The ADAMs family of proteases: new biomarkers and therapeutic targets for cancer?

The ADAMs are transmembrane proteins implicated in proteolysis and cell adhesion. Forty gene members of the family have been identified, of which 21 are believed to be functional in humans. As proteases, their main substrates are the ectodomains of other transmembrane proteins. These substrates include precursor forms of growth factors, cytokines, growth factor receptors, cytokine receptors and several different types of adhesion molecules. Although altered expression of specific ADAMs has been implicated in different diseases, their best-documented role is in cancer formation and progression. ADAMs shown to play a role in cancer include ADAM9, ADAM10, ADAM12, ADAM15 and ADAM17. Two of the ADAMs, i.e., ADAM10 and 17 appear to promote cancer progression by releasing HER/EGFR ligands. The released ligands activate HER/EGFR signalling that culminates in increased cell proliferation, migration and survival. Consistent with a causative role in cancer, several ADAMs are emerging as potential cancer biomarkers for aiding cancer diagnosis and predicting patient outcome. Furthermore, a number of selective ADAM inhibitors, especially against ADAM10 and ADAM17, have been shown to have anti-cancer effects. At least one of these inhibitors is now undergoing clinical trials in patients with breast cancer.     

Inflammasome activation: from inflammatory disease to infection

The recognition of pathogen-derived molecules by the innate immune system is mediated by a number of receptors, including members of the TLR (Toll-like receptor), RLH [RIG (retinoic acid-inducible gene)-like helicase] and the NLR (NOD-like receptor) families. NLRs in particular are also involved in the recognition of host-derived 'danger'-associated molecules which are produced under conditions of cellular stress or injury. Activation of these receptors leads to the assembly of high-molecular-mass complexes called inflammasomes which in turn leads to the generation of active caspase 1 and to the production of mature IL-1β (interleukin 1β). The discovery that NLRP3 (NLR-related protein 3) can recognize host-derived particulate matter such as uric acid and cholesterol crystals has led to this inflammasome being implicated in a number of inflammatory diseases, including gout, atherosclerosis and Type?2 diabetes. In addition, aberrant NLRP3 activation has also been observed in a number of heritable disorders now referred to as cryopyrinopathies. On the other hand, a number of studies have reported that recognition of both viral and bacterial products by NLRs is required for effective pathogen clearance. The present review discusses both aspects of NLR activation and will highlight the role of additional inflammasome complexes in sensing infection.     

Toll-like receptor 3 (TLR3) signaling requires TLR4 Interactor with leucine-rich REPeats (TRIL)

Toll-like receptors (TLRs) are a family of proteins that act as the primary sensors of microbial products. Many TLRs require accessory molecules in order to recognize these microbial products and initiate signal transduction cascades. We have identified TRIL (TLR4 interactor with leucine-rich repeats) as a novel modulator of TLR4 signaling showing high expression in the brain. We now show that TRIL also plays a role in TLR3 signaling. TRIL is expressed intracellularly in the astrocytoma cell line U373 and in the monocytic cell line THP1. TRIL co-localizes with the endosomal compartment. These data are consistent with a role for TRIL in TLR3 signaling and endosomal TLR4 signaling. TRIL was induced by the TLR3 ligand poly(I:C). Overexpression of TRIL enhanced cytokine production and interferon-stimulated response element (ISRE) luciferase activity following poly(I:C) stimulation in U373. TRIL interacted with TLR3, and this interaction was enhanced following poly(I:C) stimulation. Transient knockdown of TRIL with siRNA or stable knockdown using shRNA in U373 cells inhibited TLR3 signaling, reducing ISRE luciferase, RANTES, and type I interferon production. Knockdown of TRIL did not affect TLR2 signaling. Most accessory molecules identified to date, such as CD14, gp96, PRAT4a, and Unc93B, all play roles in multiple TLR signaling pathways, and we now show that this is also the case for TRIL.     

Activation of biliverdin-IXalpha reductase by inorganic phosphate and related anions

The N-terminal region of a 32 kDa cell-surface-binding protein, encoded by the D8L gene of vaccinia virus, shows sequence homology to CAs (carbonic anhydrases; EC 4.2.1.1). The active CAs catalyse the reversible hydration of CO2 to bicarbonate participating in many physiological processes. The CA-like domain of vaccinia protein [vaccCA (vaccinia virus CA-like protein)] contains one of the three conserved histidine residues required for co-ordination to the catalytic zinc ion and for enzyme activity. In the present study, we report the engineering of catalytically active vaccCA mutants by introduction of the missing histidine residues into the wild-type protein. The wild-type vaccCA was inactive as a catalyst and does not bind sulfonamide CA inhibitors. Its position on a phylogram with other hCAs (human CAs) shows a relationship with the acatalytic isoforms CA X and XI, suggesting that the corresponding viral gene was acquired from the human genome by horizontal gene transfer. The single mutants (vaccCA N92H/Y69H) showed low enzyme activity and low affinity for acetazolamide, a classical sulfonamide CA inhibitor. The activity of the double mutant, vaccCA N92H/Y69H, was much higher, of the same order of magnitude as that of some human isoforms, namely CA VA and CA XII. Moreover, its affinity for acetazolamide was high, comparable with that of the most efficient human isoenzyme, CA II (in the low nanomolar range). Multiplication of vaccinia virus in HeLa cells transfected with the vaccCA N92H/Y69H double mutant was approx. 2-fold more efficient than in wild-type vaccCA transfectants, suggesting that the reconstitution of the enzyme activity improved the virus life cycle.     

Direct correlation between sialic acid binding and infection of cells by two human polyomaviruses (JC virus and BK virus)

For the human polyomaviruses JC virus (JCV) and BK virus (BKV), the first step to a successful infection involves binding to sialic acid moieties located on the surfaces of host cells. By stripping and then reconstituting specific sialic acid linkages on host cells, we show that JCV uses both α(2,3)-linked and α(2,6)-linked sialic acids on N-linked glycoproteins to infect cells. For both JCV and BKV, the sialic acid linkages required for cell surface binding directly correlate with the linkages required for infection. In addition to sialic acid linkage data, these data suggest that the third sugar from the carbohydrate chain terminus is important for virus binding and infection.     

The response of fenestrations, actin, and caveolin-1 to vascular endothelial growth factor in SK Hep1 cells

To study the regulation of fenestrations by vascular endothelial growth factor in liver sinusoidal endothelial cells, SK Hep1 cells were transfected with green fluorescence protein (GFP)-actin and GFP-caveolin-1. SK Hep1 cells had pores; some of which appeared to be fenestrations (diameter 55 +/- 28 nm, porosity 2.0 +/- 1.4%), rudimentary sieve plates, bristle-coated micropinocytotic vesicles and expressed caveolin-1, von Willebrand factor, vascular endothelial growth factor receptor-2, endothelial nitric oxide synthase and clathrin, but not CD31. There was avid uptake of formaldehyde serum albumin, consistent with endocytosis. Vascular endothelial growth factor caused an increase in porosity to 4.8 +/- 2.6% (P < 0.01) and pore diameter to 104 +/- 59 nm (P < 0.001). GFP-actin was expressed throughout the cells, whereas GFP-caveolin-1 had a punctate appearance; both responded to vascular endothelial growth factor by contraction toward the nucleus over hours in parallel with the formation of fenestrations. SK Hep1 cells resemble liver sinusoidal endothelial cells, and the vascular endothelial growth factor-induced formation of fenestration-like pores is preceded by contraction of actin cytoskeleton and attached caveolin-1 toward the nucleus.