Disruption of the toxic conformation of the expanded polyglutamine stretch leads to suppression of aggregate formation and cytotoxicity

The polyglutamine (polyQ) diseases are a class of inherited neurodegenerative diseases including Huntington's disease, caused by the expansion of a polyQ stretch within each disease protein. This expansion is thought to cause a conformational change in the protein leading to aggregation of the protein, resulting in cytotoxicity. To analyze whether disrupting the toxic conformation of the polyQ protein can alter its aggregation propensity and cytotoxicity, we examined the effect of interruption of the expanded polyQ stretch by proline insertion, since prolines cause great alterations in protein conformation. Here, we show that insertion of prolines into the expanded polyQ stretch indeed disrupts its ordered secondary structure, leading to suppression of polyQ protein aggregation both in vitro and in cell culture, and reduction of cytotoxicity in correlation with the number of proline interruptions. Furthermore, we found that a short polyQ stretch with a proline interruption is able to inhibit aggregation of the expanded polyQ protein in trans. These results show that a gain in toxic conformation of the expanded polyQ protein is essential for aggregation and cytotoxicity, providing insight into establishing therapies against the polyQ diseases.     

Polyglutamine expansion mutation yields a pathological epitope linked to nucleation of protein aggregate: determinant of Huntington's disease onset

Polyglutamine (polyQ) expansion mutation causes conformational, neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. These diseases are characterized by the aggregation of misfolded proteins, such as amyloid fibrils, which are toxic to cells. Amyloid fibrils are formed by a nucleated growth polymerization reaction. Unexpectedly, the critical nucleus of polyQ aggregation was found to be a monomer, suggesting that the rate-limiting nucleation process of polyQ aggregation involves the folding of mutated protein monomers. The monoclonal antibody 1C2 selectively recognizes expanded pathogenic and aggregate-prone glutamine repeats in polyQ diseases, including Huntington's disease (HD), as well as binding to polyleucine. We have therefore assayed the in vitro and in vivo aggregation kinetics of these monomeric proteins. We found that the repeat-length-dependent differences in aggregation lag times of variable lengths of polyQ and polyleucine tracts were consistently related to the integration of the length-dependent intensity of anti-1C2 signal on soluble monomers of these proteins. Surprisingly, the correlation between the aggregation lag times of polyQ tracts and the intensity of anti-1C2 signal on soluble monomers of huntingtin precisely reflected the repeat-length dependent age-of-onset of HD patients. These data suggest that the alterations in protein surface structure due to polyQ expansion mutation in soluble monomers of the mutated proteins act as an amyloid-precursor epitope. This, in turn, leads to nucleation, a key process in protein aggregation, thereby determining HD onset. These findings provide new insight into the gain-of-function mechanisms of polyQ diseases, in which polyQ expansion leads to nucleation rather than having toxic effects on the cells.     

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.     

Monitoring polyglutamine toxicity in yeast

Experiments in yeast have significantly contributed to our understanding of general aspects of biochemistry, genetics, and cell biology. Yeast models have also delivered deep insights in to the molecular mechanism underpinning human diseases, including neurodegenerative diseases. Many neurodegenerative diseases are associated with the conversion of a protein from a normal and benign conformation into a disease-associated and toxic conformation - a process called protein misfolding. The misfolding of proteins with abnormally expanded polyglutamine (polyQ) regions causes several neurodegenerative diseases, such as Huntington's disease and the Spinocerebellar Ataxias. Yeast cells expressing polyQ expansion proteins recapitulate polyQ length-dependent aggregation and toxicity, which are hallmarks of all polyQ-expansion diseases. The identification of modifiers of polyQ toxicity in yeast revealed molecular mechanisms and cellular pathways that contribute to polyQ toxicity. Notably, several of these findings in yeast were reproduced in other model organisms and in human patients, indicating the validity of the yeast polyQ model. Here, we describe different expression systems for polyQ-expansion proteins in yeast and we outline experimental protocols to reliably and quantitatively monitor polyQ toxicity in yeast.     

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.     

Identifying polyglutamine protein species in situ that best predict neurodegeneration

Polyglutamine (polyQ) stretches exceeding a threshold length confer a toxic function to proteins that contain them and cause at least nine neurological disorders. The basis for this toxicity threshold is unclear. Although polyQ expansions render proteins prone to aggregate into inclusion bodies, this may be a neuronal coping response to more toxic forms of polyQ. The exact structure of these more toxic forms is unknown. Here we show that the monoclonal antibody 3B5H10 recognizes a species of polyQ protein in situ that strongly predicts neuronal death. The epitope selectively appears among some of the many low-molecular-weight conformational states assumed by expanded polyQ and disappears in higher-molecular-weight aggregated forms, such as inclusion bodies. These results suggest that protein monomers and possibly small oligomers containing expanded polyQ stretches can adopt a conformation that is recognized by 3B5H10 and is toxic or closely related to a toxic species.     

Physical chemistry of polyglutamine: intriguing tales of a monotonous sequence

Polyglutamine (polyQ) sequences of unknown normal function are present in a significant number of proteins, and their repeat expansion is associated with a number of genetic neurodegenerative diseases. PolyQ solution structure and properties are important not only because of the normal and abnormal biology associated with these sequences but also because they represent an interesting case of a biologically relevant homopolymer. As the common thread in expanded polyQ repeat diseases, it is important to understand the structure and properties of simple polyQ sequences. At the same time, experience has shown that sequences attached to polyQ, whether in artificial constructs or in disease proteins, can influence structure and properties. The two major contenders for the molecular source of the neurotoxicity implicit in polyQ expansion within disease proteins are a populated toxic conformation in the monomer ensemble and a toxic aggregated species. This review summarizes experimental and computational studies on the solution structure and aggregation properties of both simple and complex polyQ sequences, and their repeat-length dependence. As a representative of complex polyQ proteins, the behavior of huntingtin N-terminal fragments, such as exon-1, receives special attention.     

Reprint of "Improved spectral resolution in COSY (1)H NMR spectra of proteins via double quantum filtering"

Polyglutamine (polyQ) diseases are inherited neurodegenerative diseases characterized by the aggregation of proteins containing expanded polyQ tract. It has been shown that expanded polyQ tract-containing proteins impair the functions of other cellular proteins. However, quantitative changes of cellular proteins in cells expressing expanded polyQ tract-containing proteins have not been performed. Here, we performed proteomic analysis of cells expressing expanded polyQ tract-containing proteins, and showed that GRP78, the endoplasmic reticulum (ER) chaperone, was significantly decreased in the cells expressing enhanced green fluorescent protein with a pathological-length polyQ tract (EGFP-polyQ97), but not with a non-pathological-length polyQ tract (EGFP-polyQ24). In addition, we revealed that down-regulation of GRP78 expression resulted in increase of the aggregation of EGFP-polyQ97. Conversely, the aggregation of EGFP-polyQ97 was suppressed by the overexpression of GRP78 in the cells. Furthermore, it seemed that the decreased GRP78 expression in the cells expressing EGFP-polyQ97 was due to the enhanced protein degradation of GRP78 through the ubiquitin-proteasome pathway. These findings indicated that GRP78, which has an inhibitory effect on the aggregation of proteins containing expanded polyQ tract, may be an effective target for the treatment of polyQ diseases.     

Molecular origin of polyglutamine aggregation in neurodegenerative diseases

Expansion of polyglutamine (polyQ) tracts in proteins results in protein aggregation and is associated with cell death in at least nine neurodegenerative diseases. Disease age of onset is correlated with the polyQ insert length above a critical value of 35-40 glutamines. The aggregation kinetics of isolated polyQ peptides in vitro also shows a similar critical-length dependence. While recent experimental work has provided considerable insights into polyQ aggregation, the molecular mechanism of aggregation is not well understood. Here, using computer simulations of isolated polyQ peptides, we show that a mechanism of aggregation is the conformational transition in a single polyQ peptide chain from random coil to a parallel beta-helix. This transition occurs selectively in peptides longer than 37 glutamines. In the beta-helices observed in simulations, all residues adopt beta-strand backbone dihedral angles, and the polypeptide chain coils around a central helical axis with 18.5 +/- 2 residues per turn. We also find that mutant polyQ peptides with proline-glycine inserts show formation of antiparallel beta-hairpins in their ground state, in agreement with experiments. The lower stability of mutant beta-helices explains their lower aggregation rates compared to wild type. Our results provide a molecular mechanism for polyQ-mediated aggregation.     

Evolution and function of CAG/polyglutamine repeats in protein-protein interaction networks

Expanded runs of consecutive trinucleotide CAG repeats encoding polyglutamine (polyQ) stretches are observed in the genes of a large number of patients with different genetic diseases such as Huntington's and several Ataxias. Protein aggregation, which is a key feature of most of these diseases, is thought to be triggered by these expanded polyQ sequences in disease-related proteins. However, polyQ tracts are a normal feature of many human proteins, suggesting that they have an important cellular function. To clarify the potential function of polyQ repeats in biological systems, we systematically analyzed available information stored in sequence and protein interaction databases. By integrating genomic, phylogenetic, protein interaction network and functional information, we obtained evidence that polyQ tracts in proteins stabilize protein interactions. This happens most likely through structural changes whereby the polyQ sequence extends a neighboring coiled-coil region to facilitate its interaction with a coiled-coil region in another protein. Alteration of this important biological function due to polyQ expansion results in gain of abnormal interactions, leading to pathological effects like protein aggregation. Our analyses suggest that research on polyQ proteins should shift focus from expanded polyQ proteins into the characterization of the influence of the wild-type polyQ on protein interactions.     

Study of the conformational transition of A beta(1-42) using D-amino acid replacement analogues

A critical event in Alzheimer's disease is the transition of Abeta peptides from their soluble forms into disease-associated beta-sheet-rich conformers. Structural analysis of a complete D-amino acid replacement set of Abeta(1-42) enabled us to localize in the full-length 42-mer peptide the region responsible for the conformational switch into a beta-sheet structure. Although NMR spectroscopy of trifluoroethanol-stabilized monomeric Abeta(1-42) delineated two separated helical domains, only the destabilization of helix I, comprising residues 11-24, caused a transition to a beta-sheet structure. This conformational alpha-to-beta switch was directly accompanied by an aggregation process leading to the formation of amyloid fibrils.     

Surface plasmon resonance characterization of specific binding of polyglutamine aggregation inhibitors to the expanded polyglutamine stretch

Proteins with an abnormally expanded polyglutamine (polyQ) stretch are prone to change their conformations, leading to their aggregation, and cause inherited neurodegenerative diseases called the polyQ diseases. Although screening for polyQ aggregation inhibitors has been extensively performed, many common false-positive hits have been identified so far. In this study, we employed surface plasmon resonance (SPR) to characterize the binding specificities and affinities of polyQ aggregation inhibitors to the expanded polyQ stretch. SPR successfully detected specific binding of polyQ binding peptide 1 (QBP1) to the expanded polyQ stretch (K_d = 5.7 μM), and non-specific binding of Congo red to polyQ proteins independent of their polyQ-length. Binding affinities of polyQ aggregation inhibitors to the expanded polyQ stretch were correlated with their inhibitory effects on polyQ aggregation. We therefore conclude that SPR is a useful technique for screening for specific polyQ aggregation inhibitors as promising therapeutic candidates for the currently untreatable polyQ diseases.     

Aggregation of expanded polyglutamine domain in yeast leads to defects in endocytosis

The role of aggregation of abnormal proteins in cellular toxicity is of general importance for understanding many neurological disorders. Here, using a yeast model, we demonstrate that mutations in many proteins involved in endocytosis and actin function dramatically enhance the toxic effect of polypeptides with an expanded polyglutamine (polyQ) domain. This enhanced cytotoxicity required polyQ aggregation and was dependent on the yeast protein Rnq1 in its prion form. In wild-type cells, expression of expanded polyQ followed by its aggregation led to specific and acute inhibition of endocytosis, which preceded growth inhibition. Some components of the endocytic machinery were efficiently recruited into the polyQ aggregates. Furthermore, in cells with polyQ aggregates, cortical actin patches were delocalized and actin was recruited into the polyQ aggregates. Aggregation of polyQ in mammalian HEK293 cells also led to defects in endocytosis. Therefore, it appears that inhibition of endocytosis is a direct consequence of polyQ aggregation and could significantly contribute to cytotoxicity.     

The DNAJB6 and DNAJB8 Protein Chaperones Prevent Intracellular Aggregation of Polyglutamine Peptides

Fragments of proteins containing an expanded polyglutamine (polyQ) tract are thought to initiate aggregation and toxicity in at least nine neurodegenerative diseases, including Huntington''s disease. Because proteasomes appear unable to digest the polyQ tract, which can initiate intracellular protein aggregation, preventing polyQ peptide aggregation by chaperones should greatly improve polyQ clearance and prevent aggregate formation. Here we expressed polyQ peptides in cells and show that their intracellular aggregation is prevented by DNAJB6 and DNAJB8, members of the DNAJ (Hsp40) chaperone family. In contrast, HSPA/Hsp70 and DNAJB1, also members of the DNAJ chaperone family, did not prevent peptide-initiated aggregation. Intriguingly, DNAJB6 and DNAJB8 also affected the soluble levels of polyQ peptides, indicating that DNAJB6 and DNAJB8 inhibit polyQ peptide aggregation directly. Together with recent data showing that purified DNAJB6 can suppress fibrillation of polyQ peptides far more efficiently than polyQ expanded protein fragments in vitro, we conclude that the mechanism of DNAJB6 and DNAJB8 is suppression of polyQ protein aggregation by directly binding the polyQ tract.     

Insights into the Aggregation Mechanism of PolyQ Proteins with Different Glutamine Repeat Lengths

Polyglutamine (polyQ) diseases, including Huntington’s disease, result from the aggregation of an abnormally expanded polyQ repeat in the affected protein. The length of the polyQ repeat is essential for the disease’s onset; however, the molecular mechanism of polyQ aggregation is still poorly understood. Controlled conditions and initiation of the aggregation process are prerequisites for the detection of transient intermediate states. We present an attenuated total reflection Fourier-transform infrared spectroscopic approach combined with protein immobilization to study polyQ aggregation dependent on the polyQ length. PolyQ proteins were engineered mimicking the mammalian N-terminus fragment of the Huntingtin protein and containing a polyQ sequence with the number of glutamines below (Q11), close to (Q38), and above (Q56) the disease threshold. A monolayer of the polyQ construct was chemically immobilized on the internal reflection element of the attenuated total reflection cell, and the aggregation was initiated via enzymatic cleavage. Structural changes of the polyQ sequence were monitored by time-resolved infrared difference spectroscopy. We observed faster aggregation kinetics for the longer sequences, and furthermore, we could distinguish β-structured intermediates for the different constructs, allowing us to propose aggregation mechanisms dependent on the repeat length. Q11 forms a β-structured aggregate by intermolecular interaction of stretched monomers, whereas Q38 and Q56 undergo conformational changes to various β-structured intermediates, including intramolecular β-sheets.     

Ataxin-1 fusion partners alter polyQ lethality and aggregation

Intranuclear inclusion bodies (IBs) are the histopathologic markers of multiple protein folding diseases. IB formation has been extensively studied using fluorescent fusion products of pathogenic polyglutamine (polyQ) expressing proteins. These studies have been informative in determining the cellular targets of expanded polyQ protein as well as the methods by which cells rid themselves of IBs. The experimental thrust has been to intervene in the process of polyQ aggregation in an attempt to alleviate cytotoxicity. However new data argues against the notion that polyQ aggregation and cytotoxicity are inextricably linked processes. We reasoned that changing the protein context of a disease causing polyQ protein could accelerate its precipitation as an IB, potentially reducing its cytotoxicity. Our experimental strategy simply exploited the fact that conjoined proteins influence each others folding and aggregation properties. We fused a full-length pathogenic ataxin-1 construct to fluorescent tags (GFP and DsRed1-E5) that exist at different oligomeric states. The spectral properties of the DsRed1-E5-ataxin-1 transfectants had the additional advantage of allowing us to correlate fluorochrome maturation with cytotoxicity. Each fusion protein expressed a distinct cytotoxicity and IB morphology. Flow cytometric analyses of transfectants expressing the greatest fluorescent signals revealed that the DsRed1-E5-ataxin-1 fusion was more toxic than GFP fused ataxin-1 (31.8+/-4.5% cell death versus 12.85+/-3%), although co-transfection with the GFP fusion inhibited maturation of the DsRed1-E5 fluorochrome and diminished the toxicity of the DsRed1-E5-ataxin-1 fusion. These data show that polyQ driven aggregation can be influenced by fusion partners to generate species with different toxic properties and provide new opportunities to study IB aggregation, maturation and lethality.     

Wild type huntingtin toxicity in yeast: Implications for the role of amyloid cross-seeding in polyQ diseases

Proteins with expanded polyglutamine (polyQ) regions are prone to form amyloids, which can cause diseases in humans and toxicity in yeast. Recently, we showed that in yeast non-toxic amyloids of Q-rich proteins can induce aggregation and toxicity of wild type huntingtin (Htt) with a short non-pathogenic polyglutamine tract. Similarly to mutant Htt with an elongated N-terminal polyQ sequence, toxicity of its wild type counterpart was mediated by induced aggregation of the essential Sup35 protein, which contains a Q-rich region. Notably, polymerization of Sup35 was not caused by the initial benign amyloids and, therefore, aggregates of wild type Htt acted as intermediaries in seeding Sup35 polymerization. This exemplifies a protein polymerization cascade which can generate a network of interdependent polymers. Here we discuss cross-seeded protein polymerization as a possible mechanism underlying known interrelations between different polyQ diseases. We hypothesize that similar mechanisms may enable proteins, which possess expanded Q-rich tracts but are not associated with diseases, to promote the development of polyQ diseases.     

Protein misfolding inside cells: the case of huntingtin and Huntington's disease

Huntington's disease is one of the several neurodegenerative diseases caused by dominant mutations that expand the number of glutamine codons within an existing poly-glutamine (polyQ) repeat sequence of a gene. An expanded polyQ sequence in the huntingtin gene is known to cause the huntingtin protein to aggregate and form intracellular inclusions as disease progresses. However, the role that polyQ-induced aggregation plays in disease is yet to be fully determined. This review focuses on key questions remaining for how the expanded polyQ sequences affect the aggregation properties of the huntingtin protein and the corresponding effects on cellular machinery. The scope includes the technical challenges that remain for rigorously assessing the effects of aggregation on the cellular machinery.     

Polyglutamine and neurodegeneration: structural aspects

Polyglutamine (polyQ) diseases are inherited neurodegenerative disorders caused by proteins with expanded polyQ regions. Although the pathological mechanisms of these diseases have not yet been elucidated, the processes of protein misfolding and aggregation seem to be a direct cause of neurodegeneration. Detailed structural information on polyQ proteins is therefore essential in order to understand the mechanisms underlying pathogenesis and to design therapeutic strategies. In the past decade, several studies have investigated the structural properties of polyQ proteins and the molecular basis of aggregation and fibre formation. The results obtained in these studies are reviewed here.