Physiological Functions of the COPI Complex in Higher Plants

COPI vesicles are essential to the retrograde transport of proteins in the early secretory pathway. The COPI coatomer complex consists of seven subunits, termed α-, β-, β′-, γ-, δ-, ε-, and ζ-COP, in yeast and mammals. Plant genomes have homologs of these subunits, but the essentiality of their cellular functions has hampered the functional characterization of the subunit genes in plants. Here we have employed virus-induced gene silencing (VIGS) and dexamethasone (DEX)-inducible RNAi of the COPI subunit genes to study the in vivo functions of the COPI coatomer complex in plants. The β′-, γ-, and δ-COP subunits localized to the Golgi as GFP-fusion proteins and interacted with each other in the Golgi. Silencing of β′-, γ-, and δ-COP by VIGS resulted in growth arrest and acute plant death in Nicotiana benthamiana, with the affected leaf cells exhibiting morphological markers of programmed cell death. Depletion of the COPI subunits resulted in disruption of the Golgi structure and accumulation of autolysosome-like structures in earlier stages of gene silencing. In tobacco BY-2 cells, DEX-inducible RNAi of β′-COP caused aberrant cell plate formation during cytokinesis. Collectively, these results suggest that COPI vesicles are essential to plant growth and survival by maintaining the Golgi apparatus and modulating cell plate formation.     

Isolation and characterization of a <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> strain capable of degrading zearalenone

The worldwide contamination of cereals, oilseeds, and other crops by mycotoxin-producing moulds is a significant problem. Mycotoxins have adverse effects on humans and animals that result in illnesses and economic losses. Reduction or elimination of mycotoxin contamination in food and feed is an important issue. This study aimed to screen soil bacteria for degradation of zearalenone (ZEN). A pure culture of strain CK1 isolated from soil samples showed most capable of degradation of ZEN. Using physiological, biochemical, and 16S rRNA gene sequence analysis methods, CK1 was identified as Bacillus licheniformis. Addition of 2 ppm of ZEN in Luria–Bertani (LB) medium, B. licheniformis CK1 decreased 95.8% of ZEN after 36 h of incubation. In ZEN-contaminated corn meal medium, B. licheniformis CK1 decreased more than 98% of ZEN after 36 h of incubation. In addition, B. licheniformis CK1 was non-hemolytic, non-enterotoxin producing, and displayed high levels of extracellular xylanase, cellulase, and protease activities. These findings suggest that B. licheniformis CK1 could be used to reduce the concentrations of ZEN and improve the digestibility of nutrients in feedstuffs simultaneously.     

Structure and mechanism of Escherichia coli glutathionylspermidine amidase belonging to the family of cysteine; histidine-dependent amidohydrolases/peptidases

The bifunctional Escherichia coli glutathionylspermidine synthetase/amidase (GspSA) catalyzes both the synthesis and hydrolysis of Gsp. Its amidase domain (GspA), which catalyzes the hydrolysis of Gsp into glutathione and spermidine, plays an important role in redox sensing and protein S-thiolation. To gain insight of the regulation and catalytic mechanism of and further understand the recycling of the Gsp dimer and Gsp-S-protein adducts, we solved two crystal structures of GspA and GspSA both with the C59A mutation and bound with the substrate, Gsp. In both structures, Cys59, His131, and Glu147 form the catalytic triad, which is similar to other cysteine proteases. Comparison of the GspA_Gsp complex and apo GspSA structures indicates that on binding with Gsp, the side chains of Asn149 and Gln58 of the amidase domain are induced to move closer to the carbonyl oxygen of the cleaved amide bond of Gsp, thereby participating in catalysis. In addition, the helix-loop region of GspA, corresponding to the sequence (30)YSSLDPQEYEDDA(42), involves in regulating the substrate binding. Our previous study indicated that the thiol of Cys59 of GspA is only oxidized to sulfenic acid by H(2)O(2). When comparing the active site of GspA with those of other cysteine proteases, we found that limited space and hydrophobicity of the environment around Cys59 play an important role to inhibit its further oxidation. The structural results presented here not only elucidate the catalytic mechanism and regulation of GspA but also help us to design small molecules to inhibit or probe for the activity of GspA.     

Time-dependent botulinum neurotoxin serotype A metalloprotease inhibitors

Botulinum neurotoxins (BoNTs) are the most lethal of biological substances, and are categorized as class A biothreat agents by the Centers for Disease Control and Prevention. There are currently no drugs to treat the deadly flaccid paralysis resulting from BoNT intoxication. Among the seven BoNT serotypes, the development of therapeutics to counter BoNT/A is a priority (due to its long half-life in the neuronal cytosol and its ease of production). In this regard, the BoNT/A enzyme light chain (LC) component, a zinc metalloprotease responsible for the intracellular cleavage of synaptosomal-associated protein of 25 kDa, is a desirable target for developing post-BoNT/A intoxication rescue therapeutics. In an earlier study, we reported the high throughput screening of a library containing 70,000 compounds, and uncovered a novel class of benzimidazole acrylonitrile-based BoNT/A LC inhibitors. Herein, we present both structure–activity relationships and a proposed mechanism of action for this novel inhibitor chemotype.     

Identification of 11-amino acid peptides that disrupt Notch-mediated processes in <Emphasis Type="Italic">Drosophila</Emphasis>

Background  

The conserved Notch signaling pathway regulates cell fate decisions and maintains stem cells in multicellular organisms. Up-regulation of Notch signaling is observed in several types of cancer and is causally involved in proliferation and survival of cancer cells. Thus, it is of great interest to look for anti-Notch reagents for therapeutic purposes. In model animal Drosophila, Notch signaling restricts selection of sensory organ precursors (SOPs) during external sensory (ES) organ development. To look for novel genes that can suppress Notch signaling, we performed a gain-of-function modifier screen to look for genes that enhance the phenotype of ectopic ES organs induced by overexpression of phyllopod, a gene required for SOP specification.     

Molecular functions of the PP2A regulatory subunit Tap46 in plants

Tap42/α4 is a regulatory subunit of the protein phosphatase 2A (PP2A) family of phosphatases and plays a role in the target of rapamycin (TOR) pathway that regulates cell growth, ribosome biogenesis, translation and cell cycle progression in both yeast and mammals. We determined the cellular functions of Tap46, the plant homolog of Tap42/α4, in both Arabidopsis thaliana and Nicotiana benthamiana. Tap46 associated with the catalytic subunits of PP2A and the PP2A-like phosphatases PP4 and PP6 in vivo. Tap46 was phosphorylated by TOR in vitro, indicating that Tap46 is a direct substrate of TOR kinase. Tap46 deficiency caused cellular phenotypes that are similar to TOR-depletion phenotypes, including repression of global translation and activation of both autophagy and nitrogen recycling. Furthermore, Tap46 depletion regulated total PP2A activity in a time-dependent manner similar to TOR deficiency. These results suggest that Tap46 acts as a positive effector of the TOR signaling pathway in controlling diverse metabolic processes in plants. However, Tap46 silencing caused acute cell death, while TOR silencing only hastened senescence. Furthermore, mitotic cells with reduced Tap46 levels exhibited chromatin bridges at anaphase, while TOR depletion did not cause a similar defect. These findings suggest that Tap46 may have TOR-independent functions as well as functions related to TOR signaling in plants.Key words: acute cell death, autophagy, chromatin bridge, nitrogen mobilization, protein phosphatases, target of rapamycin (TOR)Yeast type 2A phosphatase-associated protein 42 kDa (Tap42) is a regulatory subunit that directly associates with catalytic subunits of the protein phosphatase 2A (PP2A) family of protein phosphatases to make a heterodimer and regulates the activity and substrate specificity of the intact enzyme complex.¹ Functions of Tap42 as a component of the target of rapamycin (TOR) signaling pathway have been well characterized in yeast.^1–3 Tap42-regulated phosphatase activities play a major role in signal transduction mediated by TOR. Accumulating evidence suggest that TOR regulates phosphorylation of target proteins by restraining PP2A activity through Tap42 phosphorylation.^1–3 Rapamycin inhibits TOR activity and also influences Tap42-mediated phosphatase regulation in yeast.^3–5α4, the mammalian homolog of Tap42, also associates with the catalytic subunits of PP2A, PP4 and PP6 to make a heterodimer.⁶ Rapamycin inhibits mammalian TOR (mTOR) activity, but it is not clear whether rapamycin prevents the formation of the α4/PP2Ac complex or whether α4 stimulates or represses PP2Ac activity.^7–9 Interestingly, loss of Tap42 function in Drosophila does not affect TOR-regulated activities, including cell growth, metabolism and S6 kinase activity, but results in mitotic arrest caused by spindle anomalies and subsequent activation of c-Jun N-terminal kinase signaling and apoptosis.¹⁰ Similarly, α4 deletion in mice leads to the rapid onset of apoptosis in both proliferating and differentiated cells, while rapamycin itself does not severely affect adult cells.¹¹ Furthermore, while TOR depletion causes developmental arrest and organ degeneration at the L3 stage in Caenorhabditis elegans, loss of α4 does not reproduce TOR deficiency phenotypes, but mainly leads to a fertility defect.¹² Taken together, these results suggest that the yeast Tap42/TOR paradigm is not completely conserved in higher eukaryotes and that Tap42/α4 functions may not be exclusively dependent on the Tor signaling pathway.In this study, we investigated the in vivo functions and phosphatase regulation of Tap46, the plant Tap42/α4 homolog, in relation to TOR in Nicotiana benthamiana, Arabidopsis and tobacco BY2 cells. Tap46 was shown to interact with the catalytic subunits of PP2A, PP4 and PP6 in vivo. Recombinant Tap46 protein was phosphorylated by immunoprecipitated TOR kinase and its deletion forms in vitro. Dexamethasone-induced RNAi of Tap46 caused dramatic repression of global translation and activation of both autophagy and nitrogen mobilization in the early stages of gene silencing. These phenotypes mimic those of TOR inactivation or TOR deficiency in Arabidopsis, yeast and mammals, indicating that Tap46 is a critical mediator of the Tor pathway in the regulation of these metabolic processes in plants. However, these early phenotypes of Tap46-deficient plants were soon followed by an acute and rapid programmed cell-death (PCD), while TOR silencing only led to growth retardation and premature senescence in Arabidopsis and N. benthamiana, confirming results from a previous study.¹³ The PCD caused by Tap46 deficiency is consistent with the apoptosis induced by loss of Tap42/α4 function in both Drosophila and mice.^10,11 Thus Tap42/α4/Tap46 appears to have a strong anti-apoptotic activity in higher eukaryotes. The underlying mechanisms of PCD activation caused by Tap46 depletion remain to be revealed, but it is possible that the inappropriate modulation of phosphatase activity and aberrant protein phosphorylation led to stress signaling and PCD activation.Another interesting phenotype of Tap46 deficiency is the formation of chromatin bridges in anaphase during mitosis, suggesting a role for Tap46 in plant cell mitotic progression. However, there have been no reports of anaphase bridge formation in tor mutants of any organisms. In Drosophila, loss of Tap42 function causes spindle disorganization and pre-anaphase arrest prior to the onset of apoptosis.¹⁰ In addition, Drosophila mutants with a defective regulatory subunit of PP2A exhibit an increased number of lagging chromosomes and chromatin bridges in anaphase.^14,15 Tap46 likely regulates the functions of PP2A family phosphatases during mitosis by direct association with their catalytic subunits, thereby modulating both the activity and specificity of the enzyme. Accumulating evidence reveals dynamic functions of PP2A during mitosis in both yeast and mammals: PP2A regulates kinetochore function, sister chromatid cohesion, spindle bipolarity and progression to anaphase.^15–17 Counteracting the activity of protein kinases, PP4 has also been implicated in both centrosome maturation and function during mitosis.¹⁸ Based on immunolabeling results, Tap46 was visualized as distinct spots around chromatin and mitotic spindles during mitosis in tobacco BY2 cells (Lee HS and Pai HS, unpublished results). Further studies will address the interacting partners and dynamic relocation of Tap46 during the cell cycle.Our results in this study demonstrated that Tap46 plays an important regulatory role in plant growth and metabolism; a major part of its function appears related to TOR signaling. However, we consistently observed certain phenotypic differences between Tap46-silenced and TOR-silenced Arabidopsis and N. benthamiana plants: an acute and rapid PCD occurred upon Tap46 silencing but not upon TOR silencing, despite a similar degree of gene silencing. Furthermore, we did not observe anaphase bridge formation in mitotic root-tip cells of ethanol-induced TOR RNAi Arabidopsis plants, while chromatin bridges were repeatedly observed in Tap46-silenced tobacco BY2 and Arabidopsis root-tip cells. Although an ancient Tap42/TOR paradigm observed in yeast appears to be conserved in plants, new TOR-independent functions of Tap46 might have evolved, the abrogation of which can cause massive PCD activation and anaphase bridge formation. Tap46 is a major regulator of cellular PP2A activity in plant cells by interacting with multiple phosphatase partners. Unraveling the molecular networks of Tap46 activity and interactions is essential for understanding its TOR-dependent and -independent functions in plants.     

Serological testing versus other strategies for diagnosis of active tuberculosis in India: a cost-effectiveness analysis

Background

Undiagnosed and misdiagnosed tuberculosis (TB) drives the epidemic in India. Serological (antibody detection) TB tests are not recommended by any agency, but widely used in many countries, including the Indian private sector. The cost and impact of using serology compared with other diagnostic techniques is unknown.

Methods and Findings

Taking a patient cohort conservatively equal to the annual number of serological tests done in India (1.5 million adults suspected of having active TB), we used decision analysis to estimate costs and effectiveness of sputum smear microscopy (US$3.62 for two smears), microscopy plus automated liquid culture (mycobacterium growth indicator tube [MGIT], US$20/test), and serological testing (anda-tb ELISA, US$20/test). Data on test accuracy and costs were obtained from published literature. We adopted the perspective of the Indian TB control sector and an analysis frame of 1 year. Our primary outcome was the incremental cost per disability-adjusted life year (DALY) averted. We performed one-way sensitivity analysis on all model parameters, with multiway sensitivity analysis on variables to which the model was most sensitive.If used instead of sputum microscopy, serology generated an estimated 14,000 more TB diagnoses, but also 121,000 more false-positive diagnoses, 102,000 fewer DALYs averted, and 32,000 more secondary TB cases than microscopy, at approximately four times the incremental cost (US$47.5 million versus US$11.9 million). When added to high-quality sputum smears, MGIT culture was estimated to avert 130,000 incremental DALYs at an incremental cost of US$213 per DALY averted. Serology was dominated by (i.e., more costly and less effective than) MGIT culture and remained less economically favorable than sputum smear or TB culture in one-way and multiway sensitivity analyses.

Conclusions

In India, sputum smear microscopy remains the most cost-effective diagnostic test available for active TB; efforts to increase access to quality-assured microscopy should take priority. In areas where high-quality microscopy exists and resources are sufficient, MGIT culture is more cost-effective than serology as an additional diagnostic test for TB. These data informed a recently published World Health Organization policy statement against serological tests. Please see later in the article for the Editors'' Summary