Characterization of a Serine Hydrolase Targeted by Acyl-protein Thioesterase Inhibitors in Toxoplasma gondii

In eukaryotic organisms, cysteine palmitoylation is an important reversible modification that impacts protein targeting, folding, stability, and interactions with partners. Evidence suggests that protein palmitoylation contributes to key biological processes in Apicomplexa with the recent palmitome of the malaria parasite Plasmodium falciparum reporting over 400 substrates that are modified with palmitate by a broad range of protein S-acyl transferases. Dynamic palmitoylation cycles require the action of an acyl-protein thioesterase (APT) that cleaves palmitate from substrates and conveys reversibility to this posttranslational modification. In this work, we identified candidates for APT activity in Toxoplasma gondii. Treatment of parasites with low micromolar concentrations of β-lactone- or triazole urea-based inhibitors that target human APT1 showed varied detrimental effects at multiple steps of the parasite lytic cycle. The use of an activity-based probe in combination with these inhibitors revealed the existence of several serine hydrolases that are targeted by APT1 inhibitors. The active serine hydrolase, TgASH1, identified as the homologue closest to human APT1 and APT2, was characterized further. Biochemical analysis of TgASH1 indicated that this enzyme cleaves substrates with a specificity similar to APTs, and homology modeling points toward an APT-like enzyme. TgASH1 is dispensable for parasite survival, which indicates that the severe effects observed with the β-lactone inhibitors are caused by the inhibition of non-TgASH1 targets. Other ASH candidates for APT activity were functionally characterized, and one of them was found to be resistant to gene disruption due to the potential essential nature of the protein.     

The MAN antigens are non-lamin constituents of the nuclear lamina in vertebrate cells

The characterization of the human antiserum designated MAN has led to the identification of a subset of non-lamin proteins that are exclusively located at the nuclear periphery in all vertebrate cell types examined, from human to fish. Immunoreactive protein species were whown to comprise three major polypeptides of M _r 78000, 58000 and 40000. These antigens co-partitioned with the nuclear lamina during in situ isolation of nuclear matrices from lamin A/C-positive and-negative mammlian cells. Using double immunofluorescence, the spatial relationship of MAN antigens to type-A and type-B lamins was further examined throughout the cell cycle of lamin A/C-positive mammalian cells. In interphase HeLa and 3t3 cells, MAN antigens colocalized with both types of lamins at the periphery of the nucleus, but were absent from intranuclear foci of lamin B. As HeLa cells proceeded into mitosis, MAN antigens were seen to segregate from lamins A/C and coredistribute with lamin B. Lamins A/C disassembled during late prophase/early prometaphase and reassociated with chromatin in telophase/cytokinesis. In contrast, MAN antigens and lamin B dispersed late during prometaphase and reassembled on chromosomes in anaphase. Altogether, our data suggest that MAN antigens may play key functions in the maintenance of the structural integrity of the nuclear compartment in vertebrate cells.     

Targeted disruption of Aldh1a1 (Raldh1) provides evidence for a complex mechanism of retinoic acid synthesis in the developing retina

Genetic studies have shown that retinoic acid (RA) signaling is required for mouse retina development, controlled in part by an RA-generating aldehyde dehydrogenase encoded by Aldh1a2 (Raldh2) expressed transiently in the optic vesicles. We examined the function of a related gene, Aldh1a1 (Raldh1), expressed throughout development in the dorsal retina. Raldh1(-/-) mice are viable and exhibit apparently normal retinal morphology despite a complete absence of Raldh1 protein in the dorsal neural retina. RA signaling in the optic cup, detected by using a RARE-lacZ transgene, is not significantly altered in Raldh1(-/-) embryos at embryonic day 10.5, possibly due to normal expression of Aldh1a3 (Raldh3) in dorsal retinal pigment epithelium and ventral neural retina. However, at E16.5 when Raldh3 is expressed ventrally but not dorsally, Raldh1(-/-) embryos lack RARE-lacZ expression in the dorsal retina and its retinocollicular axonal projections, whereas normal RARE-lacZ expression is detected in the ventral retina and its axonal projections. Retrograde labeling of adult Raldh1(-/-) retinal ganglion cells indicated that dorsal retinal axons project to the superior colliculus, and electroretinography revealed no defect of adult visual function, suggesting that dorsal RA signaling is unnecessary for retinal ganglion cell axonal outgrowth. We observed that RA synthesis in liver of Raldh1(-/-) mice was greatly reduced, thus showing that Raldh1 indeed participates in RA synthesis in vivo. Our findings suggest that RA signaling may be necessary only during early stages of retina development and that if RA synthesis is needed in dorsal retina, it is catalyzed by multiple enzymes, including Raldh1.     

An NS3 serine protease inhibitor abrogates replication of subgenomic hepatitis C virus RNA

The hepatitis C virus (HCV) NS3 protease is essential for polyprotein maturation and viral propagation, and it has been proposed as a suitable target for antiviral drug discovery. An N-terminal hexapeptide cleavage product of a dodecapeptide substrate identified as a weak competitive inhibitor of the NS3 protease activity was optimized to a potent and highly specific inhibitor of the enzyme. The effect of this potent NS3 protease inhibitor was evaluated on replication of subgenomic HCV RNA and compared with interferon-alpha (IFN-alpha), which is currently used in the treatment of HCV-infected patients. Treatment of replicon-containing cells with the NS3 protease inhibitor or IFN-alpha showed a dose-dependent decrease in subgenomic HCV RNA that reached undetectable levels following a 14-day treatment. Kinetic studies in the presence of either NS3 protease inhibitor or IFN-alpha also revealed similar profiles in HCV RNA decay with half-lives of 11 and 14 h, respectively. The finding that an antiviral specifically targeting the NS3 protease activity inhibits HCV RNA replication further validates the NS3 enzyme as a prime target for drug discovery and supports the development of NS3 protease inhibitors as a novel therapeutic approach for HCV infection.     

4-Alkoxy-2,6-diaminopyrimidine derivatives: inhibitors of cyclin dependent kinases 1 and 2

The cyclin dependent kinase (cdk) inhibitor NU6027, 4-cyclohexylmethoxy-5-nitroso-pyrimidine-2,6-diamine (IC(50) vs cdk1/cyclinB1=2.9+/-0.1 microM and IC(50) vs cdk2/cyclinA3=2.2+/-0.6 microM), was used as the basis for the design of a series of 4-alkoxy-2,6-diamino-5-nitrosopyrimidine derivatives. The synthesis and evaluation of 21 compounds as potential inhibitors of cyclin-dependent kinases 1 and 2 is described and the structure-activity relationships relating to NU6027 have been probed. Simple alkoxy- or cycloalkoxy-groups at the O(4)-position were tolerated, with the 4-(2-methylbutoxy)-derivative (IC(50) vs cdk1/cyclinB1=12+/-2 microM and cdk2/cyclinA3=13+/-4 microM) retaining significant activity. Substitutions at the N(6) position were not tolerated. Replacement of the 5-nitroso substituent with ketone, oxime and semicarbazone groups essentially abolished activity. However, the derivative bearing an isosteric 5-formyl group, 2,6-diamino-4-cyclohexylmethoxy-pyrimidine-5-carbaldehyde, showed modest activity (IC(50) vs cdk1/cyclinB1=35+/-3 microM and cdk2/cyclinA3=43+/-3 microM). The X-ray crystal structure of the 5-formyl compound bound to cdk2 has been determined to 2.3A resolution. The intramolecular H-bond deduced from the structure with NU6027 bound to cdk2 is not evident in the structure with the corresponding formyl compound. Thus the parent compound, 4-cyclohexylmethoxy-5-nitrosopyrimidine-2,6-diamine (NU6027), remains the optimal basis for future structure-activity studies for cyclin-dependent kinase inhibitors in this series.     

Practitioner versus participant perspectives on conservation tenders

Extensive clearing of native vegetation on rural properties throughout Australia over the last century has generated significant damage to biodiversity. Conservation tenders have been broadly used to reduce the detrimental impact of such widespread clearance. To date, Australian conservation tender research has largely been limited to program evaluations and landholder surveys. This analysis differs by comparing and contrasting the views of non-landholders involved with these programs with those of participant landholders. The non-landholder group consists of individuals with involvement in conservation tenders across Australia. By contrast, the landholder group consists of individuals with participation experience in a series of Victorian tender initiatives. Each group is surveyed to investigate the drivers of cost-effectiveness within tender programs and landholder participation. This analysis explores these two perspectives, revealing important convergences and divergences in opinion. Both practitioners and landholders indicate that programs supported by close agency–landholder relationships and offering flexibility to landholders are most likely to succeed, particularly where landholders perceive the tender instrument to be fair. Whilst practitioners emphasise the role of transaction costs issues and program characteristics in achieving cost-effective biodiversity outcomes, landholders indicate that these factors are less important to participation rates. This research is important to guide future implementation of tender programs both in Australia and internationally.     

Vitamin K-dependent carboxylase: Evidence for a semiquinone radical intermediate

Nanosecond laser flash photolysis has been used to produce and identify the vitamin K semiquinone (radical) from vitamin K dihydroquinone and to observe its formation and decay in the presence of vitamin K-dependent carboxylase (epoxidase). The activity of vitamin K-dependent carboxylase is not decreased by exposure to the laser. Absorbance of the semiquinone is proportional to enzyme concentration and is stimulated by a synthetic substrate, PheLeuGluGluIle. Stabilization of the semiquinone is observed in the presence of the enzyme. The semiquinone is rapidly destroyed in the presence of inhibitors of vitamin K-dependent carboxylase and vitamin K epoxidase.