Direct interaction of focal adhesion kinase with p190RhoGEF

Focal adhesion kinase (FAK) is a protein-tyrosine kinase that associates with multiple cell surface receptors and signaling proteins through which it can modulate the activity of several intracellular signaling pathways. FAK activity can influence the formation of distinct actin cytoskeletal structures such as lamellipodia and stress fibers in part through effects on small Rho GTPases, although the molecular interconnections of these events are not well defined. Here, we report that FAK interacts with p190RhoGEF, a RhoA-specific GDP/GTP exchange factor, in neuronal cells and in brain tissue extracts by co-immunoprecipitation and co-localization analyses. Using a two-hybrid assay and deletion mutagenesis, the binding site of the FAK C-terminal focal adhesion targeting (FAT) domain was identified within the C-terminal coiled-coil domain of p190RhoGEF. Binding was independent of a LD-like binding motif within p190RhoGEF, yet FAK association was disrupted by a mutation (Leu-1034 to Ser) that weakens the helical bundle structure of the FAK FAT domain. Neuro-2a cell binding to laminin increased endogenous FAK and p190RhoGEF tyrosine phosphorylation, and co-transfection of a dominant-negative inhibitor of FAK activity, termed FRNK, inhibited lamininstimulated p190RhoGEF tyrosine phosphorylation and p21 RhoA GTP binding. Overexpression of FAK in Neuro-2a cells increased both endogenous p190RhoGEF tyrosine phosphorylation and RhoA activity, whereas these events were inhibited by FRNK co-expression. Because insulin-like growth factor 1 treatment of Neuro-2a cells increased FAK tyrosine phosphorylation and enhanced p190RhoGEF-mediated activation of RhoA, our results support the conclusion that FAK association with p190RhoGEF functions as a signaling pathway downstream of integrins and growth factor receptors to stimulate Rho activity.     

A High Mobility Group Protein from the Dipteran Insects Ceratitis capitata and Bactrocera oleae

Nuclei from Bactrocera oleae and Ceratitis capitata larvae contain a major protein that shares most of the characteristics of vertebrate high mobility group (HMG) proteins. Proteins are extracted from nuclei with 0.35 M NaCl, are soluble in 5% perchloric acid, are relatively small (molecular weight in the range of 10–16 kDa), and have both a high basic and a high acidic amino acid content. The amino acid constitution of these proteins is similar to that of the HMGB protein family of vertebrates. The proteins cross-react with antibodies raised against the HMGD chromosomal protein of Drosophila melanogaster. The possible relatedness of these proteins to high mobility group proteins is discussed.     

Crystal structure of the human GGA1 GAT domain

GGAs are a family of vesicle-coating regulatory proteins that function in intracellular protein transport. A GGA molecule contains four domains, each mediating interaction with other proteins in carrying out intracellular transport. The GAT domain of GGAs has been identified as the structural entity that binds membrane-bound ARF, a molecular switch regulating vesicle-coat assembly. It also directly interacts with rabaptin5, an essential component of endosome fusion. A 2.8 A resolution crystal structure of the human GGA1 GAT domain is reported here. The GAT domain contains four helices and has an elongated shape with the longest dimension exceeding 80 A. Its longest helix is involved in two structural motifs: an N-terminal helix-loop-helix motif and a C-terminal three-helix bundle. The N-terminal motif harbors the most conservative amino acid sequence in the GGA GAT domains. Within this conserved region, a cluster of residues previously implicated in ARF binding forms a hydrophobic surface patch, which is likely to be the ARF-binding site. In addition, a structure-based mutagenesis-biochemical analysis demonstrates that the C-terminal three-helix bundle of this GAT domain is responsible for the rabaptin5 binding. These structural characteristics are consistent with a model supporting multiple functional roles for the GAT domain.     

Chiral detection of carotenoid assemblies

Carotenoid assemblies were produced by aqueous dilution of ethanolic solutions. UV/VIS and CD spectroscopy revealed the formation of J- and H-types of aggregates of both right- and left-handed kinds. Simulation of UV/VIS spectra of the aggregates showed characteristic differences between the two types. 6'-Epimers of capsanthol ((all-E,3R,3'S,5'R)-beta,kappa-carotene-3,3',6'-triols) formed assemblies with increased chirality in dilute solution. While the absorption of 6'R-capsanthol giving H-type aggregate does not depend on the concentration, 6'S-capsanthol yielding J-type assembly showed concentration-dependent absorption intensity. Dilute aggregate of 6'R-capsanthol is characterized by an extremely large A value of -6,600. The transformation of J- to H-type assembly was observed in the mixtures of the epimers producing an intermediate kind of aggregate. A hypothetical structure for H-type assemblies is proposed.     

GNIP, a novel protein that binds and activates glycogenin, the self-glucosylating initiator of glycogen biosynthesis

Glycogenin is a self-glucosylating protein involved in the initiation of glycogen biosynthesis. Self-glucosylation leads to the formation of an oligosaccharide chain, which, when long enough, supports the action of glycogen synthase to elongate it and form a mature glycogen molecule. To identify possible regulators of glycogenin, the yeast two-hybrid strategy was employed. By using rabbit skeletal muscle glycogenin as a bait, cDNAs encoding three different proteins were isolated from the human skeletal muscle cDNA library. Two of the cDNAs encoded glycogenin and glycogen synthase, respectively, proteins known to be interactors. The third cDNA encoded a polypeptide of unknown function and was designated GNIP (glycogenin interacting protein). Northern blot analysis revealed that GNIP mRNA is highly expressed in skeletal muscle. The gene for GNIP generates at least four isoforms by alternative splicing. The largest isoform GNIP1 contains, from NH(2)- to COOH-terminal, a RING finger, a B box, a putative coiled-coil region, and a B30.2-like motif. The previously identified protein TRIM7 (tripartite motif containing protein 7) is also derived from the GNIP gene and is composed of the RING finger, B box, and coiled-coil regions. The GNIP2 and GNIP3 isoforms consist of the coiled-coil region and B30.2-like domain. Physical interaction between GNIP2 and glycogenin was confirmed by co-immunoprecipitation, and in addition GNIP2 was shown to stimulate glycogenin self-glucosylation 3-4-fold. GNIPs may represent a novel participant in the initiation of glycogen synthesis.     

Allograft rejection by primed/memory CD8+ T cells is CD154 blockade resistant: therapeutic implications for sensitized transplant recipients

We have shown that CD8(+) CTLs are the key mediators of accelerated rejection, and that CD8(+) T cells represent the prime targets of CD154 blockade in sensitized mouse recipients of cardiac allografts. However, the current protocols require CD154 blockade at the time of sensitization, whereas delayed treatment fails to affect graft rejection in sensitized recipients. To elucidate the mechanisms of costimulation blockade-resistant rejection and to improve the efficacy of CD154-targeted therapy, we found that alloreactive CD8(+) T cells were activated despite the CD154 blockade in sensitized hosts. Comparative CD8 T cell activation study in naive vs primed hosts has shown that although both naive and primed/memory CD8(+) T cells relied on the CD28 costimulation for their activation, only naive, not primed/memory, CD8(+) T cells depend on CD154 signaling to differentiate into CTL effector cells. Adjunctive therapy was designed accordingly to deplete primed/memory CD8(+) T cells before the CD154 blockade. Indeed, unlike anti-CD154 monotherapy, transient depletion of CD8(+) T cells around the time of cardiac engraftment significantly improved the efficacy of delayed CD154 blockade in sensitized hosts. Hence, this report provides evidence for 1) differential requirement of CD154 costimulation signals for naive vs primed/memory CD8(+) T cells, and 2) successful treatment of clinically relevant sensitized recipients to achieve stable long term graft acceptance.     

FT-IR study of some seco- and apocarotenoids

FT-IR spectroscopy was applied to investigate 15 different carotenoids. The following compounds were examined: beta-carotenone (1); semi-beta-carotenon-epoxide (2); beta-apo-8'-carotenal (3); ethyl-beta-apo-8'-carotenoate (4); beta-citraurin (5); 5,6-Epoxy-beta-caroten-8'-al (6); beta-citraurin-epoxide (7); apo-10'-violaxanthal (8); persicaxanthin (9); capsylaldehyde (10); capsanthylal (11); retinol (12); retinal (13); retinoic acid (14); and bixin (15). Some characteristic functional groups (Cz.dbnd;C, Cz.dbnd;O, CHO, OH, etc.) were identified. We focused on the influence of conjugation of the keto-, aldehyde- and ester groups on the absorption of the Cz.dbnd;C bonds. This method is useful in the fast analysis of the biologically important carotenoids especially if there are small samples available.     

Involvement of LEU13 in interferon-induced refractoriness of human RSa cells to cell killing by X rays

Culture of human cells with human interferon alpha and beta (IFNA and IFNB) results in increased resistance of the cells to cell killing by X rays. To identify candidate genes responsible for the IFN-induced X-ray resistance, we searched for genes whose expression levels are increased in human RSa cells treated with IFNA, using an mRNA differential display method and Northern blotting analysis. RSa cells, which showed increased survival (assayed by colony formation) after X irradiation when they were treated with IFNA prior to irradiation, showed increased expression levels of LEU13 (IFITM1) mRNA after IFNA treatment alone. In contrast, IF(r) and F-IF(r) cells, both of which are derived from RSa cells, showed increased X-ray resistance and high constitutive LEU13 mRNA expression levels compared to the parental RSa cells. Furthermore, the IFNA-induced resistance of RSa cells to killing by X rays was suppressed by antisense oligonucleotides for LEU13 mRNA. LEU13, a leukocyte surface protein, was previously reported to mediate the actions of IFN such as inhibition of cell proliferation. The present results suggest a novel role of LEU13 different from that in the inhibition of cell proliferation, involved in IFNA-induced refractoriness of RSa cells to X rays.