Activated leukocyte cell adhesion molecule is a component of the endothelial junction involved in transendothelial monocyte migration

Transendothelial leukocyte migration is a major aspect of the innate immune response. It is essential in repair and regeneration of damaged tissues and is regulated by multiple cell adhesion molecules (CAMs) including members of the immunoglobulin (Ig) superfamily. Activated leukocyte cell adhesion molecule (ALCAM/CD166) is an Ig CAM expressed by activated monocytes and endothelial cells. Hitherto, the functional relevance of ALCAM expression by endothelial cells and activated monocytes remained unknown. In this report, we demonstrate soluble recombinant human ALCAM significantly inhibited the rate of transendothelial migration of monocyte cell lines. Direct involvement of ALCAM in transendothelial migration was evident from the robust inhibition of this process by ALCAM blocking antibodies. However, soluble recombinant ALCAM had no impact on monocyte migration or adhesion to endothelium. Localization of ALCAM specifically at cell-cell junctions in endothelial cells supported its role in transendothelial migration. This study is the first to localize ALCAM to endothelial cell junctions and demonstrate a functional relevance for co-expression of ALCAM by activated monocytes and endothelial cells.     

Resolution of multiple substrate binding sites in cytochrome P450 3A4: the stoichiometry of the enzyme-substrate complexes probed by FRET and Job's titration

To explore the mechanism of homotropic cooperativity in human cytochrome P450 3A4 (CYP3A4) we studied the interactions of the enzyme with 1-pyrenebutanol (1-PB), 1-pyrenemethylamine (PMA), and bromocriptine by FRET from the substrate fluorophore to the heme, and by absorbance spectroscopy. These approaches combined with an innovative setup of titration-by-dilution and continuous variation (Job's titration) experiments allowed us to probe the relationship between substrate binding and the subsequent spin transition caused by 1-PB or bromocriptine or the type-II spectral changes caused by PMA. The 1-PB-induced spin shift in CYP3A4 reveals prominent homotropic cooperativity, which is characterized by a Hill coefficient of 1.8 +/- 0.3 (S50 = 8.0 +/- 1.1 microM). In contrast, the interactions of CYP3A4 with bromocriptine or PMA reveal no cooperativity, exhibiting KD values of 0.31 +/- 0.08 microM and 7.1 +/- 2.3 microM, respectively. The binding of all three substrates monitored by FRET in titration-by-dilution experiments at an enzyme:substrate ratio of 1 reveals a simple bimolecular interaction with KD values of 0.16 +/- 0.09, 4.8 +/- 1.4, and 0.18 +/- 0.09 microM for 1-PB, PMA, and bromocriptine, respectively. Correspondingly, Job's titration experiments showed that the 1-PB-induced spin shift reflects the formation of a complex of the enzyme with two substrate molecules, while bromocriptine and PMA exhibit 1:1 binding stoichiometry. Combining the results of Job's titrations with the value of KD obtained in our FRET experiments, we demonstrate that the interactions of CYP3A4 with 1-PB obey a sequential binding mechanism, where the spin transition is triggered by the binding of 1-PB to the low-affinity site, which becomes possible only upon saturation of the high-affinity site.     

Both PA63 and PA83 are endocytosed within an anthrax protective antigen mixed heptamer: a putative mechanism to overcome a furin deficiency

Anthrax toxin consists of protective antigen (PA), and lethal (LF) and edema (EF) factors. A 83 kDa PA monomer (PA83) precursor binds to the cell receptor. Furin-like proprotein convertases (PCs) cleave PA83 to generate cell-bound 63 kDa protein (PA63). PA63 oligomerizes to form a ring-shaped heptamer that binds LF-EF and facilitates their entry into the cells. Several additional PCs, as opposed to furin alone, are capable of processing PA83. Following the incomplete processing of the available pool of PA83, the functional heptamer includes both PA83 and PA63. The available structures of the receptor-PA complex imply that the presence of either one or two molecules of PA83 will not impose structural limitations on the formation of the heptamer and the association of either the (PA83)(1)(PA63)(6) or (PA83)(2)(PA63)(5) heteroheptamer with LF-EF. Our data point to the intriguing mechanism of anthrax that appears to facilitate entry of the toxin into the cells which express limiting amounts of PCs and an incompletely processed PA83 pool.     

Evidence for a dimeric assembly of two titin/telethonin complexes induced by the telethonin C-terminus

The Z-disk region defines the lateral boundary of the sarcomere and requires a high level of mechanical strength to provide a stable framework for large filamentous muscle proteins. The level of complexity at this area is reflected by a large number of protein-protein interactions. Recently, we unraveled how the N-terminus of the longest filament component, the giant muscle protein titin, is assembled into an antiparallel (2:1) sandwich complex by the N-terminal titin-binding segment of the Z-disk ligand telethonin/T-cap [Zou, P., Pinotsis, N., Lange, S., Song, Y.H., Popov, A., Mavridis, I., Mayans, O.M., Gautel, M., Wilmanns, M., 2006. Palindromic assembly of the giant muscle protein titin in the sarcomeric Z-disk. Nature 439, 229-233]. In this contribution, we present structural data of a related complex of the titin N-terminus with full-length telethonin. The C-terminus of telethonin remains invisible, suggesting that it does not fold into a defined structure even in the presence of titin. In contrast to the structure with truncated telethonin, a dimer of two titin/telethonin complexes is formed within the crystal environment, potentially indicating the formation of higher oligomers. We further investigated the structure and dynamics of this assembly by small-angle X-ray scattering, circular dichroism, and in vivo complementation data. The data consistently indicate the involvement of the C-terminal part of telethonin into the assembly of two titin/telethonin complexes.     

An integrated morphological and molecular approach to a new species description in the Trypanosomatidae: the case of Leptomonas podlipaevi n. sp., a parasite of Boisea rubrolineata (Hemiptera: Rhopalidae)

Leptomonas podlipaevi n. sp., a new trypanosomatid species, is described herein based on light microscopic, ultrastructural, and molecular phylogenetic data. The organism is pleomorphic both in host and culture, with two predominant forms-a typical promastigote with a long flagellum and a shorter promastigote with a small or barely extending flagellum. Several spliced leader RNA repeat sequences obtained from the original cultures and the clonal lines representing two types of cells were all nearly identical. These sequences formed a tight cluster in the neighbor-joining tree well separated from other trypanosomatid species. Glyceraldehyde phosphate dehydrogenase gene sequences were determined for L. podlipaevi and 10 previously described trypanosomatid species. Molecular phylogenetic analysis has demonstrated that the new species is most closely related to Leptomonas seymouri and Leptomonas pyrrhocoris. The analysis has also highlighted the polyphyly of the genus Leptomonas.     

Arabidopsis AtNek2 Kinase is Essential and Associates with Microtubules

NIMA-related kinases (Neks) are a large family of serine/threonine kinases that have been linked to cell-cycle regulation in fungi and mammals. Large families of NIMA-related kinases are also conserved in plants. We demonstrate that AtNek2, a member of the NIMA-related kinase family in Arabidopsis, is a gene fundamental for plant survival and its down-regulation has a pleiotropic effect on leaf cell morphogenesis and plant development. Intracellular localization of YFP::AtNek2 showed that AtNek2 proteins co-distribute with the microtubular cytoskeleton. As a microtubular-associated protein AtNek2 might influence the dynamics of microtubules and consequently cell morphogenesis. This is supported by the observation that misexpression of AtNek2 in RNAi mutants leads to a distorted organization of cells.     

Ligand‐induced formation of transient dimers of mammalian 12/15‐lipoxygenase: A key to allosteric behavior of this class of enzymes?

Mammalian lipoxygenases (LOXs) have been implicated in cellular defense response and are important for physiological homeostasis. Since their discovery, LOXs have been believed to function as monomeric enzymes that exhibit allosteric properties. In aqueous solutions, the rabbit 12/15-LOX is mainly present as hydrated monomer but changes in the local physiochemical environment suggested a monomer-dimer equilibrium. Because the allosteric character of the enzyme can hardly be explained using a single ligand binding-site model, we proposed that the binding of allosteric effectors may shift the monomer-dimer equilibrium toward dimer formation. To test this hypothesis, we explored the impact of an allosteric effector [13(S)-hydroxyoctadeca-9(Z),11(E)-dienoic acid] on the structural properties of rabbit 12/15-LOX by small-angle X-ray scattering. Our data indicate that the enzyme undergoes ligand-induced dimerization in aqueous solution, and molecular dynamics simulations suggested that LOX dimers may be stable in the presence of substrate fatty acids. These data provide direct structural evidence for the existence of LOX dimers, where two noncovalently linked enzyme molecules might work in unison and, therefore, such mode of association might be related to the allosteric character of 12/15-LOX. Introduction of negatively charged residues (W181E + H585E and L183E + L192E) at the intermonomer interface disturbs the hydrophobic dimer interaction of the wild-type LOX, and this structural alteration may lead to functional distortion of mutant enzymes.     

A Flexible Multidomain Structure Drives the Function of the Urokinase-type Plasminogen Activator Receptor (uPAR)

The urokinase-type plasminogen activator receptor (uPAR) provides a rendezvous between proteolytic degradation of the extracellular matrix and integrin-mediated adhesion to vitronectin. These processes are, however, tightly linked because the high affinity binding of urokinase regulates the binding of uPAR to matrix-embedded vitronectin. Although crystal structures exist to define the corresponding static bi- and trimolecular receptor complexes, it is evident that the dynamic property of uPAR plays a decisive role in its function. In the present study, we combine small angle x-ray scattering, hydrogen-deuterium exchange, and surface plasmon resonance to develop a structural model describing the allosteric regulation of uPAR. We show that the flexibility of its N-terminal domain provides the key for understanding this allosteric mechanism. Importantly, our model has direct implications for understanding uPAR-assisted cell adhesion and migration as well as for translational research, including targeted intervention therapy and non-invasive tumor imaging in vivo.     

2′-OH of mRNA are critical for the binding of its codons at the 40S ribosomal P site but not at the mRNA entry site

The roles of 2′-OH groups in the binding of mRNA to human ribosomes were studied using site-directed cross-linking. We found that both mRNA and mDNA analogues bearing a cross-linker can modify ribosomal proteins (rps) S3e and S2e at the mRNA entry site independently on tRNA presence, but only mRNA analogues were capable of a tRNA^Phe-dependent binding to human ribosomes and cross-linking to rpS26e in the mRNA binding centre. Thus, 2′-OH groups of mRNA are unimportant for binding at the entry site but they are crucial for codon-anticodon interactions at the P site, implying the existence of mRNA-ribosome contacts that do not occur in bacteria.