Role of histidine interruption in mitigating the pathological effects of long polyglutamine stretches in SCA1: A molecular approach

Polyglutamine expansions, leading to aggregation, have been implicated in various neurodegenerative disorders. The range of repeats observed in normal individuals in most of these diseases is 19-36, whereas mutant proteins carry 40-81 repeats. In one such disorder, spinocerebellar ataxia (SCA1), it has been reported that certain individuals with expanded polyglutamine repeats in the disease range (Q(12)HQHQ(12)HQHQ(14/15)) but with histidine interruptions were found to be phenotypically normal. To establish the role of histidine, a comparative study of conformational properties of model peptide sequences with (Q(12)HQHQ(12)HQHQ(12)) and without (Q(42)) interruptions is presented here. Q(12)HQHQ(12)HQHQ(12) displays greater solubility and lesser aggregation propensity compared to uninterrupted Q(42) as well as much shorter Q(22). The solvent and temperature-driven conformational transitions (beta structure <--> random coil --> alpha helix) displayed by these model polyQ stretches is also discussed in the present report. The study strengthens our earlier hypothesis of the importance of histidine interruptions in mitigating the pathogenicity of expanded polyglutamine tract at the SCA1 locus. The relatively lower propensity for aggregation observed in case of histidine interrupted stretches even in the disease range suggests that at a very low concentration, the protein aggregation in normal cells, is possibly not initiated at all or the disease onset is significantly delayed. Our present study also reveals that besides histidine interruption, proline interruption in polyglutamine stretches can lower their aggregation propensity.     

Effect of tissue transglutaminase on the solubility of proteins containing expanded polyglutamine repeats

The expansion of a polyglutamine (polyQ) domain in neuronal proteins is the molecular genetic cause of at least eight neurodegenerative diseases. Proteins with a polyQ domain that is greater than 40 Q (Q40) residues form insoluble intranuclear and cytoplasmic inclusions. Expanded polyQ proteins self-associate by non-covalent interactions and become insoluble. They can also be covalently cross-linked by tissue transglutaminase (TTG), a calcium-dependent enzyme present in cells throughout the nervous system. However, it remains unclear whether TTG cross-linking directly contributes to the insolubility of the expanded polyQ proteins. Using an in vitro solubility assay, we found TTG cross-linked Q62 monomers into high molecular weight soluble complexes in a calcium-dependent reaction. Inhibition of TTG cross-linking by primary amine substrates including putrescine and biotinylated pentylamine antagonized TTG's ability to form soluble complexes. In contrast, primary amines (histamine and lysine) that were less effective inhibitors of TTG cross-linking did not inhibit Q62 from becoming insoluble. In summary, TTG can increase the solubility of expanded polyQ proteins by catalyzing intermolecular cross-links. This demonstrates directly that TTG will reduce the ability of expanded polyQ proteins from becoming insoluble. Furthermore, the effectiveness of a primary amine substrate at inhibiting formation of insoluble inclusions may be related to their ability to inhibit intermolecular cross-linking by TTG.     

Eukaryotic proteasomes cannot digest polyglutamine sequences and release them during degradation of polyglutamine-containing proteins

Long glutamine sequences (polyQ) occur in many cell proteins, and several neurodegenerative diseases result from expansion of these sequences. PolyQ-containing proteins are degraded by proteasomes, whose three active sites prefer to cleave after hydrophobic, basic, or acidic residues. We tested whether these particles can digest a polyQ chain. Eukaryotic 26S and 20S proteasomes failed to cut within stretches of 9-29Q residues in peptides. While digesting a myoglobin Q(35) fusion protein, the proteasomes spared the polyQ sequence. In contrast, archaeal proteasomes, whose 14 active sites are less specific, rapidly digested such polyQ repeats. Therefore, when degrading polyQ proteins, eukaryotic proteasomes must release aggregation-prone polyQ-containing fragments for further hydrolysis by unidentified peptidases. In polyQ diseases, such polyQ sequences (38-300Qs) exceed the lengths of normal proteasome products (2-25 residues). Occasional failure of these long undegradable sequences to exit may interfere with proteasome function and help explain why longer polyQ expansions promote early disease onset.     

PolyQ repeat expansions in ATXN2 associated with ALS are CAA interrupted repeats

Amyotrophic lateral sclerosis (ALS) is a devastating, rapidly progressive disease leading to paralysis and death. Recently, intermediate length polyglutamine (polyQ) repeats of 27-33 in ATAXIN-2 (ATXN2), encoding the ATXN2 protein, were found to increase risk for ALS. In ATXN2, polyQ expansions of ≥ 34, which are pure CAG repeat expansions, cause spinocerebellar ataxia type 2. However, similar length expansions that are interrupted with other codons, can present atypically with parkinsonism, suggesting that configuration of the repeat sequence plays an important role in disease manifestation in ATXN2 polyQ expansion diseases. Here we determined whether the expansions in ATXN2 associated with ALS were pure or interrupted CAG repeats, and defined single nucleotide polymorphisms (SNPs) rs695871 and rs695872 in exon 1 of the gene, to assess haplotype association. We found that the expanded repeat alleles of 40 ALS patients and 9 long-repeat length controls were all interrupted, bearing 1-3 CAA codons within the CAG repeat. 21/21 expanded ALS chromosomes with 3CAA interruptions arose from one haplotype (GT), while 18/19 expanded ALS chromosomes with <3CAA interruptions arose from a different haplotype (CC). Moreover, age of disease onset was significantly earlier in patients bearing 3 interruptions vs fewer, and was distinct between haplotypes. These results indicate that CAG repeat expansions in ATXN2 associated with ALS are uniformly interrupted repeats and that the nature of the repeat sequence and haplotype, as well as length of polyQ repeat, may play a role in the neurological effect conferred by expansions in ATXN2.     

Autophagy genes protect against disease caused by polyglutamine expansion proteins in Caenorhabditis elegans

Expanded polyglutamine (polyQ) proteins aggregate intracellularly in Huntington's disease and other neurodegenerative disorders. The lysosomal degradation pathway, autophagy, is known to promote clearance of polyQ protein aggregates in cultured cells. Moreover, basal autophagy in neuronal cells in mice prevents neurodegeneration by suppressing the accumulation of abnormal intracellular proteins. However, it is not yet known whether autophagy genes play a role in vivo in protecting against disease caused by mutant aggregate-prone, expanded polyQ proteins. To examine this question, we used two models of polyQ-induced toxicity in C. elegans, including the expression of polyQ40 aggregates in muscle and the expression of a human huntingtin disease fragment containing a polyQ tract of 150 residues (Htn-Q150) in ASH sensory neurons. Here, we show that genetic inactivation of autophagy genes accelerates the accumulation of polyQ40 aggregates in C. elegans muscle cells and exacerbates polyQ40-induced muscle dysfunction. Autophagy gene inactivation also increases the accumulation of Htn-Q150 aggregates in C. elegans ASH sensory neurons and results in enhanced neurodegeneration. These data provide in vivo genetic evidence that autophagy genes suppress the accumulation of polyQ aggregates and protect cells from disease caused by polyQ toxicity.     

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.     

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.     

Autophagy Genes Protect Against Disease Caused by Polyglutamine Expansion Proteins in Caenorhabditis elegans

Expanded polyglutamine (polyQ) proteins aggregate intracellularly in Huntington’s disease and other neurodegenerative disorders. The lysosomal degradation pathway, autophagy, is known to promote clearance of polyQ protein aggregates in cultured cells. Moreover, basal autophagy in neuronal cells in mice prevents neurodegeneration by suppressing the accumulation of abnormal intracellular proteins. However, it is not yet known whether autophagy genes play a role in vivo in protecting against disease caused by mutant aggregate-prone, expanded polyQ proteins. To examine this question, we used two models of polyQ-induced toxicity in C. elegans, including the expression of polyQ40 aggregates in muscle and the expression of a human huntingtin disease fragment containing a polyQ tract of 150 residues (Htn-Q150) in ASH sensory neurons. Here, we show that genetic inactivation of autophagy genes accelerates the accumulation of polyQ40 aggregates in C. elegans muscle cells and exacerbates polyQ40-induced muscle dysfunction. Autophagy gene inactivation also increases the accumulation of Htn-Q150 aggregates in C. elegans ASH sensory neurons and results in enhanced neurodegeneration. These data provide in vivo genetic evidence that autophagy genes suppress the accumulation of polyQ aggregates and protect cells from disease caused by polyQ toxicity.     

Expanded Polyglutamine-containing N-terminal Huntingtin Fragments Are Entirely Degraded by Mammalian Proteasomes

Huntington disease is a neurodegenerative disorder caused by an expanded polyglutamine (polyQ) repeat within the protein huntingtin (Htt). N-terminal fragments of the mutant Htt (mHtt) proteins containing the polyQ repeat are aggregation-prone and form intracellular inclusion bodies. Improving the clearance of mHtt fragments by intracellular degradation pathways is relevant to obviate toxic mHtt species and subsequent neurodegeneration. Because the proteasomal degradation pathway has been the subject of controversy regarding the processing of expanded polyQ repeats, we examined whether the proteasome can efficiently degrade Htt-exon1 with an expanded polyQ stretch both in neuronal cells and in vitro. Upon targeting mHtt-exon1 to the proteasome, rapid and complete clearance of mHtt-exon1 was observed. Proteasomal degradation of mHtt-exon1 was devoid of polyQ peptides as partial cleavage products by incomplete proteolysis, indicating that mammalian proteasomes are capable of efficiently degrading expanded polyQ sequences without an inhibitory effect on the proteasomal activity.     

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.     

RCC1-like repeat proteins: a pangenomic, structurally diverse new superfamily of beta-propeller domains

The beta-propeller fold is a phylogenetically widespread, common protein architecture able to support a range of different functions such as catalysis, ligand binding and transport, regulation and protein binding. Interestingly, it appears that the beta-propeller topology is also compatible with strikingly diverse sequences. Amongst this diversity, there are three large groups of proteins with related sequences and very important cellular and intercellular regulatory functions: WD, kelch, and YWTD proteins. A common characteristic between these protein families is that their sequences, while distinct, all contain internal repeats 40-45 residues long. Through a pangenomic analysis using internal repeat profiles derived from the structurally known propeller modules of the eukaryotic protein RCC1 and the related prokaryotic protein BLIP-II, we have defined a new superfamily of propeller repeats, the RCC1-like repeats (RLRs). These sequences turn out to be more phylogenetically widespread than other large groups of propeller proteins, occurring in both prokaryotic and eukaryotic genomes. Interestingly, our research showed that RLR domains with different numbers of repeats exist, ranging from 3 to 7, and possibly more. A novel, intriguing finding is the discovery of sequences with 3 repeats, as well as proteins with 10 modular units, though in the latter case it is not clear whether these are made of two 5-bladed domains or a single, novel 10-bladed propeller. In addition, the results indicate that circular permutation events may have taken place in the evolution of these proteins. It is now established that the group of RLR proteins is extremely numerous and is characterized by unique, remarkable features which place it in a position of special interest as an important superfamily of proteins in nature.     

Sir2 is induced by oxidative stress in a yeast model of Huntington disease and its activation reduces protein aggregation

Huntington disease (HD) is a neurodegenerative disorder caused by expansion of CAG trinucleotide repeats, leading to an elongated polyglutamine sequence (polyQ) in the huntingtin protein. Misfolding of mutant polyQ proteins with expanded tracts results in aggregation, causing cytotoxicity. Oxidative stress in HD has been documented in humans as important to disease progression. Using yeast cells as a model of HD, we report that when grown at high glucose concentration, cells expressing mutant polyQ do not show apparent oxidative stress. At higher cell densities, when glucose becomes limiting and cells are metabolically shifting from fermentation to respiration, protein oxidation and catalase activity increases in relation to the length of the polyQ tract. Oxidative stress, either endogenous as a result of mutant polyQ expression or exogenously generated, increases Sir2 levels. Δ sir2 cells expressing expanded polyQ lengths show signs of oxidative stress even at the early exponential phase. In a wild-type background, isonicotinamide, a Sir2 activator, decreases mutant polyQ aggregation and the stress generated by expanded polyQ. Taken together, these results describe mutant polyQ proteins as being more toxic in respiring cells, causing oxidative stress and an increase in Sir2 levels. Activation of Sir2 would play a protective role against this toxicity.     

Beta conformation of polyglutamine track revealed by a crystal structure of Huntingtin N-terminal region with insertion of three histidine residues

Huntington disease is an autosomal-dominant neurodegenerative disorder caused by a polyglutamine (polyQ) expansion (> 35Q) in the first exon (EX1) of huntingtin protein (Htt). mHtt protein is thought to adopt one or more toxic conformation(s) that are involved in pathogenic interactions in cells . However, the structure of mHtt is not known. Here, we present a near atomic resolution structure of mHtt36Q-EX1. To facilitate crystallization, three histidine residues (3H) were introduced within the Htt36Q stretch resulting in the sequence of Q₇HQHQHQ₂₇. The Htt36Q3H region adopts α-helix, loop, β-hairpin conformations. Furthermore, we observed interactions between the backbone of the Htt36Q3H β-strand with the aromatic residues mimicking putative-toxic interactions with other proteins. Our findings support previous predictions that the expanded mHtt-polyQ region adopts a β-sheet structure. Detailed structural information about mHtt improves our understanding of the pathogenic mechanisms in HD and other polyQ expansion disorders and may form the basis for rational design of small molecules that target toxic conformations of disease-causing proteins.     

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.     

Comparative structural analysis of the binding domain of follicle stimulating hormone receptor

Proteins with leucine-rich repeats (LRRs) specialize in mediating protein-protein interactions. The hormone binding portion of the receptor for follicle stimulating hormone (FSH) is an LRR protein by sequence, and the crystal structure of this domain from human FSH receptor in a complex with FSH shows that it does indeed have an LRR structure. It differs from other LRR domains, however, in being an all-beta protein composed of highly irregular repeats and having only slight overall curvature. Despite these distinctions and a superficial resemblance to beta-helical proteins, the binding domain of FSH receptor clearly is an LRR protein. The structure does consist of two parts with distinctively different curvatures. Comparison with the structures of other LRR-containing proteins shows a correlation between curvature and main-chain hydrogen bonding pattern of the parallel beta-sheet. The hormone-binding site is located at the concave surface of the receptor structure, a feature common to proteins with LRR motifs. Analysis of the ligand-binding site of LRR-containing proteins reveals that they generally utilize extensive interface area and a large number of charged residues to facilitate high-affinity protein-protein interactions.     

Glutamine repeats and neurodegenerative diseases: molecular aspects

Eight severe inherited neurodegenerative diseases are caused by expansion of glutamine repeats in the affected proteins. In every case, proteins with repeats of fewer than 38 glutamine residues are harmless, but those with repeats of more than 41 glutamine residues form toxic neuronal nuclear aggregates in the affected neurons. Similarly, proteins that have repeats of fewer than 37 glutamine residues are soluble in vitro, whereas proteins with repeats of more than 40 glutamine residues precipitate as insoluble fibres, apparently because of a structural transition associated with the increased length.     

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.