Impaired Heat Shock Response in Cells Expressing Full-Length Polyglutamine-Expanded Huntingtin

The molecular mechanisms by which polyglutamine (polyQ)-expanded huntingtin (Htt) causes neurodegeneration in Huntington's disease (HD) remain unclear. The malfunction of cellular proteostasis has been suggested as central in HD pathogenesis and also as a target of therapeutic interventions for the treatment of HD. We present results that offer a previously unexplored perspective regarding impaired proteostasis in HD. We find that, under non-stress conditions, the proteostatic capacity of cells expressing full length polyQ-expanded Htt is adequate. Yet, under stress conditions, the presence of polyQ-expanded Htt impairs the heat shock response, a key component of cellular proteostasis. This impaired heat shock response results in a reduced capacity to withstand the damage caused by cellular stress. We demonstrate that in cells expressing polyQ-expanded Htt the levels of heat shock transcription factor 1 (HSF1) are reduced, and, as a consequence, these cells have an impaired a heat shock response. Also, we found reduced HSF1 and HSP70 levels in the striata of HD knock-in mice when compared to wild-type mice. Our results suggests that full length, non-aggregated polyQ-expanded Htt blocks the effective induction of the heat shock response under stress conditions and may thus trigger the accumulation of cellular damage during the course of HD pathogenesis

Amyloid-Mediated Sequestration of Essential Proteins Contributes to Mutant Huntingtin Toxicity in Yeast

BACKGROUND: Polyglutamine expansion is responsible for several neurodegenerative disorders, among which Huntington disease is the most well-known. Studies in the yeast model demonstrated that both aggregation and toxicity of a huntingtin (htt) protein with an expanded polyglutamine region strictly depend on the presence of the prion form of Rnq1 protein ([PIN+]), which has a glutamine/asparagine-rich domain. PRINCIPAL FINDINGS: Here, we showed that aggregation and toxicity of mutant htt depended on [PIN+] only quantitatively: the presence of [PIN+] elevated the toxicity and the levels of htt detergent-insoluble polymers. In cells lacking [PIN+], toxicity of mutant htt was due to the polymerization and inactivation of the essential glutamine/asparagine-rich Sup35 protein and related inactivation of another essential protein, Sup45, most probably via its sequestration into Sup35 aggregates. However, inhibition of growth of [PIN+] cells depended on Sup35/Sup45 depletion only partially, suggesting that there are other sources of mutant htt toxicity in yeast. CONCLUSIONS: The obtained data suggest that induced polymerization of essential glutamine/asparagine-rich proteins and related sequestration of other proteins which interact with these polymers represent an essential source of htt toxicity

Off-Target Effects of Psychoactive Drugs Revealed by Genome-Wide Assays in Yeast

To better understand off-target effects of widely prescribed psychoactive drugs, we performed a comprehensive series of chemogenomic screens using the budding yeast Saccharomyces cerevisiae as a model system. Because the known human targets of these drugs do not exist in yeast, we could employ the yeast gene deletion collections and parallel fitness profiling to explore potential off-target effects in a genome-wide manner. Among 214 tested, documented psychoactive drugs, we identified 81 compounds that inhibited wild-type yeast growth and were thus selected for genome-wide fitness profiling. Many of these drugs had a propensity to affect multiple cellular functions. The sensitivity profiles of half of the analyzed drugs were enriched for core cellular processes such as secretion, protein folding, RNA processing, and chromatin structure. Interestingly, fluoxetine (Prozac) interfered with establishment of cell polarity, cyproheptadine (Periactin) targeted essential genes with chromatin-remodeling roles, while paroxetine (Paxil) interfered with essential RNA metabolism genes, suggesting potential secondary drug targets. We also found that the more recently developed atypical antipsychotic clozapine (Clozaril) had no fewer off-target effects in yeast than the typical antipsychotics haloperidol (Haldol) and pimozide (Orap). Our results suggest that model organism pharmacogenetic studies provide a rational foundation for understanding the off-target effects of clinically important psychoactive agents and suggest a rational means both for devising compound derivatives with fewer side effects and for tailoring drug treatment to individual patient genotypes

Polyglutamine Induced Misfolding of Huntingtin Exon1 is Modulated by the Flanking Sequences

Polyglutamine (polyQ) expansion in exon1 (XN1) of the huntingtin protein is linked to Huntington's disease. When the number of glutamines exceeds a threshold of approximately 36–40 repeats, XN1 can readily form amyloid aggregates similar to those associated with disease. Many experiments suggest that misfolding of monomeric XN1 plays an important role in the length-dependent aggregation. Elucidating the misfolding of a XN1 monomer can help determine the molecular mechanism of XN1 aggregation and potentially help develop strategies to inhibit XN1 aggregation. The flanking sequences surrounding the polyQ region can play a critical role in determining the structural rearrangement and aggregation mechanism of XN1. Few experiments have studied XN1 in its entirety, with all flanking regions. To obtain structural insights into the misfolding of XN1 toward amyloid aggregation, we perform molecular dynamics simulations on monomeric XN1 with full flanking regions, a variant missing the polyproline regions, which are hypothesized to prevent aggregation, and an isolated polyQ peptide (Qn). For each of these three constructs, we study glutamine repeat lengths of 23, 36, 40 and 47. We find that polyQ peptides have a positive correlation between their probability to form a β-rich misfolded state and their expansion length. We also find that the flanking regions of XN1 affect its probability to^x_page_count=28 form a β-rich state compared to the isolated polyQ. Particularly, the polyproline regions form polyproline type II helices and decrease the probability of the polyQ region to form a β-rich state. Additionally, by lengthening polyQ, the first N-terminal 17 residues are more likely to adopt a β-sheet conformation rather than an α-helix conformation. Therefore, our molecular dynamics study provides a structural insight of XN1 misfolding and elucidates the possible role of the flanking sequences in XN1 aggregation

Disease-Associated Mutant Ubiquitin Causes Proteasomal Impairment and Enhances the Toxicity of Protein Aggregates

Protein homeostasis is critical for cellular survival and its dysregulation has been implicated in Alzheimer's disease (AD) and other neurodegenerative disorders. Despite the growing appreciation of the pathogenic mechanisms involved in familial forms of AD, much less is known about the sporadic cases. Aggregates found in both familial and sporadic AD often include proteins other than those typically associated with the disease. One such protein is a mutant form of ubiquitin, UBB+1, a frameshift product generated by molecular misreading of a wild-type ubiquitin gene. UBB+1 has been associated with multiple disorders. UBB+1 cannot function as a ubiquitin molecule, and it is itself a substrate for degradation by the ubiquitin/proteasome system (UPS). Accumulation of UBB+1 impairs the proteasome system and enhances toxic protein aggregation, ultimately resulting in cell death. Here, we describe a novel model system to investigate how UBB+1 impairs UPS function and whether it plays a causal role in protein aggregation. We expressed a protein analogous to UBB+1 in yeast (Ubext) and demonstrated that it caused UPS impairment. Blocking ubiquitination of Ubext or weakening its interactions with other ubiquitin-processing proteins reduced the UPS impairment. Expression of Ubext altered the conjugation of wild-type ubiquitin to a UPS substrate. The expression of Ubext markedly enhanced cellular susceptibility to toxic protein aggregates but, surprisingly, did not induce or alter nontoxic protein aggregates in yeast. Taken together, these results suggest that Ubext interacts with more than one protein to elicit impairment of the UPS and affect protein aggregate toxicity. Furthermore, we suggest a model whereby chronic UPS impairment could inflict deleterious consequences on proper protein aggregate sequestration

Rapid selection of cyclic peptides that reduce alpha-synuclein toxicity in yeast and animal models

Phage display has demonstrated the utility of cyclic peptides as general protein ligands but cannot access proteins inside eukaryotic cells. Expanding a new chemical genetics tool, we describe the first expressed library of head-to-tail cyclic peptides in yeast (Saccharomyces cerevisiae). We applied the library to selections in a yeast model of alpha-synuclein toxicity that recapitulates much of the cellular pathology of Parkinson's disease. From a pool of 5 million transformants, we isolated two related cyclic peptide constructs that specifically reduced the toxicity of human alpha-synuclein. These expressed cyclic peptide constructs also prevented dopaminergic neuron loss in an established Caenorhabditis elegans Parkinson's model. This work highlights the speed and efficiency of using libraries of expressed cyclic peptides for forward chemical genetics in cellular models of human disease

Protein Folding Activity of the Ribosome is involved in Yeast Prion Propagation.

6AP and GA are potent inhibitors of yeast and mammalian prions and also specific inhibitors of PFAR, the protein-folding activity borne by domain V of the large rRNA of the large subunit of the ribosome. We therefore explored the link between PFAR and yeast prion [PSI(+)] using both PFAR-enriched mutants and site-directed methylation. We demonstrate that PFAR is involved in propagation and de novo formation of [PSI(+)]. PFAR and the yeast heat-shock protein Hsp104 partially compensate each other for [PSI(+)] propagation. Our data also provide insight into new functions for the ribosome in basal thermotolerance and heat-shocked protein refolding. PFAR is thus an evolutionarily conserved cell component implicated in the prion life cycle, and we propose that it could be a potential therapeutic target for human protein misfolding diseases

Selenomethionine Incorporation into Amyloid Sequences Regulates Fibrillogenesis and Toxicity

The capacity of a polypeptide chain to engage in an amyloid formation process and cause a conformational disease is contained in its sequence. Some of the sequences undergoing fibrillation contain critical methionine (Met) residues which in vivo can be synthetically substituted by selenomethionine (SeM) and alter their properties

Elevated Proteasome Capacity Extends Replicative Lifespan in Saccharomyces cerevisiae

Aging is characterized by the accumulation of damaged cellular macromolecules caused by declining repair and elimination pathways. An integral component employed by cells to counter toxic protein aggregates is the conserved ubiquitin/proteasome system (UPS). Previous studies have described an age-dependent decline of proteasomal function and increased longevity correlates with sustained proteasome capacity in centenarians and in naked mole rats, a long-lived rodent. Proof for a direct impact of enhanced proteasome function on longevity, however, is still lacking. To determine the importance of proteasome function in yeast aging, we established a method to modulate UPS capacity by manipulating levels of the UPS–related transcription factor Rpn4. While cells lacking RPN4 exhibit a decreased non-adaptable proteasome pool, loss of UBR2, an ubiquitin ligase that regulates Rpn4 turnover, results in elevated Rpn4 levels, which upregulates UPS components. Increased UPS capacity significantly enhances replicative lifespan (RLS) and resistance to proteotoxic stress, while reduced UPS capacity has opposing consequences. Despite tight transcriptional co-regulation of the UPS and oxidative detoxification systems, the impact of proteasome capacity on lifespan is independent of the latter, since elimination of Yap1, a key regulator of the oxidative stress response, does not affect lifespan extension of cells with higher proteasome capacity. Moreover, since elevated proteasome capacity results in improved clearance of toxic huntingtin fragments in a yeast model for neurodegenerative diseases, we speculate that the observed lifespan extension originates from prolonged elimination of damaged proteins in old mother cells. Epistasis analyses indicate that proteasome-mediated modulation of lifespan is at least partially distinct from dietary restriction, Tor1, and Sir2. These findings demonstrate that UPS capacity determines yeast RLS by a mechanism that is distinct from known longevity pathways and raise the possibility that interventions to promote enhanced proteasome function will have beneficial effects on longevity and age-related disease in humans

HDNetDB: A Molecular Interaction Database for Network-Oriented Investigations into Huntington’s Disease

Huntington's disease (HD) is a progressive and fatal neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin gene. Although HD is monogenic, its molecular manifestation appears highly complex and involves multiple cellular processes. The recent application of high throughput platforms such as microarrays and mass-spectrometry has indicated multiple pathogenic routes. The massive data generated by these techniques together with the complexity of the pathogenesis, however, pose considerable challenges to researchers. Network-based methods can provide valuable tools to consolidate newly generated data with existing knowledge, and to decipher the interwoven molecular mechanisms underlying HD. To facilitate research on HD in a network-oriented manner, we have developed HDNetDB, a database that integrates molecular interactions with many HD-relevant datasets. It allows users to obtain, visualize and prioritize molecular interaction networks using HD-relevant gene expression, phenotypic and other types of data obtained from human samples or model organisms. We illustrated several HDNetDB functionalities through a case study and identified proteins that constitute potential cross-talk between HD and the unfolded protein response (UPR). HDNetDB is publicly accessible at http://hdnetdb.sysbiolab.eu.CHDI Foundation [A-2666]; Portuguese Fundacao para a Ciencia e a Tecnologia [SFRH/BPD/70718/2010, SFRH/BPD/96890/2013, IF/00881/2013, UID/BIM/04773/2013 - CBMR, UID/Multi/04326/2013 - CCMAR]info:eu-repo/semantics/publishedVersio