Pharmacological tuning of heat shock protein 70 modulates polyglutamine toxicity and aggregation

Nine neurodegenerative disorders are caused by the abnormal expansion of polyglutamine (polyQ) regions within distinct proteins. Genetic and biochemical evidence has documented that the molecular chaperone, heat shock protein 70 (Hsp70), modulates polyQ toxicity and aggregation, yet it remains unclear how Hsp70 might be used as a potential therapeutic target in polyQ-related diseases. We have utilized a pair of membrane-permeable compounds that tune the activity of Hsp70 by either stimulating or by inhibiting its ATPase functions. Using these two pharmacological agents in both yeast and PC12 cell models of polyQ aggregation and toxicity, we were surprised to find that stimulating Hsp70 solubilized polyQ conformers and simultaneously exacerbated polyQ-mediated toxicity. By contrast, inhibiting Hsp70 ATPase activity protected against polyQ toxicity and promoted aggregation. These findings clarify the role of Hsp70 as a possible drug target in polyQ disorders and suggest that Hsp70 uses ATP hydrolysis to help partition polyQ proteins into structures with varying levels of proteotoxicity. Our results thus support an emerging concept in which certain kinds of polyQ aggregates may be protective, while more soluble polyQ species are toxic.     

Loss of Hsp70 in Drosophila is pleiotropic, with effects on thermotolerance, recovery from heat shock and neurodegeneration

The heat-shock response is a programmed change in gene expression carried out by cells in response to environmental stress, such as heat. This response is universal and is characterized by the synthesis of a small group of conserved protein chaperones. In Drosophila melanogaster the Hsp70 chaperone dominates the profile of protein synthesis during the heat-shock response. We recently generated precise deletion alleles of the Hsp70 genes of D. melanogaster and have used those alleles to characterize the phenotypes of Hsp70-deficient flies. Flies with Hsp70 deletions have reduced thermotolerance. We find that Hsp70 is essential to survive a severe heat shock, but is not required to survive a milder heat shock, indicating that a significant degree of thermotolerance remains in the absence of Hsp70. However, flies without Hsp70 have a lengthened heat-shock response and an extended developmental delay after a non-lethal heat shock, indicating Hsp70 has an important role in recovery from stress, even at lower temperatures. Lack of Hsp70 also confers enhanced sensitivity to a temperature-sensitive lethal mutation and to the neurodegenerative effects produced by expression of a human polyglutamine disease protein.     

Could yeast prion domains originate from polyQ/N tracts?

A significant body of evidence shows that polyglutamine (polyQ) tracts are important for various biological functions. The characteristic polymorphism of polyQ length is thought to play an important role in the adaptation of organisms to their environment. However, proteins with expanded polyQ are prone to form amyloids, which cause diseases in humans and animals and toxicity in yeast. Saccharomyces cerevisiae contain at least 8 proteins which can form heritable amyloids, called prions, and most of them are proteins with glutamine- and asparagine-enriched domains. Yeast prion amyloids are susceptible to fragmentation by the protein disaggregase Hsp104, which allows them to propagate and be transmitted to daughter cells during cell divisions. We have previously shown that interspersion of polyQ domains with some non-glutamine residues stimulates fragmentation of polyQ amyloids in yeast and that yeast prion domains are often enriched in one of these residues. These findings indicate that yeast prion domains may have derived from polyQ tracts via accumulation and amplification of mutations. The same hypothesis may be applied to polyasparagine (polyN) tracts, since they display similar properties to polyQ, such as length polymorphism, amyloid formation and toxicity. We propose that mutations in polyQ/N may be favored by natural selection thus making prion domains likely by-products of the evolution of polyQ/N.     

Genomic deletions of the Drosophila melanogaster Hsp70 genes

Homologous recombination can produce directed mutations in the genomes of a number of model organisms, including Drosophila melanogaster. One of the most useful applications has been to delete target genes to generate null alleles. In Drosophila, specific gene deletions have not yet been produced by this method. To test whether such deletions could be produced by homologous recombination in D. melanogaster we set out to delete the Hsp70 genes. Six nearly identical copies of this gene, encoding the major heat-shock protein in Drosophila, are found at two separate but closely linked loci. This arrangement has thwarted standard genetic approaches to generate an Hsp70-null fly, making this an ideal test of gene targeting. In this study, ends-out targeting was used to generate specific deletions of all Hsp70 genes, including one deletion that spanned approximately 47 kb. The Hsp70-null flies are viable and fertile. The results show that genomic deletions of varied sizes can be readily generated by homologous recombination in Drosophila.     

Polyglutamine tracts regulate autophagy

Expansions of polyglutamine (polyQ) tracts in different proteins cause 9 neurodegenerative conditions, such as Huntington disease and various ataxias. However, many normal mammalian proteins contain shorter polyQ tracts. As these are frequently conserved in multiple species, it is likely that some of these polyQ tracts have important but unknown biological functions. Here we review our recent study showing that the polyQ domain of the deubiquitinase ATXN3/ataxin-3 enables its interaction with BECN1/beclin 1, a key macroautophagy/autophagy initiator. ATXN3 regulates autophagy by deubiquitinating BECN1 and protecting it from proteasomal degradation. Interestingly, expanded polyQ tracts in other polyglutamine disease proteins compete with the shorter ATXN3 polyQ stretch and interfere with the ATXN3-BECN1 interaction. This competition results in decreased BECN1 levels and impaired starvation-induced autophagy, which phenocopies the loss of autophagic function mediated by ATXN3. Our findings describe a new autophagy-protective mechanism that may be altered in multiple neurodegenerative diseases.     

Chaperonin TRiC promotes the assembly of polyQ expansion proteins into nontoxic oligomers

Aberrant folding and fibrillar aggregation by polyglutamine (polyQ) expansion proteins are associated with cytotoxicity in Huntington's disease and other neurodegenerative disorders. Hsp70 chaperones have an inhibitory effect on fibril formation and can alleviate polyQ cytotoxicity. Here we show that the cytosolic chaperonin, TRiC, functions synergistically with Hsp70 in this process and is limiting in suppressing polyQ toxicity in a yeast model. In vitro reconstitution experiments revealed that TRiC, in cooperation with the Hsp70 system, promotes the assembly of polyQ-expanded fragments of huntingtin (Htt) into soluble oligomers of approximately 500 kDa. Similar oligomers were observed in yeast cells upon TRiC overexpression and were found to be benign, in contrast to conformationally distinct Htt oligomers of approximately 200 kDa, which accumulated at normal TRiC levels and correlated with inhibition of cell growth. We suggest that TRiC cooperates with the Hsp70 system as a key component in the cellular defense against amyloid-like protein misfolding.     

Insight into role of selection in the evolution of polyglutamine tracts in humans

Glutamine tandem repeats are common in eukaryotic proteins. Although some studies have proposed that replication slippage plays an important role in shaping these repeats, the role of natural selection in glutamine tandem repeat evolution is somewhat unclear. In this study, we identified all of the glutamine tandem repeats containing four or more glutamines in human proteins and then estimated the nonsynonymous (d(N)) and synonymous (d(S)) substitution rates for the regions flanking the glutamine tandem repeats and the proteins containing them. The results indicated that most of the proteins containing polyglutamine (polyQ) tracts of four or more glutamines have undergone purifying selection, and that the purifying selection for the regions flanking the repeats is weaker. Additionally, we observed that the conserved repeats were under stronger selection constraints than the nonconserved repeats. Interestingly, we found that there was a higher level of purifying selection for the regions flanking the polyQ tracts encoded by pure CAG codons compared with those encoded by mixed codons. Based on our findings, we propose that selection has played a more important role than was previously speculated in constraining the expansion of polyQ tracts encoded by pure codons.     

Drosophila importin alpha1 performs paralog-specific functions essential for gametogenesis

Importin alpha's mediate nuclear transport by linking nuclear localization signal (NLS)-containing proteins to importin beta1. Animal genomes encode three conserved groups of importin alpha's, alpha1's, alpha2's, and alpha3's, each of which are competent to bind classical NLS sequences. Using Drosophila melanogaster we describe the isolation and phenotypic characterization of the first animal importin alpha1 mutant. Animal alpha1's are more similar to ancestral plant and fungal alpha1-like genes than to animal alpha2 and alpha3 genes. Male and female importin alpha1 (Dalpha1) null flies developed normally to adulthood (with a minor wing defect) but were sterile with defects in gametogenesis. The Dalpha1 mutant phenotypes were rescued by Dalpha1 transgenes, but not by Dalpha2 or Dalpha3 transgenes. Genetic interactions between the ectopic expression of Dalpha1 and the karyopherins CAS and importin beta1 suggest that high nuclear levels of Dalpha1 are deleterious. We conclude that Dalpha1 performs paralog-specific activities that are essential for gametogenesis and that regulation of subcellular Dalpha1 localization may affect cell fate decisions. The initial expansion and specialization of the animal importin alpha-gene family may have been driven by the specialized needs of gametogenesis. These results provide a framework for studies of the more complex mammalian importin alpha-gene family.     

Modulation of Molecular Chaperones in Huntington’s Disease and Other Polyglutamine Disorders

Polyglutamine expansion mutations in specific proteins underlie the pathogenesis of a group of progressive neurodegenerative disorders, including Huntington’s disease, spinal and bulbar muscular atrophy, dentatorubral-pallidoluysian atrophy, and several spinocerebellar ataxias. The different mutant proteins share ubiquitous expression and abnormal proteostasis, with misfolding and aggregation, but nevertheless evoke distinct patterns of neurodegeneration. This highlights the relevance of the full protein context where the polyglutamine expansion occurs and suggests different interactions with the cellular proteostasis machinery. Molecular chaperones are key elements of the proteostasis machinery and therapeutic targets for neurodegeneration. Here, we provide a focused review on Hsp90, Hsp70, and their co-chaperones, and how their genetic or pharmacological modulation affects the proteostasis and disease phenotypes in cellular and animal models of polyglutamine disorders. The emerging picture is that, in principle, Hsp70 modulation may be more amenable for long-term treatment by promoting a more selective clearance of mutant proteins than Hsp90 modulation, which may further decrease the necessary wild-type counterparts. It seems, nevertheless, unlikely that a single Hsp70 modulator will benefit all polyglutamine diseases. Indeed, available data, together with insights from effects on tau and alpha-synuclein in models of Alzheimer’s and Parkinson’s diseases, indicates that Hsp70 modulators may lead to different effects on the proteostasis of different mutant and wild-type client proteins. Future studies should include the further development of isoform selective inhibitors, namely to avoid off-target effects on Hsp in the mitochondria, and their characterization in distinct polyglutamine disease models to account for client protein-specific differences.     

Ordered assembly of heat shock proteins, Hsp26, Hsp70, Hsp90, and Hsp104, on expanded polyglutamine fragments revealed by chemical probes

In Saccharomyces cerevisae, expanded polyglutamine (polyQ) fragments are assembled into discrete cytosolic aggregates in a process regulated by the molecular chaperones Hsp26, Hsp70, Hsp90, and Hsp104. To better understand how the different chaperones might cooperate during polyQ aggregation, we used sequential immunoprecipitations and mass spectrometry to identify proteins associated with either soluble (Q25) or aggregation-prone (Q103) fragments at both early and later times after induction of their expression. We found that Hsp26, Hsp70, Hsp90, and other chaperones interact with Q103, but not Q25, within the first 2 h. Further, Hsp70 and Hsp90 appear to be partially released from Q103 prior to the maturation of the aggregates and before the recruitment of Hsp104. To test the importance of this seemingly ordered process, we used a chemical probe to artificially enhance Hsp70 binding to Q103. This treatment retained both Hsp70 and Hsp90 on the polyQ fragment and, interestingly, limited subsequent exchange for Hsp26 and Hsp104, resulting in incomplete aggregation. Together, these results suggest that partial release of Hsp70 may be an essential step in the continued processing of expanded polyQ fragments in yeast.     

棉花粉蚧热休克蛋白基因的鉴定

热休克蛋白(heat shock proteins,Hsps)是生物体或细胞受到热胁迫后新合成的一类遗传上高度保守的蛋白,在昆虫应对外界环境因子胁迫时起着重要作用。为了系统研究棉花粉蚧Phenacoccus solenopsis Hsp基因家族,对棉花粉蚧转录组基因注释信息进行分析、获得目标序列,并应用NCBI上Blast X等软件进行比对、共鉴定出24条热激蛋白(Hsp)基因,包括3个Hsp90、8个Hsp70、2个Hsp60和11个s Hsp(small heat shock protein,s Hsp)基因。对棉花粉蚧与模式昆虫家蚕Bombyx mori、黑腹果蝇Drosophila melanogaster、赤拟谷盗Tribolium castaneum系统进化关系分析显示,昆虫的小分子量热休克蛋白s Hsp具有很强的种属特异性,Hsp70家族的保守性比s Hsp强。棉花粉蚧热激蛋白基因的鉴定为深入研究该虫Hsp与生长发育、抗逆境的相互关系奠定了基础。     

Pathogenic and non-pathogenic polyglutamine tracts have similar structural properties: towards a length-dependent toxicity gradient

Abnormally expanded polyglutamine (polyQ) tracts provide a gain of toxic functions to nine otherwise unrelated human proteins and induce progressive neurodegenerative diseases. Over the past ten years, it was suggested that only polyQ tracts longer than a specific threshold adopt a particular structure, which would be the cause of the apparent polyQ length-dependent toxicity threshold observed in polyQ diseases. We have used a combination of biochemical and biophysical approaches to compare the structural properties of polyQ of pathogenic and non-pathogenic lengths under various conditions. We observe that pathogenic and non-pathogenic polyQ, as soluble species and upon interaction with a partner, during aggregation, or as mature aggregates, display very similar structural properties. PolyQ length only influences the aggregation kinetics and, to a lesser extent, the stability of the aggregates. We thus propose that polyQ toxicity does not depend on a structural transition occurring above a specific threshold, but rather that polyQ tracts are inherently toxic sequences, whose deleterious effect gradually increases with their length. We discuss how polyQ properties and other cellular factors may explain the existence of an apparent polyQ length-dependent toxicity threshold.     

The structure of a polyQ-anti-polyQ complex reveals binding according to a linear lattice model

Huntington and related neurological diseases result from expansion of a polyglutamine (polyQ) tract. The linear lattice model for the structure and binding properties of polyQ proposes that both expanded and normal polyQ tracts in the preaggregation state are random-coil structures but that an expanded polyQ repeat contains a larger number of epitopes recognized by antibodies or other proteins. The crystal structure of polyQ bound to MW1, an antibody against polyQ, reveals that polyQ adopts an extended, coil-like structure. Consistent with the linear lattice model, multimeric MW1 Fvs bind more tightly to longer than to shorter polyQ tracts and, compared with monomeric Fv, bind expanded polyQ repeats with higher apparent affinities. These results suggest a mechanism for the toxicity of expanded polyQ and a strategy to link anti-polyQ compounds to create high-avidity therapeutics.     

Identification of anti-prion compounds as efficient inhibitors of polyglutamine protein aggregation in a zebrafish model

Several neurodegenerative diseases, including Huntington disease (HD), are associated with aberrant folding and aggregation of polyglutamine (polyQ) expansion proteins. Here we established the zebrafish, Danio rerio, as a vertebrate HD model permitting the screening for chemical suppressors of polyQ aggregation and toxicity. Upon expression in zebrafish embryos, polyQ-expanded fragments of huntingtin (htt) accumulated in large SDS-insoluble inclusions, reproducing a key feature of HD pathology. Real time monitoring of inclusion formation in the living zebrafish indicated that inclusions grow by rapid incorporation of soluble htt species. Expression of mutant htt increased the frequency of embryos with abnormal morphology and the occurrence of apoptosis. Strikingly, apoptotic cells were largely devoid of visible aggregates, suggesting that soluble oligomeric precursors may instead be responsible for toxicity. As in nonvertebrate polyQ disease models, the molecular chaperones, Hsp40 and Hsp70, suppressed both polyQ aggregation and toxicity. Using the newly established zebrafish model, two compounds of the N'-benzylidene-benzohydrazide class directed against mammalian prion proved to be potent inhibitors of polyQ aggregation, consistent with a common structural mechanism of aggregation for prion and polyQ disease proteins.     

Huntington toxicity in yeast model depends on polyglutamine aggregation mediated by a prion-like protein Rnq1

The cause of Huntington's disease is expansion of polyglutamine (polyQ) domain in huntingtin, which makes this protein both neurotoxic and aggregation prone. Here we developed the first yeast model, which establishes a direct link between aggregation of expanded polyQ domain and its cytotoxicity. Our data indicated that deficiencies in molecular chaperones Sis1 and Hsp104 inhibited seeding of polyQ aggregates, whereas ssa1, ssa2, and ydj1-151 mutations inhibited expansion of aggregates. The latter three mutants strongly suppressed the polyQ toxicity. Spontaneous mutants with suppressed aggregation appeared with high frequency, and in all of them the toxicity was relieved. Aggregation defects in these mutants and in sis1-85 were not complemented in the cross to the hsp104 mutant, demonstrating an unusual type of inheritance. Since Hsp104 is required for prion maintenance in yeast, this suggested a role for prions in polyQ aggregation and toxicity. We screened a set of deletions of nonessential genes coding for known prions and related proteins and found that deletion of the RNQ1 gene specifically suppressed aggregation and toxicity of polyQ. Curing of the prion form of Rnq1 from wild-type cells dramatically suppressed both aggregation and toxicity of polyQ. We concluded that aggregation of polyQ is critical for its toxicity and that Rnq1 in its prion conformation plays an essential role in polyQ aggregation leading to the toxicity.     

Persistence time of loss-of-function mutations at nonessential loci affecting eye color in Drosophila melanogaster

Persistence time of a mutant allele, the expected number of generations before its elimination from the population, can be estimated as the ratio of the number of segregating mutations per individual over the mutation rate per generation. We screened two natural populations of Drosophila melanogaster for mutations causing clear-cut eye phenotypes and detected 25 mutant alleles, falling into 19 complementation groups, in 1164 haploid genomes, which implies 0.021 eye mutations/genome. The de novo haploid mutation rate for the same set of loci was estimated as 2 x 10(-4) in a 10-generation mutation-accumulation experiment. Thus, the average persistence time of all mutations causing clear-cut eye phenotypes is approximately 100 generations (95% confidence interval: 61-219). This estimate shows that the strength of selection against phenotypically drastic alleles of nonessential loci is close to that against recessive lethals. In both cases, deleterious alleles are apparently eliminated by selection against heterozygous individuals, which show no visible phenotypic differences from wild type.     

S-element insertions are associated with the evolution of the Hsp70 genes in Drosophila melanogaster

The "selfish DNA" theory postulates that transposable elements (TEs) are intragenomic parasites, and that natural selection against deleterious effects associated with their presence is the main force preventing their genomic spread in natural populations. In agreement with this model, TEs in Drosophila melanogaster populations are usually found at low frequencies in most genomic locations. Only a few cases of fixation of TE insertions have been reported, usually in regions of low recombination, where selection is expected to be less effective. Here, we report a population genetics study on the apparent fixation of an S-element in a highly recombining region in two natural populations of D. melanogaster. Three similar fragments of an S-element are inserted into the 5' regions of three members of a heat shock gene family, Hsp70 (Hsp70Aa and Hsp70Ab in polytene chromosome band 87A, and Hsp70Bb in 87C). A PCR-based analysis suggests that the insertions are fixed or at high frequencies in the entire species. A population survey of the levels of nucleotide sequence variation at the insertion site in 87C in two natural populations of D. melanogaster provided evidence for reduced levels of variation in the region, normal levels of recombination, and selection, reflected in a significant departure from neutrality of the variant frequency spectrum. This was particularly strong for the S-element inverted repeats (IRs) and suggests that these are of functional significance for the host.     

A novel GTPase, CRAG, mediates promyelocytic leukemia protein-associated nuclear body formation and degradation of expanded polyglutamine protein

Polyglutamine diseases are inherited neurodegenerative diseases caused by the expanded polyglutamine proteins (polyQs). We have identified a novel guanosine triphosphatase (GTPase) named CRAG that contains a nuclear localization signal (NLS) sequence and forms nuclear inclusions in response to stress. After ultraviolet irradiation, CRAG interacted with and induced an enlarged ring-like structure of promyelocytic leukemia protein (PML) body in a GTPase-dependent manner. Reactive oxygen species (ROS) generated by polyQ accumulation triggered the association of CRAG with polyQ and the nuclear translocation of the CRAG-polyQ complex. Furthermore, CRAG promoted the degradation of polyQ at PML/CRAG bodies through the ubiquitin-proteasome pathway. CRAG knockdown by small interfering RNA in neuronal cells consistently blocked the nuclear translocation of polyQ and enhanced polyQ-mediated cell death. We propose that CRAG is a modulator of PML function and dynamics in ROS signaling and is protectively involved in the pathogenesis of polyglutamine diseases.     

Solution structure of polyglutamine tracts in GST-polyglutamine fusion proteins

Aggregation of expanded polyglutamine (polyQ) seems to be the cause of various genetic neurodegenerative diseases. Relatively little is known as yet about the polyQ structure and the mechanism that induces aggregation. We have characterised the solution structure of polyQ in a proteic context using a model system based on glutathione S-transferase fusion proteins. A wide range of biophysical techniques was applied. For the first time, nuclear magnetic resonance was used to observe directly and selectively the conformation of polyQ in the pathological range. We demonstrate that, in solution, polyQs are in a random coil conformation. However, under destabilising conditions, their aggregation behaviour is determined by the polyQ length.     

Hsp70 duplication in the Drosophila melanogaster species group: how and when did two become five?

To determine how the modern copy number (5) of hsp70 genes in Drosophila melanogaster evolved, we localized the duplication events that created the genes in the phylogeny of the melanogaster group, examined D. melanogaster genomic sequence to investigate the mechanisms of duplication, and analyzed the hsp70 gene sequences of Drosophila orena and Drosophila mauritiana. The initial two-to-four hsp70 duplication occurred 10--15 MYA, according to fixed in situ hybridization to polytene chromosomes, before the origin and divergence of the melanogaster and five other species subgroups of the melanogaster group. Analysis of more than 30 kb of flanking sequence surrounding the hsp70 gene clusters suggested that this duplication was likely a retrotransposition. For the melanogaster subgroup, Southern hybridization and an hsp70 restriction map confirmed the conserved number (4) and arrangement of hsp70 genes in the seven species other than D. melanogaster. Drosophila melanogaster is unique; tandem duplication and gene conversion at the derived cluster yielded a fifth hsp70 gene. The four D. orena hsp70 genes are highly similar and concertedly evolving. In contrast, the D. mauritiana hsp70 genes are divergent, and many alleles are nonfunctional. The proliferation, concerted evolution, and maintenance of functionality in the D. melanogaster hsp70 genes is consistent with the action of natural selection in this species.