Tropomyosin is a two-chain ␣-helical coiled coil whose periodic interactions with the F-actin helix are critical for thin filament stabilization and the regulation of muscle contraction. Here we deduce the mechanical and chemical basis of these interactions from the 2.3-Å-resolution crystal structure of the middle three of tropomyosin's seven periods. Geometrically specific bends of the coiled coil, produced by clusters of core alanines, and variable bends about gaps in the core, produced by isolated alanines, occur along the molecule. The crystal packing is notable in signifying that the functionally important fifth period includes an especially favorable protein-binding site, comprising an unusual apolar patch on the surface together with surrounding charged residues. Based on these and other results, we have constructed a specific model of the thin filament, with the N-terminal halves of each period (i.e., the so-called ''␣ zones'') of tropomyosin axially aligned with subdomain 3 of each monomer in F-actin.alanine ͉ ␣-helix ͉ cardiomyopathy ͉ coiled coil ͉ packing

The 300 kDa cation-independent mannose 6-phosphate receptor (CI-MPR) mediates the intracellular transport of newly synthesized lysosomal enzymes containing mannose 6-phosphate on their N-linked oligosaccharides. In addition to its role in lysosome biogenesis, the CI-MPR interacts with a number of different extracellular ligands at the cell surface, including latent transforming growth factor-b, insulin-like growth factor-II, plasminogen, and urokinase-type plasminogen activator receptor (uPAR), to regulate cell growth and motility. We have solved the crystal structure of the N-terminal 432 residues of the CI-MPR at 1.8 Å resolution, which encompass three out of the 15 repetitive domains of its extracytoplasmic region. The three domains, which exhibit similar topology to each other and to the 46 kDa cation-dependent mannose 6-phosphate receptor, assemble into a compact structure with the uPAR/plasminogen and the carbohydrate-binding sites situated on opposite faces of the molecule. Knowledge of the arrangement of these three domains has allowed us to propose a model of the entire extracytoplasmic region of the CI-MPR that provides a context with which to envision the numerous binding interactions carried out by this multi-faceted receptor.

The insulin-like growth factor II/mannose 6-phosphate receptor is a multifunctional receptor that binds to a diverse array of mannose 6-phosphate (Man-6-P) modified proteins as well as nonglycosylated ligands. Previous studies have mapped its two Man-6-P binding sites to a minimum of three domains, 1-3 and 7-9, within its 15-domain extracytoplasmic region. Since the primary amino acid determinants of carbohydrate recognition by the insulin-like growth factor II/mannose 6-phosphate receptor are predicted by sequence alignment to the cation-dependent mannose 6-phosphate receptor to reside within domains 3 and 9, constructs encoding either domain 3 alone or domain 9 alone were expressed in a Pichia pastoris expression system and tested for their ability to bind several carbohydrate ligands, including Man-6-P, pentamannosyl phosphate, the lysosomal enzyme, ␤-glucuronidase, and the carbohydrate modifications (mannose 6-sulfate and Man-6-P methyl ester) found on Dictyostelium discoideum lysosomal enzymes. Although both constructs were functional in ligand binding and dissociation, these studies demonstrate the ability of domain 9 alone to fold into a high affinity (K d ‫؍‬ 0.3 ؎ 0.1 nM) carbohydrate-recognition domain whereas the domain 3 alone construct is capable of only low affinity binding (K d ϳ 500 nM) toward ␤-glucuronidase, suggesting that residues in adjacent domains (domains 1 and/or 2) are important, either directly or indirectly, for optimal binding by domain 3.

Antibody responses to T cell-independent type 2 (TI-2) antigens (Ag), such as bacterial capsular polysaccharides, are critical for host defense. In mice, B-1b cells expressing a CD11b+FSChiCD21lo/-CD19hi phenotype play a key role in producing antibodies against T cell-independent type 2 (TI-2) antigens (Ag). In primates, a distinct IgM+CD27+ “memory” B cell population is thought to generate TI-2 antibody responses and evidence for a B-1b-like cell population participating in these responses is lacking. Herein, we demonstrate that nonhuman primates (NHP; African green monkeys and cynomolgus macaques) harbor serosal B cells expressing a CD11b+FSChiCD21lo/-CD80+/-CD19hi phenotype, constitutively active Stat3, and increased reactivity with phosphorylcholine, similar to murine peritoneal B-1a and B-1b cell populations. Similar to what is observed for murine B-1b cells, NHP CD11b+FSChiCD21lo/-CD19hi B cells dominate the Ag-specific B cell response and antibody production against the TI-2 Ag, TNP-Ficoll. Although Ag-specific IgM+ B cells expressing CD27 were not detected prior to immunization, Ag-specific CD11b+CD19hi B cells expressed and maintained an IgM+IgDloCD27+CD80+ phenotype following immunization. Thus, the murine and NHP B cell populations responding to TNP-Ficoll are highly similar, with the main exception being that Ag-specific NHP B-1-like cells express CD27 following TI-2 Ag encounter. Therefore, murine B-1b and primate IgM+CD27+ “memory” B cell subsets proposed to produce TI-2 antibody responses may be highly related if not identical. Overall, these data not only support that B-1-like cells are present in NHP, but provide evidence that these cells perform the same functions attributed to murine B-1b cells.

Despite the emergence of the PD-1:PD-1 ligand (PD-L) regulatory axis as a promising target for treating multiple human diseases, remarkably little is known about how this pathway regulates responses to extracellular bacterial infections. We found that PD-1−/− mice, as well as wild type mice treated with a PD-1 blocking antibody, exhibited significantly increased survival against lethal Streptococcus pneumoniae infection following either priming with low-dose pneumococcal respiratory infection or S. pneumoniae-capsular polysaccharide immunization. Enhanced survival in mice with disrupted PD-1:PD-L interactions was explained by significantly increased proliferation, isotype switching, and IgG production by pneumococcal capsule-specific B cells. Both PD-1 ligands, B7-H1 and B7-DC, contributed to PD-1-mediated suppression of protective capsule-specific IgG. Importantly, PD-1 was induced on capsule-specific B cells and suppressed IgG production and protection against pneumococcal infection in a B cell-intrinsic manner. These results provide the first demonstration of a physiologic role for B cell-intrinsic PD-1 expression in vivo. In summary, our study reveals that B cell-expressed PD-1 plays a central role in regulating protection against S. pneumoniae, and thereby represents a promising target for bolstering immunity to encapsulated bacteria.

The efficacy of different vaccines in protecting elderly individuals against Streptococcus pneumoniae infections is not clear. In the current study, aged mice (22-25 months old) exhibited significantly increased susceptibility to respiratory infection with serotype 3 S. pneumoniae relative to younger adult mice, regardless of whether mice were naive or immunized with native pneumococcal polysaccharide (PPS; Pneumovax23) or protein-PPS conjugate (Prevnar-13) vaccines. Nonetheless, Pneumovax-immunized aged mice developed limited bacteremia following respiratory challenge and exhibited significantly increased survival following systemic challenge relative to Prevnar-immune aged mice and young mice that had received either vaccine. This was explained by >10-fold increases in PPS-specific immunoglobulin G (IgG) levels in Pneumovax-immunized aged mice relative to other groups. Remarkably, PPS3-specific B-cell expansion, IgG switching, plasmablast differentiation, and spleen and bone marrow antibody-secreting cell frequencies were 10-fold higher in aged mice following Pneumovax immunization relative to young mice, due to significantly increased B-1b cell participation. In summary, this study highlights (1) the need to devise strategies to enhance respiratory immunity in aged populations, (2) the diverse responses young and aged populations generate to Pneumovax vs Prevnar vaccines, and (3) the potential value of exploiting B-1b cell responses in aged individuals for increased vaccine efficacy.

B-1 cells produce natural antibodies which provide an integral first line of defense against pathogens while also performing important homeostatic housekeeping functions. In this study, we demonstrate programmed cell death 1 ligand 2 (PD-L2) regulates the production of natural antibodies against phosphorylcholine (PC). Naïve PD-L2-deficient (PD-L2−/−) mice produced significantly more PC-reactive IgM and IgA. This afforded PD-L2−/− mice with selectively enhanced protection against PC-expressing non-typeable Haemophilus influenzae (NTHi), but not PC-negative NTHi, relative to wild type mice. PD-L2−/− mice had significantly increased PC-specific CD138+ splenic plasmablasts bearing a B-1a phenotype, and produced PC-reactive Abs largely of the T15 idiotype. Importantly, PC-reactive B-1 cells expressed PD-L2 and irradiated chimeras demonstrated B cell-intrinsic PD-L2 expression regulated PC-specific Ab production. In addition to increased PC-specific IgM, naïve PD-L2−/− mice and irradiated chimeras reconstituted with PD-L2−/− B cells had significantly higher levels of IL-5 – a potent stimulator of B-1 cell Ab production. PDL2 mAb blockade of wild type B-1 cells in culture significantly increased CD138 and Blimp1 expression and PC-specific IgM, but did not affect proliferation. PDL2 mAb blockade significantly increased IL-5+ T cells in culture. Both IL-5 neutralization and STAT5 inhibition blunted the effects of PDL2 mAb blockade on B-1 cells. Thus, B-1 cell-intrinsic PD-L2 expression inhibits IL-5 production by T cells and thereby limits natural Ab production by B-1 cells. These findings have broad implications for the development of therapeutic strategies aimed at altering natural Ab levels critical for protection against infectious disease, autoimmunity, allergy, cancer, and atherosclerosis.

ERK-dependent signaling is key to many pathways through which extracellular signals are transduced into cell-fate decisions. One conundrum is the way in which disparate signals induce specific responses through a common, ERK-dependent kinase cascade. While studies have revealed intricate ways of controlling ERK signaling through spatiotemporal localization and phosphorylation dynamics, additional modes of ERK regulation undoubtedly remain to be discovered. We hypothesized that fine-tuning of ERK signaling could occur by cysteine oxidation. We report that ERK is actively and directly oxidized by signal-generated H2O2 during proliferative signaling, and that ERK oxidation occurs downstream of a variety of receptor classes tested in four cell lines. Furthermore, within the tested cell lines and proliferative signals, we observed that both activation loop-phosphorylated and non-phosphorylated ERK undergo sulfenylation in cells and that dynamics of ERK sulfenylation is dependent on the cell growth conditions prior to stimulation. We also tested the effect of endogenous ERK oxidation on kinase activity and report that phosphotransfer reactions are reversibly inhibited by oxidation by as much as 80 to 90%, underscoring the importance of considering this additional modification when assessing ERK activation in response to extracellular signals.