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.     

Mapping of the epitope of monoclonal antibody 2B4 to the proline-rich region of human Huntingtin, a region critical for aggregation and toxicity

Huntington's disease is a neurodegenerative disease caused by a polyglutamine (polyQ) expansion in Huntingtin, which provokes aggregation of a proteolytic amino-terminal fragment of the affected protein encompassing the polyQ expansion. Accumulation of mutant Huntingtin somehow triggers cellular dysfunction and leads to a progressive degeneration of striatal neurons. Despite considerable efforts, the function of Huntingtin as well as the precise molecular mechanisms by which the expanded polyQ elicits cellular dysfunction remain unclear. In addition, no treatment is available to prevent, cure, or even slow down the progression of this devastating disorder. Antibodies are valuable tools to understand protein function and disease mechanisms. Here, we have identified the epitope recognized by the mAb 2B4, a broadly used antibody generated against the amino-terminal region of Huntingtin, which detects both aggregated and soluble Huntingtin. The 2B4 antibody specifically recognizes amino acids 50-64 of human Huntingtin but not the murine homologous region. Furthermore, the 2B4 epitope resides within the proline-rich region of Huntingtin, which is critical for polyQ aggregation and toxicity. These properties suggest that the 2B4 antibody might be useful in antibody-based therapeutic strategies.     

Genome-wide screen for modifiers of ataxin-3 neurodegeneration in Drosophila

Spinocerebellar ataxia type-3 (SCA3) is among the most common dominantly inherited ataxias, and is one of nine devastating human neurodegenerative diseases caused by the expansion of a CAG repeat encoding glutamine within the gene. The polyglutamine domain confers toxicity on the protein Ataxin-3 leading to neuronal dysfunction and loss. Although modifiers of polyglutamine toxicity have been identified, little is known concerning how the modifiers function mechanistically to affect toxicity. To reveal insight into spinocerebellar ataxia type-3, we performed a genetic screen in Drosophila with pathogenic Ataxin-3-induced neurodegeneration and identified 25 modifiers defining 18 genes. Despite a variety of predicted molecular activities, biological analysis indicated that the modifiers affected protein misfolding. Detailed mechanistic studies revealed that some modifiers affected protein accumulation in a manner dependent on the proteasome, whereas others affected autophagy. Select modifiers of Ataxin-3 also affected tau, revealing common pathways between degeneration due to distinct human neurotoxic proteins. These findings provide new insight into molecular pathways of polyQ toxicity, defining novel targets for promoting neuronal survival in human neurodegenerative disease.     

多聚谷氨酰胺延伸蛋白募集细胞内转录因子及对基因转录调控的影响

多聚谷氨酰胺（polyglutamine,PolyQ）疾病是由特定基因序列中CAG三核苷酸的不稳定重复扩增所引发的一类神经退行性疾病。至今已发现9种类型的PolyQ疾病,其中多数疾病的致病蛋白质在转录调控中发挥着重要的病理作用。PolyQ蛋白中谷氨酰胺的异常重复延伸会引发蛋白质错误折叠并在细胞中积聚形成包涵体。积聚的蛋白质可通过自身结构域、泛素修饰和RNA等介导的相互作用,有效地募集细胞内的转录因子、泛素接头或受体蛋白,以及分子伴侣等组分到包涵体中。这些组分在细胞中的可溶性比例减少,使得机体内的转录调控系统功能受损,造成转录失调从而诱发疾病。因此,研究异常延伸的PolyQ蛋白对细胞内转录因子及其他组分的募集作用,可在分子水平上解释神经退行性疾病的发病机制,从而为临床应用提供潜在的预防和治疗方法。     

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.     

Diseases of unstable repeat expansion: mechanisms and common principles

The list of developmental and degenerative diseases that are caused by expansion of unstable repeats continues to grow, and is now approaching 20 disorders. The pathogenic mechanisms that underlie these disorders involve either loss of protein function or gain of function at the protein or RNA level. Common themes have emerged within and between these different classes of disease; for example, among disorders that are caused by gain-of-function mechanisms, altered protein conformations are central to pathogenesis, leading to changes in protein activity or abundance. In all these diseases, the context of the expanded repeat and the abundance, subcellular localization and interactions of the proteins and RNAs that are affected have key roles in disease-specific phenotypes.     

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.     

The structural impact of a polyglutamine tract is location-dependent

Polyglutamine (polyQ) expansion leads to protein aggregation and neurodegeneration in Huntington's disease and eight other inherited neurological conditions. Expansion of the polyQ tract beyond a threshold of 37 glutamines leads to the formation of toxic nuclear aggregates. This suggests that polyQ expansion causes a conformational change within the protein, the nature of which is unclear. There is a trend in the disease proteins that the polyQ tract is located external to but not within a structured domain. We have created a model polyQ protein in which the repeat location mimics the flexible environment of the polyQ tract in the disease proteins. Our model protein recapitulates the aggregation features observed with the clinical proteins and allows structural characterization. With the use of NMR spectroscopy and a range of biophysical techniques, we demonstrate that polyQ expansion into the pathological range has no effect on the structure, dynamics, and stability of a domain adjacent to the polyQ tract. To explore the clinical significance of repeat location, we engineered a variant of the model protein with a polyQ tract within the domain, a location that does not mimic physiological context, demonstrating significant destabilization and structural perturbation. These different effects highlight the importance of repeat location. We conclude that protein misfolding within the polyQ tract itself is the driving force behind the key characteristics of polyQ disease, and that structural perturbation of flanking domains is not required.     

Disruption of axonal transport by loss of huntingtin or expression of pathogenic polyQ proteins in Drosophila

We tested whether proteins implicated in Huntington's and other polyglutamine (polyQ) expansion diseases can cause axonal transport defects. Reduction of Drosophila huntingtin and expression of proteins containing pathogenic polyQ repeats disrupt axonal transport. Pathogenic polyQ proteins accumulate in axonal and nuclear inclusions, titrate soluble motor proteins, and cause neuronal apoptosis and organismal death. Expression of a cytoplasmic polyQ repeat protein causes adult retinal degeneration, axonal blockages in larval neurons, and larval lethality, but not neuronal apoptosis or nuclear inclusions. A nuclear polyQ repeat protein induces neuronal apoptosis and larval lethality but no axonal blockages. We suggest that pathogenic polyQ proteins cause neuronal dysfunction and organismal death by two non-mutually exclusive mechanisms. One mechanism requires nuclear accumulation and induces apoptosis; the other interferes with axonal transport. Thus, disruption of axonal transport by pathogenic polyQ proteins could contribute to early neuropathology in Huntington's and other polyQ expansion diseases.     

Expression of extended polyglutamine sequentially activates initiator and effector caspases

To date, eight neurodegenerative disorders, including Huntington's disease and dentatorubral-pallidoluysian atrophy, have been identified to be caused by expansion of a CAG repeat coding for a polyglutamine (polyQ) stretch. It is, however, unclear how polyQ expansion mediates neuronal cell death observed in these disorders. Here, we have established a tetracycline-regulated expression system producing 19 and 56 repeats of glutamine fused with green fluorescent protein. Induced expression of the 56 polyQ, but not of the 19 polyQ stretch caused marked nuclear aggregation and apoptotic morphological changes of the nucleus. In vitro enzyme assays and Western blotting showed that polyQ56 expression sequentially activated initiator and effector caspases, such as caspase-8 or -9, and caspase-3, respectively. Furthermore, using cell-permeable fluorogenic substrate, the activation of caspase-3-like proteases was demonstrated in intact cells with aggregated polyQ. This is the first direct evidence that the expression of extended polyQ activates caspases and together with the previous findings that some of the products of genes responsible for CAG repeat diseases are substrates of caspase-3 indicates an important role of caspases in the pathogenesis of these diseases.     

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.     

Analyzing the aggregation of polyglutamine-expansion proteins and its modulation by molecular chaperones

Polyglutamine (polyQ)-expansion proteins cause protein aggregation in the cytosol and nucleus of neuronal cells, leading to neurodegenerative diseases. For example, expansion of the polyQ tract (>40 repeats) in huntingtin (htt) proteins leads to Huntington disease, while polyQ-expanded ataxins cause several types of ataxias. PolyQ-rich inclusions are found in neuronal cells of patients, suggesting that polyQ disease is caused by protein misfolding. However, the mechanisms by which polyQ-expansion proteins exert neuronal toxicity are largely unknown. Here, we review experimental procedures to analyze the roles of molecular chaperones in preventing polyQ aggregation and toxicity as well as to measure the characteristics and dynamics of polyQ aggregation, particularly focusing on cellular models and dynamic imaging of fluorescently-labeled polyQ-expansion proteins and their modulation by chaperones.     

Pathogenic mechanisms of a polyglutamine-mediated neurodegenerative disease, spinocerebellar ataxia type 1

Spinocerebellar ataxia type 1 (SCA1) is one of nine inherited neurodegenerative diseases caused by the expansion of a CAG trinucleotide repeat encoding a polyglutamine tract. SCA1 patients lose motor coordination and develop slurred speech, spasticity, and cognitive impairments. Difficulty with coordinating swallowing and breathing eventually causes death. Genetic evidence indicates that the disease mutation induces a toxic gain of function in the SCA1 encoded protein ATXN1. The discovery that residues in ATXN1 outside of the polyglutamine tract are crucial for pathogenesis hinted that alterations in the normal function of this protein are linked to its toxicity. Biochemical and genetic studies provide evidence that the polyglutamine expansion enhances interactions that are normally regulated by phosphorylation at Ser(776) and a subsequent alteration in its interaction with other cellular proteins. Moreover, the finding that other ATXN1 interactions are decreased in disease suggests that the polyglutamine expansion contributes to disease by both a gain-of-function mechanism and partial loss of function.     

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.     

Does Huntingtin play a role in selective macroautophagy?

The accumulation of protein aggregates in neurons appears to be a basic feature of neurodegenerative disease. In huntington disease (HD), a progressive and ultimately fatal neurodegenerative disorder caused by an expansion of the polyglutamine repeat within the protein huntingtin (Htt), the immediate proximal cause of disease is well understood. However, the cellular mechanisms which modulate the rate at which fragments of Htt containing polyglutamine accumulate in neurons is a central issue in the development of approaches to modulate the rate and extent of neuronal loss in this disease. We have recently found that Htt is phosphorylated by the kinase IKK on serine (s) 13, activating its phosphorylation on S16 and its acetylation and poly-SUMOylation, modifications that modulate its clearance by the proteasome and lysosome in cells.¹ In the discussion here I suggest that Htt may have a normal function in the lysosomal mechanism of selective macroautophagy involved in its own degradation which may share some similarity with the yeast cytoplasm to vacuole targeting (Cvt) pathway. Pharmacologic activation of this pathway may be useful early in disease progression to treat HD and other neurodegenerative diseases characterized by the accumulation of disease proteins.Key words: Huntington disease, Huntingtin, polyglutamine, autophagy, IKKAn age-related reduction in protein clearance mechanisms has been implicated in the pathogenesis of neurodegenerative diseases including the polyglutamine (polyQ) repeat diseases, Alzheimer disease (AD), Parkinson disease (PD) and Amyotrophic Lateral Sclerosis (ALS). These diseases are each associated with the accumulation of insoluble protein aggregates in diseased neurons. Huntington Disease (HD), caused by an expansion of the polyQ repeat in the protein Huntingtin (Htt), is one such disease of aging in which mutant Htt inclusions form in striatal and cortical neurons as disease progresses. Clarification of the mechanisms of Htt clearance is paramount to finding therapeutic targets to treat HD that may be broadly useful in the treatment of these currently incurable neurodegenerative diseases.     

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.     

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.     

Interaction with polyglutamine-expanded huntingtin alters cellular distribution and RNA processing of huntingtin yeast two-hybrid protein A (HYPA)

Huntington disease (HD) is an autosomal inherited disorder that causes the deterioration of brain cells. The polyglutamine (polyQ) expansion of huntingtin (Htt) is implicated in the pathogenesis of HD via interaction with an RNA splicing factor, Htt yeast two-hybrid protein A/forming-binding protein 11 (HYPA/FBP11). Besides the pathogenic polyQ expansion, Htt also contains a proline-rich region (PRR) located exactly in the C terminus to the polyQ tract. However, how the polyQ expansion influences the PRR-mediated protein interaction and how this abnormal interaction leads to the biological consequence remain elusive. Our NMR structural analysis indicates that the PRR motif of Htt cooperatively interacts with the tandem WW domains of HYPA through domain chaperoning effect of WW1 on WW2. The polyQ-expanded Htt sequesters HYPA to the cytosolic location and then significantly reduces the efficiency of pre-mRNA splicing. We propose that the toxic gain-of-function of the polyQ-expanded Htt that causes dysfunction of cellular RNA processing contributes to the pathogenesis of HD.