Critical role of the proline-rich region in Huntingtin for aggregation and cytotoxicity in yeast

Nine neurodegenerative diseases, such as Huntington, are caused by a polyglutamine (poly(Q)) expansion in otherwise unrelated proteins. Although poly(Q) expansion causes aggregation of the affected proteins, the protein context might determine the selective neuronal vulnerability found in each disease. Here we have report that, although expression of Huntingtin derivatives with a pathological poly(Q) expansion are innocuous in yeast, deletion of the flanking proline-rich region alters the shape and number of poly(Q) inclusions and unmasks toxic properties. Strikingly, deletion of Hsp104 increases the size of inclusions formed by expanded poly(Q) lacking the proline-rich region and abolishes toxicity. Overexpression of the chaperones Hsp104 or Hsp70 rescues growth defects in affected cells without resolving inclusions. However, aggregates formed by nontoxic Huntingtin derivatives or by toxic derivatives cured by chaperones are physically distinct from aggregates formed by toxic proteins. This study identifies the proline-rich region in Huntingtin as a profound cis-acting modulator of expanded poly(Q) toxicity and distinguishes between aggregates of toxic or non-toxic proteins.     

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.     

Overexpression of a conserved HSP40 chaperone reduces toxicity of several neurodegenerative disease proteins

TDP-43 and FUS are DNA/RNA binding proteins associated with neuronal inclusions in amyotrophic lateral sclerosis (ALS) patients. Other neurodegenerative diseases are also characterized by neuronal protein aggregates, e.g. Huntington's disease, associated with polyglutamine (polyQ) expansions in the protein huntingtin. Here we discuss our recent paper establishing similarities between aggregates of TDP-43 that have short glutamine and asparagine (Q/N)-rich modules and are soluble in detergents, with those of polyQ and PIN4C that have large Q/N-rich domains and are detergent-insoluble. We also present new, similar data for FUS. Together, we show that like overexpression of polyQ or PIN4C, overexpression of FUS or TDP-43 causes inhibition of the ubiquitin proteasome system (UPS) and toxicity, both of which are mitigated by overexpression of the Hsp40 chaperone Sis1. Also, in all cases toxicity is enhanced by the [PIN⁺] prion. In addition, we show that the Sis1 mammalian homolog DNAJBI reduces toxicity arising from overexpressed FUS and TDP-43 respectively in human embryonic kidney cells and primary rodent neurons. The common properties of these proteins suggest that heterologous aggregates may enhance the toxicity of a variety of disease-related aggregating proteins, and further that chaperones and the UPS may be key therapeutic targets for diseases characterized by protein inclusions.     

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。     

RNA-binding protein TLS is a major nuclear aggregate-interacting protein in huntingtin exon 1 with expanded polyglutamine-expressing cells

Formation of intracellular aggregates is the hallmark of polyglutamine (polyQ) diseases. We analyzed the components of purified nuclear polyQ aggregates by mass spectrometry. As a result, we found that the RNA-binding protein translocated in liposarcoma (TLS) was one of the major components of nuclear polyQ aggregate-interacting proteins in a Huntington disease cell model and was also associated with neuronal intranuclear inclusions of R6/2 mice. In vitro study revealed that TLS could directly bind to truncated N-terminal huntingtin (tNhtt) aggregates but could not bind to monomer GST-tNhtt with 18, 42, or 62Q, indicating that the tNhtt protein acquired the ability to sequester TLS after forming aggregates. Thioflavin T assay and electron microscopic study further supported the idea that TLS bound to tNhtt-42Q aggregates at the early stage of tNhtt-42Q amyloid formation. Immunohistochemistry showed that TLS was associated with neuronal intranuclear inclusions of Huntington disease human brain. Because TLS has a variety of functional roles, the sequestration of TLS to polyQ aggregates may play a role in diverse pathological changes in the brains of patients with polyQ diseases.     

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.     

多聚谷氨酰胺延伸蛋白募集细胞内正常蛋白质或RNA的分子机制

多聚谷氨酰胺(PolyQ)疾病,是一类由编码蛋白质的基因中CAG三核苷酸重复序列的异常延伸所引发的神经退行性疾病.CAG三核苷酸重复序列导致所编码蛋白质的PolyQ序列的异常延伸,使蛋白质发生错误折叠和积聚,并在细胞内形成包涵体.包涵体的形成是神经退行性疾病的一个重要特征.PolyQ蛋白在积聚过程中,可以将细胞内与其特异相互作用的蛋白质或RNA募集到包涵体中.被募集的其他蛋白质或RNA不仅自身的可溶性组分减少,而且由于被"挟持"到包涵体中其在细胞内的有效组分也相应地减少,从而影响其正常的生物功能.根据特异相互作用的模式,我们将募集作用分为以下几种类型:蛋白质(含Poly Q蛋白)的共积聚;特定结构域或模体介导的募集作用(包括泛素等修饰介导的募集作用);RNA介导的募集作用;以及对分子伴侣蛋白的募集作用.PolyQ延伸蛋白的积聚和对其他组分的募集可能是引发细胞毒性和神经退行性病变的重要原因.     

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.     

Fibrillar structure and charge determine the interaction of polyglutamine protein aggregates with the cell surface

The pathogenesis of most neurodegenerative diseases, including transmissible diseases like prion encephalopathy, inherited disorders like Huntington disease, and sporadic diseases like Alzheimer and Parkinson diseases, is intimately linked to the formation of fibrillar protein aggregates. It is becoming increasingly appreciated that prion-like intercellular transmission of protein aggregates can contribute to the stereotypical spread of disease pathology within the brain, but the mechanisms underlying the binding and uptake of protein aggregates by mammalian cells are largely uninvestigated. We have investigated the properties of polyglutamine (polyQ) aggregates that endow them with the ability to bind to mammalian cells in culture and the properties of the cell surface that facilitate such uptake. Binding and internalization of polyQ aggregates are common features of mammalian cells and depend upon both trypsin-sensitive and trypsin-resistant saturable sites on the cell surface, suggesting the involvement of cell surface proteins in this process. polyQ aggregate binding depends upon the presence of a fibrillar amyloid-like structure and does not depend upon electrostatic interaction of fibrils with the cell surface. Sequences in the huntingtin protein that flank the amyloid-forming polyQ tract also influence the extent to which aggregates are able to bind to cell surfaces.     

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.     

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.     

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.     

Identification of anti-prion compounds as efficient inhibitors of polyglutamine protein aggregation in a zebrafish model

Several neurodegenerative diseases, including Huntington disease (HD), are associated with aberrant folding and aggregation of polyglutamine (polyQ) expansion proteins. Here we established the zebrafish, Danio rerio, as a vertebrate HD model permitting the screening for chemical suppressors of polyQ aggregation and toxicity. Upon expression in zebrafish embryos, polyQ-expanded fragments of huntingtin (htt) accumulated in large SDS-insoluble inclusions, reproducing a key feature of HD pathology. Real time monitoring of inclusion formation in the living zebrafish indicated that inclusions grow by rapid incorporation of soluble htt species. Expression of mutant htt increased the frequency of embryos with abnormal morphology and the occurrence of apoptosis. Strikingly, apoptotic cells were largely devoid of visible aggregates, suggesting that soluble oligomeric precursors may instead be responsible for toxicity. As in nonvertebrate polyQ disease models, the molecular chaperones, Hsp40 and Hsp70, suppressed both polyQ aggregation and toxicity. Using the newly established zebrafish model, two compounds of the N'-benzylidene-benzohydrazide class directed against mammalian prion proved to be potent inhibitors of polyQ aggregation, consistent with a common structural mechanism of aggregation for prion and polyQ disease proteins.     

Cathepsins L and Z are critical in degrading polyglutamine-containing proteins within lysosomes

In neurodegenerative diseases caused by extended polyglutamine (polyQ) sequences in proteins, aggregation-prone polyQ proteins accumulate in intraneuronal inclusions. PolyQ proteins can be degraded by lysosomes or proteasomes. Proteasomes are unable to hydrolyze polyQ repeat sequences, and during breakdown of polyQ proteins, they release polyQ repeat fragments for degradation by other cellular enzymes. This study was undertaken to identify the responsible proteases. Lysosomal extracts (unlike cytosolic enzymes) were found to rapidly hydrolyze polyQ sequences in peptides, proteins, or insoluble aggregates. Using specific inhibitors against lysosomal proteases, enzyme-deficient extracts, and pure cathepsins, we identified cathepsins L and Z as the lysosomal cysteine proteases that digest polyQ proteins and peptides. RNAi for cathepsins L and Z in different cell lines and adult mouse muscles confirmed that they are critical in degrading polyQ proteins (expanded huntingtin exon 1) but not other types of aggregation-prone proteins (e.g. mutant SOD1). Therefore, the activities of these two lysosomal cysteine proteases are important in host defense against toxic accumulation of polyQ proteins.     

Inefficient degradation of truncated polyglutamine proteins by the proteasome

Accumulation of mutant proteins into misfolded species and aggregates is characteristic for diverse neurodegenerative diseases including the polyglutamine diseases. While several studies have suggested that polyglutamine protein aggregates impair the ubiquitin-proteasome system, the molecular mechanisms underlying the interaction between polyglutamine proteins and the proteasome have remained elusive. In this study, we use fluorescence live-cell imaging to demonstrate that the proteasome is sequestered irreversibly within aggregates of overexpressed N-terminal mutant Huntingtin fragment or simple polyglutamine expansion proteins. Moreover, by direct targeting of polyglutamine proteins for proteasomal degradation, we observe incomplete degradation of these substrates both in vitro and in vivo. Thus, our data reveal that intrinsic properties of the polyglutamine proteins prevent their efficient degradation and clearance. Additionally, fluorescence resonance energy transfer is detected between the proteasome and aggregated polyglutamine proteins indicative of a close and stable interaction. We propose that polyglutamine-containing proteins are kinetically trapped within proteasomes, which could explain their deleterious effects on cellular function over time.     

Intracellular aggregation of polypeptides with expanded polyglutamine domain is stimulated by stress-activated kinase MEKK1

Abnormal proteins, which escape chaperone-mediated refolding or proteasome-dependent degradation, aggregate and form inclusion bodies (IBs). In several neurodegenerative diseases, such IBs can be formed by proteins with expanded polyglutamine (polyQ) domains (e.g., huntingtin). This work studies the regulation of intracellular IB formation using an NH(2)-terminal fragment of huntingtin with expanded polyQ domain. We demonstrate that the active form of MEKK1, a protein kinase that regulates several stress-activated signaling cascades, stimulates formation of the IBs. This function of MEKK1 requires kinase activity, as the kinase-dead mutant of MEKK1 cannot stimulate this process. Exposure of cells to UV irradiation or cisplatin, both of which activate MEKK1, also augmented the formation of IBs. The polyQ-containing huntingtin fragment exists in cells in two distinct forms: (a) in a discrete soluble complex, and (b) in association with insoluble fraction. MEKK1 strongly stimulated recruitment of polyQ polypeptides into the particulate fraction. Notably, a large portion of the active form of MEKK1 was associated with the insoluble fraction, concentrating in discrete sites, and polyQ-containing IBs always colocalized with them. We suggest that MEKK1 is involved in a process of IB nucleation. MEKK1 also stimulated formation of IBs with two abnormal polypeptides lacking the polyQ domain, indicating that this kinase has a general effect on protein aggregation.     

Human receptor for activated protein kinase C1 associates with polyglutamine aggregates and modulates polyglutamine toxicity

Formation of SDS-insoluble protein aggregates in affected neurons is a cellular pathological feature of polyglutamine (polyQ) disease. We identified a multi-WD-domain protein, receptor for activated protein kinase C1 (RACK1), as a novel polyQ aggregate component from a Drosophila transgenic polyQ disease model. We showed that WD domains were crucial determinants for the recruitment of RACK1 to polyQ aggregates. Over-expression of the human RACK1 protein suppressed polyQ-induced neurodegeneration in vivo. This is the first report to demonstrate the involvement of WD-domain proteins in polyQ pathogenesis, and the proteomic approach described here can be applied to the investigation of other protein aggregation disorders including Alzheimer’s and Parkinson’s diseases.     

A Highlights from MBoC Selection: Praja1 ubiquitin ligase facilitates degradation of polyglutamine proteins and suppresses polyglutamine-mediated toxicity

A network of chaperones and ubiquitin ligases sustain intracellular proteostasis and is integral in preventing aggregation of misfolded proteins associated with various neurodegenerative diseases. Using cell-based studies of polyglutamine (polyQ) diseases, spinocerebellar ataxia type 3 (SCA3) and Huntington’s disease (HD), we aimed to identify crucial ubiquitin ligases that protect against polyQ aggregation. We report here that Praja1 (PJA1), a Ring-H2 ubiquitin ligase abundantly expressed in the brain, is diminished when polyQ repeat proteins (ataxin-3/huntingtin) are expressed in cells. PJA1 interacts with polyQ proteins and enhances their degradation, resulting in reduced aggregate formation. Down-regulation of PJA1 in neuronal cells increases polyQ protein levels vis-a-vis their aggregates, rendering the cells vulnerable to cytotoxic stress. Finally, PJA1 suppresses polyQ toxicity in yeast and rescues eye degeneration in a transgenic Drosophila model of SCA3. Thus, our findings establish PJA1 as a robust ubiquitin ligase of polyQ proteins and induction of which might serve as an alternative therapeutic strategy in handling cytotoxic polyQ aggregates.