Structural basis for TRIM72 oligomerization during membrane damage repair.

Tripartite Motif Protein 72 (TRIM72, also named MG53) mediates membrane damage repair through membrane fusion and exocytosis. During injury, TRIM72 molecules form intermolecular disulfide bonds in response to the oxidative environment and TRIM72 oligomers are proposed to connect vesicles to the plasma membrane and promote membrane fusion in conjunction with other partners like dysferlin and caveolin. However, the detailed mechanism of TRIM72 oligomerization and action remains unclear. Here we present the crystal structure of TRIM72 B-box-coiled-coil-SPRY domains (BCC-SPRY), revealing the molecular basis of TRIM72 oligomerization, which is closely linked to disulfide bond formation. Through structure-guided mutagenesis, we have identified and characterized key residues that are important for the membrane repair function of TRIM72. Our results also demonstrate that TRIM72 interacts with several kinds of negatively charged lipids in addition to phosphatidylserine. Our work provides a structural foundation for further mechanistic studies as well as the clinical application of TRIM72.

Cadherin repeat 5 mutation associated with Bt resistance in a field-derived strain of pink bollworm.

Evolution of resistance by pests reduces the benefits of transgenic crops that produce insecticidal proteins from Bacillus thuringiensis (Bt). Here we analyzed resistance to Bt toxin Cry1Ac in a field-derived strain of pink bollworm (Pectinophora gossypiella), a global pest of cotton. We discovered that the r14 allele of the pink bollworm cadherin gene (PgCad1) has a 234-bp insertion in exon 12 encoding a mutant PgCad1 protein that lacks 36 amino acids in cadherin repeat 5 (CR5). A strain homozygous for this allele had 237-fold resistance to Cry1Ac, 1.8-fold cross-resistance to Cry2Ab, and developed from neonate to adult on Bt cotton producing Cry1Ac. Inheritance of resistance to Cry1Ac was recessive and tightly linked with r14. PgCad1 transcript abundance in midgut tissues did not differ between resistant and susceptible larvae. Toxicity of Cry1Ac to transformed insect cells was lower for cells expressing r14 than for cells expressing wild-type PgCad1. Wild-type PgCad1 was transported to the cell membrane, whereas PgCad1 produced by r14 was not. In larval midgut tissue, PgCad1 protein occurred primarily on the brush border membrane only in susceptible larvae. The results imply r14 mediates pink bollworm resistance to Cry1Ac by reduced translation, increased degradation, and/or mislocalization of cadherin.

GATAe transcription factor is involved in Bacillus thuringiensis Cry1Ac toxin receptor gene expression inducing toxin susceptibility.

The insecticidal Cry toxins produced by Bacillus thuringiensis (Bt) are powerful tools for insect control. Cry toxin receptors such as cadherin (CAD), ABCC2 transporter and alkaline phosphatase (ALP), located on insect midgut cells, are needed for Cry toxicity. Although insect cell lines are useful experimental models for elucidating toxin action mechanism, most of them show low expression of Cry-receptors genes. The GATA transcription factor family plays important roles in regulating development and differentiation of intestine stem cells. Here, we investigated whether GATAs transcription factors are involved in the expression of Cry1Ac-receptors genes, using multiple insect cell lines. Four GATA genes were identified in the transcriptome of the midgut tissue from the lepidopteran larvae Helicoverpa armigera. These HaGATA genes were transiently expressed in three lepidopteran cell lines, Spodoptera frugiperda Sf9, H. armigera QB-Ha-E5 and Trichoplusia ni Hi5. Analysis of transcription activity using transcriptional gene-fusions showed that only H. armigera GATAe (HaGATAe) significantly increased the transcription of CAD, ABCC2 and ALP receptors genes in all insect cell lines. Key DNA regions for HaGATAe regulation were identified in the promoter sequence of these Cry-receptors genes by using promoter deletion mapping. The transient expression of HaGATAe in these cell lines, conferred sensitivity to Cry1Ac toxin, although in Hi5 cells the susceptibility to Cry1Ac was lower than in other two cell lines. High sensitivity to Cry1Ac correlated with simultaneous transcription of ABCC2 and CAD genes in Sf9 and QB-Ha-E5 cells. Our results reveal that HaGATAe enhances transcription of several lepidopteran Cry1Ac receptor genes in cultured insect cells.

The Cadherin Cry1Ac Binding-Region is Necessary for the Cooperative Effect with ABCC2 Transporter Enhancing Insecticidal Activity of Bacillus thuringiensis Cry1Ac Toxin.

Bacillus thuringiensis Cry1Ac toxin binds to midgut proteins, as cadherin (CAD) and ABCC2 transporter, to form pores leading to larval death. In cell lines, co-expression of CAD and ABCC2 enhance Cry1Ac toxicity significantly, but the mechanism remains elusive. Here, we show that the expression of Helicoverpa armigera CAD (HaCAD-GFP) in Hi5 cells induces susceptibility to Cry1Ac and enhanced Cry1Ac toxicity when co-expressed with H. armigera ABCC2 (HaABCC2-GFP), since Cry1Ac toxicity increased 735-fold compared to Hi5 cells expressing HaCAD-GFP alone or 28-fold compared to HaABCC2-GFP alone. In contrast, the expression of the Spodoptera litura CAD (SlCAD-GFP) in Hi5 cells did not induce susceptibility to Cry1Ac nor it potentiated Cry1Ac toxicity with HaABCC2-GFP. To identify the CAD regions involved in the enhancement of Cry1Ac toxicity with ABCC2, the different CAD domains were replaced between SlCAD-GFP and HaCad-GFP proteins, and cytotoxicity assays were performed in Hi5 cells in the absence or presence of HaABCC2-GFP. The HaCAD toxin-binding region (TB), specifically the CAD repeat-11, was necessary to enhance Cry1Ac toxicity with ABCC2. We propose that CAD TB is involved in recruiting Cry1Ac to localize it in a good position for its interaction with the ABCC2, resulting in efficient toxin membrane insertion enhancing Cry1Ac toxicity.

Transposon insertion causes cadherin mis-splicing and confers resistance to Bt cotton in pink bollworm from China.

Transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt) are cultivated extensively, but rapid evolution of resistance by pests reduces their efficacy. We report a 3,370-bp insertion in a cadherin gene associated with resistance to Bt toxin Cry1Ac in the pink bollworm (Pectinophora gossypiella), a devastating global cotton pest. We found the allele (r15) harboring this insertion in a field population from China. The insertion is a miniature inverted repeat transposable element (MITE) that contains two additional transposons and produces two mis-spliced transcript variants (r15A and r15B). A strain homozygous for r15 had 290-fold resistance to Cry1Ac, little or no cross-resistance to Cry2Ab, and completed its life cycle on Bt cotton producing Cry1Ac. Inheritance of resistance was recessive and tightly linked with r15. For transformed insect cells, susceptibility to Cry1Ac was greater for cells producing the wild-type cadherin than for cells producing the r15 mutant proteins. Recombinant cadherin protein occurred on the cell surface in cells transformed with the wild-type or r15A sequences, but not in cells transformed with the r15B sequence. The similar resistance of pink bollworm to Cry1Ac in laboratory- and field-selected insects from China, India and the U.S. provides a basis for developing international resistance management practices.

Pink Bollworm Resistance to Bt Toxin Cry1Ac Associated with an Insertion in Cadherin Exon 20.

Insecticidal proteins from Bacillus thuringiensis (Bt) are widely used to control insect pests, but their efficacy is reduced when pests evolve resistance. We report on a novel allele (r16) of the cadherin gene (PgCad1) in pink bollworm (Pectinophora gossypiella) associated with resistance to Bt toxin Cry1Ac, which is produced by transgenic cotton. The r16 allele isolated from a field population in China has 1545 base pairs of a degenerate transposon inserted in exon 20 of PgCad1, which generates a mis-spliced transcript containing a premature stop codon. A strain homozygous for r16 had 300-fold resistance to Cry1Ac, 2.6-fold cross-resistance to Cry2Ab, and completed its life cycle on transgenic Bt cotton producing Cry1Ac. Inheritance of Cry1Ac resistance was recessive and tightly linked with r16. Compared with transfected insect cells expressing wild-type PgCad1, cells expressing r16 were less susceptible to Cry1Ac. Recombinant cadherin protein was transported to the cell membrane in cells transfected with the wild-type PgCad1 allele, but not in cells transfected with r16. Cadherin occurred on brush border membrane vesicles (BBMVs) in the midgut of susceptible larvae, but not resistant larvae. These results imply that the r16 allele mediates Cry1Ac resistance in pink bollworm by interfering with the localization of cadherin.

A single amino acid polymorphism in ABCC2 loop 1 is responsible for differential toxicity of Bacillus thuringiensis Cry1Ac toxin in different Spodoptera (Noctuidae) species.

Bacillus thuringiensis Cry toxins exert their toxicity by forming membrane pores after binding with larval midgut membrane proteins known as receptors. Spodoptera litura and Spodoptera frugiperda belong to the same genus, but S. litura is tolerant to Cry1Ac, while S. frugiperda is susceptible. The mechanism involved in the differential toxicity of Cry1Ac to these insect species is not understood. Amino acid sequences analysis of ABCC2, a well-recognized Cry1Ac receptor, from both species showed high sequence identity. Hi5 insect cells expressing SfABCC2 from S. frugiperda were 65-fold more susceptible than those expressing the SlABCC2 from S. litura. Substitution of fragments, point mutations and deletions between the ABCC2 of the two species revealed that ABCC2 amino acid Q125 from SfABCC2 or E125 from SlABCC2 was key factor for the differential Cry1Ac toxicity to Hi5 cells expressing these receptors. Consistently with this, cells expressing Helicoverpa armigera HaABCC2Q122-GFP, were more susceptible to Cry1Ac than cells expressing HaABCC2E122-GFP mutant. Q125 or E125 is located in a predicted exposed loop 1 region of ABCC2 indicating that this region could be important for Cry1Ac binding. These findings identified a single amino acid residue located in loop 1 of ABCC2 transporter as responsible for the different levels of susceptibility to Cry1Ac among various lepidopteran species.

Resistance to Bacillus thuringiensis linked with a cadherin transmembrane mutation affecting cellular trafficking in pink bollworm from China.

Evolution of pest resistance reduces the efficacy of insecticidal proteins from the gram-positive bacterium Bacillus thuringiensis (Bt) used widely in sprays and transgenic crops. In some previously studied strains of three major lepidopteran pests, resistance to Bt toxin Cry1Ac is associated with mutations disrupting the extracellular or cytoplasmic domains of cadherin proteins that bind Cry1Ac in the midgut of susceptible larvae. Here we report the first case of a cadherin transmembrane mutation associated with insect resistance to Bt. We discovered this mutation in a strain of the devastating global cotton pest, the pink bollworm (Pectinophora gossypiella), derived from a field population in the Yangtze River Valley of China. The mutant allele analyzed here has a 207 base pair deletion and encodes a cadherin protein lacking its transmembrane domain. Relative to a susceptible strain, a strain homozygous for this allele had 220-fold resistance to Cry1Ac and 2.1-fold cross-resistance to Cry2Ab. On transgenic cotton plants producing Cry1Ac, no susceptible larvae survived, but the resistant strain completed its life cycle. Inheritance of resistance to Cry1Ac was autosomal, recessive and tightly linked with the cadherin gene. Transportation of cadherin protein to the cell membrane and susceptibility to Cry1Ac occurred in transfected insect cells expressing the wild type cadherin allele, but not in transfected insect cells expressing the mutant cadherin allele. The results imply that the mutant allele analyzed here confers resistance to Cry1Ac by disrupting cellular trafficking of cadherin.

FOXA transcriptional factor modulates insect susceptibility to Bacillus thuringiensis Cry1Ac toxin by regulating the expression of toxin-receptor ABCC2 and ABCC3 genes.

Cry toxins produced by Bacillus thuringiensis (Bt) are insecticidal proteins widely used in insect control. Recently, it was shown that ATP-binding cassette transporter proteins (ABC) such as ABCC2, ABCC3, ABCG1 and ABCA2 are implicated in the insecticidal action of Cry toxins as putative receptors. However, the transcriptional regulators involved in the expression of ABC transporter genes remain unknown. Sequence analysis of promoter regions of ABCC2 gene from Helicoverpa armigera and ABCC3 gene from Spodoptera litura Sl-HP cultured cells, revealed the potential participation of Forkhead box protein A (FOXA), a transcription factor that regulates the expression of genes through remodeling chromatin. To determine if FOXA was involved in regulating expression of ABCC2 and ABCC3 genes, the expression of FOXA, ABCC2 and ABCC3 was compared in Sl-HP cells that are sensitive to Cry1Ac toxin with those in S. frugiperda Sf9 cells that are not sensitive to the toxin. Expression levels of those genes were significantly higher in Sl-HP than in Sf9 cells. Transient expression of FOXA in Sf9 cells activated ABCC2 and ABCC3 transcription, which directly correlated with enhanced Cry1Ac-susceptibility in these cells. Silencing of FOXA gene expression by RNAi in H. armigera larvae resulted in a decreased expression of ABCC2 and ABCC3 without affecting expression of other Cry toxin receptor genes such as alkaline phosphatase, aminopeptidase or cadherin. Silencing of FOXA gene expression also resulted in a Cry1Ac-tolerant phenotype since lower mortality and higher pupation rate were observed in diet containing Cry1Ac protoxin in comparison with the control group. These results demonstrate that FOXA up-regulates expression of the Cry1Ac-toxin receptor ABCC2 and ABCC3 genes, and that lower FOXA expression correlates with tolerance to Cry toxin in cell lines and in lepidopteran larvae.

NMR structure and function of Helicoverpa armigera sterol carrier protein-2, an important insecticidal target from the cotton bollworm.

The cotton bollworm, Helicoverpa armigera, has developed strong resistance to many insecticides. Sterol Carrier Protein-2 (SCP-2) is an important non-specific lipid transfer protein in insects and appears to be a potential new target. In order to elucidate the structure and function of Helicoverpa armigera SCP-2 (HaSCP-2), NMR spectroscopy, docking simulations, mutagenesis and bioassays were performed. HaSCP-2 composed of five α-helices and four stranded ß-sheets. The folds of α-helices and ß-sheets interacted together to form a hydrophobic cavity with putative entrance and exit openings, which served as a tunnel for accommodating and transporting of lipids. Several sterols and fatty acids could interact with HaSCP-2 via important hydrophobic sites, which could be potential targets for insecticides. Mutagenesis experiments indicated Y51, F53, F89, F110, I117 and Q131 may be the key functional sites. HaSCP-2 showed high cholesterol binding activity and SCP-2 inhibitors (SCPIs) could inhibit the biological activity of HaSCP-2. SCPI-treated larvae at young stage showed a significant decrease of cholesterol uptake in vivo. Our study describes for the first time a NMR structure of SCP-2 in lepidopteran H. armigera and reveals its important function in cholesterol uptake, which facilitates the screening of effective insecticides targeting the insect cholesterol metabolism.

A rapid UPLC method for simultaneous determination of eleven components in 'Ge-Gen-Qin-Lian' decoction.

BACKGROUND: 'Ge-Gen-Qin-Lian' Decoction derived from 'Shang-Han-Lun' compiled by Zhang Zhongjing. It is widely used in the treatment of acute gastroenteritis, bacillary dysentery, virus diarrhea. This paper describes a sensitive and specific assay for the determination of the 11-marker compounds using ultra performance liquid chromatography (UPLC). OBJECTIVE: To develop an UPLC method for simultaneous determination of 11 bioactive compounds in 'Ge-Gen-Qin-Lian' preparations. MATERIALS AND METHODS: The chromatography analysis was performed on an Agilent Proshell 120 EC-C18 column (4.6 × 50 mm, 2.7 µm) at 30°C with a gradient elution of methanol, 0.5% formic acid and 0.5% ammonium acetate at a flow rate 1.0 ml/min and UV detected at 270 nm. RESULTS: All calibration curves showed good linear regression (r ≥ 0.9993) within tested ranges. Limits of detection (LOD) and limits of quantification (LOQ) fell in the range between 0.0691-1.04 µg/ml and 0.23-3.43 µg/ml, respectively. The mean recovery of each herbal medicine ranged from 96.60 to 102.11%. CONCLUSION: The method was validated for repeatability, precision, stability, accuracy, and selectivity. The validated method was successfully applied to simultaneous analysis of these active components in 'Ge-Gen-Qin-Lian' decoction.