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.     

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.     

RACK1 modulates polyglutamine-induced neurodegeneration by promoting ERK degradation in Drosophila

Polyglutamine diseases are neurodegenerative diseases caused by the expansion of polyglutamine (polyQ) tracts within different proteins. Although multiple pathways have been found to modulate aggregation of the expanded polyQ proteins, the mechanisms by which polyQ tracts induced neuronal cell death remain unknown. We conducted a genome-wide genetic screen to identify genes that suppress polyQ-induced neurodegeneration when mutated. Loss of the scaffold protein RACK1 alleviated cell death associated with the expression of polyQ tracts alone, as well as in models of Machado-Joseph disease (MJD) and Huntington’s disease (HD), without affecting proteostasis of polyQ proteins. A genome-wide RNAi screen for modifiers of this rack1 suppression phenotype revealed that knockdown of the E3 ubiquitin ligase, POE (Purity of essence), further suppressed polyQ-induced cell death, resulting in nearly wild-type looking eyes. Biochemical analyses demonstrated that RACK1 interacts with POE and ERK to promote ERK degradation. These results suggest that RACK1 plays a key role in polyQ pathogenesis by promoting POE-dependent degradation of ERK, and implicate RACK1/POE/ERK as potent drug targets for treatment of polyQ diseases.     

Mutant huntingtin promotes the fibrillogenesis of wild-type huntingtin: a potential mechanism for loss of huntingtin function in Huntington's disease

Aggregation of huntingtin (htt) in neuronal inclusions is associated with the development of Huntington's disease (HD). Previously, we have shown that mutant htt fragments with polyglutamine (polyQ) tracts in the pathological range (>37 glutamines) form SDS-resistant aggregates with a fibrillar morphology, whereas wild-type htt fragments with normal polyQ domains do not aggregate. In this study we have investigated the co-aggregation of mutant and wild-type htt fragments. We found that mutant htt promotes the aggregation of wild-type htt, causing the formation of SDS-resistant co-aggregates with a fibrillar morphology. Conversely, mutant htt does not promote the fibrillogenesis of the polyQ-containing protein NOCT3 or the polyQ-binding protein PQBP1, although these proteins are recruited into inclusions containing mutant htt aggregates in mammalian cells. The formation of mixed htt fibrils is a highly selective process that not only depends on polyQ tract length but also on the surrounding amino acid sequence. Our data suggest that mutant and wild-type htt fragments may also co-aggregate in neurons of HD patients and that a loss of wild-type htt function may contribute to HD pathogenesis.     

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.     

The rate of polyQ-mediated aggregation is dramatically affected by the number and location of surrounding domains

The nine polyglutamine (polyQ) neurodegenerative diseases are caused in part by a gain-of-function mechanism involving protein misfolding, the deposition of β-sheet-rich aggregates and neuronal toxicity. While previous experimental evidence suggests that the polyQ-induced misfolding mechanism is context dependent, the properties of the host protein, including the domain architecture and location of the polyQ tract, have not been investigated. Here, we use variants of a model polyQ-containing protein to systematically determine the effect of the location and number of flanking folded domains on polyQ-mediated aggregation. Our data indicate that when a pathological-length polyQ tract is present between two domains, it aggregates more slowly than the same-length tract in a terminal location within the protein. We also demonstrate that increasing the number of flanking domains decreases the polyQ protein's aggregation rate. Our experimental data, together with a bioinformatic analysis of all human proteins possessing polyQ tracts, suggest that repeat location and protein domain architecture affect the disease susceptibility of human polyQ proteins.     

Proteins with Intrinsically Disordered Domains Are Preferentially Recruited to Polyglutamine Aggregates

Intracellular protein aggregation is the hallmark of several neurodegenerative diseases. Aggregates formed by polyglutamine (polyQ)-expanded proteins, such as Huntingtin, adopt amyloid-like structures that are resistant to denaturation. We used a novel purification strategy to isolate aggregates formed by human Huntingtin N-terminal fragments with expanded polyQ tracts from both yeast and mammalian (PC-12) cells. Using mass spectrometry we identified the protein species that are trapped within these polyQ aggregates. We found that proteins with very long intrinsically-disordered (ID) domains (≥100 amino acids) and RNA-binding proteins were disproportionately recruited into aggregates. The removal of the ID domains from selected proteins was sufficient to eliminate their recruitment into polyQ aggregates. We also observed that several neurodegenerative disease-linked proteins were reproducibly trapped within the polyQ aggregates purified from mammalian cells. Many of these proteins have large ID domains and are found in neuronal inclusions in their respective diseases. Our study indicates that neurodegenerative disease-associated proteins are particularly vulnerable to recruitment into polyQ aggregates via their ID domains. Also, the high frequency of ID domains in RNA-binding proteins may explain why RNA-binding proteins are frequently found in pathological inclusions in various neurodegenerative diseases.     

Bax-inhibiting peptide protects cells from polyglutamine toxicity caused by Ku70 acetylation

Polyglutamine (polyQ) diseases, such as Huntington's disease and Machado-Joseph disease (MJD), are caused by gain of toxic function of abnormally expanded polyQ tracts. Here, we show that expanded polyQ of ataxin-3 (Q79C), a gene that causes MJD, stimulates Ku70 acetylation, which in turn dissociates the proapoptotic protein Bax from Ku70, thereby promoting Bax activation and subsequent cell death. The Q79C-induced cell death was significantly blocked by Ku70 or Bax-inhibiting peptides (BIPs) designed from Ku70. Furthermore, expression of SIRT1 deacetylase and the addition of a SIRT1 agonist, resveratrol, reduced Q79C toxicity. In contrast, mimicking acetylation of Ku70 abolished the ability of Ku70 to suppress Q79C toxicity. These results indicate that Bax and Ku70 acetylation play important roles in Q79C-induced cell death, and that BIP may be useful in the development of therapeutics for 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.     

Disease-associated polyglutamine stretches in monomeric huntingtin adopt a compact structure

Abnormal polyglutamine (polyQ) tracts are the only common feature in nine proteins that each cause a dominant neurodegenerative disorder. In Huntington's disease, tracts longer than 36 glutamines in the protein huntingtin (htt) cause degeneration. In situ, monoclonal antibody 3B5H10 binds to different htt fragments in neurons in proportion to their toxicity. Here, we determined the structure of 3B5H10 Fab to 1.9?? resolution by X-ray crystallography. Modeling demonstrates that the paratope forms a groove suitable for binding two β-rich polyQ strands. Using small-angle X-ray scattering, we confirmed that the polyQ epitope recognized by 3B5H10 is a compact two-stranded hairpin within monomeric htt and is abundant in htt fragments unbound to antibody. Thus, disease-associated polyQ stretches preferentially adopt compact conformations. Since 3B5H10 binding predicts degeneration, this compact polyQ structure may be neurotoxic.     

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.     

Analysis of the two p97/VCP/Cdc48p proteins of Caenorhabditis elegans and their suppression of polyglutamine-induced protein aggregation

A class of inherited neurodegenerative diseases including Huntington's disease is caused by polyglutamine (polyQ) expansion in the responsible proteins. Pathology is typically associated with polyQ expansions of greater than 40 residues, and the longer the length of the expansion, the earlier the onset of disease. It has been reported that p97/VCP/Cdc48p, a member of AAA family of proteins, can bind to longer polyQ tracts. In Caenorhabditis elegans, two p97/VCP/Cdc48p homologues, C41C4.8 and C06A1.1, have been identified. Our results indicate that these p97/VCP/Cdc48p homologues have essential but redundant functions in C. elegans. To provide a model system for investigating the molecular basis of pathogenesis, we have expressed polyQ expansions fused to green fluorescent protein in the body wall muscle cells of C. elegans. When the repeats are longer than 40, discrete cytoplasmic aggregates are formed and these appear at an early stage of embryogenesis. The formation of aggregates was partially suppressed by co-expression of either C41C4.8 or C06A1.1. These results suggest that these p97/VCP/Cdc48p homologues, AAA chaperones, may play a protective role in polyQ aggregation.     

A Coarse-Grained Model for Polyglutamine Aggregation Modulated by Amphipathic Flanking Sequences

The aggregation of proteins with expanded polyglutamine (polyQ) tracts is directly relevant to the formation of neuronal intranuclear inclusions in Huntington’s disease. In vitro studies have uncovered the effects of flanking sequences as modulators of the driving forces and mechanisms of polyQ aggregation in sequence segments associated with HD. Specifically, a seventeen-residue amphipathic stretch (N17) that is directly N-terminal to the polyQ tract in huntingtin decreases the overall solubility, destabilizes nonfibrillar aggregates, and accelerates fibril formation. Published results from atomistic simulations showed that the N17 module reduces the frequency of intermolecular association. Our reanalysis of these simulation results demonstrates that the N17 module also reduces interchain entanglements between polyQ domains. These two effects, which are observed on the smallest lengthscales, are incorporated into phenomenological pair potentials and used in coarse-grained Brownian dynamics simulations to investigate their impact on large-scale aggregation. We analyze the results from Brownian dynamics simulations using the framework of diffusion-limited cluster aggregation. When entanglements prevail, which is true in the absence of N17, small spherical clusters and large linear aggregates form on distinct timescales, in accord with in vitro experiments. Conversely, when entanglements are quenched and a barrier to intermolecular associations is introduced, both of which are attributable to N17, the timescales for forming small species and large linear aggregates become similar. Therefore, the combination of a reduction of interchain entanglements through homopolymeric polyQ and barriers to intermolecular associations appears to be sufficient for providing a minimalist phenomenological rationalization of in vitro observations regarding the effects of N17 on polyQ aggregation.     

Evidence for a recruitment and sequestration mechanism in Huntington's disease.

Polyglutamine (polyQ) extension in the coding sequence of mutant huntingtin causes neuronal degeneration associated with the formation of insoluble polyQ aggregates in Huntington's disease. We constructed an array of CAG/CAA triplet repeats, coding for a range of 25-300 glutamine residues, which was used to generate expression constructs with minimal flanking sequence. Normal-length (25 glutamine residues) polyQ did not aggregate when transfected alone. Remarkably, when co-transfected with extended (100-300 glutamine residues) polyQ tracts, normal-length polyQ-containing peptides were trapped in insoluble detergent-resistant aggregates. Aggregates formed in the cytoplasm but were visible in the nucleus only when a strong nuclear localization signal was present. Intermolecular interactions between polyQ tracts mediated the localization of heterogeneous aggregates into the nucleolus by nucleolin protein. Our results suggest that extended polyQ can interact with cellular polyQ-containing proteins, transport them to ectopic cellular locations, and form heterogeneous polyQ aggregates. We provide evidence for a recruitment mechanism for pathogenesis in the polyQ neurodegenerative disorders. In susceptible cells, extended polyQ tracts in huntingtin might interact with and sequester or deplete certain endogenous polyQ-containing cellular proteins.     

A Coarse-Grained Model for Polyglutamine Aggregation Modulated by Amphipathic Flanking Sequences

The aggregation of proteins with expanded polyglutamine (polyQ) tracts is directly relevant to the formation of neuronal intranuclear inclusions in Huntington’s disease. In vitro studies have uncovered the effects of flanking sequences as modulators of the driving forces and mechanisms of polyQ aggregation in sequence segments associated with HD. Specifically, a seventeen-residue amphipathic stretch (N17) that is directly N-terminal to the polyQ tract in huntingtin decreases the overall solubility, destabilizes nonfibrillar aggregates, and accelerates fibril formation. Published results from atomistic simulations showed that the N17 module reduces the frequency of intermolecular association. Our reanalysis of these simulation results demonstrates that the N17 module also reduces interchain entanglements between polyQ domains. These two effects, which are observed on the smallest lengthscales, are incorporated into phenomenological pair potentials and used in coarse-grained Brownian dynamics simulations to investigate their impact on large-scale aggregation. We analyze the results from Brownian dynamics simulations using the framework of diffusion-limited cluster aggregation. When entanglements prevail, which is true in the absence of N17, small spherical clusters and large linear aggregates form on distinct timescales, in accord with in vitro experiments. Conversely, when entanglements are quenched and a barrier to intermolecular associations is introduced, both of which are attributable to N17, the timescales for forming small species and large linear aggregates become similar. Therefore, the combination of a reduction of interchain entanglements through homopolymeric polyQ and barriers to intermolecular associations appears to be sufficient for providing a minimalist phenomenological rationalization of in vitro observations regarding the effects of N17 on polyQ aggregation.     

Aggregation of polyQ‐extended proteins is promoted by interaction with their natural coiled‐coil partners

Polyglutamine (polyQ) diseases are genetically inherited neurodegenerative disorders. They are caused by mutations that result in polyQ expansions of particular proteins. Mutant proteins form intranuclear aggregates, induce cytotoxicity and cause neuronal cell death. Protein interaction data suggest that polyQ regions modulate interactions between coiled‐coil (CC) domains. In the case of the polyQ disease spinocerebellar ataxia type‐1 (SCA1), interacting proteins with CC domains further enhance aggregation and toxicity of mutant ataxin‐1 (ATXN1). Here, we suggest that CC partners interacting with the polyQ region of a mutant protein, increase its aggregation while partners that interact with a different region reduce the formation of aggregates. Computational analysis of genetic screens revealed that CC‐rich proteins are highly enriched among genes that enhance pathogenicity of polyQ proteins, supporting our hypothesis. We therefore suggest that blocking interactions between mutant polyQ proteins and their CC partners might constitute a promising preventive strategy against neurodegeneration.     

The Role of Interruptions in polyQ in the Pathology of SCA1

At least nine dominant neurodegenerative diseases are caused by expansion of CAG repeats in coding regions of specific genes that result in abnormal elongation of polyglutamine (polyQ) tracts in the corresponding gene products. When above a threshold that is specific for each disease the expanded polyQ repeats promote protein aggregation, misfolding and neuronal cell death. The length of the polyQ tract inversely correlates with the age at disease onset. It has been observed that interruption of the CAG tract by silent (CAA) or missense (CAT) mutations may strongly modulate the effect of the expansion and delay the onset age. We have carried out an extensive study in which we have complemented DNA sequence determination with cellular and biophysical models. By sequencing cloned normal and expanded SCA1 alleles taken from our cohort of ataxia patients we have determined sequence variations not detected by allele sizing and observed for the first time that repeat instability can occur even in the presence of CAG interruptions. We show that histidine interrupted pathogenic alleles occur with relatively high frequency (11%) and that the age at onset inversely correlates linearly with the longer uninterrupted CAG stretch. This could be reproduced in a cellular model to support the hypothesis of a linear behaviour of polyQ. We clarified by in vitro studies the mechanism by which polyQ interruption slows down aggregation. Our study contributes to the understanding of the role of polyQ interruption in the SCA1 phenotype with regards to age at disease onset, prognosis and transmission.