new opportunities to develop therapeutics that target the pathological functions of these cytokines.

Induction of the alternative NF-κB pathway by lymphotoxin αß (LTαß) relies on internalization of LTß receptor.

Several tumor necrosis factor receptor (TNFR) family members activate both the classical and the alternative NF-κB pathways. However, how a single receptor engages these two distinct pathways is still poorly understood. Using lymphotoxin ß receptor (LTßR) as a prototype, we showed that activation of the alternative, but not the classical, NF-κB pathway relied on internalization of the receptor. Further molecular analyses revealed a specific cytosolic region of LTßR essential for its internalization, TRAF3 recruitment, and p100 processing. Interestingly, we found that dynamin-dependent, but clathrin-independent, internalization of LTßR appeared to be required for the activation of the alternative, but not the classical, NF-κB pathway. In vivo, ligand-induced internalization of LTßR in mesenteric lymph node stromal cells correlated with induction of alternative NF-κB target genes. Thus, our data shed light on LTßR cellular trafficking as a process required for specific biological functions of NF-κB.

Allosteric regulation of the ubiquitin:NIK and ubiquitin:TRAF3 E3 ligases by the lymphotoxin-beta receptor.

The lymphotoxin-beta receptor (LTbetaR) activates the NF-kappaB2 transcription factors, p100 and RelB, by regulating the NF-kappaB-inducing kinase (NIK). Constitutive proteosomal degradation of NIK limits NF-kappaB activation in unstimulated cells by the ubiquitin:NIK E3 ligase comprised of subunits TNFR-associated factors (TRAF)3, TRAF2, and cellular inhibitor of apoptosis (cIAP). However, the mechanism releasing NIK from constitutive degradation remains unclear. We found that insertion of a charge-repulsion mutation in the receptor-binding crevice of TRAF3 ablated binding of both LTbetaR and NIK suggesting a common recognition site. A homologous mutation in TRAF2 inhibited cIAP interaction and blocked NIK degradation. Furthermore, the recruitment of TRAF3 and TRAF2 to the ligated LTbetaR competitively displaced NIK from TRAF3. Ligated LTbetaR complexed with TRAF3 and TRAF2 redirected the specificity of the ubiquitin ligase reaction to polyubiquitinate TRAF3 and TRAF2, leading to their proteosomal degradation. Stimulus-dependent degradation of TRAF3 required the RING domain of TRAF2, but not of TRAF3, implicating TRAF2 as a key E3 ligase in TRAF turnover. The combined action of competitive displacement of NIK and TRAF degradation halted NIK turnover, and promoted its association with IKKalpha and signal transmission. These results indicate the LTbetaR modifies the ubiquitin:NIK E3 ligase, and also acts as an allosteric regulator of the ubiquitin:TRAF E3 ligase.