Structure of CD20 in complex with the therapeutic monoclonal antibody rituximab.

Cluster of differentiation 20 (CD20) is a B cell membrane protein that is targeted by monoclonal antibodies for the treatment of malignancies and autoimmune disorders but whose structure and function are unknown. Rituximab (RTX) has been in clinical use for two decades, but how it activates complement to kill B cells remains poorly understood. We obtained a structure of CD20 in complex with RTX, revealing CD20 as a compact double-barrel dimer bound by two RTX antigen-binding fragments (Fabs), each of which engages a composite epitope and an extensive homotypic Fab:Fab interface. Our data suggest that RTX cross-links CD20 into circular assemblies and lead to a structural model for complement recruitment. Our results further highlight the potential relevance of homotypic Fab:Fab interactions in targeting oligomeric cell-surface markers

Genetically encoded photocross-linkers determine the biological binding site of exendin-4 peptide in the N-terminal domain of the intact human glucagon-like peptide-1 receptor (GLP-1R)

The glucagon-like peptide-1 receptor (GLP-1R) is a key therapeutic target in the management of type II diabetes mellitus, with actions including regulation of insulin biosynthesis and secretion, promotion of satiety, and preservation of β-cell mass. Like most class B G protein-coupled receptors (GPCRs), there is limited knowledge linking biological activity of the GLP-1R with the molecular structure of an intact, full-length, and functional receptor·ligand complex. In this study, we have utilized genetic code expansion to site-specifically incorporate the photoactive amino acid p-azido-l-phenylalanine (azF) into N-terminal residues of a full-length functional human GLP-1R in mammalian cells. UV-mediated photolysis of azF was then carried out to induce targeted photocross-linking to determine the proximity of the azido group in the mutant receptor with the peptide exendin-4. Cross-linking data were compared directly with the crystal structure of the isolated N-terminal extracellular domain of the GLP-1R in complex with exendin(9–39), revealing both similarities as well as distinct differences in the mode of interaction. Generation of a molecular model to accommodate the photocross-linking constraints highlights the potential influence of environmental conditions on the conformation of the receptor·peptide complex, including folding dynamics of the peptide and formation of dimeric and higher order oligomeric receptor multimers. These data demonstrate that crystal structures of isolated receptor regions may not give a complete reflection of peptide/receptor interactions and should be combined with additional experimental constraints to reveal peptide/receptor interactions occurring in the dynamic, native, and full-length receptor state

Mechanism of intramembrane proteolysis investigated with purified rhomboid proteases

Intramembrane proteases have the unusual property of cleaving peptide bonds within the lipid bilayer, an environment not obviously suited to a water-requiring hydrolysis reaction. These enzymes include site-2 protease, γ-secretase/presenilin, signal peptide peptidase and the rhomboids, and they have a wide range of cellular functions. All have multiple transmembrane domains and, because of their high hydrophobicity, have been difficult to purify. We have now developed an in vitro assay to monitor rhomboid activity in the detergent solubilised state. This has allowed us to isolate for the first time a highly pure rhomboid with catalytic activity. Our results suggest that detergent-solubilised rhomboid activity mimics its activity in biological membranes in many aspects. Analysis of purified mutant proteins suggests that rhomboids use a serine protease catalytic dyad instead of the previously proposed triad. This analysis also suggests that other conserved residues participate in subsidiary functions like ligand binding and water supply. We identify a motif shared between rhomboids and the recently discovered derlins, which participate in translocation of misfolded membrane proteins

Invariant natural killer T (iNKT) cell exhaustion in sarcoidosis

Invariant natural killer T (iNKT) cells are integral components of immune responses during many chronic diseases, yet their surface phenotypes, subset distribution, and polyfunctional capacity in this environment are largely unknown. Therefore, using flow cytometry, we determined iNKT cell phenotypic and functional characteristics in subjects with chronic inflammatory disease sarcoidosis and matched controls. We found that sarcoidosis subjects displayed lower iNKT-cell frequencies, which correlated with lung fibrosis, C-reactive protein levels, and other measures of clinical disease. The CD4(-) CD8(-) (double negative, DN) iNKT-cell population was selectively lower in diseased individuals and the remaining DN iNKT cells exhibited higher frequencies of the activation markers CD69 and CD56. Functionally, both total IFN-γ(+) and the dual-functional IFN-γ(+) TNF-α(+) iNKT cells were decreased in sarcoidosis subjects and these functional defects correlated with total iNKT-cell circulating frequencies. As the loss of polyfunctionality can reflect functional exhaustion, we measured the surface antigens programmed death-1 receptor and CD57 and found that levels inversely correlated with dual-functional iNKT-cell percentages. These findings reveal that, similar to traditional T cells, iNKT cells may also undergo functional exhaustion, and that circulating iNKT-cell frequencies reflect these defects. Programmed death-1 receptor antagonists may therefore be attractive therapeutic candidates for sarcoidosis and other iNKT-cell-mediated chronic diseases

Activation of Notch signaling pathway precedes heart regeneration in zebrafish

Several vertebrates display the ability to regenerate parts of their body after amputation. During this process, differentiated cells reenter the cell cycle and proliferate to generate a mass of undifferentiated cells. Repatterning mechanisms act on these cells to eventually shape a regenerated tissue or organ that replaces the amputated one. Experiments with regenerating limbs/fins in newts and zebrafish have shown that members of the Msx family of homeodomain-containing transcription factors play key roles during blastema formation and patterning. Here we show that adult zebrafish have a remarkable capacity to regenerate the heart in a process that involves up-regulation of msxB and msxC genes. We present evidence indicating that heart regeneration involves the execution of a specific genetic program, rather than redeployment of a cardiac development program. Preceding Msx activation, there is a marked increase in the expression of notch1b and deltaC, which we show are also up-regulated during fin regeneration. These data suggest a role for the Notch pathway in the activation of the regenerative response. Taken together, our results underscore the use of zebrafish as a model for investigating the process of regeneration in particular and the biology of stem cells in general. Advances in these fields will undoubtedly aid in the implementation of strategies for regenerative medicine

Structural Analysis of Bacterial ABC Transporter Inhibition by an Antibody Fragment

SummaryBacterial ATP-binding cassette (ABC) importers play critical roles in nutrient acquisition and are potential antibacterial targets. However, structural bases for their inhibition are poorly defined. These pathways typically rely on substrate binding proteins (SBPs), which are essential for substrate recognition, delivery, and transporter function. We report the crystal structure of a Staphylococcus aureus SBP for Mn(II), termed MntC, in complex with FabC1, a potent antibody inhibitor of the MntABC pathway. This pathway is essential and highly expressed during S. aureus infection and facilitates the import of Mn(II), a critical cofactor for enzymes that detoxify reactive oxygen species (ROS). Structure-based functional studies indicate that FabC1 sterically blocks a structurally conserved surface of MntC, preventing its interaction with the MntB membrane importer and increasing wild-type S. aureus sensitivity to oxidative stress by more than 10-fold. The results define an SBP blocking mechanism as the basis for ABC importer inhibition by an engineered antibody fragment