B cell homeostasis has been shown to critically depend on BAFF, the B cell activation factor from the tumor necrosis factor (TNF) family. Although BAFF is already known to bind two receptors, BCMA and TACI, we have identified a third receptor for BAFF that we have termed BAFF-R. BAFF-R binding appears to be highly specific for BAFF, suggesting a unique role for this ligand-receptor interaction. Consistent with this, the BAFF-R locus is disrupted in A/WySnJ mice, which display a B cell phenotype qualitatively similar to that of the BAFF-deficient mice. Thus, BAFF-R appears to be the principal receptor for BAFF-mediated mature B cell survival.

A proliferation-inducing ligand (APRIL) is a ligand of the tumor necrosis factor (TNF) family that stimulates tumor cell growth in vitro and in vivo. Expression of APRIL is highly upregulated in many tumors including colon and prostate carcinomas. Here we identify B cell maturation antigen (BCMA) and transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI), two predicted members of the TNF receptor family, as receptors for APRIL. APRIL binds BCMA with higher affinity than TACI. A soluble form of BCMA, which inhibits the proliferative activity of APRIL in vitro, decreases tumor cell proliferation in nude mice. Growth of HT29 colon carcinoma cells is blocked when mice are treated once per week with the soluble receptor. These results suggest an important role for APRIL in tumorigenesis and point towards a novel anticancer strategy.

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The oligomeric state of BAFF (B cell activing factor), a tumor necrosis factor (TNF) family cytokine that plays a critical role in B cell development and survival, has been the subject of recent debate. Myc-tagged BAFF starting at residue Gln136 was previously reported to crystallize as trimers at pH 4.5, whereas a histidine-tagged construct of BAFF, starting at residue Ala134, formed a virus-like cluster containing 60 monomers when crystallized at pH 9.0. The formation of the BAFF 60-mer was pH dependent, requiring pH >or= 7.0. More recently, 60-mer formation was suggested to be artificially induced by the histidine tag, and it was proposed that BAFF, like all other TNF family members, is trimeric. We report here that a construct of BAFF with no amino-terminal tag (Ala134-BAFF) can form a 60-mer in solution. Using size exclusion chromatography and static light scattering to monitor trimer to 60-mer ratios in BAFF preparations, we find that 60-mer formation is pH-dependent and requires histidine 218 within the DE loop of BAFF. Biacore measurements established that the affinity of Ala134-BAFF for the BAFF receptor BAFFR/BR3 is similar to that of myc-Gln136-BAFF, which is exclusively trimeric in solution. However, Ala134-BAFF is more efficacious than myc-Gln136-BAFF in inducing B cell proliferation in vitro. We additionally show that BAFF that is processed and secreted by 293T cells transfected with full-length BAFF, or by a histiocytic lymphoma cell line (U937) that expresses BAFF endogenously, forms a pH-dependent 60-mer in solution. Our results indicate that the formation of the 60-mer in solution by the BAFF extracellular domain is an intrinsic property of the protein, and therefore that this more active form of BAFF may be physiologically relevant.

IntroductionThe mammalian hedgehog (Hh) protein family (sonic, desert, and indian) are relatives of a Drosophila hedgehog protein that regulates segmental polarity during development and are expressed at many sites in the developing mouse embryo (1). Within the nervous system, sonic hedgehog protein (SHh) is associated with development and patterning of the central nervous system (2-5). In contrast, the morphogenic effects of desert hedgehog protein (DHh) are restricted to the peripheral nervous system and appear to focus specifically on cellular elements of the epi-and perineurial sheaths (6). Deletion of the dhh gene that codes for DHh in mice results in disruption of the fascicular structure of peripheral nerves, with development of a thin and disorganized perineurial sheath and an increase in blood-nerve barrier permeability that is associated with decreased connexin-43 expression by perineurial cells (6). A similar modification of nerve structure was associated with a homozygous missense mutation of the DHH gene in a human diagnosed with "minifascicular neuropathy" (7). These findings point to a role for Hh proteins in the development of the peripheral nervous system.Recent findings in acute peripheral-nerve injury models have revealed a novel function for hedgehog proteins in adult nerve regeneration and repair. The shh mRNA was strikingly upregulated in the peripheral nerve after crush injury (8), and exogenous SHh protein administration enhanced the speed of nerve recovery (9). The Hh signaling pathway is also upregulated soon after injury in a model of surgically induced hind limb ischemia, and exogenous SHh protein administration augments blood-flow recovery and limb salvage (10). Emerging evidence therefore suggests that signaling pathways initiated by Hh proteins are involved in the response to a range of peripheral-nerve lesions and participate in the repair and recovery process. Hedgehog proteins modulate development and patterning of the embryonic nervous system. As expression of desert hedgehog and the hedgehog receptor, patched-1, persist in the postnatal and adult peripheral nerves, the hedgehog pathway may have a role in maturation and maintenance of the peripheral nervous system in normal and disease states. We measured desert hedgehog expression in the peripheral nerve of maturing diabetic rats and found that diabetes caused a significant reduction in desert hedgehog mRNA. Treating diabetic rats with a sonic hedgehog-IgG fusion protein fully restored motor-and sensory-nerve conduction velocities and maintained the axonal caliber of large myelinated fibers. Diabetes-induced deficits in retrograde transport of nerve growth factor and sciatic-nerve levels of calcitonin gene-related product and neuropeptide Y were also ameliorated by treatment with the sonic hedgehog-IgG fusion protein, as was thermal hypoalgesia in the paw. These studies implicate disruption of normal hedgehog function in the etiology of diabetes-induced peripheral-nerve dysfunction and indicate that delivery of exogenous hedge...

The structure reveals that 5c8 neutralizes CD40L function by sterically blocking CD40 binding. The antigenic epitope is localized in a region of the surface that is likely to be structurally perturbed as a result of genetic mutations that cause hyper-IgM syndrome. The symmetric trimeric arrangement of the Fab fragments in the complex results in a geometry that facilitates the formation of large clusters of complexes on the cell surface.

BIO8898 is one of several synthetic organic molecules that have recently been reported to inhibit receptor binding and function of the constitutively trimeric tumor necrosis factor (TNF) family cytokine CD40 ligand (CD40L, a.k.a. CD154). Small molecule inhibitors of protein-protein interfaces are relatively rare, and their discovery is often very challenging, therefore to understand how BIO8898 achieves this feat we characterized its mechanism of action using biochemical assays and X-ray crystallography. BIO8898 inhibited soluble CD40L binding to CD40-Ig with a potency of IC50 ~25 μM, and inhibited CD40L-dependent cell apoptosis in a cellular assay. A co-crystal structure of BIO8898 with CD40L revealed that one inhibitor molecule binds per protein trimer. Surprisingly, the compound binds not at the surface of the protein, but by intercalating deeply between two subunits of the homotrimeric cytokine, disrupting a constitutive protein-protein interface and breaking the protein’s three-fold symmetry. The compound forms several hydrogen bonds with the protein, within an otherwise hydrophobic binding pocket. In addition to the translational splitting of the trimer, binding of BIO8898 was accompanied by additional local and longer-range conformational perturbations of the protein, both in the core and in a surface loop. Binding of BIO8898 is reversible, and the resulting complex is stable and does not lead to detectable dissociation of the protein trimer. Our results suggest that a set of core aromatic residues that are conserved across a subset of TNF family cytokines might represent a generic hot-spot for the induced-fit binding of trimer-disrupting small molecules.

IntroductionThe mammalian hedgehog (Hh) protein family (sonic, desert, and indian) are relatives of a Drosophila hedgehog protein that regulates segmental polarity during development and are expressed at many sites in the developing mouse embryo (1). Within the nervous system, sonic hedgehog protein (SHh) is associated with development and patterning of the central nervous system (2-5). In contrast, the morphogenic effects of desert hedgehog protein (DHh) are restricted to the peripheral nervous system and appear to focus specifically on cellular elements of the epi-and perineurial sheaths (6). Deletion of the dhh gene that codes for DHh in mice results in disruption of the fascicular structure of peripheral nerves, with development of a thin and disorganized perineurial sheath and an increase in blood-nerve barrier permeability that is associated with decreased connexin-43 expression by perineurial cells (6). A similar modification of nerve structure was associated with a homozygous missense mutation of the DHH gene in a human diagnosed with "minifascicular neuropathy" (7). These findings point to a role for Hh proteins in the development of the peripheral nervous system.Recent findings in acute peripheral-nerve injury models have revealed a novel function for hedgehog proteins in adult nerve regeneration and repair. The shh mRNA was strikingly upregulated in the peripheral nerve after crush injury (8), and exogenous SHh protein administration enhanced the speed of nerve recovery (9). The Hh signaling pathway is also upregulated soon after injury in a model of surgically induced hind limb ischemia, and exogenous SHh protein administration augments blood-flow recovery and limb salvage (10). Emerging evidence therefore suggests that signaling pathways initiated by Hh proteins are involved in the response to a range of peripheral-nerve lesions and participate in the repair and recovery process. Hedgehog proteins modulate development and patterning of the embryonic nervous system. As expression of desert hedgehog and the hedgehog receptor, patched-1, persist in the postnatal and adult peripheral nerves, the hedgehog pathway may have a role in maturation and maintenance of the peripheral nervous system in normal and disease states. We measured desert hedgehog expression in the peripheral nerve of maturing diabetic rats and found that diabetes caused a significant reduction in desert hedgehog mRNA. Treating diabetic rats with a sonic hedgehog-IgG fusion protein fully restored motor-and sensory-nerve conduction velocities and maintained the axonal caliber of large myelinated fibers. Diabetes-induced deficits in retrograde transport of nerve growth factor and sciatic-nerve levels of calcitonin gene-related product and neuropeptide Y were also ameliorated by treatment with the sonic hedgehog-IgG fusion protein, as was thermal hypoalgesia in the paw. These studies implicate disruption of normal hedgehog function in the etiology of diabetes-induced peripheral-nerve dysfunction and indicate that delivery of exogenous hedge...