Engineering of an Iranian Bordetella pertussis strain producing inactive pertussis toxin.

Introduction. Differences between the genomic and virulence profile of Bordetella pertussis circulating strains and vaccine strains are considered as one of the important reasons for the resurgence of whooping cough (pertussis) in the world. Genetically inactivated B. pertussis is one of the new strategies to generate live-attenuated vaccines against whooping cough.Aim. The aim of this study was to construct a B. pertussis strain based on a predominant profile of circulating Iranian isolates that produces inactivated pertussis toxin (PTX).Methodology. The B. pertussis strain BPIP91 with predominant genomic and virulence pattern was selected from the biobank of the Pasteur Institute of Iran. A BPIP91 derivative with R9K and E129G alterations in the S1 subunit of PTX (S1mBPIP91) was constructed by the site-directed mutagenesis and homologous recombination. Genetic stability and antigen expression of S1mBPIP91 were tested by serially in vitro passages and immunoblot analyses, respectively. The reduction in toxicity of S1mBPIP91 was determined by Chinese hamster ovary (CHO) cell clustering.Results. All constructs and S1mBPIP91 were confirmed via restriction enzyme analysis and DNA sequencing. The engineered mutations in S1mBPIP91 were stable after 20 serial in vitro passages. The production of virulence factors was also confirmed in S1mBPIP91. The CHO cell-clustering test demonstrated the reduction in PTX toxicity in S1mBPIP91.Conclusion. A B. pertussis of the predominant genomic and virulence lineage in Iran was successfully engineered to produce inactive PTX. This attenuated strain will be useful to further studies to develop both whole cell and acellular pertussis vaccines.

Overriding FUS autoregulation in mice triggers gain-of-toxic dysfunctions in RNA metabolism and autophagy-lysosome axis.

Mutations in coding and non-coding regions of FUS cause amyotrophic lateral sclerosis (ALS). The latter mutations may exert toxicity by increasing FUS accumulation. We show here that broad expression within the nervous system of wild-type or either of two ALS-linked mutants of human FUS in mice produces progressive motor phenotypes accompanied by characteristic ALS-like pathology. FUS levels are autoregulated by a mechanism in which human FUS downregulates endogenous FUS at mRNA and protein levels. Increasing wild-type human FUS expression achieved by saturating this autoregulatory mechanism produces a rapidly progressive phenotype and dose-dependent lethality. Transcriptome analysis reveals mis-regulation of genes that are largely not observed upon FUS reduction. Likely mechanisms for FUS neurotoxicity include autophagy inhibition and defective RNA metabolism. Thus, our results reveal that overriding FUS autoregulation will trigger gain-of-function toxicity via altered autophagy-lysosome pathway and RNA metabolism function, highlighting a role for protein and RNA dyshomeostasis in FUS-mediated toxicity.

Nanobody interaction unveils structure, dynamics and proteotoxicity of the Finnish-type amyloidogenic gelsolin variant.

AGel amyloidosis, formerly known as familial amyloidosis of the Finnish-type, is caused by pathological aggregation of proteolytic fragments of plasma gelsolin. So far, four mutations in the gelsolin gene have been reported as responsible for the disease. Although D187N is the first identified variant and the best characterized, its structure has been hitherto elusive. Exploiting a recently-developed nanobody targeting gelsolin, we were able to stabilize the G2 domain of the D187N protein and obtained, for the first time, its high-resolution crystal structure. In the nanobody-stabilized conformation, the main effect of the D187N substitution is the impairment of the calcium binding capability, leading to a destabilization of the C-terminal tail of G2. However, molecular dynamics simulations show that in the absence of the nanobody, D187N-mutated G2 further misfolds, ultimately exposing its hydrophobic core and the furin cleavage site. The nanobody's protective effect is based on the enhancement of the thermodynamic stability of different G2 mutants (D187N, G167R and N184K). In particular, the nanobody reduces the flexibility of dynamic stretches, and most notably decreases the conformational entropy of the C-terminal tail, otherwise stabilized by the presence of the Ca2+ ion. A Caenorhabditis elegans-based assay was also applied to quantify the proteotoxic potential of the mutants and determine whether nanobody stabilization translates into a biologically relevant effect. Successful protection from G2 toxicity in vivo points to the use of C. elegans as a tool for investigating the mechanisms underlying AGel amyloidosis and rapidly screen new therapeutics.

TDP-43 causes neurotoxicity and cytoskeletal dysfunction in primary cortical neurons.

TDP-43-mediated proteinopathy is a key factor in the pathology of amyotrophic lateral sclerosis (ALS). A potential underlying mechanism is dysregulation of the cytoskeleton. Here we investigate the effects of expressing TDP-43 wild-type and M337V and Q331K mutant isoforms on cytoskeletal integrity and function, using rat cortical neurons in vitro. We find that TDP-43 protein becomes mislocalised in axons over 24-72 hours in culture, with protein aggregation occurring at later timepoints (144 hours). Quantitation of cell viability showed toxicity of both wild-type and mutant constructs which increased over time, especially of the Q331K mutant isoform. Analysis of the effects of TDP-43 on axonal integrity showed that TDP-43-transfected neurons had shorter axons than control cells, and that growth cone sizes were smaller. Axonal transport dynamics were also impaired by transfection with TDP-43 constructs. Taken together these data show that TDP-43 mislocalisation into axons precedes cell death in cortical neurons, and that cytoskeletal structure and function is impaired by expression of either TDP-43 wild-type or mutant constructs in vitro. These data suggest that dysregulation of cytoskeletal and neuronal integrity is an important mechanism for TDP-43-mediated proteinopathy.

MIF inhibits the formation and toxicity of misfolded SOD1 amyloid aggregates: implications for familial ALS.

Mutations in superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease caused by the progressive loss of motor neurons in the brain and spinal cord. It has been suggested that toxicity of mutant SOD1 results from its misfolding, however, it is yet unclear why misfolded SOD1 accumulates specifically within motor neurons. We recently demonstrated that macrophage migration inhibitory factor (MIF)-a multifunctional protein with cytokine/chemokine activity and cytosolic chaperone-like properties-inhibits the accumulation of misfolded SOD1. Here, we show that MIF inhibits mutant SOD1 nuclear clearance when overexpressed in motor neuron-like NSC-34 cells. In addition, MIF alters the typical SOD1 amyloid aggregation pathway in vitro, and, instead, promotes the formation of disordered aggregates, as measured by Thioflavin T (ThT) assay and transmission electron microscopy (TEM) imaging. Moreover, we report that MIF reduces the toxicity of misfolded SOD1 by directly interacting with it, and that the chaperone function and protective effect of MIF in neuronal cultures do not require its intrinsic catalytic activities. Importantly, we report that the locked-trimeric MIFN110C mutant, which exhibits strongly impaired CD74-mediated cytokine functions, has strong chaperone activity, dissociating, for the first time, these two cellular functions. Altogether, our study implicates MIF as a potential therapeutic candidate in the treatment of ALS.

Nuclear export of misfolded SOD1 mediated by a normally buried NES-like sequence reduces proteotoxicity in the nucleus.

Over 170 different mutations in the gene encoding SOD1 all cause amyotrophic lateral sclerosis (ALS). Available studies have been primarily focused on the mechanisms underlying mutant SOD1 cytotoxicity. How cells defend against the cytotoxicity remains largely unknown. Here, we show that misfolding of ALS-linked SOD1 mutants and wild-type (wt) SOD1 exposes a normally buried nuclear export signal (NES)-like sequence. The nuclear export carrier protein CRM1 recognizes this NES-like sequence and exports misfolded SOD1 to the cytoplasm. Antibodies against the NES-like sequence recognize misfolded SOD1, but not native wt SOD1 both in vitro and in vivo. Disruption of the NES consensus sequence relocalizes mutant SOD1 to the nucleus, resulting in higher toxicity in cells, and severer impairments in locomotion, egg-laying, and survival in Caenorhabditis elegans. Our data suggest that SOD1 mutants are removed from the nucleus by CRM1 as a defense mechanism against proteotoxicity of misfolded SOD1 in the nucleus.

Distinct cellular toxicity of two mutant huntingtin mRNA variants due to translation regulation.

Huntington's disease (HD) is a neurodegenerative disorder caused by CAG repeat expansion within exon1 of the HTT gene. The gene generates two mRNA variants that carry either a short or long 3' untranslated region (3'UTR) while encoding the same protein. It remains unknown whether the two mRNA variants play distinct roles in HD pathogenesis. We found that the long HTT 3'UTR was capable of guiding mRNA to neuronal dendrites, suggesting that some long-form HTT mRNA is transported to dendrites for local protein synthesis. To assay roles of two HTT mRNA variants in cell bodies, we expressed mRNA harboring HTT exon1 containing 23x or 145x CAGs with the short or long 3'UTR. We found that mutant mRNA containing the short 3'UTR produced more protein aggregates and caused more apoptosis in both cultured neurons and HEK293 cells, compared with mutant mRNA containing the long 3'UTR. Although the two 3'UTRs did not affect mRNA stability, we detected higher levels of protein synthesis from mRNA containing the short 3'UTR than from mRNA containing the long 3'UTR. These results indicate that the long HTT 3'UTR suppresses translation. Thus, short-form mutant HTT mRNA will be more efficient in producing toxic protein than its long-form counterpart.

F199E substitution reduced toxicity of Clostridium perfringens epsilon toxin by depriving the receptor binding capability.

Epsilon toxin (ETX), a potent toxin, is produced by types B and D strains of Clostridium perfringens, which could cause severe diseases in humans and domestic animals. Mutant rETXF199E was previously demonstrated to be a good vaccine candidate. However, the mechanism concerned remains unknown. To clarify how F199E substitution reduced ETX toxicity, we performed a series of experiments. The results showed that the cell-binding and pore-forming ability of rETXF199E was almost abolished. We speculated that F199E substitution reduced toxicity by depriving the receptor binding capability of ETX, which contributed to the hypothesis that domain I of ETX is responsible for cell binding. In addition, our data suggested that ETX could cause Ca2+ release from intracellular Ca2+ stores, which may underlie an alternate pathway leading to cell death. Furthermore, ETX induced crenation of the MDCK cells was observed, with sags and crests first appearing on the surface of condensed MDCK cells, according to scanning electron microscopy. The data also demonstrated the safety and potentiality of rETXF199E as a vaccine candidate for humans. In summary, findings of this work potentially contribute to a better understanding of the pathogenic mechanism of ETX and the development of vaccine against diseases caused by ETX, using mutant proteins.

Distinct mechanisms of mutant huntingtin toxicity in different yeast strains.

Expansion of polyglutamine stretches in several proteins causes neurodegenerative amyloidoses, including Huntington disease. In yeast, mutant huntingtin (mHtt) with a stretch of 103 glutamine residues (HttQ103) forms toxic aggregates. A range of yeast strains have been used to elucidate the mechanisms of mHtt toxicity, and have revealed perturbations of various unrelated processes. HttQ103 aggregates can induce aggregation of cellular proteins, many of which contain glutamine/asparagine-rich regions, including Sup35 and Def1. In the strain 74-D694 HttQ103, toxicity is related to aggregation-mediated depletion of soluble Sup35 and its interacting partner Sup45. Def1 was also implicated in mHtt toxicity, since its lack detoxified HttQ103 in another yeast strain, BY4741. Here we show that in BY4742, deletion of DEF1 lowers HttQ103 toxicity and decreases the amount of its polymers, but does not affect copolymerization of Sup35. Furthermore, in contrast to 74-D694, increasing the levels of soluble Sup35 and Sup45 does not alleviate toxicity of HttQ103 in BY4742. These data demonstrate a difference in the mechanisms underlying mHtt toxicity in different yeast strains and suggest that in humans with Huntington disease, neurons of different brain compartments and cells in other tissues can also be damaged by different mechanisms.

Control of the structural landscape and neuronal proteotoxicity of mutant Huntingtin by domains flanking the polyQ tract.

Many neurodegenerative diseases are linked to amyloid aggregation. In Huntington's disease (HD), neurotoxicity correlates with an increased aggregation propensity of a polyglutamine (polyQ) expansion in exon 1 of mutant huntingtin protein (mHtt). Here we establish how the domains flanking the polyQ tract shape the mHtt conformational landscape in vitro and in neurons. In vitro, the flanking domains have opposing effects on the conformation and stabilities of oligomers and amyloid fibrils. The N-terminal N17 promotes amyloid fibril formation, while the C-terminal Proline Rich Domain destabilizes fibrils and enhances oligomer formation. However, in neurons both domains act synergistically to engage protective chaperone and degradation pathways promoting mHtt proteostasis. Surprisingly, when proteotoxicity was assessed in rat corticostriatal brain slices, either flanking region alone sufficed to generate a neurotoxic conformation, while the polyQ tract alone exhibited minimal toxicity. Linking mHtt structural properties to its neuronal proteostasis should inform new strategies for neuroprotection in polyQ-expansion diseases.

Inhibition of DNA Methyltransferases Blocks Mutant Huntingtin-Induced Neurotoxicity.

Although epigenetic abnormalities have been described in Huntington's disease (HD), the causal epigenetic mechanisms driving neurodegeneration in HD cortex and striatum remain undefined. Using an epigenetic pathway-targeted drug screen, we report that inhibitors of DNA methyltransferases (DNMTs), decitabine and FdCyd, block mutant huntingtin (Htt)-induced toxicity in primary cortical and striatal neurons. In addition, knockdown of DNMT3A or DNMT1 protected neurons against mutant Htt-induced toxicity, together demonstrating a requirement for DNMTs in mutant Htt-triggered neuronal death and suggesting a neurodegenerative mechanism based on DNA methylation-mediated transcriptional repression. Inhibition of DNMTs in HD model primary cortical or striatal neurons restored the expression of several key genes, including Bdnf, an important neurotrophic factor implicated in HD. Accordingly, the Bdnf promoter exhibited aberrant cytosine methylation in mutant Htt-expressing cortical neurons. In vivo, pharmacological inhibition of DNMTs in HD mouse brains restored the mRNA levels of key striatal genes known to be downregulated in HD. Thus, disturbances in DNA methylation play a critical role in mutant Htt-induced neuronal dysfunction and death, raising the possibility that epigenetic strategies targeting abnormal DNA methylation may have therapeutic utility in HD.

The multigene families of actinoporins (part II): Strategies for heterologous production in Escherichia coli.

The sea anemone venom contains pore-forming proteins (PFP) named actinoporins, due to their purification from organisms belonging to Actiniaria order and its ability to form pores in sphingomyelin-containing membranes. Actinoporins are generally basic, monomeric and single-domain small proteins (∼20 kDa) that are classified as α-type PFP since the pore formation in membranes occur through α-helical elements. Different actinoporin isoforms have been isolated from most of the anemones species, as was analyzed in the first part of this review. Several actinoporin full-length genes have been identified from genomic-DNA libraries or messenger RNA. Since the actinoporins lack carbohydrates and disulfide bridges, their expression in bacterial systems is suitable. The actinoporins heterologous expression in Escherichia coli simplifies their production, replaces the natural source reducing the ecological damage in anemone populations, and allows the production of site-specific mutants for the study of the structure-function relationship. In this second part of the review, the strategies for heterologous production of actinoporins in Escherichia coli are analyzed, as well as the different approaches used for their purification. The activity of the recombinant proteins with respect to the wild-type is also reviewed.

Molecular insights into cell toxicity of a novel familial amyloidogenic variant of ß2-microglobulin.

The first genetic variant of ß2 -microglobulin (b2M) associated with a familial form of systemic amyloidosis has been recently described. The mutated protein, carrying a substitution of Asp at position 76 with an Asn (D76N b2M), exhibits a strongly enhanced amyloidogenic tendency to aggregate with respect to the wild-type protein. In this study, we characterized the D76N b2M aggregation path and performed an unprecedented analysis of the biochemical mechanisms underlying aggregate cytotoxicity. We showed that, contrarily to what expected from other amyloid studies, early aggregates of the mutant are not the most toxic species, despite their higher surface hydrophobicity. By modulating ganglioside GM1 content in cell membrane or synthetic lipid bilayers, we confirmed the pivotal role of this lipid as aggregate recruiter favouring their cytotoxicity. We finally observed that the aggregates bind to the cell membrane inducing an alteration of its elasticity (with possible functional unbalance and cytotoxicity) in GM1-enriched domains only, thus establishing a link between aggregate-membrane contact and cell damage.

Nonnative SOD1 trimer is toxic to motor neurons in a model of amyotrophic lateral sclerosis.

Since the linking of mutations in the Cu,Zn superoxide dismutase gene (sod1) to amyotrophic lateral sclerosis (ALS) in 1993, researchers have sought the connection between SOD1 and motor neuron death. Disease-linked mutations tend to destabilize the native dimeric structure of SOD1, and plaques containing misfolded and aggregated SOD1 have been found in the motor neurons of patients with ALS. Despite advances in understanding of ALS disease progression and SOD1 folding and stability, cytotoxic species and mechanisms remain unknown, greatly impeding the search for and design of therapeutic interventions. Here, we definitively link cytotoxicity associated with SOD1 aggregation in ALS to a nonnative trimeric SOD1 species. We develop methodology for the incorporation of low-resolution experimental data into simulations toward the structural modeling of metastable, multidomain aggregation intermediates. We apply this methodology to derive the structure of a SOD1 trimer, which we validate in vitro and in hybridized motor neurons. We show that SOD1 mutants designed to promote trimerization increase cell death. Further, we demonstrate that the cytotoxicity of the designed mutants correlates with trimer stability, providing a direct link between the presence of misfolded oligomers and neuron death. Identification of cytotoxic species is the first and critical step in elucidating the molecular etiology of ALS, and the ability to manipulate formation of these species will provide an avenue for the development of future therapeutic strategies.

Cell and Context-Dependent Effects of the Heat Shock Protein DNAJB6 on Neuronal Survival.

Previous studies performed in cell lines have shown that the heat shock protein, DNAJB6, protects against the proteotoxic effects of mutant huntingtin (mut-Htt) via direct interaction with mut-Htt. However, these studies were performed primarily using in vitro models and cell lines. We report that when expressed in primary neurons, DNAJB6 induces cell death. Neurotoxicity is observed with both the DNAJB6a isoform, which is strictly nuclear, and the DNAJB6b isoform, which is predominantly cytoplasmic, suggesting that neurotoxicity is mediated in the nucleus. However, when co-expressed in primary neurons with mut-Htt, DNAJB6 protects against mut-Htt neurotoxicity. This suggests that the contrasting effect of DNAJB6 on neuronal viability depends on the presence or absence of proteotoxic stress. Neurotoxicity of DNAJB6 cannot be prevented by inhibition of glycogen synthase kinase 3 beta (GSK3ß) or c-Jun N-terminal kinase (JNK) but is prevented by pharmacological inhibition of cyclin-dependent kinases (CDKs). Expression of dominant-negative forms of CDK2 or CDK4, or of p21(CIP1), the physiological inhibitor of CDKs, also inhibits DNAJB6 neurotoxicity. DNAJB6 neurotoxicity can also be inhibited by histone deacetylase-4 (HDAC4), which interacts with DNAJB6 and which has previously been described to inhibit cell cycle progression. These results conclude that neurotoxicity resulting from elevated DNAJB6 is cell cycle dependent.

Parkin Protects Against Misfolded SOD1 Toxicity by Promoting Its Aggresome Formation and Autophagic Clearance.

Mutations in Cu/Zn superoxide dismutase (SOD1) cause autosomal dominant amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease with no effective treatment. Despite ample evidence indicating involvement of mutation-induced SOD1 protein misfolding and aggregation in ALS pathogenesis, the molecular mechanisms that control cellular management of misfolded, aggregation-prone SOD1 mutant proteins remain unclear. Here, we report that parkin, an E3 ubiquitin-protein ligase which is linked to Parkinson's disease, is a novel regulator of cellular defense against toxicity induced by ALS-associated SOD1 mutant proteins. We find that parkin mediates K63-linked polyubiquitination of SOD1 mutants in cooperation with the UbcH13/Uev1a E2 enzyme and promotes degradation of these misfolded SOD1 proteins by the autophagy-lysosome system. In response to strong proteotoxic stress associated with proteasome impairment, parkin promotes sequestration of misfolded and aggregated SOD1 proteins to form perinuclear aggresomes, regulates positioning of lysosomes around misfolded SOD1 aggresomes, and facilitates aggresome clearance by autophagy. Our findings reveal parkin-mediated cytoprotective mechanisms against misfolded SOD1 toxicity and suggest that enhancing parkin-mediated cytoprotection may provide a novel therapeutic strategy for treating ALS.

ENC1 Modulates the Aggregation and Neurotoxicity of Mutant Huntingtin Through p62 Under ER Stress.

Huntington's disease (HD) is a devastating neurodegenerative disorder, which is caused by the expression and aggregation of polyQ-expanded mutant huntingtin protein (mtHTT). While toxic mtHTT aggregates are primarily eliminated through autophagy, autophagy dysfunction is often observed in HD pathogenesis. Here, we show that ectodermal-neural cortex 1 (ENC1), a novel binding partner of sequestosome 1 (p62), negatively regulates autophagy under endoplasmic reticulum (ER) stress. We found that ER stress significantly increases the expression of ENC1 via inositol-requiring enzyme 1 (IRE1)-TNF receptor-associated factor 2 (TRAF2)-c-Jun N-terminal kinase (JNK) pathway. Ectopic expression of ENC1 alone induces the accumulation of detergent-resistant mtHTT aggregates and downregulation of ENC1 alleviates ER stress-induced mtHTT aggregation. Simultaneously, ER stress-induced impairment of autophagy flux is ameliorated by downregulation of ENC1. From immunoprecipitation and immunocytochemical assays, we found that ENC1 binds to p62 through its BTB and C-terminal Kelch (BACK) domain and this interaction is enhanced under ER stress. In particular, ENC1 preferentially interacts with the phosphorylated p62 at Ser403 during ER stress. Interestingly, ENC1 colocalizes with mtHTT aggregates and its C-terminal Kelch domain is required for interfering with the access of p62 to ubiquitinated mtHTT aggregates, thus inhibiting cargo recognition of p62. Accordingly, knockdown of ENC1 expression enhances colocalization of p62 with mtHTT aggregates. Consequently, ENC1 knockdown relieves death of neuronal cells expressing mtHTT under ER stress. These results suggest that ENC1 interacts with the phosphorylated p62 to impair autophagic degradation of mtHTT aggregates and affects cargo recognition failure under ER stress, leading to the accumulation and neurotoxicity of mtHTT aggregates.

RTP801 Is Involved in Mutant Huntingtin-Induced Cell Death.

RTP801 expression is induced by cellular stress and has a pro-apoptotic function in non-proliferating differentiated cells such as neurons. In several neurodegenerative disorders, including Parkinson's disease and Alzheimer's disease, elevated levels of RTP801 have been observed, which suggests a role for RTP801 in neuronal death. Neuronal death is also a pathological hallmark in Huntington's disease (HD), an inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. Currently, the exact mechanisms underlying mutant huntingtin (mhtt)-induced toxicity are still unclear. Here, we investigated whether RTP801 is involved in (mhtt)-induced cell death. Ectopic exon-1 mhtt elevated RTP801 mRNA and protein levels in nerve growth factor (NGF)-differentiated PC12 cells and in rat primary cortical neurons. In neuronal PC12 cells, mhtt also contributed to RTP801 protein elevation by reducing its proteasomal degradation rate, in addition to promoting RTP801 gene expression. Interestingly, silencing RTP801 expression with short hairpin RNAs (shRNAs) blocked mhtt-induced cell death in NGF-differentiated PC12 cells. However, RTP801 protein levels were not altered in the striatum of Hdh(Q7/Q111) and R6/1 mice, two HD models that display motor deficits but not neuronal death. Importantly, RTP801 protein levels were elevated in both neural telencephalic progenitors differentiated from HD patient-derived induced pluripotent stem cells and in the putamen and cerebellum of human HD postmortem brains. Taken together, our results suggest that RTP801 is a novel downstream effector of mhtt-induced toxicity and that it may be relevant to the human disease.

The Toxic Effects of Pathogenic Ataxin-3 Variants in a Yeast Cellular Model.

Ataxin-3 (AT3) is a deubiquitinating enzyme that triggers an inherited neurodegenerative disorder, spinocerebellar ataxia type 3, when its polyglutamine (polyQ) stretch close to the C-terminus exceeds a critical length. AT3 variants carrying the expanded polyQ are prone to associate with each other into amyloid toxic aggregates, which are responsible for neuronal death with ensuing neurodegeneration. We employed Saccharomyces cerevisiae as a eukaryotic cellular model to better clarify the mechanism by which AT3 triggers the disease. We expressed three variants: one normal (Q26), one expanded (Q85) and one truncated for a region lying from the beginning of its polyQ stretch to the end of the protein (291Δ). We found that the expression of the expanded form caused reduction in viability, accumulation of reactive oxygen species, imbalance of the antioxidant defense system and loss in cell membrane integrity, leading to necrotic death. The truncated variant also exerted a qualitatively similar, albeit milder, effect on cell growth and cytotoxicity, which points to the involvement of also non-polyQ regions in cytotoxicity. Guanidine hydrochloride, a well-known inhibitor of the chaperone Hsp104, almost completely restored wild-type survival rate of both 291Δ- and Q85-expressing strains. This suggests that AT3 aggregation and toxicity is mediated by prion forms of yeast proteins, as this chaperone plays a key role in their propagation.

A deletion mutant ndv200 of the Bacillus thuringiensis vip3BR insecticidal toxin gene is a prospective candidate for the next generation of genetically modified crop plants resistant to lepidopteran insect damage.

MAIN CONCLUSION: Ectopic expression of a deletion mutant ( ndv200 ) of Bacillus thuringiensis vip3BR gene in tobacco plant provided almost complete protection against major crop pests cotton boll worm ( Helicoverpa armigera ), black cut worm ( Agrotis ipsilon ) and cotton leaf worm ( Spodoptera littoralis ). Whereas vip3BR transgenic tobacco plant failed to protect themselves from these insects and showed resistance towards cotton leaf worm only. An analogous form of the Bacillus thuringiensis vip3Aa insecticidal toxin gene, named vip3BR, was identified and characterized, and exhibited similar attributes to the well-known Vip3Aa toxin. Vip3BR possessed broad-spectrum lepidopteran-specific insecticidal properties effective against most major crop pests of the Indian subcontinent. A Vip3BR toxin protein N-terminal deletion mutant, Ndv200, showed increased insecticidal potency relative to the native toxin, which conferred efficacy against four major crop pests, including cotton boll worm (Helicoverpa armigera), black cut worm (Agrotis ipsilon), cotton leaf worm (Spodoptera littoralis), and rice yellow stem borer (Scirpophaga incertulas). Ligand blot analysis indicated the Ndv200 toxin recognized the same larval midgut receptors as the native Vip3BR toxin, but differed from receptors recognized by Cry1A toxins. In the present study, we tested the prospect of the vip3BR and ndv200 toxin gene as candidate in development of insect-resistant genetically engineered crop plants by generating transgenic tobacco plant. The study revealed that the ndv200 mutant of vip3BR insecticidal toxin gene is a strong and prospective candidate for the next generation of genetically modified crop plants resistant to lepidopteran insects.