Atg22 recycles amino acids to link the degradative and recycling functions of autophagy

In response to stress conditions (such as nutrient limitation or accumulation of damaged organelles) and certain pathological situations, eukaryotic cells use autophagy as a survival mechanism. During nutrient stress the main purpose of autophagy is to degrade cytoplasmic materials within the lysosome/vacuole lumen and generate an internal nutrient pool that is recycled back to the cytosol. This study elucidates a molecular mechanism for linking the degradative and recycling roles of autophagy. We show that in contrast to published studies, Atg22 is not directly required for the breakdown of autophagic bodies within the lysosome/vacuole. Instead, we demonstrate that Atg22, Avt3, and Avt4 are partially redundant vacuolar effluxers, which mediate the efflux of leucine and other amino acids resulting from autophagic degradation. The release of autophagic amino acids allows the maintenance of protein synthesis and viability during nitrogen starvation. We propose a "recycling" model that includes the efflux of macromolecules from the lysosome/vacuole as the final step of autophagy.     

Discovery of novel inhibitors of Bcl-xL using multiple high-throughput screening platforms

Bcl-xL is a member of the Bcl-2 family of proteins that are implicated to play a vital role in several diseases including cancer. Bcl-xL suppresses apoptosis; thus the inhibition of Bcl-xL function could restore the apoptotic process. To identify antagonists of Bcl-xL function, two ultra-high-throughput screens were implemented. An activity assay utilized fluorescence polarization, based on the binding of fluorescein-labeled peptide [the BH3 domain of BAD protein (F-Bad 6)] to Bcl-xL. A 384-well plate assay with mixtures of 10 drug compounds per well, combined with a fast plate reader, resulted in a throughput of 46,080 data points/day. Utilizing this screening format, 370,400 compounds were screened in duplicate and 425 inhibitors with an IC(50) below 100 microM were identified. The second assay format, affinity selection/mass spectrometry (ASMS), used ultrafiltration to separate Bcl-xL binders from nonbinders in mixtures of 2400 compounds. The bound species were subsequently separated from the protein and analyzed by flow injection electrospray mass spectrometry. Utilizing the ASMS format, 263,382 compounds were screened in duplicate and 29 binders with affinities below 100 microM were identified. Two novel classes of Bcl-xL inhibitors were identified by both methods and confirmed to bind (13)C-labeled Bcl-xL using heteronuclear magnetic resonance spectroscopy.     

Double-stranded RNA interference of a rice PI/GLO paralog, OsMADS2, uncovers its second-whorl-specific function in floral organ patterning

Unlike many eudicot species, grasses have duplicated PI/GLO-like genes. Functional analysis of one of the rice PI/GLO paralogs, OsMADS2, is reported here. Our data demonstrate its essential role in lodicule development and implicate the second PI/GLO paralog, OsMADS4, to suffice for stamen specification. We provide the first evidence for differential contributions of grass PI/GLO paralogs in patterning second- and third-whorl floral organs.     

Changes in lipid peroxidation and free radical scavengers in kidney of hypothyroid and hyperthyroid rats

Kidney weight was significantly decreased in hypothyroidism (induced by Na131I administration) and increased in hyperthyroidism (induced by thyroxine treatment) as compared to control in female Wistar rats. The tissue lipid peroxidation level remained unchanged in hyperthyroid rats but significantly increased in hypothyroid rats. Superoxide dismutase was decreased in both experimental groups but more so in hyperthyroid rats. Catalase was reduced significantly in hyperthyroid rats but remained unaffected in hypothyroid rats. Tissue glutathione peroxidase (GPx) activity was increased while reduced glutathione levels remained unaltered in both hypothyroid and hyperthyroid rats. Plasma GPx activity was significantly low in both the hypothyroid and hyperthyroid rats. The results suggest alterations in the oxidative stress in hypothyroid and hyperthyroid rat kidneys with concomitant changes of free radical scavengers.     

Polar localization of CheA2 in Rhodobacter sphaeroides requires specific Che homologs

Rhodobacter sphaeroides is a motile bacterium that has multiple chemotaxis genes organized predominantly in three major operons (cheOp(1), cheOp(2), and cheOp(3)). The chemoreceptor proteins are clustered at two distinct locations, the cell poles and in one or more cytoplasmic clusters. One intriguing possibility is that the physically distinct chemoreceptor clusters are each composed of a defined subset of specific chemotaxis proteins, including the chemoreceptors themselves plus specific CheW and CheA proteins. Here we report the subcellular localization of one such protein, CheA(2), under aerobic and photoheterotrophic growth conditions. CheA(2) is predominantly clustered and localized at the cell poles under both growth conditions. Furthermore, its localization is dependent upon one or more genes in cheOp(2) but not those of cheOp(1) or cheOp(3). In E. coli, the polar localization of CheA depends upon CheW. The R. sphaeroides cheOp(2) contains two cheW genes. Interestingly, CheW(2) is required under both aerobic and photoheterotrophic conditions, whereas CheW(3) is not required under aerobic conditions but appears to play a modest role under photoheterotrophic conditions. This suggests that R. sphaeroides contains at least two distinct chemotaxis complexes, possibly composed of proteins dedicated for each subcellular location. Furthermore, the composition of these spatially distinct complexes may change under different growth conditions.     

Putting thought to paper: a microARCS protease screen

In micro-arrayed compound screening (microARCS), an agarose gel is used as a reaction vessel that maintains humidity and compound location as well as being a handling system for reagent addition. Two or more agarose gels may be used to bring test compounds, targets, and reagents together, relying on the pore size of the gel matrix to regulate diffusion of reactants. It is in the microenvironment of the agarose matrix that all the components of an enzymatic reaction interact and result in inhibitable catalytic activity. In an effort to increase the throughput of microARCS-based screens, reduce the effort involved in manipulating agarose gels, and reduce costs, blotter paper was used rather than a second agarose gel to introduce a substrate to a gel containing a target enzyme. In this assay, the matrix of the blotter paper did not prevent the substrate from diffusing into the enzyme gel. The compound density of the microARCS format, the ease of manipulating sheets of paper for reagent addition, and a scheduled protocol for running multiple gels allowed for a throughput capacity of more than 200,000 tests per hour. A protease assay was developed and run in the microARCS format at a rate of 200,000 tests per hour using blotter paper to introduce the substrate. Picks in the primary screen were retested in the microARCS format at a density of 384 compounds per sheet. IC(50) values were confirmed in a 96-well plate format. The screen identified several small molecule inhibitors of the enzyme. The details of the screening format and the analysis of the hits from the screen are presented.     

The discovery of acylated beta-amino acids as potent and orally bioavailable VLA-4 antagonists

Acylated beta-amino acids are described as potent, specific and orally bioavailable antagonists of VLA-4. The initial lead was identified from a combinatorial library. Subsequent optimization using a traditional medicinal chemistry approach led to significant improvement in potency (up to 8-fold) while maintaining good pharmacokinetic properties.     

Application of Micro Arrayed Compound Screening (microARCS) to identify inhibitors of caspase-3

Micro Arrayed Compound Screening (microARCS) is a miniaturized ultra-high-throughput screening platform developed at Abbott Laboratories. In this format, 8,640 discrete compounds are spotted and dried onto a polystyrene sheet, which has the same footprint as a 96-well plate. A homogeneous time-resolved fluorescence assay format (LANCE) was applied to identify the inhibitors of caspase-3 using a peptide substrate labeled with a fluorescent europium chelate and a dabcyl quencher. The caspase-3 enzyme was cast into a thin agarose gel, which was placed on a sheet containing test compounds. A second gel containing caspase substrate was then laid above the enzyme gel to initiate the reaction. Caspase-3 cleaves the substrate and separates the europium from the quencher, giving rise to a time-resolved fluorescent signal, which was detected using a ViewLux charge-coupled device imaging system. Potential inhibitors of caspase-3 appeared as dark spots on a bright fluorescent background. Results from the microARCS assay format were compared to those from a conventional 96-well plate-screening format.     

Pentoxifylline functions as an adjuvant in vivo to enhance T cell immune responses by inhibiting activation-induced death

Modalities for inducing long-lasting immune responses are essential components of vaccine design. Most currently available immunological adjuvants empirically used for this purpose cause some inflammation, limiting clinical acceptability. We show that pentoxifylline (PF), a phosphodiesterase (PDE) inhibitor in common clinical use, enhances long-term persistence of T cell responses, including protective responses to a bacterial immunogen, Salmonella typhimurium, via a cAMP-dependent protein kinase A-mediated effect on T cells if given to mice for a brief period during immunization. PF inhibits activation-mediated loss of superantigen-reactive CD4 as well as CD8 T cells in vivo without significantly affecting their activation, and inhibits activation-induced death and caspase induction in stimulated CD4 as well as CD8 T cells in vitro without preventing the induction of activation markers. Consistent with this ability to prevent activation-induced death in not only CD4 but also CD8 T cells, PF also enhances the persistence of CD8 T cell responses in vivo. Thus, specific inhibition of activation-induced T cell apoptosis transiently during immune priming is likely to enhance the persistence of CD4 and CD8 T cell responses to vaccination, and pharmacological modulators of the cAMP pathway already in clinical use can be used for this purpose as immunological adjuvants.