An assay system for measuring the acute production of sphingosine 1-phosphate in intact monolayers

Sphingosine kinase (SK) is a signaling enzyme that phosphorylates sphingosine to produce sphingosine 1-phosphate. Sphingosine and sphingosine 1-phosphate (S1P) belong to a class of bioactive sphingolipid metabolites that are critical in a number of cellular processes, yet often have opposing biological functions. The intracellular localization of sphingosine kinase has been demonstrated in multiple studies to be a critical aspect of its signaling function. To date, assays of sphingosine kinase activity have been developed for measuring activity in lysates, where the effects of localization are lost. Here we outline a system in which the rate of production of S1P can be measured in intact cells using exogenously added radiolabeled ATP instead of tritiated sphingosine. The surprising ability of ATP to enter unpermeabilized monolayers is one aspect that makes this assay simple, efficient, and inexpensive, yet sensitive enough to measure endogenous enzyme activity. The assay is well behaved in terms of kinetics and substrate dependence. Overall, this assay is ideal for future studies to identify changes in S1P production in intact cells such as those that result from the differential intracellular targeting of sphingosine kinase.     

The compartmentalization and translocation of the sphingosine kinases: mechanisms and functions in cell signaling and sphingolipid metabolism

Members of the sphingosine kinase (SK) family of lipid signaling enzymes, comprising SK1 and SK2 in humans, are receiving considerable attention for their roles in a number of physiological and pathophysiological processes. The SKs are considered signaling enzymes based on their production of the potent lipid second messenger sphingosine-1-phosphate, which is the ligand for a family of five G-protein-linked receptors. Both SK1 and SK2 are intracellular enzymes and do not possess obvious membrane anchor domains within their primary sequences. The native substrates (sphingosine and dihydrosphingosine) are lipids, as are the corresponding products, and therefore would have a propensity to be membrane associated, suggesting that specific membrane localization of the SKs could affect both access to substrate and localized production of product. Here, we consider the emerging picture of the SKs as enzymes localized to specific intracellular sites, sometimes by agonist-dependent translocation, the mechanism targeting these enzymes to those sites, and the functional consequence of that localization. Not only is the signaling output of the SKs affected by subcellular localization, but the role of these enzymes as metabolic regulators of sphingolipid metabolism may be impacted as well.     

Methionine aminopeptidase-2 blockade reduces chronic collagen-induced arthritis: potential role for angiogenesis inhibition

The enzyme methionine aminopeptidase-2 (MetAP-2) is thought to play an important function in human endothelial cell proliferation, and as such provides a valuable target in both inflammation and cancer. Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with increased synovial vascularity, and hence is a potential therapeutic target for angiogenesis inhibitors. We examined the use of PPI-2458, a selective non-reversible inhibitor of MetAP-2, in disease models of RA, namely acute and chronic collagen-induced arthritis (CIA) in mice. Whilst acute CIA is a monophasic disease, CIA induced with murine collagen type II manifests as a chronic relapsing arthritis and mimics more closely the disease course of RA. Our study showed PPI-2458 was able to reduce clinical signs of arthritis in both acute and chronic CIA models. This reduction in arthritis was paralleled by decreased joint inflammation and destruction. Detailed mechanism of action studies demonstrated that PPI-2458 inhibited human endothelial cell proliferation and angiogenesis in vitro, without affecting production of inflammatory cytokines. Furthermore, we also investigated release of inflammatory cytokines and chemokines from human RA synovial cell cultures, and observed no effect of PPI-2458 on spontaneous expression of cytokines and chemokines, or indeed on the angiogenic molecule vascular endothelial growth factor (VEGF). These results highlight MetAP-2 as a good candidate for therapeutic intervention in RA.     

Human macrophage migration inhibitory factor: a proven immunomodulatory cytokine?

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory mediator with the ability to induce various immunomodulatory responses and override glucocorticoid-driven immunosuppression. Some of these functions have been linked to the unusual enzymatic properties of the protein, namely tautomerase and oxidoreductase activities. However, there are conflicting reports regarding the functional role of these enzymatic properties in normal physiological homeostasis and disease progression. Therefore, we have produced a highly pure, virtually endotoxin-free recombinant MIF preparation and fully characterized this using a variety of biochemical and biophysical approaches. The recombinant protein, with demonstrable enzymatic activity, was then used to systematically examine the biological activity of MIF. Surprisingly, treatment with MIF alone failed to induce cytokine expression, with the exception of IL-8. However, co-treatment of lipopolysaccharide (LPS) in conjunction with MIF produced synergistic secretion of tumor necrosis factor-alpha, interleukin (IL)-1, and IL-8 compared with LPS alone. The potentiating effect of MIF was seen at physiologically relevant concentrations. These data suggest that MIF has no conventional cytokine activity but, rather, acts to modulate and amplify the response to LPS.     

A Splice-Isoform of Vesicle-associated Membrane Protein-1 (VAMP-1) Contains a Mitochondrial Targeting Signal

Screening of a library derived from primary human endothelial cells revealed a novel human isoform of vesicle-associated membrane protein-1 (VAMP-1), a protein involved in the targeting and/or fusion of transport vesicles to their target membrane. We have termed this novel isoform VAMP-1B and designated the previously described isoform VAMP-1A. VAMP-1B appears to be an alternatively spliced form of VAMP-1. A similar rat splice variant of VAMP-1 (also termed VAMP-1B) has recently been reported. Five different cultured cell lines, from different lineages, all contained VAMP-1B but little or no detectable VAMP-1A mRNA, as assessed by PCR. In contrast, brain mRNA contained VAMP-1A but no VAMP-1B. The VAMP-1B sequence encodes a protein identical to VAMP-1A except for the carboxy-terminal five amino acids. VAMP-1 is anchored in the vesicle membrane by a carboxy-terminal hydrophobic sequence. In VAMP-1A the hydrophobic anchor is followed by a single threonine, which is the carboxy-terminal amino acid. In VAMP-1B the predicted hydrophobic membrane anchor is shortened by four amino acids, and the hydrophobic sequence is immediately followed by three charged amino acids, arginine-arginine-aspartic acid. Transfection of human endothelial cells with epitope-tagged VAMP-1B demonstrated that VAMP-1B was targeted to mitochondria whereas VAMP-1A was localized to the plasma membrane and endosome-like structures. Analysis of C-terminal mutations of VAMP-1B demonstrated that mitochondrial targeting depends both on the addition of positive charge at the C terminus and a shortened hydrophobic membrane anchor. These data suggest that mitochondria may be integrated, at least at a mechanistic level, to the vesicular trafficking pathways that govern protein movement between other organelles of the cell.     

Primaquine blocks transport by inhibiting the formation of functional transport vesicles. Studies in a cell-free assay of protein transport through the Golgi apparatus.

The lysosomotropic amine primaquine has previously been shown to inhibit both secretory and recycling processes of cells in culture. We have used a cell-free assay that reconstitutes glycoprotein transport through the Golgi apparatus to investigate the mechanism of action of primaquine. In this assay, primaquine inhibits protein transport at a half-maximal concentration of 50 microM, similar to the concentration previously reported to disrupt protein secretion in cultured cells. Kinetic analysis of primaquine inhibition indicates that its point of action is at an early step in the vesicular transport mechanism. Primaquine does not inhibit the fusion of vesicles already attached to their target membranes. Primaquine irreversibly inactivates the membranes that form transport vesicles (donor), but not the membranes that are the destination of those vesicles (acceptor). Morphological data indicate that primaquine inhibits the budding of vesicles from the donor membranes. Once formed, the vesicles are refractile to primaquine action, and their attachment to and fusion with acceptor membranes proceeds unimpeded. In addition to illuminating the mechanism of action of primaquine, this study suggests that the selective action of this agent will make it a useful tool in the study of the formation of transport vesicles.