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Anticarsia gemmatalis (velvetbean caterpillar) and Chrysodeixis includens (soybean looper) are two important defoliation pests of soybeans. In the present study, we have investigated the susceptibility and brush border membrane-binding properties of both species to Bacillus thuringiensis Cry1Ea toxin. Bioassays performed in first-instar larvae demonstrated potent activity against both soybean pests in terms of mortality or practical mortality. Competition-binding studies carried out with 125Iodine-labelled Cry1Ea, demonstrated the presence of specific binding sites on the midgut brush border membrane vesicles (BBMV) of both insect species. Heterologous competition-binding experiments indicated that Cry1Ea does not share binding sites with Cry1Ac or Cry1Fa in either soybean pest. This study contributes to the knowledge of Cry1Ea toxicity and midgut binding sites in A. gemmatalis and C. includens and sheds light on the cross-resistance potential of Cry1Ea with other Bt proteins aimed at controlling lepidopteran pests in soybeans.

Insect pests are a major cause of crop losses worldwide, with an estimated economic cost of $470 billion annually. Biotechnological tools have been introduced to control such insects without the need for chemical pesticides; for instance, the development of transgenic plants harbouring genes encoding insecticidal proteins. The Vip3 (vegetative insecticidal protein 3) family proteins from Bacillus thuringiensis convey toxicity to species within the Lepidoptera, and have wide potential applications in commercial agriculture. Vip3 proteins are proposed to exert their insecticidal activity through pore formation, though to date there is no mechanistic description of how this occurs on the membrane. Here we present cryo-EM structures of a Vip3 family toxin in both inactive and activated forms in conjunction with structural and functional data on toxin–membrane interactions. Together these data demonstrate that activated Vip3Bc1 complex is able to insert into membranes in a highly efficient manner, indicating that receptor binding is the likely driver of Vip3 specificity., The Vip3 family proteins from Bacillus thuringiensis are thought to exert their insecticidal activity through pore formation. Here authors present cryo-EM structures of a Vip3 family toxin in both inactive and activated forms and show the activated Vip3Bc1 in its pore forming conformation on the membrane.

Vip3A proteins are important for the control of spodopteran pests in crops, including Spodoptera frugiperda (fall armyworm). Native Vip3Ab1 controls S. frugiperda, but it is ineffective against S. eridania (southern armyworm), a major pest of soybean in South America. Recently, a Vip3Ab1 chimera with a modified C-terminus was described, Vip3Ab1-740, which has increased potency against S. eridania while maintaining activity against S. frugiperda. As S. frugiperda and S. eridania are differentially susceptible to Vip3Ab1, experiments were conducted to identify and understand the mechanism by which this expanded potency is conferred. The role of protein stability, processing, and in vivo effects of Vip3Ab1 and Vip3Ab1-740 in both of these species was investigated. Biochemical characterization of the midgut fluids of these two species indicated no obvious differences in the composition and activity of digestive enzymes, which protease inhibitor studies indicated were likely serine proteases. Histological examination demonstrated that both proteins cause midgut disruption in S. frugiperda, while only Vip3Ab1-740 affects S. eridania. Immunolocalization indicated that both proteins were present in the midgut of S. frugiperda, but only Vip3Ab1-740 was detected in the midgut of S. eridania. We conclude that the gain of toxicity of Vip3Ab1-740 to S. eridania is due to an increase in protein stability in the midgut, which was conferred by C-terminal modification.

In this work, we characterized 2 novel insecticidal proteins; Vip3Ab1 and Vip3Bc1. These proteins display unique insecticidal spectra and have differential rates of processing by lepidopteran digestive enzymes. Furthermore, we have found that both proteins exist as tetramers in their native state before and after proteolysis. In addition, we expressed truncated forms and protein chimeras to gain a deeper understanding of toxin specificity and stability. Our study confirms a role for the C-terminal 65 kDa domain in directing insect specificity. Importantly, these data also indicate a specific interaction between the 20 kDa amino terminus and 65 kDa carboxy terminus, after proteolytic processing. We demonstrate the C-terminal 65 kDa to be labile in native proteolytic conditions in absence of the 20 kDa N-terminus. Thus, the 20 kDa fragment functions to provide stability to the C-terminal domain, which is necessary for lethal toxicity against lepidopteran insects.

Vegetative insecticidal proteins (Vips) from Bacillus thuringiensis (Bt) are unique from crystal (Cry) proteins found in Bt parasporal inclusions as they are secreted during the bacterial vegetative growth phase and bind unique receptors to exert their insecticidal effects. We previously demonstrated that large modifications of the Vip3 C-terminus could redirect insecticidal spectrum but results in an unstable protein with no lethal activity. In the present work, we have generated a new Vip3 protein, Vip3Ab1-740, via modest modification of the Vip3Ab1 C-terminus. Vip3Ab1-740 is readily processed by midgut fluid enzymes and has lethal activity towards Spodoptera eridania, which is not observed with the Vip3Ab1 parent protein. Importantly, Vip3Ab1-740 does retain the lethal activity of Vip3Ab1 against other important lepidopteran pests. Furthermore, transgenic plants expressing Vip3Ab1-740 are protected against S. eridania, Spodoptera frugiperda, Helicoverpa zea, and Pseudoplusia includens. Thus, these studies demonstrate successful engineering of Vip3 proteins at the C-terminus to broaden insecticidal spectrum, which can be employed for functional expression in planta.

CSF-1 is the well-known ligand for CSF-1R, which plays a vital role in monocyte–macrophage generation, survival, and function. IL-34 is a newly discovered cytokine that also signals through CSF-1R. Although there are limited data for downstream signaling and pathway activation for CSF-1, none are published, to date, for expression profiles of IL-34. The objective of this study was to characterize and compare the signaling pathways downstream of the CSF-1R receptor, based on these two ligands. This was accomplished through transcriptional profiling and pathway analysis of CD14 + human monocytes differentiated with each ligand. Additionally, cells were treated with a CSF-1R inhibitor GW2580 to establish that observations associated with each ligand were CSF-1R mediated. Gene expression profiles were generated for each condition using Agilent 4x44K Whole Human Genome Microarrays. Overall profiles generated by each cytokine were similar (∼75% of genes) with a dampened effect noted on some pathways (∼25% of genes) with IL-34. One key difference observed, between the two cytokines was in the repression of CCR2 message. A similar divergence in protein level was established by FACS analysis. The differential effect on CCR2 expression has major implications for monocyte/macrophage biology including homeostasis and function. Further study of IL-34 effects on monocyte/macrophage biology will shed light on the specific role each ligand plays and the context in which these roles are important. To our knowledge, this study is the first to illustrate downstream transcriptional profiles and pathways of IL-34 in comparison with CSF-1 and identify notable differences in CCR2 expression.

Objective Fibronectin fragments are thought to play a critical role in the initiation and progression of cartilage degradation in arthritis. In a recent study, fibronectin neoepitopes resulting from cleavage of intact fibronectin at the Ala271/Val272 scissile bond, generating an ∼30-kd fragment with the new C-terminus VRAA271 and an ∼50–85-kd fragment with the new N-terminus 272VYQP, were identified in osteoarthritis (OA) cartilage. The present study was undertaken to isolate the enzymes responsible for this cleavage from human OA chondrocytes. Methods Fibronectin-degrading activity in human OA chondrocyte–conditioned medium (OACCM) was purified using conventional chromatography. A fluorescent peptide was developed based on the fibronectin scissile bond 269RAAVal272, and this peptide was used to track fibronectinase activity during purification. Western blotting with antibodies that detect the fibronectin neoepitopes VRAA271 and 272VYQP was used to confirm cleavage of intact fibronectin by the enzymatically active fractions. Mass spectrometry was used to identify the proteins found in the fibronectinase-enriched fractions, with further confirmation by Western blotting. In addition, a recombinant enzyme identified by mass spectrometry was tested by Western blotting and dimethylmethylene blue assay for its ability to produce fibronectin neoepitopes in OA cartilage. Results Purification of OACCM by chromatography resulted in isolation of a fibronectin-degrading enzyme, and mass spectrometry identified ADAM-8 as the fibronectinase present in these preparations. Furthermore, treatment of OA cartilage with recombinant human ADAM-8 promoted cartilage catabolism. Conclusion The results of this study identify ADAM-8 as a fibronectinase in human OA chondrocytes. Because ADAM-8 is capable of producing the fibronectin neoepitopes VRAA271 and 272VYQP in human OA cartilage, this enzyme may be an important mediator of cartilage catabolism.

ADAMTS-4 and ADAMTS-5 are aggrecanases responsible for the breakdown of cartilage aggrecan in osteoarthritis. Multiple ADAMTS-4 cleavage sites have been described in several matrix proteins including aggrecan, versican, and brevican, but no concise predictive cleavage motif has been identified for this protease. By screening a 13-mer peptide library with a diversity of 10(8), we have identified the ADAMTS-4 cleavage motif E-(AFVLMY)-X(0,1)-(RK)-X(2,3)-(ST)-(VYIFWMLA), with Glu representing P1. Several 13-mer peptides containing this motif, including DVQEFRGVTAVIR and HNEFRQRETYMVF, were shown to be substrates for ADAMTS-4. These peptides were found to be specific substrates for ADAMTS-4 as they were not cleaved by ADAMTS-5. Modification of these peptides with donor (6-FAM) and acceptor (QSY-9) molecules resulted in the development of fluorescence-based substrates with a Km of approximately 35 microM. Furthermore, the role of Glu at P1 and Phe at P1' in binding and catalysis was studied by exploring substitution of these amino acids with the D-isomeric forms. Substitution of P1 with dGlu was tolerable for binding, but not catalysis, whereas substitution of P1' with dPhe precluded both binding and catalysis. Similarly, replacement of Glu with Asp at P1 abolished recognition and cleavage of the peptide. Finally, BLAST results of the ADAMTS-4 cleavage motif identified matrilin-3 as a new substrate for ADAMTS-4. When tested, recombinant ADAMTS-4 effectively cleaved intact matrilin-3 at the predicted motif at Glu435/Ala436 generating two species of 45 and 5 kDa.

Objective Fibronectin fragments are present at high concentrations in the cartilage of patients with rheumatoid arthritis and patients with osteoarthritis (OA) and have been shown to promote cartilage catabolism in human cartilage cultures, suggesting that fibronectin fragments participate in the initiation and progression of arthritic disease. This study was undertaken to 1) identify the major fibronectin fragments in human OA cartilage and confirm their ability to elicit cartilage catabolism, 2) identify the cleavage sites in fibronectin and generate the corresponding neoepitope antibodies, and 3) explore the utility of fibronectin neoepitopes as biomarkers. Methods Fibronectin fragments were purified from human OA cartilage using affinity chromatography; their N-termini were then identified by sequencing. Bovine nasal cartilage was treated with affinity-purified fibronectin fragments and assayed for aggrecan breakdown by monitoring the release of glycosaminoglycans and the aggrecan neoepitope 1771AGEG. Fibronectin neoepitopes were detected by Western blotting in cytokine-treated media of human cartilage explants, and by immunohistochemical analyses of human OA cartilage. Results Multiple fibronectin fragments were isolated from human OA cartilage, and all contained the N-terminus 272VYQP. These fragments induced aggrecanase-mediated cartilage catabolism in bovine cartilage explants. Fibronectin fragments with the N-terminus 272VYQP and fragments with the C-terminus VRAA271 were detected following cytokine treatment of human cartilage extracts. These neoepitopes localized with areas of aggrecan loss in OA cartilage. Conclusion Human OA cartilage contains fibronectin fragments with catabolic activity and a major cleavage site within fibronectin. This study is the first to characterize fibronectin neoepitopes in OA cartilage, suggesting that they may represent a novel biomarker of arthritis.

Summary A disintegrin and metalloproteinase 8 (ADAM8), a catalytically active member of the ADAMs family of enzymes, is expressed primarily on immune cells and thus probably involved in inflammatory responses. ADAM8 is also produced by chondrocytes, and recombinant ADAM8 can induce cartilage catabolism. We therefore decided to test the role of ADAM8 in autoimmune inflammatory arthritis using transgenic mice expressing catalytically inactive ADAM8. Transgenic DBA/1J mice expressing an inactivating point mutation in the ADAM8 gene to change Glu330 to Gln330 (ADAM8EQ) were generated to evaluate the proteolytic function of ADAM8 in an lipopolysaccharide-synchronized collagen-induced arthritis (LPS-CIA) model of autoimmune arthritis. The systemic inflammatory reaction to LPS was also evaluated in these mice. Expression profiling of paw joints from wild-type mice revealed that ADAM8 mRNA levels increased at the onset of clinical arthritis and correlated well with cellular macrophage markers. When subjected to LPS-CIA, ADAM8EQ mice demonstrated decreased incidence and severity of clinical arthritis compared to wild-type mice. Histological examination of paw joints from ADAM8EQ mice confirmed marked attenuation of synovial inflammation, cartilage degradation and bone resorption when compared to wild-type mice. However, transgenic mice and wild-type mice responded similarly to LPS-induced systemic inflammation with regard to mortality, organ weights, neutrophil sequestration and serum cytokine/chemokine production. We conclude that ADAM8 proteolytic activity plays a key role in the development of experimental arthritis and may thus be an attractive target for the treatment of arthritic disorders while minimizing risk of immunocompromise.

A disintegrin and metalloprotease-8 (ADAM8) is thought to play a role in cancer and inflammatory diseases such as allergy, arthritis, and asthma. Despite the implication of ADAM8 in these diseases, the functional role of ADAM8 catalytic activity remains unclear. In this report, we demonstrate that an early critical autolytic event, we have termed pre-processing, is accelerated at acidic pH (pH 5.5) while autolytic activation is abrogated under the same conditions. Likewise, we found that pre-processing is hindered and autolytic activation is facilitated in neutral pH conditions, and thus demonstrates a pH-dependent shift in substrate selectivity. This finding is further supported by two peptide substrates corresponding to the pre-processing and C-terminal scissile bonds that were preferentially cleaved at acidic and neutral pH, respectively. Lastly, we found fibronectin cleavage to be attenuated at pH 5.5, while two novel substrates, brevican, and vitronectin, were readily cleaved in neutral or acidic conditions.

Members of the ADAM (a disintegrin and metalloproteinase) family of proteins possess a multidomain architecture which permits functionalities as adhesion molecules, signalling intermediates and proteolytic enzymes. ADAM8 is found on immune cells and is induced by multiple pro-inflammatory stimuli suggesting a role in inflammation. Here we describe an activation mechanism for recombinant human ADAM8 that is independent from classical PC (pro-protein convertase)-mediated activation. N-terminal sequencing revealed that, unlike other ADAMs, ADAM8 undergoes pre-processing at Glu158, which fractures the Pro (pro-segment)-domain before terminal activation takes place to remove the putative cysteine switch (Cys167). ADAM8 lacking the DIS (disintegrin) and/or CR (cysteine-rich) and EGF (epidermal growth factor) domains displayed impaired ability to complete this event. Thus pre-processing of the Pro-domain is co-ordinated by DIS and CR/EGF domains. Furthermore, by placing an EK (enterokinase) recognition motif between the Pro- and catalytic domains of multiple constructs, we were able to artificially remove the pro-segment prior to pre-processing. In the absence of pre-processing of the Pro-domain a marked decrease in specific activity was observed with the autoactivated enzyme, suggesting that the Pro-domain continued to associate and inhibit active enzyme. Thus, pre-processing of the Pro-domain of human ADAM8 is important for enzyme maturation by preventing re-association of the pro-segment with the catalytic domain. Given the observed necessity of DIS and CR/EGF for pre-processing, we conclude that these domains are crucial for the proper activation and maturation of human ADAM8.

Aging is associated with changes in T cells including involution of the thymus gland and an imbalance in the proportion of naive (CD44lo) and memory (CD44hi) T cells in the periphery. Reversal of these changes may improve immunity in the aged. We sought to determine whether 4 months of moderately intense treadmill running (EXC; 5 days/week, 45 min/day, 13-22 m/min) in 2 month (Y) and 18 month (O) old male Balb/c mice would alter T lymphocyte profiles in the thymus and spleen when compared to sedentary controls (CON). Splenocytes and thymocytes were harvested 24-48 h after the last exercise session and analyzed using immunofluorescence and flow cytometry. While there were significant age-related changes (lower cell number, altered subsets) in the thymuses of O when compared to Y mice, exercise training failed to affect any of these measures in mice of either age. Aged mice exhibited a significantly (p < .05) higher percentage of splenic memory cells and a lower percentage of naive cells in both the CD4 and CD8 T cell subsets. Interestingly, exercise training significantly (p < .05) increased the percentage of naive and decreased the percentage of memory cells in both the CD4+ (69.6+/-1.7% naive and 30.4+/-1.7% memory for OCON vs. 75.0+/-1.5% naive and 25.0+/-1.5% memory in OEXC) and CD8+ (60.0+/-2.6% naive and 40.0+/-2.6% memory in OCON vs. 76.7+/-2.7% naive and 23.3+/-2.7% memory in OEXC) T cells subsets in O, but not Y, mice. This effect was due to a decrease in the absolute number of memory cells and not an increase in the absolute number of naive cells. We conclude that 4 months of EXC has little restorative effect on the thymus in aged mice, but can restore the percentages of naive and memory cells in the spleen towards that of young mice, perhaps due to removal of memory cells.