The "linker" region (amino acids 38-47) of the disintegrin elegantin is a novel inhibitory domain of integrin alpha5beta1-dependent cell adhesion on fibronectin: evidence for the negative regulation of fibronectin synergy site biological activity

Disintegrins are a family of potent inhibitors of cell-cell and cell-matrix adhesion. In this study we have identified a region of the disintegrin elegantin, termed the "linker domain" (amino acids 38-47), with inhibitory activity toward alpha(5)beta(1)-mediated cell adhesion on fibronectin (Fn). Using a chimeric structure-function approach in which sequences of the functionally distinct disintegrin kistrin were introduced into the elegantin template at targeted sites, a loss of inhibitory function toward alpha(5)beta(1)-mediated adhesion on Fn was observed when the elegantin linker domain was substituted. Subsequent analysis comparing the inhibitory efficacies of the panel of elegantin-kistrin chimeras toward CHO alpha(5) cell adhesion on recombinant Fn III(6-10) fragments showed that the loss of inhibitory activity associated with the disruption of the elegantin linker domain was dependent upon the presence of a functional Fn III(9) synergy site within the Fn III(6-10) substrate. This suggested that the elegantin linker domain inhibits primarily the activity of the Fn synergy domain in promoting alpha(5)beta(1) integrin-mediated cell adhesion. Construction of a cyclic peptide corresponding to the entire region of the elegantin linker domain showed that this domain has intrinsic alpha(5)beta(1) inhibitory activity comparable with the activity of the RGDS peptide. These data demonstrate a novel biological function for a disintegrin domain that antagonizes integrin-mediated cell adhesion.     

Ablation of myosin-binding protein-C accelerates force development in mouse myocardium

Myosin-binding protein-C (MyBP-C) is a thick filament-associated protein that binds tightly to myosin. Given that cMyBP-C may act to modulate cooperative activation of the thin filament by constraining the availability of myosin cross-bridges for binding to actin, we investigated the role of MyBP-C in the regulation of cardiac muscle contraction. We assessed the Ca(2+) sensitivity of force (pCa(50)) and the activation dependence of the rate of force redevelopment (k(tr)) in skinned myocardium isolated from wild-type (WT) and cMyBP-C null (cMyBP-C(-/-)) mice. Mechanical measurements were performed at 22 degrees C in the absence and presence of a strong-binding, nonforce-generating analog of myosin subfragment-1 (NEM-S1). In the absence of NEM-S1, maximal force and k(tr) and the pCa(50) of isometric force did not differ between WT and cMyBP-C(-/-) myocardium; however, ablation of cMyBP-C-accelerated k(tr) at each submaximal force. Treatment of WT and cMyBP-C(-/-) myocardium with 3 muM NEM-S1 elicited similar increases in pCa(50,) but the effects of NEM-S1 to increase k(tr) at submaximal forces and thereby markedly reduce the activation dependence of k(tr) occurred to a greater degree in cMyBP-C(-/-) myocardium. Together, these results support the idea that cMyBP-C normally acts to constrain the interaction between myosin and actin, which in turn limits steady-state force development and the kinetics of cross-bridge interaction.     

A two component chitin-binding protein from French bean -- association of a proline-rich protein with a cysteine-rich polypeptide

A 42kDa chitin-binding proline-rich protein (PRP) from French bean has been previously characterised through its involvement in plant-pathogen interactions. It is located at the plasmalemma-wall interface, intercellular spaces and binds to the pathogen Colletotrichum lindemuthianum in vitro and in planta. It is also present in cell wall appositions formed in response to an hrp mutant of Xanthomonas campestris. We now show that the 42kDa protein is composed of two components, a 25kDa polypeptide member of the PRP family of legumes and a 6.8kDa cysteine-rich peptide with high similarity to snakin-2 from potato. Snakins bind to pathogens and are antimicrobial. Molecular cloning of the longest PRP corresponding to the N-terminal sequence of the purified protein and the 6.8kDa component is reported. The cognate mRNAs show coordinate expression. The two-component protein complex has already been shown to be involved in binding and immobilising pathogens through oxidative cross-linking of the PRP components but could also function as a two-component chitin-receptor involved in plant-pathogen interactions through antimicrobial activity and/or signalling.     

Role of immunoproteasomes in cross-presentation

The evidence that proteasomes are involved in the processing of cross-presented proteins is indirect and based on the in vitro use of proteasome inhibitors. It remains, therefore, unclear whether cross-presentation of MHC class I peptide epitopes can occur entirely within phagolysosomes or whether it requires proteasome degradation. To address this question, we studied in vivo cross-presentation of an immunoproteasome-dependent epitope. First, we demonstrated that generation of the immunodominant HY Uty(246-254) epitope is LMP7 dependent, resulting in the lack of rejection of male LMP7-deficient (LMP7(-/-)) skin grafts by female LMP7(-/-) mice. Second, we ruled out an altered Uty(246-254)-specific T cell repertoire in LMP7(-/-) female mice and demonstrated efficient Uty(246-254) presentation by re-expressing LMP7 in male LMP7(-/-) cells. Finally, we observed that LMP7 expression significantly enhanced cross-priming of Uty(246-254)-specific T cells in vivo. The observations that male skin grafts are not rejected by LMP7(-/-) female mice and that presentation of a proteasome-dependent peptide is not efficiently rescued by alternative cross-presentation pathways provide strong evidence that proteasomes play an important role in cross-priming events.     

TNF-alpha blockade down-regulates the CD40/CD40L pathway in the mucosal microcirculation: a novel anti-inflammatory mechanism of infliximab in Crohn's disease

The CD40/CD40 ligand (CD40L) pathway is involved in Crohn's disease (CD) pathogenesis. In the patients' circulation, soluble CD40L (sCD40L) levels are elevated and surface CD40L is increased in platelets and T cells, whereas in the intestine CD40 is overexpressed in the microvasculature and CD40L in platelets and T cells. The therapeutic effects of infliximab in CD are attributed to its systemic anti-TNF-alpha action, but because TNF-alpha modulates both CD40 and CD40L, we investigated whether infliximab affects the CD40/CD40L pathway in the intestine. Eighteen CD patients were evaluated before and after infliximab therapy. Plasma sCD40L was measured by ELISA and platelet and peripheral blood T cell (PBT) CD40L expression by flow cytometry. Microvascular CD40 and VCAM-1 expression were assessed in mucosal biopsies by immunohistochemistry and by flow cytometry in human intestinal microvascular endothelial cells (HIMEC). Cell cultures were performed in the presence and absence of infliximab. Infliximab treatment significantly reduced plasma sCD40L levels and eliminated CD40 and VCAM-1 from mucosal microvessels. In vitro infliximab prevented TNF-alpha-induced CD40 and VCAM-1 expression by HIMEC, and reduced PBT, but not platelet, surface CD40L expression and sCD40L release. In addition, infliximab decreased T cell-induced VCAM-1 expression in HIMEC by down-regulating CD40L in T cells and promoting T cells apoptosis. These findings point to a novel mechanism of action of infliximab, i.e., the disruption of CD40/CD40L-dependent cognate interactions between intestinal microvessels and T cells. Thus, in addition to neutralizing TNF-alpha and inducing T cell death, the therapeutic effects of infliximab in CD appear to be also mediated by inhibition of vascular inflammation in the gut.     

Role for RAD18 in homologous recombination in DT40 cells

RAD18 is an E3 ubiquitin ligase that catalyzes the monoubiquitination of PCNA, a modification central to DNA damage bypass and postreplication repair in both yeast and vertebrates. Although current evidence suggests that homologous recombination provides an essential backup in vertebrate rad18 mutants, we show that in chicken DT40 cells this is not the case and that RAD18 plays a role in the recombination reaction itself. Gene conversion tracts in the immunoglobulin locus of rad18 cells are shorter and are associated with an increased frequency of deletions and duplications. rad18 cells also exhibit reduced efficiency of gene conversion induced by targeted double-strand breaks in a reporter construct. Blocking an early stage of the recombination reaction by disruption of XRCC3 not only suppresses immunoglobulin gene conversion but also prevents the aberrant immunoglobulin gene rearrangements associated with RAD18 deficiency, reverses the elevated sister chromatid exchange of the rad18 mutant, and reduces its sensitivity to DNA damage. Together, these data suggest that homologous recombination is toxic in the absence of RAD18 and show that, in addition to its established role in postreplication repair, RAD18 is also required for the orderly completion of gene conversion.     

The retinoblastoma protein regulates pericentric heterochromatin

The retinoblastoma protein (pRb) has been proposed to regulate cell cycle progression in part through its ability to interact with enzymes that modify histone tails and create a repressed chromatin structure. We created a mutation in the murine Rb1 gene that disrupted pRb's ability to interact with these enzymes to determine if it affected cell cycle control. Here, we show that loss of this interaction slows progression through mitosis and causes aneuploidy. Our experiments reveal that while the LXCXE binding site mutation does not disrupt pRb's interaction with the Suv4-20h histone methyltransferases, it dramatically reduces H4-K20 trimethylation in pericentric heterochromatin. Disruption of heterochromatin structure in this chromosomal region leads to centromere fusions, chromosome missegregation, and genomic instability. These results demonstrate the surprising finding that pRb uses the LXCXE binding cleft to control chromatin structure for the regulation of events beyond the G(1)-to-S-phase transition.     

Tertiary structure prediction of SARS coronavirus helicase

SARS coronavirus, SCV, has been recently responsible of a sudden and widespread infection which caused almost 800 victims. The limited amount of SCV protein structural information is partially responsible of the lack of specific drugs against the virus. Coronavirus helicases are very conserved and peculiar proteins which have been proposed as suitable targets for antiviral drugs, such as bananins, which have been recently shown to inhibit the SCV helicase in vitro. Here, the quaternary structure of SCV helicase has been predicted, which will provide a solid foundation for the rational design of other antiviral helicase inhibitors.