Cysteine protease gene expression and proteolytic activity during senescence of Alstroemeria petals

The functional life of the flower is terminated by senescence and/or abscission. Multiple processes contribute to produce the visible signs of petal wilting and inrolling that typify senescence, but one of the most important is that of protein degradation and remobilization. This is mediated in many species through protein ubiquitination and the action of specific protease enzymes. This paper reports the changes in protein and protease activity during development and senescence of Alstroemeria flowers, a Liliaceous species that shows very little sensitivity to ethylene during senescence and which shows perianth abscission 8-10 d after flower opening. Partial cDNAs of ubiquitin (ALSUQ1) and a putative cysteine protease (ALSCYP1) were cloned from Alstroemeria using degenerate PCR primers and the expression pattern of these genes was determined semi-quantitatively by RT-PCR. While the levels of ALSUQ1 only fluctuated slightly during floral development and senescence, there was a dramatic increase in the expression of ALSCYP1 indicating that this gene may encode an important enzyme for the proteolytic process in this species. Three papain class cysteine protease enzymes showing different patterns of activity during flower development were identified on zymograms, one of which showed a similar expression pattern to the cysteine protease cDNA.

Physical and functional characterisation of apoptotic cell-derived extracellular vesicles

Damaged, aged or unwanted cells are removed from the body by an active process known as apoptosis. This highly orchestrated programme results in the exposure of 'flags' at the dying cell surface and the release of attractive signals to recruit phagocytes. Together these changes ensure efficient phagocytic removal of dying cells and prevention of inflammatory and autoimmune disorders. Extracellular vesicles (EV) are released from a variety of cells (both viable and apoptotic) and they serve as a novel means of intercellular communication. They range in size: 70-100nm ('exosomes') through 100-1000nm ('microparticles') to large vesicles released from dying cells ('apoptotic bodies'). Release of apoptotic cell-derived extracellular vesicles (acdEV) of less than 1000nm is an important mechanism by which phagocytes are attracted to sites of cell death. Using a variety of approaches we characterize the release, physical characteristics and function of acdEV. Using fluorescence microscopy we demonstrate release of ICAM-3 on acdEV from dying leukocytes and, through the use of resistive pulse technology (qNano, IZON Science), we accurately size and quantitate acdEV release. The function of acdEV is revealed through the use of both horizontal chemotaxis assays (Dunn chambers) and vertical transwell migration assays (Cell-IQ, CM Technologies). These assays reveal potent chemoattractive capacity of acdEV and associated ICAM-3. Additionally we demonstrate an additional novel function of acdEV as anti-inflammatory immune-modulators. These data support an integrated approach to the physical and functional analyses of EV.

Characterisation of ICAM-3 and extracellular vesicles in the clearance of apoptotic cells

Apoptotic cell clearance by phagocytes is a vital part of programmed cell death that prevents dying cells from undergoing necrosis which may lead to inflammatory and autoimmune disorders. Apoptotic cells (AC) are removed by phagocytes, in a process that involves 'find me' and 'eat me' signals that facilitate the synapsing and engulfment of cell corpses. Extracellular vesicles (EV) are shed during apoptosis and promote phagocyte recruitment. Binding of AC is achieved by multiple ligand-receptor interactions. One interesting AC associated ligand is ICAM-3, a highly glycosylated adhesion molecule of the IgSF family, expressed on human leukocytes. On viable cells ICAM-3 participates in initiating immune responses, whereas on AC we show it attracts phagocytes through EV and aids in the binding of AC to the phagocytes. This project aims to characterize the role of ICAM-3 and EV in the clearance of AC and to identify the mechanisms that underlie their function in apoptotic cell clearance. Human B cells induced to apoptosis by UV irradiation were observed during their progression from viable to apoptotic via flow cytometry. The involvement of ICAM-3 in mediating interaction between AC and MØ was assessed. The ability of ICAM3 on EV to mediate chemoattraction was observed using chemotaxis assays. Additionally the anti-inflammatory effect was assessed using LPS-induced TNF-α production that suggested it may have anti-inflammatory effects. Future work in this project will assess the role of ICAM3 on EV from different phases of apoptosis to exert functional effects both in vitro and in vivo.

Characterization of neutrophil function in Papillon-Lefèvre syndrome

Papillon-Lefévre syndrome is a rare, inherited, autosomal-recessive disease, characterized by palmoplantar keratosis and severe prepubertal periodontitis, leading to premature loss of all teeth. Papillon-Lefévre syndrome is caused by a mutation in the cathepsin C gene, resulting in complete loss of activity and subsequent failure to activate immune response proteins. Periodontitis in Papillon-Lefévre syndrome is thought to arise from failure to eliminate periodontal pathogens as a result of cathepsin C deficiency, although mechanistic pathways remain to be elucidated. The aim of this study was to characterize comprehensively neutrophil function in Papillon-Lefévre syndrome. Peripheral blood neutrophils were isolated from 5 patients with Papillon-Lefévre syndrome, alongside matched healthy control subjects. For directional chemotactic accuracy, neutrophils were exposed to the chemoattractants MIP-1α and fMLP and tracked by real-time videomicroscopy. Reactive oxygen species generation was measured by chemiluminescence. Neutrophil extracellular trap formation was assayed fluorometrically, and proinflammatory cytokine release was measured following overnight culture of neutrophils with relevant stimuli. Neutrophil serine protease deficiencies resulted in a reduced ability of neutrophils to chemotax efficiently and an inability to generate neutrophil extracellular traps. Neutrophil extracellular trap-bound proteins were also absent in Papillon-Lefévre syndrome, and Papillon-Lefévre syndrome neutrophils released higher levels of proinflammatory cytokines in unstimulated and stimulated conditions, and plasma cytokines were elevated. Notably, neutrophil chemoattractants MIP-1α and CXCL8 were elevated in Papillon-Lefévre syndrome neutrophils, as was reactive oxygen species formation. We propose that relentless recruitment and accumulation of hyperactive/reactive neutrophils (cytokines, reactive oxygen species) with increased tissue transit times into periodontal tissues, alongside a reduced antimicrobial capacity, create a locally destructive chronic inflammatory cycle in Papillon-Lefévre syndrome.

The extracellular surface of the GLP-1 receptor is a molecular trigger for biased agonism

Ligand-directed signal bias offers opportunities for sculpting molecular events, with the promise of better, safer therapeutics. Critical to the exploitation of signal bias is an understanding of the molecular events coupling ligand binding to intracellular signaling. Activation of class B G protein-coupled receptors is driven by interaction of the peptide N terminus with the receptor core. To understand how this drives signaling, we have used advanced analytical methods that enable separation of effects on pathway-specific signaling from those that modify agonist affinity and mapped the functional consequence of receptor modification onto three-dimensional models of a receptor-ligand complex. This yields molecular insights into the initiation of receptor activation and the mechanistic basis for biased agonism. Our data reveal that peptide agonists can engage different elements of the receptor extracellular face to achieve effector coupling and biased signaling providing a foundation for rational design of biased agonists.

Extracellular Nm23H1 stimulates neurite outgrowth from dorsal root ganglia neurons in vitro independently of nerve growth factor supplementation or its nucleoside diphosphate kinase activity

The nucleoside diphosphate (NDP) kinase, Nm23H1, is a highly expressed during neuronal development, whilst induced over-expression in neuronal cells results in increased neurite outgrowth. Extracellular Nm23H1 affects the survival, proliferation and differentiation of non-neuronal cells. Therefore, this study has examined whether extracellular Nm23H1 regulates nerve growth. We have immobilised recombinant Nm23H1 proteins to defined locations of culture plates, which were then seeded with explants of embryonic chick dorsal root ganglia (DRG) or dissociated adult rat DRG neurons. The substratum-bound extracellular Nm23H1 was stimulatory for neurite outgrowth from chick DRG explants in a concentration-dependent manner. On high concentrations of Nm23H1, chick DRG neurite outgrowth was extensive and effectively limited to the location of the Nm23H1, i.e. neuronal growth cones turned away from adjacent collagen-coated substrata. Nm23H1-coated substrata also significantly enhanced rat DRG neuronal cell adhesion and neurite outgrowth in comparison to collagen-coated substrata. These effects were independent of NGF supplementation. Recombinant Nm23H1 (H118F), which does not possess NDP kinase activity, exhibited the same activity as the wild-type protein. Hence, a novel neuro-stimulatory activity for extracellular Nm23H1 has been identified in vitro, which may function in developing neuronal systems. © 2010 Elsevier Inc.