N-glycosylation of mouse TRAIL-R and human TRAIL-R1 enhances TRAIL-induced death.

APO2L/TRAIL (TNF-related apoptosis-inducing ligand) induces death of tumor cells through two agonist receptors, TRAIL-R1 and TRAIL-R2. We demonstrate here that N-linked glycosylation (N-glyc) plays also an important regulatory role for TRAIL-R1-mediated and mouse TRAIL receptor (mTRAIL-R)-mediated apoptosis, but not for TRAIL-R2, which is devoid of N-glycans. Cells expressing N-glyc-defective mutants of TRAIL-R1 and mouse TRAIL-R were less sensitive to TRAIL than their wild-type counterparts. Defective apoptotic signaling by N-glyc-deficient TRAIL receptors was associated with lower TRAIL receptor aggregation and reduced DISC formation, but not with reduced TRAIL-binding affinity. Our results also indicate that TRAIL receptor N-glyc impacts immune evasion strategies. The cytomegalovirus (CMV) UL141 protein, which restricts cell-surface expression of human TRAIL death receptors, binds with significant higher affinity TRAIL-R1 lacking N-glyc, suggesting that this sugar modification may have evolved as a counterstrategy to prevent receptor inhibition by UL141. Altogether our findings demonstrate that N-glyc of TRAIL-R1 promotes TRAIL signaling and restricts virus-mediated inhibition

Molecular basis of Lys11-polyubiquitin specificity in the deubiquitinase Cezanne

The post-translational modification of proteins with polyubiquitin regulates virtually all aspects of cell biology. Eight distinct chain linkage types in polyubiquitin co-exist and are independently regulated in cells. This ‘ubiquitin code’ determines the fate of the modified protein1. Deubiquitinating enzymes of the Ovarian Tumour (OTU) family regulate cellular signalling by targeting distinct linkage types within polyubiquitin2, and understanding their mechanisms of linkage specificity gives fundamental insights into the ubiquitin system. We here reveal how the deubiquitinase Cezanne/OTUD7B specifically targets Lys11-linked polyubiquitin. Crystal structures of Cezanne alone and in complex with mono- and Lys11-linked diubiquitin, in combination with hydrogen-deuterium exchange mass spectrometry, enable reconstruction of the enzymatic cycle in exquisite detail. An intricate mechanism of ubiquitin-assisted conformational changes activate the enzyme, and while all chain types interact with the enzymatic S1 site, only Lys11-linked chains can bind productively across the active site and stimulate catalytic turnover. Our work highlights the fascinating plasticity of deubiquitinases, and indicates that new conformational states can occur when a true substrate, such as diubiquitin, is bound at the active site

TNFAIP3 Maintains Intestinal Barrier Function and Supports Epithelial Cell Tight Junctions

Tight junctions between intestinal epithelial cells mediate the permeability of the intestinal barrier, and loss of intestinal barrier function mediated by TNF signaling is associated with the inflammatory pathophysiology observed in Crohn's disease and celiac disease. Thus, factors that modulate intestinal epithelial cell response to TNF may be critical for the maintenance of barrier function. TNF alpha-induced protein 3 (TNFAIP3) is a cytosolic protein that acts in a negative feedback loop to regulate cell signaling induced by Toll-like receptor ligands and TNF, suggesting that TNFAIP3 may play a role in regulating the intestinal barrier. To investigate the specific role of TNFAIP3 in intestinal barrier function we assessed barrier permeability in TNFAIP3−/− mice and LPS-treated villin-TNFAIP3 transgenic mice. TNFAIP3−/− mice had greater intestinal permeability compared to wild-type littermates, while villin-TNFAIP3 transgenic mice were protected from increases in permeability seen within LPS-treated wild-type littermates, indicating that barrier permeability is controlled by TNFAIP3. In cultured human intestinal epithelial cell lines, TNFAIP3 expression regulated both TNF-induced and myosin light chain kinase-regulated tight junction dynamics but did not affect myosin light chain kinase activity. Immunohistochemistry of mouse intestine revealed that TNFAIP3 expression inhibits LPS-induced loss of the tight junction protein occludin from the apical border of the intestinal epithelium. We also found that TNFAIP3 deubiquitinates polyubiquitinated occludin. These in vivo and in vitro studies support the role of TNFAIP3 in promoting intestinal epithelial barrier integrity and demonstrate its novel ability to maintain intestinal homeostasis through tight junction protein regulation

Interactions between Hedgehog proteins and their binding partners come into view

Hedgehog (Hh) proteins are secreted signaling molecules that mediate essential tissue-patterning events during embryonic development and function in tissue homeostasis and regeneration throughout life. Hh signaling is regulated by multiple mechanisms, including covalent lipid modification of the Hh protein and interactions with multiple protein and glycan partners. Unraveling the nature and effects of these interactions has proven challenging, but recent structural and biophysical studies of Hh proteins and active fragments of heparin, Ihog, Cdo, Boc, Hedgehog-interacting protein (Hhip), Patched (Ptc), and the monoclonal antibody 5E1 have added a new level of molecular detail to our understanding of how Hh signal response and distribution are regulated within tissues. We review these results and discuss their implications for understanding Hh signaling in normal and disease states

Structural basis for SHOC2 modulation of RAS signalling.

The RAS-RAF pathway is one of the most commonly dysregulated in human cancers1-3. Despite decades of study, understanding of the molecular mechanisms underlying dimerization and activation4 of the kinase RAF remains limited. Recent structures of inactive RAF monomer5 and active RAF dimer5-8 bound to 14-3-39,10 have revealed the mechanisms by which 14-3-3 stabilizes both RAF conformations via specific phosphoserine residues. Prior to RAF dimerization, the protein phosphatase 1 catalytic subunit (PP1C) must dephosphorylate the N-terminal phosphoserine (NTpS) of RAF11 to relieve inhibition by 14-3-3, although PP1C in isolation lacks intrinsic substrate selectivity. SHOC2 is as an essential scaffolding protein that engages both PP1C and RAS to dephosphorylate RAF NTpS11-13, but the structure of SHOC2 and the architecture of the presumptive SHOC2-PP1C-RAS complex remain unknown. Here we present a cryo-electron microscopy structure of the SHOC2-PP1C-MRAS complex to an overall resolution of 3 Å, revealing a tripartite molecular architecture in which a crescent-shaped SHOC2 acts as a cradle and brings together PP1C and MRAS. Our work demonstrates the GTP dependence of multiple RAS isoforms for complex formation, delineates the RAS-isoform preference for complex assembly, and uncovers how the SHOC2 scaffold and RAS collectively drive specificity of PP1C for RAF NTpS. Our data indicate that disease-relevant mutations affect complex assembly, reveal the simultaneous requirement of two RAS molecules for RAF activation, and establish rational avenues for discovery of new classes of inhibitors to target this pathway

IL-17s adopt a cystine knot fold: structure and activity of a novel cytokine, IL-17F, and implications for receptor binding

The proinflammatory cytokine interleukin 17 (IL-17) is the founding member of a family of secreted proteins that elicit potent cellular responses. We report a novel human IL-17 homolog, IL-17F, and show that it is expressed by activated T cells, can stimulate production of other cytokines such as IL-6, IL-8 and granulocyte colony-stimulating factor, and can regulate cartilage matrix turnover. Unexpectedly, the crystal structure of IL-17F reveals that IL-17 family members adopt a monomer fold typical of cystine knot growth factors, despite lacking the disulfide responsible for defining the canonical ‘knot’ structure. IL-17F dimerizes in a parallel manner like neurotrophins, and features an unusually large cavity on its surface. Remarkably, this cavity is located in precisely the same position where nerve growth factor binds its high affinity receptor, TrkA, suggesting further parallels between IL-17s and neurotrophins with respect to receptor recognition

BAFF binds to the tumor necrosis factor receptor-like molecule B cell maturation antigen and is important for maintaining the peripheral B cell population.

The tumor necrosis factor (TNF) family member B cell activating factor (BAFF) binds B cells and enhances B cell receptor-triggered proliferation. We find that B cell maturation antigen (BCMA), a predicted member of the TNF receptor family expressed primarily in mature B cells, is a receptor for BAFF. Although BCMA was previously localized to the Golgi apparatus, BCMA was found to be expressed on the surface of transfected cells and tonsillar B cells. A soluble form of BCMA, which inhibited the binding of BAFF to a B cell line, induced a dramatic decrease in the number of peripheral B cells when administered in vivo. Moreover, culturing splenic cells in the presence of BAFF increased survival of a percentage of the B cells. These results are consistent with a role for BAFF in maintaining homeostasis of the B cell population