Defining the core apoptosis pathway in the mosquito disease vector Aedes aegypti: the roles of iap1, ark, dronc, and effector caspases

To date, our knowledge of apoptosis regulation in insects comes almost exclusively from the model organism Drosophila melanogaster. In contrast, despite the identification of numerous genes that are presumed to regulate apoptosis in other insects based on sequence homology, little has been done to examine the molecular pathways that regulate apoptosis in other insects, including medically important disease vectors. In D. melanogaster, the core apoptosis pathway consists of the caspase negative regulator DIAP1, IAP antagonists, the initiator caspase Dronc and its activating protein Ark, and the effector caspase DrICE. Here we have studied the functions of several genes from the mosquito disease vector Aedes aegypti that share homology with the core apoptosis genes in D. melanogaster. Silencing of the iap1 gene in the A. aegypti cell line Aag2 caused spontaneous apoptosis, indicating that IAP1 plays a role in cell survival similar to that of DIAP1. Silencing A. aegypti ark or dronc completely inhibited apoptosis triggered by several different apoptotic stimuli. However, individual silencing of the effector caspases CASPS7 or CASPS8, which are the closest relatives to DrICE, only partially inhibited apoptosis, and silencing both CASPS7 and CASPS8 together did not have a significant additional effect. Our results suggest that the core pathway that regulates apoptosis in A. aegypti is similar to that of D. melanogaster, but that more than one effector caspase is involved in apoptosis in A. aegypti. This is interesting in light of the fact that the caspase family has expanded in mosquitoes compared to D. melanogaster.     

Prediction of a caspase-like fold in Tannerella forsythia virulence factor PrtH

Tannerella forsythia is a bacterial pathogen involved in periodontal disease. A cysteine protease PrtH has been characterized in this bacterium as a virulence factor. PrtH has the activity of detaching adherent cells from substratum, and the level of PrtH is associated with periodontal attachment loss. No reports exist on the structure, active site, and catalytic mechanism of PrtH. Using comparative sequence and structural analyses, we have identified homologs of PrtH in a number of bacterial and archaeal species. PrtH was found to be remotely related to caspases and other proteases with a caspase-like fold, such as gingipains from another periodontal pathogen Porphyromonas gingivalis. Our results offer structural and mechanistic insights into PrtH and its homologs, and help classification of this protease family.     

Naturally Occurring Triggers that Induce Apoptosis-Like Programmed Cell Death in Plasmodium berghei Ookinetes

Several protozoan parasites have been shown to undergo a form of programmed cell death that exhibits morphological features associated with metazoan apoptosis. These include the rodent malaria parasite, Plasmodium berghei. Malaria zygotes develop in the mosquito midgut lumen, forming motile ookinetes. Up to 50% of these exhibit phenotypic markers of apoptosis; as do those grown in culture. We hypothesised that naturally occurring signals induce many ookinetes to undergo apoptosis before midgut traversal. To determine whether nitric oxide and reactive oxygen species act as such triggers, ookinetes were cultured with donors of these molecules. Exposure to the nitric oxide donor SNP induced a significant increase in ookinetes with condensed nuclear chromatin, activated caspase-like molecules and translocation of phosphatidylserine that was dose and time related. Results from an assay that detects the potential-dependent accumulation of aggregates of JC-1 in mitochondria suggested that nitric oxide does not operate via loss of mitochondrial membrane potential. L-DOPA (reactive oxygen species donor) also caused apoptosis in a dose and time dependent manner. Removal of white blood cells significantly decreased ookinetes exhibiting a marker of apoptosis in vitro. Inhibition of the activity of nitric oxide synthase in the mosquito midgut epithelium using L-NAME significantly decreased the proportion of apoptotic ookinetes and increased the number of oocysts that developed. Introduction of a nitric oxide donor into the blood meal had no effect on mosquito longevity but did reduce prevalence and intensity of infection. Thus, nitric oxide and reactive oxygen species are triggers of apoptosis in Plasmodium ookinetes. They occur naturally in the mosquito midgut lumen, sourced from infected blood and mosquito tissue. Up regulation of mosquito nitric oxide synthase activity has potential as a transmission blocking strategy.     

A VPE-like protease NtTPE8 exclusively expresses in the integumentary tapetum and is involved in seed development

Programmed cell death(PCD) is an essential process for development, and shows conserved cytological features in both plants and animals. Caspases are well-known critical components of the PCD machinery in animals. However, currently few typical counterparts have been identified in plants and only several caspase-like proteases are known to be involved in plant PCD, indicating the existence of great challenge for confirming new caspase-like proteases and elucidating the mechanisms regulating plant PCD. Here, we report a novel cysteine protease, NtTPE8, which was extracted from tobacco seeds and confirmed as a new caspase-like protease.Recombinant NtTPE8 exhibited legumain and caspase-like proteolytic activities, both of which could be inhibited by the pan-caspase inhibitor(Z-VAD-FMK). Notably, NtTPE8 possessed several caspase activities and the capacity to cleave the cathepsin H substrate FVR, indicating a unique character of NtTPE8. NtTPE8 was exclusively expressed in the integumentary tapetum and thus, is the first specific molecular marker reported to date for this cell type. Downregulation of NtTPE8 caused seed abortion, via disturbing early embryogenesis, indicating its critical role in embryogenesis and seed development. In conclusion, we identified a novel caspase-like cysteine protease, NtTPE8,exclusively expressed in the integumentary tapetum that is involved in seed development.     

Crystal structure of the yeast metacaspase yca1

Yca1, the only metacaspase in Saccharomyces cerevisiae, is thought to be a clan CD cysteine protease that includes the caspase subfamily. Although yeast is a single cell eukaryote, it can undergo a cell death process reminiscent of apoptosis. Yca1 has been reported to play an important role in the regulation of such apoptotic process. However, the structure and functional mechanism of Yca1 remain largely enigmatic. In this study, we report the crystal structure of the Yca1 metacaspase at 1.7 Å resolution, confirming a caspase-like fold. In sharp contrast to canonical caspases, however, Yca1 exists as a monomer both in solution and in the crystals. Canonical caspase contains six β-strands, with strand β6 pairing up with β6 of another caspase molecule to form a homodimerization interface. In Yca1, an extra pair of antiparallel β-strands forms a continuous β-sheet with the six caspase-common β-strands, blocking potential dimerization. Yca1 was reported to undergo autocatalytic processing in yeast; overexpression in bacteria also led to autoprocessing of Yca1 into two fragments. Unexpectedly, we found that both the autocatalytic processing and the proteolytic activity of Yca1 are greatly facilitated by the presence of calcium (Ca²⁺), but not other divalent cations. Our structural and biochemical characterization identifies Yca1 as a Ca²⁺-activated cysteine protease that may cleave specific substrates during stress response in yeast.     

The antifungal plant defensin RsAFP2 from radish induces apoptosis in a metacaspase independent way in Candida albicans

We show that the antifungal plant defensin Raphanus sativus antifungal protein 2 (RsAFP2) from radish induces apoptosis and concomitantly triggers activation of caspases or caspase-like proteases in the human pathogen Candida albicans. Furthermore, we demonstrate that deletion of C. albicans metacaspase 1, encoding the only reported (putative) caspase in C. albicans, significantly affects caspase activation by the apoptotic stimulus acetic acid, but not by RsAFP2. To our knowledge, this is the first report on the induction of apoptosis with concomitant caspase activation by a defensin in this pathogen. Moreover, our data point to the existence of at least two different types of caspases or caspase-like proteases in C. albicans.     

Mechanistic Details of Glutathione Biosynthesis Revealed by Crystal Structures of Saccharomyces cerevisiae Glutamate Cysteine Ligase

Glutathione is a thiol-disulfide exchange peptide critical for buffering oxidative or chemical stress, and an essential cofactor in several biosynthesis and detoxification pathways. The rate-limiting step in its de novo biosynthesis is catalyzed by glutamate cysteine ligase, a broadly expressed enzyme for which limited structural information is available in higher eukaryotic species. Structural data are critical to the understanding of clinical glutathione deficiency, as well as rational design of enzyme modulators that could impact human disease progression. Here, we have determined the structures of Saccharomyces cerevisiae glutamate cysteine ligase (ScGCL) in the presence of glutamate and MgCl₂ (2.1 Å; R = 18.2%, R_free = 21.9%), and in complex with glutamate, MgCl₂, and ADP (2.7 Å; R = 19.0%, R_free = 24.2%). Inspection of these structures reveals an unusual binding pocket for the α-carboxylate of the glutamate substrate and an ATP-independent Mg²⁺ coordination site, clarifying the Mg²⁺ dependence of the enzymatic reaction. The ScGCL structures were further used to generate a credible homology model of the catalytic subunit of human glutamate cysteine ligase (hGCLC). Examination of the hGCLC model suggests that post-translational modifications of cysteine residues may be involved in the regulation of enzymatic activity, and elucidates the molecular basis of glutathione deficiency associated with patient hGCLC mutations.     

Barcoded Asaia bacteria enable mosquito in vivo screens and identify novel systemic insecticides and inhibitors of malaria transmission

This work addresses the need for new chemical matter in product development for control of pest insects and vector-borne diseases. We present a barcoding strategy that enables phenotypic screens of blood-feeding insects against small molecules in microtiter plate-based arrays and apply this to discovery of novel systemic insecticides and compounds that block malaria parasite development in the mosquito vector. Encoding of the blood meals was achieved through recombinant DNA-tagged Asaia bacteria that successfully colonised Aedes and Anopheles mosquitoes. An arrayed screen of a collection of pesticides showed that chemical classes of avermectins, phenylpyrazoles, and neonicotinoids were enriched for compounds with systemic adulticide activity against Anopheles. Using a luminescent Plasmodium falciparum reporter strain, barcoded screens identified 48 drug-like transmission-blocking compounds from a 400-compound antimicrobial library. The approach significantly increases the throughput in phenotypic screening campaigns using adult insects and identifies novel candidate small molecules for disease control.

This study presents a barcoding strategy that enables high-throughput phenotypic screens of blood-feeding insects against small molecules in microtiter plate-based arrays and applies this to the discovery of novel systemic insecticides and compounds that block malaria parasite development in the mosquito vector.     

Identification of paracaspases and metacaspases: two ancient families of caspase-like proteins, one of which plays a key role in MALT lymphoma

Caspases are cysteine proteases essential to apoptosis. We have identified two families of caspase-like proteins, Paracaspases (found in metazoans and Dictyostelium) and metacaspases (found in plants, fungi, and protozoa). Metazoan paracaspase prodomains contain a death domain and immunoglobulin domains. Several plant metacaspase prodomains contain zinc finger motifs resembling those in the plant hypersensitive response/cell death protein Isd-1. The human paracaspase prodomain binds Bcl10, a protein involved in the t(1;14)(p22;q32) translocation of mucosa-associated lymphoid tissue (MALT) lymphoma. Another MALT lymphoma translocation, t(11;18)(q21;q21), fuses the IAP-2 gene to the MLT1/MALT1 locus, which encodes the human paracaspase. We find that this fusion activates NF-kappaB and that the caspase domain is required for this function, since mutation of the conserved catalytic cysteine attenuates NF-kappaB activation.     

Which acetylcholinesterase functions as the main catalytic enzyme in the Class Insecta?

Most insects possess two different acetylcholinesterases (AChEs) (i.e., AChE1 and AChE2; encoded by ace1 and ace2 genes, respectively). Between the two AChEs, AChE1 has been proposed as a major catalytic enzyme based on its higher expression level and frequently observed point mutations associated with insecticide resistance. To investigate the evolutionary distribution of AChE1 and AChE2, we determined which AChE had a central catalytic function in several insect species across 18 orders. The main catalytic activity in heads was determined by native polyacrylamide gel electrophoresis in conjunction with Western blotting using AChE1- and AChE2-specific antibodies. Of the 100 insect species examined, 67 species showed higher AChE1 activity; thus, AChE1 was considered as the main catalytic enzyme. In the remaining 33 species, ranging from Palaeoptera to Hymenoptera, however, AChE2 was predominantly expressed as the main catalytic enzyme. These findings challenge the common notion that AChE1 is the only main catalytic enzyme in insects with the exception of Cyclorrhapha, and further demonstrate that the specialization of AChE2 as the main enzyme or the replacement of AChE1 function with AChE2 were rather common events, having multiple independent origins during insect evolution. It was hypothesized that the generation of multiple AChE2 isoforms by alternative splicing allowed the loss of ace1 during the process of functional replacement of AChE1 with AChE2 in Cyclorrhapha. However, the presence of AChE2 as the main catalytic enzyme in higher social Hymenoptera provides a case for the functional replacement of AChE1 with AChE2 without the loss of ace1. The current study will provide valuable insights into the evolution of AChE: which AChE has been specialized as the main catalytic enzyme and to become the main target for insecticides in different insect species.     

Rapid identification of female Culexmosquito species using Expert System in the South East Asian region

Rapid identification of mosquito (vector) species is critical for vector control and disease management. Pictorial keys of mosquito species are currently used for the identification of new mosquito species. However, this approach is not very effective. Here, we describe the use of an ID3 algorithm (part of artificial intelligence) for the rapid identification of the South East Asian female Culex mosquito species.

Availability     

STAT3 Decoy Oligodeoxynucleotides-Loaded Solid Lipid Nanoparticles Induce Cell Death and Inhibit Invasion in Ovarian Cancer Cells

Recent advances in the synthesis of multi-functional nanoparticles have opened up tremendous opportunities for the targeted delivery of genes of interest. Cationic solid lipid nanoparticles (SLN) can efficiently bind nucleic acid molecules and transfect genes in vitro. Few reports have combined SLN with therapy using decoy oligodeoxynucleotides (ODN). In the present study, we prepared SLN to encapsulate STAT3 decoy ODN; then, the properties and in vitro behavior of SLN-STAT3 decoy ODN complexes were investigated. SLN-STAT3 decoy ODN complexes were efficiently taken up by human ovarian cancer cells and significantly suppressed cell growth. Blockage of the STAT3 pathway by SLN-STAT3 decoy ODN complexes resulted in an evident induction of cell death, including apoptotic and autophagic death. The mechanism involved the increased expression of cleaved caspase 3, Bax, Beclin-1 and LC3-II and reduced expression of Bcl-2, pro-caspase 3, Survivin, p-Akt and p-mTOR. In addition, SLN-STAT3 decoy ODN complexes inhibited cell invasion by up-regulating E-cadherin expression and down-regulating Snail and MMP-9 expression. These findings confirmed that SLN as STAT3 decoy ODN carriers can induce cell death and inhibit invasion of ovarian cancer cells. We propose that SLN represent a potential approach for targeted gene delivery in cancer therapy.     

ٍSome biologically active microorganisms have the potential to suppress mosquito larvae (Culex pipiens,Diptera: Culicidae)

Malaria is a disease caused by protozoan species of the genus Plasmodium. It is widespread and becoming a challenge in several African countries in the tropical and subtropical regions. In 2010, a report was published showing that over 1.2 million death cases were occurred globally due to malaria in just one year. The transmission of the disease from one person to another occurs via the bite of the Anopheles female. It is known that Plasmodium ovale, P. vivax, P. malariae, P. falciparum, and P. knowlesi are the highly infective malaria species. The problem of this disease is the absence of any effective medical treatment or vaccine, making the mosquito control is the only feasible way for disease prevention. Pesticides are currently the most widely used method for mosquito control, despite its well-known negative effects, including health hazards on human, the increasing insecticidal resistance, and the negative impact on the environment and beneficial organisms. Biological control (also called: biocontrol) of insects has been a promising method to overcome the negative effects of using chemical insecticides, as it depends on just using the natural enemies of pests to either minimize their populations or eradicate them. This article provides an overview of the recent and effective biological means to control malaria, such as bacteria, fungi, viruses, larvivorous fish, toxorhynchites larva and nematodes. In addition, the importance, advantages, and disadvantages of the biocontrol methods will be discussed in comparison with the traditionally used chemical methods of malaria control with special reference to nanotechnology as a novel method for insects’ control.     

Oxidation as a Post-Translational Modification that Regulates Autophagy

The toxicity associated with accumulation of reactive oxygen species (ROS) has led to the evolution of various defense strategies to overcome oxidative stress, including autophagy. This pathway is involved in the removal and degradation of damaged mitochondria and oxidized proteins. At low levels, however, ROS act as signal transducers in various intracellular pathways. In a recent study we described the role of ROS as signaling molecules in starvation-induced autophagy. We showed that starvation stimulates formation of ROS, specifically H₂O₂, in the mitochondria. Furthermore, we identified the cysteine protease HsAtg4 as a direct target for oxidation by H₂O₂, and specified a cysteine residue located near the HsAtg4 catalytic site as critical for this regulation. Here we focus on Atg4, the target of regulation, and discuss possible mechanisms for the regulation of this enzyme in the autophagic process.

Addendum to:

Reactive Oxygen Species Are Essential for Autophagy and Specifically Regulate the Activity of Atg4

R. Scherz-Shouval, E. Shvets, E. Fass, H. Shorer, L. Gil and Z. Elazar

EMBO J 2007; doi: 10.1038/sj.emboj.7601623     

Caspase-like activity in the seedlings of Pisum sativum eliminates weaker shoots during early vegetative development by induction of cell death

Activation of aspartate-specific cysteine proteases (caspases) plays a crucial role in programmed cell death (PCD) in animals. Although to date caspases have not been identified in plants, caspase-like activity was described in tobacco during a hypersensitive response to pathogens and in Arabidopsis and tomato cell cultures during chemical-induced PCD. Caspase-like activity was also detected in the course of plant development during petal senescence and endosperm PCD. It is shown here that caspase-like proteases play a crucial role in the developmental cell death of secondary shoots of pea seedlings that emerge after removal of the epicotyl. Caspase-like activity was induced in senescing secondary shoots, but not in dominant growing shoots, in contrast to the papain-like cysteine protease activity that was stronger in the dominant shoot. Revitalization of the senescing shoot by cutting of the dominant shoot reduced the caspase-like activity. Injection of caspase or cysteine protease inhibitors into the remaining epicotyl tissue suppressed the death of the secondary shoots, producing seedlings with two equal shoots. These results suggest that shoot selection in pea seedlings is controlled by PCD, through the activation of caspase-like proteases.