Soluble proteins induce strong CD8+ T cell and antibody responses through electrostatic association with simple cationic or anionic lipopeptides that target TLR2

The low immunogenicity exhibited by most soluble proteins is generally due to the absence of molecular signatures that are recognized by the immune system as dangerous. In this study, we show that electrostatic binding of synthetic branched cationic or anionic lipopeptides that contain the TLR-2 agonist Pam(2)Cys markedly enhance a protein's immunogenicity. Binding of a charged lipopeptide to oppositely charged protein Ags resulted in the formation of stable complexes and occurs at physiologic pH and salt concentrations. The induction of cell-mediated responses is dependent on the electrostatic binding of lipopeptide to the protein, with no CD8(+) T cells being elicited when protein and lipopeptide possessed the same electrical charge. The CD8(+) T cells elicited after vaccination with lipopeptide-protein Ag complexes produced proinflammatory cytokines, exhibited in vivo lytic activity, and protected mice from challenge with an infectious chimeric influenza virus containing a single OVA epitope as part of the influenza neuraminidase protein. Induction of a CD8(+) T cell response correlated with the ability of lipopeptide to facilitate Ag uptake by DCs followed by trafficking of Ag-bearing cells into draining lymph nodes. Oppositely charged but not similarly charged lipopeptides were more effective in DC uptake and trafficking. Very high protein-specific Ab titers were also achieved by vaccination with complexes composed of oppositely charged lipopeptide and protein, whereas vaccination with similarly charged constituents resulted in significant but lower Ab titers. Regardless of whether similarly or oppositely charged lipopeptides were used in the induction of Ab, vaccination generated dominant IgG1 isotype Abs rather than IgG2a.     

Simultaneous formation of right- and left-handed anti-parallel coiled-coil interfaces by a coil2 fragment of human lamin A

The elementary building block of all intermediate filaments (IFs) is a dimer featuring a central α-helical rod domain flanked by the N- and C-terminal end domains. In nuclear IF proteins (lamins), the rod domain consists of two coiled-coil segments, coil1 and coil2, that are connected by a short non-helical linker. Coil1 and the C-terminal part of coil2 contain the two highly conserved IF consensus motifs involved in the longitudinal assembly of dimers. The previously solved crystal structure of a lamin A fragment (residues 305-387) corresponding to the second half of coil2 has yielded a parallel left-handed coiled coil. Here, we present the crystal structure and solution properties of another human lamin A fragment (residues 328-398), which is largely overlapping with fragment 305-387 but harbors a short segment of the tail domain. Unexpectedly, no parallel coiled coil forms within the crystal. Instead, the α-helices are arranged such that two anti-parallel coiled-coil interfaces are formed. The most significant interface has a right-handed geometry, which is accounted for by a characteristic 15-residue repeat pattern that overlays with the canonical heptad repeat pattern. The second interface is a left-handed anti-parallel coiled coil based on the predicted heptad repeat pattern. In solution, the fragment reveals only a weak dimerization propensity. We speculate that the C-terminus of coil2 might unzip, thereby allowing for a right-handed coiled-coil interface to form between two laterally aligned dimers. Such an interface might co-exist with a heterotetrameric left-handed coiled-coil assembly, which is expected to be responsible for the longitudinal A_CN contact.     

Nuclear SIRT1 activity, but not protein content, regulates mitochondrial biogenesis in rat and human skeletal muscle

The domestic cat (Felis catus), a carnivore, naturally eats a very low carbohydrate diet. In contrast, the dog (Canis familiaris), a carno-omnivore, has a varied diet. This study was performed to determine the expression of the intestinal brush border membrane sodium/glucose cotransporter, SGLT1, sweet receptor, T1R2/T1R3, and disaccharidases in these species adapted to contrasting diets. The expression (this includes function) of SGLT1, sucrase, maltase and lactase were determined using purified brush border membrane vesicles and by quantitative immunohistochemistry of fixed tissues. The pattern of expression of subunits of the sweet receptor T1R2 and T1R3 was assessed using fluorescent immunohistochemistry. In proximal, middle, and distal small intestine, SGLT1 function in dogs was 1.9- to 2.3-fold higher than in cats (P = 0.037, P = 0.0011, P = 0.027, respectively), and SGLT1 protein abundance followed an identical pattern. Both cats and dogs express T1R3 in a subset of intestinal epithelial cells, and dogs, but not cats, express T1R2. In proximal and middle regions, there were 3.1- and 1.6-fold higher lactase (P = 0.006 and P = 0.019), 4.4- and 2.9-fold higher sucrase (both P < 0.0001), and 4.6- and 3.1-fold higher maltase activity (P = 0.0026 and P = 0.0005), respectively, in the intestine of dogs compared with cats. Dogs have a potential higher capacity to digest and absorb carbohydrates than cats. Cats may suffer from carbohydrate malabsorption following ingestion of high-carbohydrate meals. However, dogs have a digestive ability to cope with diets containing significant levels of carbohydrate.     

Deficiency of sorting nexin 27 (SNX27) leads to growth retardation and elevated levels of N-methyl-D-aspartate receptor 2C (NR2C)

Phox (PX) domain-containing sorting nexins (SNXs) are emerging as important regulators of endocytic trafficking. Sorting nexin 27 (SNX27) is unique, as it contains a PDZ (Psd-95/Dlg/ZO1) domain. We show here that SNX27 is primarily targeted to the early endosome by interaction of its PX domain with PtdIns(3)P. Although targeted ablation of the SNX27 gene in mice did not significantly affect growth and survival during embryonic development, SNX27 plays an essential role in postnatal growth and survival. N-Methyl-d-aspartate (NMDA) receptor 2C (NR2C) was identified as a novel SNX27-interacting protein, and this interaction is mediated by the PDZ domain of SNX27 and the C-terminal PDZ-binding motif of NR2C. Increased NR2C expression levels, together with impaired NR2C endocytosis in SNX27(-/-) neurons, indicate that SNX27 may function to regulate endocytosis and/or endosomal sorting of NR2C. This is consistent with a role of SNX27 as a general regulator for sorting of membrane proteins containing a PDZ-binding motif, and its absence may alter the trafficking of these proteins, leading to growth and survival defects.     

An Examination of Resveratrol's Mechanisms of Action in Human Tissue: Impact of a Single Dose In Vivo and Dose Responses in Skeletal Muscle Ex Vivo

The current study tested the hypothesis that a single, moderate dose of RSV would activate the AMPK/SIRT1 axis in human skeletal muscle and adipose tissue. Additionally, the effects of RSV on mitochondrial respiration in PmFBs were examined. Eight sedentary men (23.8±2.4 yrs; BMI: 32.7±7.1) reported to the lab on two occasions where they were provided a meal supplemented with 300 mg of RSV or a placebo. Blood samples, and a muscle biopsy were obtained in the fasted state and again, with the addition of an adipose tissue biopsy, two hours post-prandial. The effect of RSV on mitochondrial respiration was examined in PmFBs taken from muscle biopsies from an additional eight men (23.4±5.4 yrs; BMI: 24.4±2.8). No effect of RSV was observed on nuclear SIRT1 activity, acetylation of p53, or phosphorylation of AMPK, ACC or PKA in either skeletal muscle or adipose tissue. A decrease in post absorptive insulin levels was accompanied by elevated skeletal muscle phosphorylation of p38 MAPK, but no change in either skeletal muscle or adipose tissue insulin signalling. Mitochondrial respiration in PmFBs was rapidly inhibited by RSV at 100–300 uM depending on the substrate examined. These results question the efficacy of a single dose of RSV at altering skeletal muscle and adipose tissue AMPK/SIRT1 activity in humans and suggest that RSV mechanisms of action in humans may be associated with altered cellular energetics resulting from impaired mitochondrial ATP production.     

Dothistroma pini, a Forest Pathogen, Contains Homologs of Aflatoxin Biosynthetic Pathway Genes

Homologs of aflatoxin biosynthetic genes have been identified in the pine needle pathogen Dothistroma pini. D. pini produces dothistromin, a difuranoanthraquinone toxin with structural similarity to the aflatoxin precursor versicolorin B. Previous studies with purified dothistromin suggest a possible role for this toxin in pathogenicity. By using an aflatoxin gene as a hybridization probe, a genomic D. pini clone was identified that contained four dot genes with similarity to genes in aflatoxin and sterigmatocystin gene clusters with predicted activities of a ketoreductase (dotA), oxidase (dotB), major facilitator superfamily transporter (dotC), and thioesterase (dotD). A D. pini dotA mutant was made by targeted gene replacement and shown to be severely impaired in dothistromin production, confirming that dotA is involved in dothistromin biosynthesis. Accumulation of versicolorin A (a precursor of aflatoxin) by the dotA mutant confirms that the dotA gene product is involved in an aflatoxin-like biosynthetic pathway. Since toxin genes have been found to be clustered in fungi in every case analyzed so far, it is speculated that the four dot genes may comprise part of a dothistromin biosynthetic gene cluster. A fifth gene, ddhA, is not a homolog of aflatoxin genes and could be at one end of the dothistromin cluster. These genes will allow comparative biochemical and genetic studies of the aflatoxin and dothistromin biosynthetic pathways and may also lead to new ways to control Dothistroma needle blight.     

Mast cell alpha-chymase reduces IgE recognition of birch pollen profilin by cleaving antibody-binding epitopes.

In sensitized individuals birch pollen induces an allergic response characterized by IgE-dependent mast cell degranulation of mediators, such as alpha-chymase and other serine proteases. In birch and other plant pollens, a major allergen is profilin. In mammals, profilin homologues are found in an intracellular form bound to cytoskeletal or cytosolic proteins or in a secreted form that may initiate signal transduction. IgE specific to birch profilin also binds human profilin I. This cross-reactivity between airborne and endogenous proteins may help to sustain allergy symptoms. The current work demonstrates that cultured mast cells constitutively secrete profilin I, which is susceptible to degranulation-dependent proteolysis. Coincubation of chymase-rich BR mastocytoma cells with Ala-Ala-Pro-Phe-chloromethylketone (a chymase inhibitor) blocks profilin cleavage, which does not occur in degranulated HMC-1 mast cells, which are rich in tryptase, but chymase deficient. These data implicate chymase as the serine protease cleaving secreted mast cell profilin. Sequencing of chymase-cleaved profilins reveals hydrolysis at Tyr(6)-Val(7) and Trp(35)-Ala(36) in birch profilin and at Trp(32)-Ala(33) in human profilin, with all sites lying within IgE-reactive epitopes. IgE immunoblotting studies with sera from birch pollen-allergic individuals demonstrate that cleavage by chymase attenuates binding of birch profilin to IgE. Thus, destruction of IgE-binding epitopes by exocytosed chymase may limit further mast cell activation by this class of common plant allergens, thereby limiting the allergic responses in sensitized individuals.     

The impact of pollen consumption on honey bee (Apis mellifera) digestive physiology and carbohydrate metabolism

Carbohydrate‐active enzymes play an important role in the honey bee (Apis mellifera) due to its dietary specialization on plant‐based nutrition. Secretory glycoside hydrolases (GHs) produced in worker head glands aid in the processing of floral nectar into honey and are expressed in accordance with age‐based division of labor. Pollen utilization by the honey bee has been investigated in considerable detail, but little is known about the metabolic fate of indigestible carbohydrates and glycosides in pollen biomass. Here, we demonstrate that pollen consumption stimulates the hydrolysis of sugars that are toxic to the bee (xylose, arabinose, mannose). GHs produced in the head accumulate in the midgut and persist in the hindgut that harbors a core microbial community composed of approximately 10⁸ bacterial cells. Pollen consumption significantly impacted total and specific bacterial abundance in the digestive tract. Bacterial isolates representing major fermentative gut phylotypes exhibited primarily membrane‐bound GH activities that may function in tandem with soluble host enzymes retained in the hindgut. Additionally, we found that plant‐originating β‐galactosidase activity in pollen may be sufficient, in some cases, for probable physiological activity in the gut. These findings emphasize the potential relative contributions of host, bacteria, and pollen enzyme activities to carbohydrate breakdown, which may be tied to gut microbiome dynamics and associated host nutrition.