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Control of integrin activation and signaling plays crucial roles in cell adhesion, spreading and migration. Here, we report that selective breakage of two conserved disulfide bonds located at the knees of integrin alpha(4)C589-C594 and beta(7)C494-C526 activated alpha(4)beta(7). This activated integrin had a unique structure that was different from the typical extended conformation of active integrin. In addition, these activated alpha(4)beta(7) integrins spontaneously clustered on the cell membrane and triggered integrin downstream signaling independent of ligand binding. Although these disulfide bonds were not broken during alpha(4)beta(7) activation by inside-out signaling or Mn2+, they could be specifically reduced by 0.1 mM dithiothreitol, a reducing strength that could be produced in vivo under certain conditions. Our findings reveal a novel mechanism of integrin activation under specific reducing conditions by which integrin can signal and promote cell spreading in the absence of ligand.

Many immune system receptors signal through cytoplasmic tyrosine-based motifs (ITAMs), but how receptor ligation results in ITAM phosphorylation remains unknown. Live-cell imaging studies showed a close interaction of the CD3epsilon cytoplasmic domain of the T cell receptor (TCR) with the plasma membrane through fluorescence resonance energy transfer between a C-terminal fluorescent protein and a membrane fluorophore. Electrostatic interactions between basic CD3epsilon residues and acidic phospholipids enriched in the inner leaflet of the plasma membrane were required for binding. The nuclear magnetic resonance structure of the lipid-bound state of this cytoplasmic domain revealed deep insertion of the two key tyrosines into the hydrophobic core of the lipid bilayer. Receptor ligation thus needs to result in unbinding of the CD3epsilon ITAM from the membrane to render these tyrosines accessible to Src kinases. Sequestration of key tyrosines into the lipid bilayer represents a previously unrecognized mechanism for control of receptor activation.

The role of autoantibodies in the pathogenesis of multiple sclerosis (MS) and other demyelinating diseases is controversial, in part because widely used western blotting and ELISA methods either do not permit the detection of conformation-sensitive antibodies or do not distinguish them from conformation-independent antibodies. We developed a sensitive assay based on self-assembling radiolabeled tetramers that allows discrimination of antibodies against folded or denatured myelin oligodendrocyte glycoprotein (MOG) by selective unfolding of the antigen domain. The tetramer radioimmunoassay (RIA) was more sensitive for MOG autoantibody detection than other methodologies, including monomer-based RIA, ELISA or fluorescent-activated cell sorting (FACS). Autoantibodies from individuals with acute disseminated encephalomyelitis (ADEM) selectively bound the folded MOG tetramer, whereas sera from mice with experimental autoimmune encephalomyelitis induced with MOG peptide immunoprecipitated only the unfolded tetramer. MOG-specific autoantibodies were identified in a subset of ADEM but only rarely in adult-onset MS cases, indicating that MOG is a more prominent target antigen in ADEM than MS.

The T cell receptor (TCR) alphabeta heterodimer communicates ligand binding to the cell interior via noncovalently associated CD3gammaepsilon, CD3deltaepsilon, and zetazeta dimers. While structures of extracellular components of the TCR-CD3 complex are known, the transmembrane (TM) domains that mediate assembly have eluded structural characterization. Incorporation of the zetazeta signaling module is known to require one basic TCRalpha and two zetazeta aspartic acid TM residues. We report the NMR structure of the zetazeta(TM) dimer, a left-handed coiled coil with substantial polar contacts. Mutagenesis experiments demonstrate that three polar positions are critical for zetazeta dimerization and assembly with TCR. The two aspartic acids create a single structural unit at the zetazeta interface stabilized by extensive hydrogen bonding, and there is evidence for a structural water molecule (or molecules) within close proximity. This structural unit, representing only the second transmembrane dimer interface solved to date, serves as a paradigm for the assembly of all modules involved in TCR signaling.

We have developed a single-molecule imaging technique that uses quantum dot-labeled peptide-major histocompatibility complex (pMHC) ligands to study CD4+ T cell functional sensitivity. We found that naive T cells, T cell blasts and memory T cells could all be triggered by a single pMHC to secrete tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2) cytokines with a rate of ~1,000, ~10,000 and ~10,000 molecules/min respectively and that additional pMHCs did not augment secretion, indicating a digital response pattern. We also found that a single pMHC localized to the immunological synapse induced the slow formation of a long-lasting T cell receptor (TCR) cluster, consistent with a serial engagement mechanism. These data show that scaling up CD4+ T cell cytokine responses involves increasingly efficient T cell recruitment rather than greater cytokine production per cell.

O-superfamily conotoxins include several families that have diverse pharmacological activity on Na+, K+ or Ca2+ channels. These superfamily toxins have been mainly found in fish-hunting and mollusk-hunting Conus species. Here, we reported two novel O-superfamily conotoxins, vx6a and vx6b, purified from a worm-hunting cone snail, Conus vexillum. Though their cysteine framework and signal peptides share high similarity with those of other members of O-superfamily, the mature vx6a and vx6b both have a low sequence homology with others. To test the biological function of vx6a, the toxin was chemically synthesized and then tested on the locust dorsal unpaired median (DUM) neuron system which containing various ion channels. Although no any activity on ion channels was found on the DUM neuron system, vx6a could clearly elicit a series of symptoms in mouse via intracranial injection, such as quivering, climbing, scratching, barrel rolling and paralysis of limbs at different dose.

International audience; The gamma-KTx-type scorpion toxins specific for K+ channels were found to interact with ERG channels on the turret region, while alpha-KTx3.2 Agitoxin-2 binds to the pore region of the Shaker K+ channel, and alpha-KTx5.3 BmP05 binds to the intermediate region of the small-conductance calcium-activated K-channel (SK(Ca)). In order to explore the critical residues for gamma-KTx binding, we determined the NMR structure of native gamma-KTx1.1 (CnErg1), a 42 amino acid residues scorpion toxin isolated from the venom of the Mexican scorpion Centruro?s noxius Hoffmann, and we used computational evolutionary trace (ET) analysis to predict possible structural and functional features of interacting surfaces. The 1H-NMR three-dimensional solution structure of native ergtoxin (CnErg1) was solved using a total of 452 distance constraints, 13 3J(NH-Halpha) and 10 hydrogen bonds. The structure is characterized by 2 segments of alpha-helices and a triple-stranded antiparallel beta-sheet stabilized by 4 disulfide bridges. The ET and structural analysis provided indication of the presence of two important amino acid residue clusters, one hydrophobic and the other hydrophilic, that should be involved in the surface contact between the toxin and the channel. Some features of the proposed interacting surface are discussed.

BmBKTx1 is a novel short chain toxin purified from the venom of the Asian scorpion Buthus martensi Karsch. It is composed of 31 residues and is structurally related to SK toxins. However, when tested on the cloned rat SK2 channel, it only partially inhibited rSK2 currents, even at a concentration of 1 microm. To screen for other possible targets, BmBKTx1 was then tested on isolated metathoracic dorsal unpaired median neurons of Locusta migratoria, in which a wide variety of ion channels are expressed. The results suggested that BmBKTx1 could specifically block voltage-gated Ca(2+)-activated K(+) currents (BK-type). This was confirmed by testing the BmBKTx1 effect on the alpha subunits of BK channels of the cockroach (pSlo), fruit fly (dSlo), and human (hSlo), heterologously expressed in HEK293 cells. The IC(50) for channel blocking by BmBKTx1 was 82 nm for pSlo and 194 nm for dSlo. Interestingly, BmBKTx1 hardly affected hSlo currents, even at concentrations as high as 10 microm, suggesting that the toxin might be insect specific. In contrast to most other scorpion BK blockers that also act on the Kv1.3 channel, BmBKTx1 did not affect this channel as well as other Kv channels. These results show that BmBKTx1 is a novel kind of blocker of BK-type Ca(2+)-activated K(+) channels. As the first reported toxin active on the Drosophila Slo channel dSlo, it will also greatly facilitate studying the physiological role of BK channels in this model organism.

BmBKTx1 is a 31-amino acid peptide identified from the venom of the Chinese scorpion Buthus martensi Karsch, blocking high-conductance calcium-activated potassium channels. Sequence homology analysis indicates that BmBKTx1 is a new subfamily of short-chain alpha-KTx toxins of the potassium channel, which we term alpha-KTx19. Synthetic BmBKTx1 was prepared by using solid-phase peptide synthesis. Two-dimensional NMR spectroscopy techniques were used to determine the solution structure of BmBKTx1. The results show that the BmBKTx1 forms a typical cysteine-stabilized alpha/beta scaffold adopted by most short-chain scorpion toxins. The structure of BmBKTx1 consists of a two-stranded antiparallel beta-sheet (residues 20-29) and an alpha-helix (residues 5-15). The three-dimensional structure of BmBKTx1 was also compared with those of two function-related scorpion toxins, charybdotoxin (ChTx) and BmTx1, and their structural and functional implications are discussed.

Recent genome-wide association (GWA) studies have identified two intergenic single nucleotide polymorphisms (SNPs) (rs11833579 and rs12425791) on chromosome 12p13 and within 11 kb of the NINJ2 gene that were significantly associated with stroke in Caucasians. However, the validity of the association has remained controversial. We performed genetic association analyses in three independent cohorts, including total of 3042 cases and 2973 controls. No significant association between these two SNPs and ischemic stroke was detected by meta-analysis after adjustment for cardiovascular risk factors under the additive model. Our data does not support that the common variants on 12p13 are major contributors of ischemic stroke in the Chinese Han population.