Autotaxin (ATX) is a prometastatic enzyme initially isolated from the conditioned medium of human melanoma cells that stimulates a myriad of biological activities, including angiogenesis and the promotion of cell growth, survival, and differentiation through the production of lysophosphatidic acid (LPA). ATX increases the aggressiveness and invasiveness of transformed cells, and ATX levels directly correlate with tumor stage and grade in several human malignancies. To study the role of ATX in the pathogenesis of malignant melanoma, we developed antibodies and small-molecule inhibitors against recombinant human protein. Immunohistochemistry of paraffinembedded human tissue shows that ATX levels are markedly increased in human primary and metastatic melanoma relative to benign nevi. Chemical screens identified several small-molecule inhibitors with binding constants ranging from nanomolar to low micromolar. Cell migration and invasion assays with melanoma cell lines show that ATX markedly stimulates melanoma cell migration and invasion, an effect suppressed by ATX inhibitors. The migratory phenotype can be rescued by the addition of the enzymatic product of ATX, LPA, confirming that the observed inhibition is linked to suppression of LPA production by ATX. Chemical analogues of the inhibitors show structure-activity relationships important for ATX inhibition and indicate pathways for their optimization. These studies suggest that ATX is an approachable molecular target for the rational design of chemotherapeutic agents directed against malignant melanoma. [Mol Cancer Ther 2008;7(10):3352 -62]

Profilins promote actin polymerization by exchanging ADP for ATP on monomeric actin, and delivering ATP-actin to growing filament barbed ends. Apicomplexan protozoa like Toxoplasma gondii invade host cells using an actin-dependent gliding motility. Toll-like receptor 11 (TLR11) generates an innate immune response upon sensing T. gondii profilin (TgPRF). The crystal structure of TgPRF reveals a parasite-specific surface motif consisting of an acidic loop, followed by a long β-hairpin. A series of structure-based profilin mutants show that TLR11 recognition of the acidic loop is responsible for most of the IL-12 secretion response to TgPRF in peritoneal macrophages. Deletion of both the acidic loop and the β-hairpin completely abrogates IL-12 secretion. Insertion of the T. gondii acidic loop and β-hairpin into yeast profilin is sufficient to generate TLR11-dependent signaling. Substitution of the acidic loop in TgPRF with the homologous loop from the apicomplexan parasite C. parvum does not affect TLR11-dependent IL-12 secretion, while substitution with the acidic loop from P. falciparum results in reduced but significant IL-12 secretion. We conclude that the parasite-specific motif in TgPRF is the key molecular pattern recognized by TLR11. Unlike other profilins, TgPRF slows nucleotide exchange on monomeric rabbit actin, and binds rabbit actin weakly. The putative TgPRF actin-binding surface includes the β-hairpin, and diverges widely from the actin-binding surfaces of vertebrate profilins.

Autotaxin (ATX) is a secreted lysophospholipase D that hydrolyzes lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA), initiating signaling cascades leading to cancer metastasis, wound healing, and angiogenesis. Knowledge of the pathway and kinetics of LPA synthesis by ATX is critical for developing quantitative physiological models of LPA signaling. We measured the individual rate constants and pathway of the LPA synthase cycle of ATX using the fluorescent lipid substrates FS-3 and 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl))-LPC. FS-3 binds rapidly (k 1 >500 M ؊1 s ؊1) and is hydrolyzed slowly (k 2 ‫؍‬ 0.024 s ؊1). Release of the first hydrolysis product is random and rapid (>1 s Autotaxin (ATX),4 also known as nucleotide pyrophosphatase/phosphodiesterase 2 (NPP2), was identified as a secreted autocrine motility-stimulating factor in melanoma cell cultures (1) and has subsequently been shown to play critical roles in angiogenesis, apoptosis, cancer metastasis, development, neuropathic pain, and wound healing (reviewed in Refs. 2-5). ATX displays both nucleotide phosphodiesterase activity (6) and a robust lysophospholipase D activity (lyso-PLD (7)). ATX phosphodiesterase activity is weak and is not considered relevant for in vivo function (7-9). Rather, the physiological activities of ATX have been attributed to synthesis of lysophosphatidic acid (LPA), a growth factor/chemokine that binds several endothelial differential gene family receptors (LPA1-5) (reviewed in Ref. 4), and initiates a variety of signaling cascades (4, 5) from lysophosphatidylcholine. ATX is the primary source of plasma LPA synthesis (10, 11).The plasma LPC concentration (50 -200 M) is comparable with the K M value for steady-state LPC hydrolysis by ATX (7,(12)(13)(14)(15). ATX binds LPA product more strongly than LPC substrate (12, 16), which has led to the hypothesis that product feedback inhibition regulates ATX activity and LPA production in vivo (16). However, rapid degeneration of serum LPA by lipid phosphate phosphohydrolase 1 (LPP1) (17, 18) would diminish LPA product inhibition of ATX.Rapid LPA degradation upon release from ATX also limits the effective target area of newly synthesized LPA, such that LPA signaling is restricted to within the diffusional area of the ATX⅐lipid complex from substrate binding locations. If LPC binding, hydrolysis, and LPA product release are rapid, LPA release and downstream signaling would be local (i.e. limited to sites of LPC binding). If, however, LPC substrate binding were more rapid than LPA release and bound LPA/LPC were inaccessible to degradation by LPP1, ATX with bound LPC/LPA could diffuse, thereby spreading LPA signaling to distal sites and cells. Recent in vivo studies show that competitive inhibition of ATX accelerates LPA degradation (19), consistent with the possibility of global ATX/LPA signaling via exclusion from LPP1.In this study, we measured the individual rate constants and pathway of the LPA synthase cycle of ATX using the fluorescent lipid substrates and LPC...

Triacetic acid lactone (TAL) is a potential platform chemical that can be produced in yeast. To evaluate the potential for industrial yeast strains to produce TAL, the g2ps1 gene encoding 2-pyrone synthase was transformed into 13 industrial yeast strains of varied genetic background. TAL production varied 63-fold between strains when compared in batch culture with glucose. Ethanol, acetate, and glycerol were also tested as potential carbon sources. Batch cultures with ethanol medium produced the highest titers. Therefore, fed-batch cultivation with ethanol feed was assayed for TAL production in bioreactors, producing our highest TAL titer, 5.2 g/L. Higher feed rates resulted in a loss of TAL and subsequent production of additional TAL side products. Finally, TAL efflux was measured and TAL is actively exported from S. cerevisiae cells. Percent yield for all strains was low, indicating that further metabolic engineering of the strains is required.

Switching the motor off: Privileged chemical scaffolds were used as a starting point for the development of a specific chemical inhibitor 1 of myosin V, a key motor protein required for intracellular transport (see picture; ADP=adenosine diphosphate, ATP=adenosine triphosphate). The potency of 1, which does not compete directly with nucleotide binding, is comparable to that of other known motor‐protein inhibitors used as probes in chemical biology.

The psychrotolerant foodborne pathogen Listeria monocytogenes withstands the stress of low temperatures and can proliferate in refrigerated food. Bacteria adapt to growth at low temperatures by increasing the production of fatty acids that increase membrane fluidity. The mechanism of homeoviscous increases in unsaturated fatty acid amounts in bacteria that predominantly contain straight-chain fatty acids is relatively well understood. By contrast the analogous mechanism in branched-chain fatty acid-containing bacteria, such as L. monocytogenes, is poorly understood. L. monocytogenes grows at low temperatures by altering its membrane composition to increase membrane fluidity, primarily by decreasing the length of fatty acid chains and increasing the anteiso to iso fatty acid ratio. FabH, the initiator of fatty acid biosynthesis, has been identified as the primary determinant of membrane fatty acid composition, but the extent of this effect has not been quantified. In this study, previously determined FabH steady-state parameters and substrate concentrations were used to calculate expected fatty acid compositions at 30°C and 10°C. FabH substrates 2-methylbutyryl-CoA, isobutyryl-CoA, and isovaleryl-CoA produce the primary fatty acids in L. monocytogenes, i.e., anteiso-odd, iso-even, and iso-odd fatty acids, respectively. In vivo concentrations of CoA derivatives were measured, but not all were resolved completely. In this case, estimates were calculated from overall fatty acid composition and FabH steady-state parameters. These relative substrate concentrations were used to calculate the expected fatty acid compositions at 10°C. Our model predicted a higher level of anteiso lipids at 10°C than was observed, indicative of an additional step beyond FabH influencing fatty acid composition at low temperatures. The potential for control of low temperature growth by feeding compounds that result in the production of butyryl-CoA, the precursor of SCFAs that rigidify the membrane and are incompatible with growth at low temperatures, is recognized.

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The contractile and enzymatic activities of myosin VI are regulated by calcium binding to associated calmodulin light chains. We have used transient phosphorescence anisotropy (TPA) to monitor the microsecond rotational dynamics of erythrosin iodoacetamide-labeled actin with strongly-bound myosin VI (MVI) and to evaluate the effect of MVI-bound calmodulin (CaM) light chain on actin filament dynamics. MVI binding lowers the amplitude but accelerates actin filament microsecond dynamics in a Ca2+ - and CaM-dependent manner, as indicated from an increase in the final anisotropy and a decrease in the correlation time of TPA decays. MVI with bound apo-CaM or Ca2+ - CaM weakly affects actin filament microsecond dynamics, relative to other myosins (e.g. muscle myosin II and myosin Va). CaM dissociation from bound MVI damps filament rotational dynamics (i.e. increases the torsional rigidity), such that the perturbation is comparable to that induced by other characterized myosins. Analysis of individual actin filament shape fluctuations imaged by fluorescence microscopy reveals a correlated effect on filament bending mechanics. These data support a model in which Ca2+ - dependent CaM binding to the IQ domain of MVI is linked to an allosteric reorganization of the actin-binding site(s), which alters the structural dynamics and the mechanical rigidity of actin filaments. Such modulation of filament dynamics may contribute to the Ca2+ – and CaM–dependent regulation of myosin VI motility and ATP utilization.