Dual function of cysteine rich domain (CRD) 1 of TNF receptor type 1: Conformational stabilization of CRD2 and control of receptor responsiveness

The proinflammatory cytokine Tumor Necrosis Factor (TNF) exists as a homotrimer, capable of binding three receptor molecules. However, signal competent ligand/receptor complexes form large clusters, likely to be stabilized by additional molecular interactions. Both TNF receptors, TNFR1 and TNFR2, contain four cysteine rich domains (CRD) in their extracellular parts. Previous work showed that the membrane distal CRD1 carries a homophilic interaction domain. Here, we investigated the functional role of CRD1 and its two submodules, A1_CRD1 and B2_CRD1, in a TNFR1-Fas chimera model system. Removal of CRD1 abolishes TNF binding. In line with these data, molecular dynamics simulations suggest that B2_CRD1 of TNFR1 serves as a scaffold to stabilize CRD2 in a conformation necessary for high affinity ligand binding. Deletion of only the N-terminal half of CRD1 (ΔA1_CRD1) of TNFR1 marginally affects ligand binding but abrogates responsiveness towards soluble TNF and reduces effectiveness as a dominant negative inhibitor of wild type TNFR1. A TNFR1-derived molecule containing the CRD1 from TNFR2 also shows reduced responsiveness to soluble TNF. These data strongly suggest that CRD1 is not only crucially involved in multimerization of unligated receptors, but is also directly involved in formation of signal competent ligand/receptor clusters, thereby controlling receptor responsiveness.     

Progranulin is required for proper ER stress response and inhibits ER stress-mediated apoptosis through TNFR2

Progranulin (PGRN) was reported to be a stress-response factor in response to hypoxia and acidosis. Here we present evidences demonstrating that PGRN is also an endoplasmic reticulum (ER) stress responsive factor: PGRN expression was induced and its activation of Erk1/2 and Akt signaling enhanced in response to ER stress; Normal ER stress response was lost in PGRN deficient cells and PGRN deficient cells became hypersusceptible to ER stress-induced apoptosis; additionally, recombinant PGRN could rescue the defects in ER-stress responses seen in PGRN deficient cells. Mechanistic studies indicated that PGRN/TNFR2 was critical for PGRN mediated regulation of ER stress response: similar to PGRN, the expression of TNFR2, but not TNFR1, was also induced in the course of ER stress; in addition, the association between PGRN and TNFR2 was markedly enhanced following ER stress; More importantly, PGRN protection of ER stress induced apoptosis was abolished when TNFR2 signaling was blocked. In addition, the 2nd and 3rd cysteine-rich domains (CRD) in the extracellular portion of TNFR2 (CRD2CRD3), known to directly bind to PGRN, disturbed the interaction of PGRN with TNFR2, and in turn abolished PGRN-mediated activation of Erk1/2 and Akt signaling and protection against apoptosis in response to ER-stress. Collectively, PGRN plays an important role in ER stress and regulates ER stress response through interacting with TNFR2. This study provides new insight into PGRN regulation of stress response and may also present PGRN as a potential molecular target for treating stress-associated disorders.     

Progranulin: a promising therapeutic target for rheumatoid arthritis

Progranulin (PGRN) is an autocrine growth factor with multiple functions. This review provides updates about the interplays of PGRN with extracellular matrix proteins, proteolytic enzymes, inflammatory cytokines, and cell surface receptors in cartilage and arthritis, with a special focus on the interaction between PGRN and TNF receptors (TNFR) and its implications in inflammatory arthritis. The paper also highlights Atsttrin, an engineered protein composed of three PGRN fragments that prevents inflammation in several inflammatory arthritis models. Identification of PGRN as a ligand of TNFR and an antagonist of TNFα signaling, together with the discovery of Atsttrin, not only betters our understanding of the pathogenesis of arthritis, but also provides new therapeutic interventions for various TNFα-mediated pathologies and conditions, including rheumatoid arthritis.     

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.     

Expression and characterization of a variant of TACI (CRD2-shortTACIFc) in Pichia pastoris

TACI is a member of the tumor necrosis factor receptor superfamily and serves as a key regulator of B cell function. The extracellular domain of a typical TNFR contains multiple copies of CRD, which bind in the monomermonomer interfaces of a trimeric ligand. TACI binds to two ligands, APRIL and BAFF, with high affinity and contains two CRD in its extracellular regions, while BCMA and BR3, contain a single or partial CRD for binding the two ligands. However, TACI can be classified as a single CRD receptor because the amino-terminal CRD1 doesn't contribute to ligand binding. To obtain a new variant of TACI possessing higher affinities for binding, we fused a repeat sequence of CRD2 to the N-terminus of the short form of TACI. The new APRIL antagonist peptide, CRD2-shortTACI-Fc, was designed based on the modeling 3-D complex structure of TACI and APRIL. As expected, the purified recombinant CRD2-shortTACI-Fc fusion protein could bind to APRIL in vitro and demonstrated dose-dependent inhibition of APRIL-induced proliferative activity in Raji cells. We found that CRD2-shortTACI-Fc, has a higher affinity for binding to ligands than short-TACI-Fc, which contains a single CRD2.     

生长因子Progranulin的结合受体和生物学功能

生长因子颗粒素蛋白前体（progranulin, PGRN）广泛存在于动物和植物组织中.研究证明,哺乳动物的PGRN是一个多功能分子,在组织/器官发育、细胞分化、肿瘤发生发展、炎症应答以及神经退行性疾病中均具有重要的作用.PGRN发挥生物学功能需要和多种结合蛋白相互结合,例如sortilin、Toll样受体9（TLR9）、肿瘤坏死因子受体（TNFR）及分泌性淋巴细胞蛋白酶抑制因子（SLPI）等. 本文将对PGRN的结合受体和生物学功能进行综述.     

ATROSAB,a humanized antagonistic anti-tumor necrosis factor receptor one-specific antibody

Tumor necrosis factor (TNF) signals through TNFR1 and TNFR2, two membrane receptors, and TNFR1 is known to be the major pathogenic mediator of chronic and acute inflammatory diseases. Present clinical intervention is based on neutralization of the ligand TNF. Selective inhibition of TNF receptor 1 (TNFR1) provides an alternative opportunity to neutralize the pro-inflammatory activity of TNF while maintaining the advantageous immunological responses mediated by TNFR2, including immune regulation, tissue homeostasis and neuroprotection. We recently humanized a mouse anti-human TNFR1 monoclonal antibody exhibiting TNFR1-neutralizing activity. This humanized antibody has been converted into an IgG1 molecule (ATROSAB) containing a modified Fc region previously demonstrated to have greatly reduced effector functions. Purified ATROSAB produced in CHO cells showed strong binding to human and rhesus TNFR1-Fc fusion protein and mouse embryonic fibroblasts transfected with a recombinant TNFR1 fusion protein with an affinity identical to the parental mouse antibody H398. Using chimeric human/mouse TNFR1 molecules, the epitope of ATROSAB was mapped to the N-terminal region (amino acid residues 1–70) comprising the first cysteine-rich domain (CRD1) and the A1 sub-domain of CRD2. In vitro, ATROSAB inhibited typical TNF-mediated responses like apoptosis induction and activation of NFκB-dependent gene expression such as IL-6 and IL-8 production. These findings open the way to further analyze the therapeutic activity of ATROSAB in relevant disease models in non-human primates.Key words: humanized IgG, antagonistic antibody, tumor necrosis factor receptor 1, epitope mapping     

Antagonistic TNF Receptor One-Specific Antibody (ATROSAB): Receptor Binding and In Vitro Bioactivity

Background

Selective inhibition of TNFR1 signaling holds the potential to greatly reduce the pro-inflammatory activity of TNF, while leaving TNFR2 untouched, thus allowing for cell survival and tissue homeostasis. ATROSAB is a humanized antagonistic anti-TNFR1 antibody developed for the treatment of inflammatory diseases.

Methodology/Principal Findings

The epitope of ATROSAB resides in the N-terminal region of TNFR1 covering parts of CRD1 and CRD2. By site-directed mutagenesis, we identified Arg68 and His69 of TNFR1 as important residues for ATROSAB binding. ATROSAB inhibited binding of ¹²⁵I-labeled TNF to HT1080 in the subnanomolar range. Furthermore, ATROSAB inhibited release of IL-6 and IL-8 from HeLa and HT1080 cells, respectively, induced by TNF or lymphotoxin alpha (LTα). Different from an agonistic antibody (Htr-9), which binds to a region close to the ATROSAB epitope but elicits strong TNFR1 activation, ATROSAB showed a negligible induction of IL-6 and IL-8 production over a broad concentration range. We further verified that ATROSAB, comprising mutations within the Fc region known to abrogate complement fixation and antibody-mediated cellular effector functions, indeed lacks binding activity for C1q, FcγRI (CD64), FcγRIIB (CD32b), and FcγRIII (CD16) disabling ADCC and CDC.

Conlusions/Significance

The data corroborate ATROSAB’s unique function as a TNFR1-selective antagonist efficiently blocking both TNF and LTα action. In agreement with recent studies of TNFR1 complex formation and activation, we suggest a model of the underlying mechanism of TNFR1 inhibition by ATROSAB.     

Ligand-induced internalization of TNF receptor 2 mediated by a di-leucin motif is dispensable for activation of the NFκB pathway

Endocytosis is an important mechanism to regulate tumor necrosis factor (TNF) signaling. In contrast to TNF receptor 1 (TNFR1; CD120a), the relevance of receptor internalization for signaling as well as the fate and route of internalized TNF receptor 2 (TNFR2; CD120b) is poorly understood. To analyze the dynamics of TNFR2 signaling and turnover at the plasma membrane we established a human TNFR2 expressing mouse embryonic fibroblast cell line in a TNFR1^−/−/TNFR2^−/− background. TNF stimulation resulted in a decrease of constitutive TNFR2 ectodomain shedding. We hypothesized that reduced ectodomain release is a result of TNF/TNFR2 complex internalization. Indeed, we could demonstrate that TNFR2 was internalized together with its ligand and cytoplasmic binding partners. Upon endocytosis the TNFR2 signaling complex colocalized with late endosome/lysosome marker Rab7 and entered the lysosomal degradation pathway. Furthermore, we identified a di-leucin motif in the cytoplasmic part of TNFR2 suggesting clathrin-dependent internalization of TNFR2. Internalization defective TNFR2 mutants are capable to signal, i.e. activate NFκB, demonstrating that the di-leucin motif dependent internalization is dispensable for this response. We therefore propose that receptor internalization primarily serves as a negative feed-back to limit TNF responses via TNFR2.     

ATROSAB,a humanized antagonistic anti-tumor necrosis factor receptor one-specific antibody

Tumor necrosis factor (TNF) signals through two membrane receptors, TNFR1 and TNFR2, and TNFR1 is known to be the major pathogenic mediator of chronic and acute inflammatory diseases. Present clinical intervention is based on neutralization of the ligand TNF. Selective inhibition of TNF receptor 1 (TNFR1) provides an alternative opportunity to neutralize the pro-inflammatory activity of TNF while maintaining the advantageous immunological responses mediated by TNFR2, including immune regulation, tissue homeostasis and neuroprotection. We recently humanized a mouse anti-human TNFR1 monoclonal antibody exhibiting TNFR1-neutralizing activity. This humanized antibody has been converted into an IgG1 molecule (ATROSAB) containing a modified Fc region previously demonstrated to have greatly reduced effector functions. Purified ATROSAB, produced in CHO cells, showed strong binding to human and rhesus TNFR1-Fc fusion protein and mouse embryonic fibroblasts transfected with a recombinant TNFR1 fusion protein with an affinity identical to the parental mouse antibody H398. Using chimeric human/mouse TNFR1 molecules, the epitope of ATROSAB was mapped to the N-terminal region (amino acid residues 1-70) comprising the first cysteine-rich domain (CRD1) and the A1 sub-domain of CRD2. In vitro, ATROSAB inhibited typical TNF-mediated responses like apoptosis induction and activation of NFκB-dependent gene expression such as IL-6 and IL-8 production. These findings open the way to further analyze the therapeutic activity of ATROSAB in relevant disease models in non-human primates.     

Are different stoichiometries feasible for complexes between lymphotoxin-alpha and tumor necrosis factor receptor 1?

Background

Tumor necrosis factors, TNF and lymphotoxin-?? (LT), are cytokines that bind to two receptors, TNFR1 and TNFR2 (TNF-receptor 1 and 2) to trigger their signaling cascades. The exact mechanism of ligand-induced receptor activation is still unclear. It is generally assumed that three receptors bind to the homotrimeric ligand to trigger a signaling event. Recent evidence, though, has raised doubts if the ligand:receptor stoichiometry should indeed be 3:3 for ligand-induced cellular response. We used molecular dynamics simulations, elastic network models, as well as MM/PBSA to analyze this question.

Results

Applying MM/PBSA methodology to different stoichiometric complexes of human LT-(TNFR1)_n=1,2,3 the free energy of binding in these complexes has been estimated by single-trajectory and separate-trajectory methods. Simulation studies rationalized the favorable binding energy in the LT-(TNFR1)₁ complex, as evaluated from single-trajectory analysis to be an outcome of the interaction of cysteine-rich domain 4 (CRD4) and the ligand. Elastic network models (ENMs) help to associate the difference in the global fluctuation of the receptors in these complexes. Functionally relevant transformation associated with these complexes reveal the difference in the dynamics of the receptor when free and in complex with LT.

Conclusions

MM/PBSA predicts complexes with a ligand-receptor molar ratio of 3:1 and 3:2 to be energetically favorable. The high affinity associated with LT-(TNFR1)₁ is due to the interaction between the CRD4 domain with LT. The global dynamics ascertained from ENMs have highlighted the differential dynamics of the receptor in different states.     

Azadirachtin Interacts with the Tumor Necrosis Factor (TNF) Binding Domain of Its Receptors and Inhibits TNF-induced Biological Responses

The role of azadirachtin, an active component of a medicinal plant Neem (Azadirachta indica), on TNF-induced cell signaling in human cell lines was investigated. Azadirachtin blocks TNF-induced activation of nuclear factor κB (NF-κB) and also expression of NF-κB-dependent genes such as adhesion molecules and cyclooxygenase 2. Azadirachtin inhibits the inhibitory subunit of NF-κB (IκBα) phosphorylation and thereby its degradation and RelA (p65) nuclear translocation. It blocks IκBα kinase (IKK) activity ex vivo, but not in vitro. Surprisingly, azadirachtin blocks NF-κB DNA binding activity in transfected cells with TNF receptor-associated factor (TRAF)2, TNF receptor-associated death domain (TRADD), IKK, or p65, but not with TNFR, suggesting its effect is at the TNFR level. Azadirachtin blocks binding of TNF, but not IL-1, IL-4, IL-8, or TNF-related apoptosis-inducing ligand (TRAIL) with its respective receptors. Anti-TNFR antibody or TNF protects azadirachtin-mediated down-regulation of TNFRs. Further, in silico data suggest that azadirachtin strongly binds in the TNF binding site of TNFR. Overall, our data suggest that azadirachtin modulates cell surface TNFRs thereby decreasing TNF-induced biological responses. Thus, azadirachtin exerts an anti-inflammatory response by a novel pathway, which may be beneficial for anti-inflammatory therapy.     

Hepatitis C Virus Core Protein Binds to the Cytoplasmic Domain of Tumor Necrosis Factor (TNF) Receptor 1 and Enhances TNF-Induced Apoptosis

The hepatitis C virus (HCV) core protein is known to be a multifunctional protein, besides being a component of viral nucleocapsids. Previously, we have shown that the core protein binds to the cytoplasmic domain of lymphotoxin β receptor, which is a member of tumor necrosis factor receptor (TNFR) family. In this study, we demonstrated that the core protein also binds to the cytoplasmic domain of TNFR 1. The interaction was demonstrated both by glutathione S-transferase fusion protein pull-down assay in vitro and membrane flotation method in vivo. Both the in vivo and in vitro binding required amino acid residues 345 to 407 of TNFR 1, which corresponds to the “death domain” of this receptor. We have further shown that stable expression of the core protein in a mouse cell line (BC10ME) or human cell lines (HepG2 and HeLa cells) sensitized them to TNF-induced apoptosis, as determined by the TNF cytotoxicity or annexin V apoptosis assay. The presence of the core protein did not alter the level of TNFR 1 mRNA in the cells or expression of TNFR 1 on the cell surface, suggesting that the sensitization of cells to TNF by the viral core protein was not due to up-regulation of TNFR 1. Furthermore, we observed that the core protein blocked the TNF-induced activation of RelA/NF-κB in murine BC10ME cells, thus at least partially accounting for the increased sensitivity of BC10ME cells to TNF. However, NF-κB activation was not blocked in core protein-expressing HeLa or HepG2 cells, implying another mechanism of TNF sensitization by core protein. These results together suggest that the core protein can promote cell death during HCV infection via TNF signaling pathways possibly as a result of its interaction with the cytoplasmic tail of TNFR 1. Therefore, TNF may play a role in HCV pathogenesis.     

Structural basis for tumor necrosis factor blockade with the therapeutic antibody golimumab

Tumor necrosis factor α (TNFα) is a proinflammatory cytokine, and elevated levels of TNFα in serum are associated with various autoimmune diseases, including rheumatoid arthritis (RA), ankylosing spondylitis (AS), Crohn's disease (CD), psoriasis, and systemic lupus erythaematosus. TNFα performs its pleiotropic functions by binding to two structurally distinct transmembrane receptors, TNF receptor (TNFR) 1 and TNFR2. Antibody‐based therapeutic strategies that block excessive TNFα signaling have been shown to be effective in suppressing such harmful inflammatory conditions. Golimumab (Simponi®) is an FDA‐approved fully human monoclonal antibody targeting TNFα that has been widely used for the treatment of RA, AS, and CD. However, the structural basis underlying the inhibitory action of golimumab remains unclear. Here, we report the crystal structure of the Fv fragment of golimumab in complex with TNFα at a resolution of 2.73 Å. The resolved structure reveals that golimumab binds to a distinct epitope on TNFα that does not overlap with the binding residues of TNFR2. Golimumab exerts its inhibitory effect by preventing binding of TNFR1 and TNFR2 to TNFα by steric hindrance. Golimumab does not induce conformational changes in TNFα that could affect receptor binding. This mode of action is specific to golimumab among the four anti‐TNFα therapeutic antibodies currently approved for clinical use.     

Polyubiquitinated Tristetraprolin Protects from TNF-induced,Caspase-mediated Apoptosis

Binding of TNF to its receptor (TNFR1) elicits the spatiotemporal assembly of two signaling complexes that coordinate the balance between cell survival and cell death. We have shown previously that, following TNF treatment, the mRNA decay protein tristetraprolin (TTP) is Lys-63-polyubiquitinated by TNF receptor-associated factor 2 (TRAF2), suggesting a regulatory role in TNFR signaling. Here we demonstrate that TTP interacts with TNFR1 in a TRAF2-dependent manner, thereby initiating the MEKK1/MKK4-dependent activation of JNK activities. This regulatory function toward JNK activation but not NF-κB activation depends on lysine 105 of TTP, which we identified as the corresponding TRAF2 ubiquitination site. Disabling TTP polyubiquitination results in enhanced TNF-induced apoptosis in cervical cancer cells. Together, we uncover a novel aspect of TNFR1 signaling where TTP, in alliance with TRAF2, acts as a balancer of JNK-mediated cell survival versus death.     

Membrane Tumor Necrosis Factor (TNF) Induces p100 Processing via TNF Receptor-2 (TNFR2)

Tumor necrosis factor (TNF) elicits its biological activities by stimulation of two receptors, TNFR1 and TNFR2, both belonging to the TNF receptor superfamily. Whereas TNFR1-mediated signal transduction has been intensively studied and is understood in detail, especially with respect to activation of the classical NFκB pathway, cell death induction, and MAP kinase signaling, TNFR2-associated signal transduction is poorly defined. Here, we demonstrate in various tumor cell lines and primary T-cells that TNFR2, but not TNFR1, induces activation of the alternative NFκB pathway. In accord with earlier findings demonstrating that only membrane TNF, but not soluble TNF, properly activates TNFR2, we further show by use of TNFR1- and TNFR2-specific mutants of soluble TNF and membrane TNF that soluble ligand trimers fail to activate the alternative NFκB pathway. In accord with the known inhibitory role of TRAF2 in the alternative NFκB pathway, TNFR2-, but not TNFR1-specific TNF induced depletion of cytosolic TRAF2. Thus, we identified activation of the alternative NFκB pathway as a TNF signaling effect that can be specifically assigned to TNFR2 and membrane TNF.     

TNFR1 and TNFR2 regulate the extrinsic apoptotic pathway in myeloma cells by multiple mechanisms

The huge majority of myeloma cell lines express TNFR2 while a substantial subset of them failed to show TNFR1 expression. Stimulation of TNFR1 in the TNFR1-expressing subset of MM cell lines had no or only a very mild effect on cellular viability. Surprisingly, however, TNF stimulation enhanced cell death induction by CD95L and attenuated the apoptotic effect of TRAIL. The contrasting regulation of TRAIL- and CD95L-induced cell death by TNF could be traced back to the concomitant NFκB-mediated upregulation of CD95 and the antiapoptotic FLIP protein. It appeared that CD95 induction, due to its strength, overcompensated a rather moderate upregulation of FLIP so that the net effect of TNF-induced NFκB activation in the context of CD95 signaling is pro-apoptotic. TRAIL-induced cell death, however, was antagonized in response to TNF because in this context only the induction of FLIP is relevant. Stimulation of TNFR2 in myeloma cells leads to TRAF2 depletion. In line with this, we observed cell death induction in TNFR1-TNFR2-costimulated JJN3 cells. Our studies revealed that the TNF-TNF receptor system adjusts the responsiveness of the extrinsic apoptotic pathway in myeloma cells by multiple mechanisms that generate a highly context-dependent net effect on myeloma cell survival.     

BAFF/BLyS receptor 3 comprises a minimal TNF receptor-like module that encodes a highly focused ligand-binding site

BAFF/BLyS, a member of the tumor necrosis family (TNF) superfamily of ligands, is a crucial survival factor for B cells. BAFF binds three receptors, TACI, BCMA, and BR3, with signaling through BR3 being essential for promoting B cell function. Typical TNF receptor (TNFR) family members bind their cognate ligands through interactions with two cysteine-rich domains (CRDs). However, the extracellular domain (ECD) of BR3 consists of only a partial CRD, with cysteine spacing distinct from other modules described previously. Herein, we report the solution structure of the BR3 ECD. A core region of only 19 residues adopts a stable structure in solution. The BR3 fold is analogous to the first half of a canonical TNFR CRD but is stabilized by an additional noncanonical disulfide bond. BAFF-binding determinants were identified by shotgun alanine-scanning mutagenesis of the BR3 ECD expressed on phage. Several of the key BAFF-binding residues are presented from a beta-turn that we have shown previously to be sufficient for ligand binding when transferred to a structured beta-hairpin scaffold [Kayagaki, N., Yan, M., Seshasayee, D., Wang, H., Lee, W., French, D. M., Grewal, I. S., Cochran, A. G., Gordon, N. C., Yin, J., Starovasnik, M. A, and Dixit, V. M. (2002) Immunity 10, 515-524]. Outside of the turn, mutagenesis identifies additional hydrophobic contacts that enhance the BAFF-BR3 interaction. The crystal structure of the minimal hairpin peptide, bhpBR3, in complex with BAFF reveals intimate packing of the six-residue BR3 turn into a cavity on the ligand surface. Thus, BR3 binds BAFF through a highly focused interaction site, unprecedented in the TNFR family.     

Exploring CRD mobility during RAS/RAF engagement at the membrane

During the activation of mitogen-activated protein kinase (MAPK) signaling, the RAS-binding domain (RBD) and cysteine-rich domain (CRD) of RAF bind to active RAS at the plasma membrane. The orientation of RAS at the membrane may be critical for formation of the RAS-RBDCRD complex and subsequent signaling. To explore how RAS membrane orientation relates to the protein dynamics within the RAS-RBDCRD complex, we perform multiscale coarse-grained and all-atom molecular dynamics (MD) simulations of KRAS4b bound to the RBD and CRD domains of RAF-1, both in solution and anchored to a model plasma membrane. Solution MD simulations describe dynamic KRAS4b-CRD conformations, suggesting that the CRD has sufficient flexibility in this environment to substantially change its binding interface with KRAS4b. In contrast, when the ternary complex is anchored to the membrane, the mobility of the CRD relative to KRAS4b is restricted, resulting in fewer distinct KRAS4b-CRD conformations. These simulations implicate membrane orientations of the ternary complex that are consistent with NMR measurements. While a crystal structure-like conformation is observed in both solution and membrane simulations, a particular intermolecular rearrangement of the ternary complex is observed only when it is anchored to the membrane. This configuration emerges when the CRD hydrophobic loops are inserted into the membrane and helices α3–5 of KRAS4b are solvent exposed. This membrane-specific configuration is stabilized by KRAS4b-CRD contacts that are not observed in the crystal structure. These results suggest modulatory interplay between the CRD and plasma membrane that correlate with RAS/RAF complex structure and dynamics, and potentially influence subsequent steps in the activation of MAPK signaling.     

Generation and Characterization of Small Single Domain Antibodies Inhibiting Human Tumor Necrosis Factor Receptor 1

The cytokine TNF is a well known drug target for several inflammatory diseases such as Crohn disease. Despite the great success of TNF blockers, therapy could be improved because of high costs and side effects. Selective inhibition of TNF receptor (TNFR) 1 signaling holds the potential to greatly reduce the pro-inflammatory activity of TNF, thereby preserving the advantageous immunomodulatory signals mediated by TNFR2. We generated a selective human TNFR1 inhibitor based on Nanobody (Nb) technology. Two anti-human TNFR1 Nbs were linked with an anti-albumin Nb to generate Nb Alb-70-96 named “TNF Receptor-One Silencer” (TROS). TROS selectively binds and inhibits TNF/TNFR1 and lymphotoxin-α/TNFR1 signaling with good affinity and IC₅₀ values, both of which are in the nanomolar range. Surface plasmon resonance analysis reveals that TROS competes with TNF for binding to human TNFR1. In HEK293T cells, TROS strongly reduces TNF-induced gene expression, like IL8 and TNF, in a dose-dependent manner; and in ex vivo cultured colon biopsies of CD patients, TROS inhibits inflammation. Finally, in liver chimeric humanized mice, TROS antagonizes inflammation in a model of acute TNF-induced liver inflammation, reflected in reduced human IL8 expression in liver and reduced IL6 levels in serum. These results demonstrate the considerable potential of TROS and justify the evaluation of TROS in relevant disease animal models of both acute and chronic inflammation and eventually in patients.