Bacterial Growth Inhibition Screen (BGIS): harnessing recombinant protein toxicity for rapid and unbiased interrogation of protein function.

In two proof-of-concept studies, we established and validated the Bacterial Growth Inhibition Screen (BGIS), which explores recombinant protein toxicity in Escherichia coli as a largely overlooked and alternative means for basic characterization of functional eukaryotic protein domains. By applying BGIS, we identified an unrecognized RNA-interacting domain in the DEK oncoprotein (this study) and successfully combined BGIS with random mutagenesis as a screening tool for loss-of-function mutants of the DNA modulating domain of DEK [1]. Collectively, our findings shed new light on the phenomenon of recombinant protein toxicity in E. coli. Given the easy and rapid implementation and wide applicability, BGIS will extend the repertoire of basic methods for the identification, analysis and unbiased manipulation of proteins.

Differential toxicity of ataxin-3 isoforms in Drosophila models of Spinocerebellar Ataxia Type 3.

The most commonly inherited dominant ataxia, Spinocerebellar Ataxia Type 3 (SCA3), is caused by a CAG repeat expansion that encodes an abnormally long polyglutamine (polyQ) repeat in the disease protein ataxin-3, a deubiquitinase. Two major full-length isoforms of ataxin-3 exist, both of which contain the same N-terminal portion and polyQ repeat, but differ in their C-termini; one (denoted here as isoform 1) contains a motif that binds ataxin-3's substrate, ubiquitin, whereas the other (denoted here as isoform 2) has a hydrophobic tail. Most SCA3 studies have focused on isoform 1, the predominant version in mammalian brain, yet both isoforms are present in brain and a better understanding of their relative pathogenicity in vivo is needed. We took advantage of the fruit fly, Drosophila melanogaster to model SCA3 and to examine the toxicity of each ataxin-3 isoform. Our assays reveal isoform 1 to be markedly more toxic than isoform 2 in all fly tissues. Reduced toxicity from isoform 2 is due to much lower protein levels as a result of its expedited degradation. Additional studies indicate that isoform 1 is more aggregation-prone than isoform 2 and that the C-terminus of isoform 2 is critical for its enhanced proteasomal degradation. According to our results, although both full-length, pathogenic ataxin-3 isoforms are toxic, isoform 1 is likely the primary contributor to SCA3 due to its presence at higher levels. Isoform 2, as a result of rapid degradation that is dictated by its tail, is unlikely to be a key player in this disease. Our findings provide new insight into the biology of this ataxia and the cellular processing of the underlying disease protein.

Molecular Dissection of FUS Points at Synergistic Effect of Low-Complexity Domains in Toxicity.

RNA-binding protein aggregation is a pathological hallmark of several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). To gain better insight into the molecular interactions underlying this process, we investigated FUS, which is mutated and aggregated in both ALS and FTLD. We generated a Drosophila model of FUS toxicity and identified a previously unrecognized synergistic effect between the N-terminal prion-like domain and the C-terminal arginine-rich domain to mediate toxicity. Although the prion-like domain is generally considered to mediate aggregation of FUS, we find that arginine residues in the C-terminal low-complexity domain are also required for maturation of FUS in cellular stress granules. These data highlight an important role for arginine-rich domains in the pathology of RNA-binding proteins.

Identification of Tau Toxicity Modifiers in the Drosophila Eye.

Drosophila is a powerful model to study human diseases thanks to its genetic tools and ease of screening. Human genes can be expressed in targeted organs and their toxicity assessed on easily scorable external phenotypes that can be used as readout to perform genetic screen of toxicity modifiers. In this chapter, I describe how to express human Tau protein in the Drosophila eye, assess protein expression by western blot, assess Tau toxicity by quantifying the size of the Tau-induced rough eye, and perform a genetic screen of modifiers of Tau toxicity in the Drosophila eye.

Differential Toxicity of Nuclear RNA Foci versus Dipeptide Repeat Proteins in a Drosophila Model of C9ORF72 FTD/ALS.

Dipeptide repeat (DPR) proteins are toxic in various models of FTD/ALS with GGGGCC (G4C2) repeat expansion. However, it is unclear whether nuclear G4C2 RNA foci also induce neurotoxicity. Here, we describe a Drosophila model expressing 160 G4C2 repeats (160R) flanked by human intronic and exonic sequences. Spliced intronic 160R formed nuclear G4C2 sense RNA foci in glia and neurons about ten times more abundantly than in human neurons; however, they had little effect on global RNA processing and neuronal survival. In contrast, highly toxic 36R in the context of poly(A)(+) mRNA were exported to the cytoplasm, where DPR proteins were produced at >100-fold higher level than in 160R flies. Moreover, the modest toxicity of intronic 160R expressed at higher temperature correlated with increased DPR production, but not RNA foci. Thus, nuclear RNA foci are neutral intermediates or possibly neuroprotective through preventing G4C2 RNA export and subsequent DPR production.

Impaired retrograde transport by the Dynein/Dynactin complex contributes to Tau-induced toxicity.

The gene mapt codes for the microtubule-associated protein Tau. The R406W amino acid substitution in Tau is associated with frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) characterized by Tau-positive filamentous inclusions. These filamentous Tau inclusions are present in a group of neurodegenerative diseases known as tauopathies, including Alzheimer's disease (AD). To gain more insights into the pathomechanism of tauopathies, we performed an RNAi-based large-scale screen in Drosophila melanogaster to identify genetic modifiers of Tau[R406W]-induced toxicity. A collection of RNAi lines, putatively silencing more than 7000 genes, was screened for the ability to modify Tau[R406W]-induced toxicity in vivo. This collection covered more than 50% of all protein coding fly genes and more than 90% of all fly genes known to have a human ortholog. Hereby, we identified 62 genes that, when silenced by RNAi, modified Tau-induced toxicity specifically. Among these 62 modifiers were three subunits of the Dynein/Dynactin complex. Analysis on segmental nerves of fly larvae showed that pan neural Tau[R406W] expression and concomitant silencing of Dynein/Dynactin complex members synergistically caused strong pathological changes within the axonal compartment, but only minor changes at synapses. At the larval stage, these alterations did not cause locomotion deficits, but became evident in adult flies. Our data suggest that Tau-induced detrimental effects most likely originate from axonal rather than synaptic dysfunction and that impaired retrograde transport intensifies detrimental effects of Tau in axons. In conclusion, our findings contribute to the elucidation of disease mechanisms in tauopathies like FTDP-17 or AD.

Aggregate formation prevents dTDP-43 neurotoxicity in the Drosophila melanogaster eye.

TDP-43 inclusions are an important histopathological feature in various neurodegenerative disorders, including Amyotrophic Lateral Sclerosis and Fronto-Temporal Lobar Degeneration. However, the relation of these inclusions with the pathogenesis of the disease is still unclear. In fact, the inclusions could be toxic themselves, induce loss of function by sequestering TDP-43 or a combination of both. Previously, we have developed a cellular model of aggregation using the TDP-43 Q/N rich amino acid sequence 331-369 repeated 12 times (12xQ/N) and have shown that these cellular inclusions are capable of sequestering the endogenous TDP-43 both in non-neuronal and neuronal cells. We have tested this model in vivo in the Drosophila melanogaster eye. The eye structure develops normally in the absence of dTDP-43, a fact previously seen in knock out fly strains. We show here that expression of EGFP 12xQ/N does not alter the structure of the eye. In contrast, TBPH overexpression is neurotoxic and causes necrosis and loss of function of the eye. More important, the neurotoxicity of TBPH can be abolished by its incorporation to the insoluble aggregates induced by EGFP 12xQ/N. This data indicates that aggregation is not toxic per se and instead has a protective role, modulating the functional TBPH available in the tissue. This is an important indication for the possible pathological mechanism in action on ALS patients.

Expression of a toll signaling regulator serpin in a mycoinsecticide for increased virulence.

Serpins are ubiquitously distributed serine protease inhibitors that covalently bind to target proteases to exert their activities. Serpins regulate a wide range of activities, particularly those in which protease-mediated cascades are active. The Drosophila melanogaster serpin Spn43Ac negatively controls the Toll pathway that is activated in response to fungal infection. The entomopathogenic fungus Beauveria bassiana offers an environmentally friendly alternative to chemical pesticides for insect control. However, the use of mycoinsecticides remains limited in part due to issues of efficacy (low virulence) and the recalcitrance of the targets (due to strong immune responses). Since Spn43Ac acts to inhibit Toll-mediated activation of defense responses, we explored the feasibility of a new strategy to engineer entomopathogenic fungi with increased virulence by expression of Spn43Ac in the fungus. Compared to the 50% lethal dose (LD50) for the wild-type parent, the LD50 of B. bassiana expressing Spn43Ac (strain Bb::S43Ac-1) was reduced ~3-fold, and the median lethal time against the greater wax moth (Galleria mellonella) was decreased by ~24%, with the more rapid proliferation of hyphal bodies being seen in the host hemolymph. In vitro and in vivo assays showed inhibition of phenoloxidase (PO) activation in the presence of Spn43Ac, with Spn43Ac-mediated suppression of activation by chymotrypsin, trypsin, laminarin, and lipopolysaccharide occurring in the following order: chymotrypsin and trypsin>laminarin>lipopolysaccharide. Expression of Spn43Ac had no effect on the activity of the endogenous B. bassianaderived cuticle-degrading protease (CDEP-1). These results expand our understanding of Spn43Ac function and confirm that suppression of insect immune system defenses represents a feasible approach to engineering entomopathogenic fungi for greater efficacy.

Triplet repeat-derived siRNAs enhance RNA-mediated toxicity in a Drosophila model for myotonic dystrophy.

More than 20 human neurological and neurodegenerative diseases are caused by simple DNA repeat expansions; among these, non-coding CTG repeat expansions are the basis of myotonic dystrophy (DM1). Recent work, however, has also revealed that many human genes have anti-sense transcripts, raising the possibility that human trinucleotide expansion diseases may be comprised of pathogenic activities due both to a sense expanded-repeat transcript and to an anti-sense expanded-repeat transcript. We established a Drosophila model for DM1 and tested the role of interactions between expanded CTG transcripts and expanded CAG repeat transcripts. These studies revealed dramatically enhanced toxicity in flies co-expressing CTG with CAG expanded repeats. Expression of the two transcripts led to novel pathogenesis with the generation of dcr-2 and ago2-dependent 21-nt triplet repeat-derived siRNAs. These small RNAs targeted the expression of CAG-containing genes, such as Ataxin-2 and TATA binding protein (TBP), which bear long CAG repeats in both fly and man. These findings indicate that the generation of triplet repeat-derived siRNAs may dramatically enhance toxicity in human repeat expansion diseases in which anti-sense transcription occurs.

Genetic screen identifies serpin5 as a regulator of the toll pathway and CHMP2B toxicity associated with frontotemporal dementia.

Frontotemporal dementia (FTD) is the most common form of dementia before 60 years of age. Rare pathogenic mutations in CHMP2B, which encodes a component of the endosomal sorting complex required for transport (ESCRT-III), are associated with FTD linked to chromosome 3 (FTD3). Animal models of FTD3 have not yet been reported, and what signaling pathways are misregulated by mutant CHMP2B in vivo is unknown. Here we report the establishment of a Drosophila model of FTD3 and show the genetic interactions between mutant CHMP2B and other components of ESCRT. Through an unbiased genome-wide screen, we identified 29 modifier loci and found that serpin5 (Spn5), a largely uncharacterized serine protease inhibitor, suppresses the melanization phenotype induced by mutant CHMP2B in the fly eye. We also found that Spn5 is a negative regulator of the Toll pathway and functions extracellularly, likely by blocking the proteolytic activation of Spaetzle, the Toll receptor ligand. Moreover, Spn5 inhibited activation of the Toll pathway by mutant CHMP2B. Our findings identify Spn5 as a regulator of the Toll pathway and CHMP2B toxicity and show that the Toll pathway is a major signaling pathway misregulated by mutant CHMP2B in vivo. This fly model will be useful to further dissect genetic pathways that are potentially relevant to the pathogenesis and treatment of FTD.

A Drosophila model of mutant human parkin-induced toxicity demonstrates selective loss of dopaminergic neurons and dependence on cellular dopamine.

Mutations in human parkin have been identified in familial Parkinson's disease and in some sporadic cases. Here, we report that expression of mutant but not wild-type human parkin in Drosophila causes age-dependent, selective degeneration of dopaminergic (DA) neurons accompanied by a progressive motor impairment. Overexpression or knockdown of the Drosophila vesicular monoamine transporter, which regulates cytosolic DA homeostasis, partially rescues or exacerbates, respectively, the degenerative phenotypes caused by mutant human parkin. These results support a model in which the vulnerability of DA neurons to parkin-induced neurotoxicity results from the interaction of mutant parkin with cytoplasmic dopamine.

An ectopic expression screen reveals the protective and toxic effects of Drosophila seminal fluid proteins.

In Drosophila melanogaster, seminal fluid regulates the reproductive and immune responses of mated females. Some seminal fluid proteins may provide protective functions to mated females, such as antimicrobial activity and/or stimulation of antimicrobial gene expression levels, while others appear to have negative effects, contributing to a "cost of mating." To identify seminal proteins that could participate in these phenomena, we used a systemic ectopic expression screen to test the effects on unmated females of proteins normally produced by the male accessory gland (Acps). Of the 21 ectopically expressed Acps that we tested for ability to assist in the clearance of a bacterial infection with Serratia marcescens, 3 Acps significantly reduced the bacterial counts of infected females, suggesting a protective role. Of the 23 Acps that we tested for toxicity, 3 were toxic, including one that has been implicated in the cost of mating in another study. We also tested ectopic expression females for other Acp-induced effects, but found no additional Acps that affected egg laying or receptivity upon ectopic expression.

Parkin suppresses dopaminergic neuron-selective neurotoxicity induced by Pael-R in Drosophila.

Parkin, an E3 ubiquitin ligase that degrades proteins with aberrant conformations, is associated with autosomal recessive juvenile Parkinsonism (AR-JP). The molecular basis of selective neuronal death in AR-JP is unknown. Here we show in an organismal system that panneuronal expression of Parkin substrate Pael-R causes age-dependent selective degeneration of Drosophila dopaminergic (DA) neurons. Coexpression of Parkin degrades Pael-R and suppresses its toxicity, whereas interfering with endogenous Drosophila Parkin function promotes Pael-R accumulation and augments its toxicity. Furthermore, overexpression of Parkin can mitigate alpha-Synuclein-induced neuritic pathology and suppress its toxicity. Our study implicates Parkin as a central player in the molecular pathway of Parkinson's disease (PD) and suggests that manipulating Parkin expression may provide a novel avenue of PD therapy.

"In vivo" toxicity of a truncated version of the Drosophila Rst-IrreC protein is dependent on the presence of a glutamine-rich region in its intracellular domain

The roughest-irregular chiasm C ( rst-irreC) gene of Drosophila melanogaster encodes a transmembrane glycoprotein containing five immunoglobulin-like domains in its extracellular portion and an intracytoplasmic tail rich in serine and threonine as well some conserved motifs suggesting signal transduction activity. In the compound eye, loss-of-function rst-irreC mutants lack the characteristic wave of programmed cell death happening in early pupa and which is essential for the elimination of the surplus interommatidial cells. Here we report an investigation on the role played by the Rst-irreC molecule in triggering programmed cell death. "In vivo" transient expression assays showed that deletion of the last 80 amino acids of the carboxyl terminus produces a form of the protein that is highly toxic to larvae. This toxicity is suppressed if an additional 47 amino acid long, glutamine-rich region ("opa-like domain"), is also removed from the protein. The results suggest the possibility that the opa-like domain and the carboxyl terminus act in concert to modulate rst-irreC function in apoptosis, and we discuss this implication in the context of the general mechanisms causing glutamine-rich neurodegenerative diseases in humans

The Drosophila melanogaster seminal fluid protein Acp62F is a protease inhibitor that is toxic upon ectopic expression.

Drosophila melanogaster seminal fluid proteins stimulate sperm storage and egg laying in the mated female but also cause a reduction in her life span. We report here that of eight Drosophila seminal fluid proteins (Acps) and one non-Acp tested, only Acp62F is toxic when ectopically expressed. Toxicity to preadult male or female Drosophila occurs upon one exposure, whereas multiple exposures are needed for toxicity to adult female flies. Of the Acp62F received by females during mating, approximately 10% enters the circulatory system while approximately 90% remains in the reproductive tract. We show that in the reproductive tract, Acp62F localizes to the lumen of the uterus and the female's sperm storage organs. Analysis of Acp62F's sequence, and biochemical assays, reveals that it encodes a trypsin inhibitor with sequence and structural similarities to extracellular serine protease inhibitors from the nematode Ascaris. In light of previous results demonstrating entry of Acp62F into the mated female's hemolymph, we propose that Acp62F is a candidate for a molecule to contribute to the Acp-dependent decrease in female life span. We propose that Acp62F's protease inhibitor activity exerts positive protective functions in the mated female's reproductive tract but that entry of a small amount of this protein into the female's hemolymph could contribute to the cost of mating.