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

Small Heat Shock Proteins Potentiate Amyloid Dissolution by Protein Disaggregases from Yeast and Humans

The authors define how small heat-shock proteins synergize to regulate the assembly and disassembly of a beneficial prion, and then they exploit this knowledge to identify the human amyloid depolymerase

Correlation of Inter-Locus Polyglutamine Toxicity with CAG•CTG Triplet Repeat Expandability and Flanking Genomic DNA GC Content

Dynamic expansions of toxic polyglutamine (polyQ)-encoding CAG repeats in ubiquitously expressed, but otherwise unrelated, genes cause a number of late-onset progressive neurodegenerative disorders, including Huntington disease and the spinocerebellar ataxias. As polyQ toxicity in these disorders increases with repeat length, the intergenerational expansion of unstable CAG repeats leads to anticipation, an earlier age-at-onset in successive generations. Crucially, disease associated alleles are also somatically unstable and continue to expand throughout the lifetime of the individual. Interestingly, the inherited polyQ length mediating a specific age-at-onset of symptoms varies markedly between disorders. It is widely assumed that these inter-locus differences in polyQ toxicity are mediated by protein context effects. Previously, we demonstrated that the tendency of expanded CAG•CTG repeats to undergo further intergenerational expansion (their ‘expandability’) also differs between disorders and these effects are strongly correlated with the GC content of the genomic flanking DNA. Here we show that the inter-locus toxicity of the expanded polyQ tracts of these disorders also correlates with both the expandability of the underlying CAG repeat and the GC content of the genomic DNA flanking sequences. Inter-locus polyQ toxicity does not correlate with properties of the mRNA or protein sequences, with polyQ location within the gene or protein, or steady state transcript levels in the brain. These data suggest that the observed inter-locus differences in polyQ toxicity are not mediated solely by protein context effects, but that genomic context is also important, an effect that may be mediated by modifying the rate at which somatic expansion of the DNA delivers proteins to their cytotoxic state

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

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

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

Conformational Targeting of Fibrillar Polyglutamine Proteins in Live Cells Escalates Aggregation and Cytotoxicity

Misfolding- and aggregation-prone proteins underlying Parkinson's, Huntington's and Machado-Joseph diseases, namely alpha-synuclein, huntingtin, and ataxin-3 respectively, adopt numerous intracellular conformations during pathogenesis, including globular intermediates and insoluble amyloid-like fibrils. Such conformational diversity has complicated research into amyloid-associated intracellular dysfunction and neurodegeneration. To this end, recombinant single-chain Fv antibodies (scFvs) are compelling molecular tools that can be selected against specific protein conformations, and expressed inside cells as intrabodies, for investigative and therapeutic purposes.Using atomic force microscopy (AFM) and live-cell fluorescence microscopy, we report that a human scFv selected against the fibrillar form of alpha-synuclein targets isomorphic conformations of misfolded polyglutamine proteins. When expressed in the cytoplasm of striatal cells, this conformation-specific intrabody co-localizes with intracellular aggregates of misfolded ataxin-3 and a pathological fragment of huntingtin, and enhances the aggregation propensity of both disease-linked polyglutamine proteins. Using this intrabody as a tool for modulating the kinetics of amyloidogenesis, we show that escalating aggregate formation of a pathologic huntingtin fragment is not cytoprotective in striatal cells, but rather heightens oxidative stress and cell death as detected by flow cytometry. Instead, cellular protection is achieved by suppressing aggregation using a previously described intrabody that binds to the amyloidogenic N-terminus of huntingtin. Analogous cytotoxic results are observed following conformational targeting of normal or polyglutamine-expanded human ataxin-3, which partially aggregate through non-polyglutamine domains.These findings validate that the rate of aggregation modulates polyglutamine-mediated intracellular dysfunction, and caution that molecules designed to specifically hasten aggregation may be detrimental as therapies for polyglutamine disorders. Moreover, our findings introduce a novel antibody-based tool that, as a consequence of its general specificity for fibrillar conformations and its ability to function intracellularly, offers broad research potential for a variety of human amyloid diseases

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

Stress granules, RNA-binding proteins and polyglutamine diseases: too much aggregation?

Stress granules (SGs) are membraneless cell compartments formed in response to different stress stimuli, wherein translation factors, mRNAs, RNA-binding proteins (RBPs) and other proteins coalesce together. SGs assembly is crucial for cell survival, since SGs are implicated in the regulation of translation, mRNA storage and stabilization and cell signalling, during stress. One defining feature of SGs is their dynamism, as they are quickly assembled upon stress and then rapidly dispersed after the stress source is no longer present. Recently, SGs dynamics, their components and their functions have begun to be studied in the context of human diseases. Interestingly, the regulated protein self-assembly that mediates SG formation contrasts with the pathological protein aggregation that is a feature of several neurodegenerative diseases. In particular, aberrant protein coalescence is a key feature of polyglutamine (PolyQ) diseases, a group of nine disorders that are caused by an abnormal expansion of PolyQ tract-bearing proteins, which increases the propensity of those proteins to aggregate. Available data concerning the abnormal properties of the mutant PolyQ disease-causing proteins and their involvement in stress response dysregulation strongly suggests an important role for SGs in the pathogenesis of PolyQ disorders. This review aims at discussing the evidence supporting the existence of a link between SGs functionality and PolyQ disorders, by focusing on the biology of SGs and on the way it can be altered in a PolyQ disease context.ALG-01-0145-FEDER-29480, SFRH/BD/133192/2017, SFRH/BD/133192/2017, SFRH/BD/148533/2019info:eu-repo/semantics/publishedVersio