Aggregation kinetics of interrupted polyglutamine peptides

Abnormally expanded polyglutamine domains are associated with at least nine neurodegenerative diseases, including Huntington's disease. Expansion of the glutamine region facilitates aggregation of the impacted protein, and aggregation has been linked to neurotoxicity. Studies of synthetic peptides have contributed substantially to our understanding of the mechanism of aggregation because the underlying biophysics of polyglutamine-mediated association can be probed independent of their context within a larger protein. In this report, interrupting residues were inserted into polyglutamine peptides (Q20), and the impact on conformational and aggregation properties was examined. A peptide with two alanine residues formed laterally aligned fibrillar aggregates that were similar to the uninterrupted Q20 peptide. Insertion of two proline residues resulted in soluble, nonfibrillar aggregates, which did not mature into insoluble aggregates. In contrast, insertion of a β-turn template _DPG rapidly accelerated aggregation and resulted in a fibrillar aggregate morphology with little lateral alignment between fibrils. These results are interpreted to indicate that (a) long-range nonspecific interactions lead to the formation of soluble oligomers, while maturation of oligomers into fibrils requires conformational conversion and (b) that soluble oligomers dynamically interact with each other, while insoluble aggregates are relatively inert. Kinetic analysis revealed that the increase in aggregation caused by the _DPG insert is inconsistent with the nucleation-elongation mechanism of aggregation featuring a monomeric β-sheet nucleus. Rather, the data support a mechanism of polyglutamine aggregation by which monomers associate into soluble oligomers, which then undergo slow structural rearrangement to form sedimentable aggregates.     

Examining Polyglutamine Peptide Length: A Connection between Collapsed Conformations and Increased Aggregation

Abnormally expanded polyglutamine domains in proteins are associated with several neurodegenerative diseases, of which the best known is Huntington's. Expansion of the polyglutamine domain facilitates aggregation of the affected protein, and several studies directly link aggregation to neurotoxicity. The age of onset of disease is inversely correlated with the length of the polyglutamine domain; this correlation motivates an examination of the role of the length of the domain on aggregation. In this investigation, peptides containing 8 to 24 glutamines were synthesized, and their conformational and aggregation properties were examined. All peptides lacked secondary structure. Fluorescence resonance energy transfer studies revealed that the peptides became increasingly collapsed as the number of glutamine residues increased. The effective persistence length was estimated to decrease from ∼ 11 to ∼ 7 Å as the number of glutamines increased from 8 to 24. A comparison of our data with theoretical results suggests that phosphate-buffered saline is a good solvent for Q8 and Q12, a theta solvent for Q16, and a poor solvent for Q20 and Q24. By dynamic light scattering, we observed that Q16, Q20, and Q24, but not Q8 or Q12, immediately formed soluble aggregates upon dilution into phosphate-buffered saline at 37 °C. Thus, Q16 stands at the transition point between good and poor solvent and between stable and aggregation-prone peptide. Examination of aggregates by transmission electron microscopy, along with kinetic assays for sedimentation, provided evidence indicating that soluble aggregates mature into sedimentable aggregates. Together, the data support a mechanism of aggregation in which monomer collapse is accompanied by formation of soluble oligomers; these soluble species lack regular secondary structure but appear morphologically similar to the sedimentable aggregates into which they eventually mature.     

Amino acid sequence requirements of peptides that inhibit polyglutamine-protein aggregation and cell death.

Proteins with expanded polyglutamine domains cause eight inherited neurodegenerative diseases including Huntington's disease. In a previous paper, we identified peptides that inhibit polyglutamine protein aggregation and cell death and now describe the amino acid sequence requirements necessary for these activities. The original 11 amino acid polyglutamine (Q) Binding Peptide 1(QBP1; SNWKWWPGIFD) can be shortened to 8 amino acids (WKWWPGIF) without loss of ability to inhibit polyglutamine aggregation. Three determinants are responsible for inhibition: a tryptophan-rich motif (WKWW), a spacer amino acid and the tripeptide GIF. GIF can be replaced by a repeat of the tryptophan-rich motif, but the spacer remains necessary. We also demonstrate concordance between peptide activity in the in vitro assay and a cellular assay of polyglutamine aggregation and cell death. Polyglutamine binding peptides targeted for intracellular delivery by fusion to TAT retain the ability to inhibit polyglutamine aggregation and cell death in transfected COS 7 cells.     

Inhibition of polyglutamine protein aggregation and cell death by novel peptides identified by phage display screening

Proteins with expanded polyglutamine domains cause eight inherited neurodegenerative diseases, including Huntington's, but the molecular mechanism(s) responsible for neuronal degeneration are not yet established. Expanded polyglutamine domain proteins possess properties that distinguish them from the same proteins with shorter glutamine repeats. Unlike proteins with short polyglutamine domains, proteins with expanded polyglutamine domains display unique protein interactions, form intracellular aggregates, and adopt a novel conformation that can be recognized by monoclonal antibodies. Any of these polyglutamine length-dependent properties could be responsible for the pathogenic effects of expanded polyglutamine proteins. To identify peptides that interfere with pathogenic polyglutamine interactions, we screened a combinatorial peptide library expressed on M13 phage pIII protein to identify peptides that preferentially bind pathologic-length polyglutamine domains. We identified six tryptophan-rich peptides that preferentially bind pathologic-length polyglutamine domain proteins. Polyglutamine-binding peptide 1 (QBP1) potently inhibits polyglutamine protein aggregation in an in vitro assay, while a scrambled sequence has no effect on aggregation. QBP1 and a tandem repeat of QBP1 also inhibit aggregation of polyglutamine-yellow fluorescent fusion protein in transfected COS-7 cells. Expression of QBP1 potently inhibits polyglutamine-induced cell death. Selective inhibition of pathologic interactions of expanded polyglutamine domains with themselves or other proteins may be a useful strategy for preventing disease onset or for slowing progression of the polyglutamine repeat diseases.     

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

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

Synthesis of pathological and nonpathological human exon 1 huntingtin

Huntington's disease (HD) is a neurodegenerative disorder that affects approximately 1 in 10 000 individuals. The underlying gene mutation was identified as a CAG‐triplet repeat expansion in the gene huntingtin. The CAG sequence codes for glutamine, and in HD, an expansion of the polyglutamine (poly‐Q) stretch above 35 glutamine residues results in pathogenicity. It has been demonstrated in various animal models that only the expression of exon 1 huntingtin, a 67‐amino acid‐long polypeptide plus a variable poly‐Q stretch, is sufficient to cause full HD‐like pathology. Therefore, a deeper understanding of exon 1 huntingtin, its structure, aggregation mechanism and interaction with other proteins is crucial for a better understanding of the disease. Here, we describe the synthesis of a 109‐amino acid‐long exon 1 huntingtin peptide including a poly‐Q stretch of 42 glutamines. This microwave‐assisted solid phase peptide synthesis resulted in milligram amounts of peptide with high purity. We also synthesized a nonpathogenic version of exon 1 huntingtin (90‐amino acid long including a poly‐Q stretch of 23 glutamine residues) using the same strategy. In circular dichroism spectroscopy, both polypeptides showed weak alpha‐helical properties with the longer peptide showing a higher helical degree. These model peptides have great potential for further biomedical analyses, e.g. for large‐scale pre‐screenings for aggregation inhibitors, further structural analyses as well as protein–protein interaction studies. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Biodegradable delivery system containing a peptide inhibitor of polyglutamine aggregation: a step toward therapeutic development in Huntington's disease

Huntington's and eight other neurodegenerative diseases occur because of CAG repeat expansion mutation culminating into an expanded polyglutamine tract in respective protein. In Huntington's disease (HD), a number of CAG repeats beyond normal repeat length (>36) lead to the formation of mutant protein, the proteolytic cleavage of which induces aggregation in polyglutamine length‐dependent manner. The neurodegeneration in this disease is linked to aggregation, and its inhibition is a potential approach for therapeutic development. Although peptides and other molecules have been developed for inhibiting aggregation, peptides in general are susceptible to degradation in vivo conditions. To understand their clinical significance, they also need to be delivered through blood–brain barrier. Here, for the first time, we have synthesized poly‐d ,l ‐lactide‐co‐glycolide nanoparticles containing a polyglutamine aggregation inhibitor peptide PGQ₉[P²], by nanoprecipitation method. This process yielded less than 200 nm spherical nanoparticles with uniform distribution. Characterization studies by infrared spectroscopy‐based and HPLC‐based assays show the presence of PGQ₉[P²] in nanoparticles. In vitro release kinetics demonstrates that nanoparticles release PGQ₉[P²] by erosion and diffusion processes. When the PGQ₉[P²]‐loaded nanoparticles are incubated with aggregation‐prone Q₃₅P₁₀ peptide, representing N‐terminal part of Huntingtin protein, it arrests the elongation phase of Q₃₅P₁₀ aggregation. These findings propose the first step toward delivery of a peptide inhibitor against polyglutamine aggregation in HD. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

An accurate model of polyglutamine

Polyglutamine repeats in proteins are highly correlated with amyloid formation and neurological disease. To better understand the molecular basis of glutamine repeat diseases, structural analysis of polyglutamine peptides as soluble monomers, oligomers, and insoluble amyloid fibrils is necessary. In this study, fluorescence resonance energy transfer (FRET) experiments and molecular dynamics simulations using different theoretical models of polyglutamine were conducted. This study demonstrates that a previously proposed simple C(α)C(β) model of polyglutamine, denoted as FCO, accurately reproduced the present FRET results and the results of previously published FRET, triplet-state quenching, and fluorescence correlation studies. Other simple C(α)C(β) models with random coil and extended β-strand parameters, and all-atom models with parm96 and parm99SB force fields, did not match the FRET result well. The FCO is an intrinsically disordered model with a high-effective persistence length producing extended peptides at short lengths (Q(N) < 10). Because of an increasing number of attractive Q-Q interactions at longer lengths, the FCO model becomes increasingly more compact at lengths between Q(N) ～ 10-16 and is as compact as many folded proteins at Q(N) > 16.     

Length-dependent aggregation of uninterrupted polyalanine peptides

Polyalanine (polyA) is the third-most prevalent homopeptide repeat in eukaryotes, behind polyglutamine and polyasparagine. Abnormal expansion of the polyA repeat is linked to at least nine human diseases, and the disease mechanism likely involves enhanced length-dependent aggregation. Because of the simplicity of its side chain, polyA has been a favorite target of computational studies, and because of their tendency to fold into α-helix, peptides containing polyA-rich domains have been a popular experimental subject. However, experimental studies on uninterrupted polyA are very limited. We synthesized polyA peptides containing uninterrupted sequences of 7 to 25 alanines (A7 to A25) and characterized their length-dependent conformation and aggregation properties. The peptides were primarily disordered, with a modest component of α-helix that increased with increasing length. From measurements of mean distance spanned by the polyA segment, we concluded that physiological buffers are neutral solvents for shorter polyA peptides and poor solvents for longer peptides. At moderate concentration and near-physiological temperature, polyA assembled into soluble oligomers, with a sharp transition in oligomer physical properties between A19 and A25. With A19, oligomers were large, contained only a small fraction of the total peptide mass, and slowly grew into loose clusters, while A25 rapidly and completely assembled into small stable oligomers of ~7 nm radius. At high temperatures, A19 assembled into fibrils, but A25 precipitated as dense, micrometer-sized particles. A comparison of these results to those obtained with polyglutamine peptides of similar design sheds light on the role of the side chain in regulating conformation and aggregation.     

The composition of the polyglutamine‐containing proteins influences their co‐aggregation properties

The sequestration of crucial cellular proteins into insoluble aggregates formed by the polypeptides containing expanded polyglutamine tracts has been proposed to be the key mechanism responsible for the abnormal cell functioning in the so‐called polyglutamine diseases. To evaluate to what extent the ability of polyglutamine sequences to recruit other proteins into the intracellular aggregates depends on the composition of the aggregating peptide, we analysed the co‐aggregation properties of the N‐terminal fragment of huntingtin fused with unrelated non‐aggregating and/or self‐aggregating peptides. We show that the ability of the mutated N‐terminal huntingtin fragment to sequester non‐related proteins can be significantly increased by fusion with the non‐aggregating reporter protein [GFP (green fluorescence protein)]. By contrast, fusion with the self‐aggregating C‐terminal fragment of the CFTR (cystic fibrosis transmembrane conductance regulator) dramatically reduces the sequestration of related non‐fused huntingtin fragments. We also demonstrate that the co‐aggregation of different non‐fused N‐terminal huntingtin fragments depends on their length, with long fragments of the wild‐type huntingtin not only excluded from the nuclear inclusions, but also very inefficiently sequestered into the cytoplasmic aggregates formed by the short fragments of mutant protein. Additionally, our results suggest that atypical intracellular aggregation patterns, which include unusual distribution and/or morphology of protein aggregates, are associated with altered ability of accumulating proteins to co‐aggregate with other peptides.     

Elongation kinetics of polyglutamine peptide fibrils: a quartz crystal microbalance with dissipation study

Abnormally expanded polyglutamine domains in proteins are associated with several neurodegenerative diseases, including Huntington's disease. Expansion of the polyglutamine (polyQ) domain facilitates aggregation of the affected protein, and several studies directly link aggregation to neurotoxicity. Studies of synthetic polyQ peptides have contributed substantially to our understanding of the mechanism of aggregation. In this report, polyQ fibrils were immobilized onto a sensor, and their elongation by polyQ peptides of various length and conformation was examined using quartz crystal microbalance with dissipation monitoring (QCM-D). The rate of elongation increased as the peptide length increased from 8 to 24 glutamines (Q8, Q20, and Q24). Monomer conformation affected elongation rates: insertion of a β-turn template d-Pro-Gly in the center of the peptide increased elongation rates several-fold, while insertion of Pro-Pro dramatically slowed elongation. Dissipation measurements of the QCM-D provided qualitative information about mechanical properties of the elongating fibrils. These data showed clear differences in the characteristics of the elongating aggregates, depending on the specific identity of the associating polyQ peptide. Elongation rates were sensitive to the pH and ionic strength of the buffer. Comparison of QCM-D data with those obtained by optical waveguide lightmode spectroscopy revealed that very little water was associated with the elongation of fibrils by the peptide containing d-Pro-Gly, but a significant amount of water was associated when the fibrils were elongated by Q20. Together, the data indicate that elongation of polyQ fibrils can occur without full consolidation to the fibril structure, resulting in variations to the aggregate structure during elongation.     

Reconsidering the mechanism of polyglutamine peptide aggregation

There are at least nine neurodegenerative diseases associated with proteins that contain an unusually expanded polyglutamine domain, the best known of which is Huntington's disease. In all of these diseases, the mutant protein aggregates into neuronal inclusions; it is generally, although not universally, believed that protein aggregation is an underlying cause of the observed neuronal degeneration. In an effort to examine the role of polyglutamine in facilitating protein aggregation, investigators have used synthetic polyglutamine peptides as model systems. Analysis of kinetic data led to the conclusions that aggregation follows a simple nucleation-elongation mechanism characterized by a significant lag time, during which the peptide is monomeric, and that the nucleus is a monomer in a thermodynamically unfavorable conformation [Chen, S. M., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 11884-11889]. We re-examined this hypothesis by measuring the aggregation kinetics of the polyglutamine peptide K2Q23K2, using sedimentation, static and dynamic light scattering, and size exclusion chromatography. Our data show that during the lag time in sedimentation kinetics, there is substantial organization of the peptide into soluble linear aggregates. These aggregates have no regular secondary structure as measured by circular dichroism but have particle dimensions and morphologies similar to those of mature insoluble aggregates. The soluble aggregates constitute approximately 30% of the total peptide mass, form rapidly, and continue to grow over a period of hours to days, eventually precipitating. Once insoluble aggregates form, loss of monomer from the solution phase continues. Our data support an assembly mechanism for polyglutamine peptide more complex than that previously proposed.     

Slow amyloid nucleation via α-helix-rich oligomeric intermediates in short polyglutamine-containing huntingtin fragments

The 17-amino-acid N-terminal segment (htt(NT)) that leads into the polyglutamine (polyQ) segment in the Huntington's disease protein huntingtin (htt) dramatically increases aggregation rates and changes the aggregation mechanism, compared to a simple polyQ peptide of similar length. With polyQ segments near or above the pathological repeat length threshold of about 37, aggregation of htt N-terminal fragments is so rapid that it is difficult to tease out mechanistic details. We describe here the use of very short polyQ repeat lengths in htt N-terminal fragments to slow this disease-associated aggregation. Although all of these peptides, in addition to htt(NT) itself, form α-helix-rich oligomeric intermediates, only peptides with Q(N) of eight or longer mature into amyloid-like aggregates, doing so by a slow increase in β-structure. Concentration-dependent circular dichroism and analytical ultracentrifugation suggest that the htt(NT) sequence, with or without added glutamine residues, exists in solution as an equilibrium between disordered monomer and α-helical tetramer. Higher order, α-helix rich oligomers appear to be built up via these tetramers. However, only htt(NT)Q(N) peptides with N=8 or more undergo conversion into polyQ β-sheet aggregates. These final amyloid-like aggregates not only feature the expected high β-sheet content but also retain an element of solvent-exposed α-helix. The α-helix-rich oligomeric intermediates appear to be both on- and off-pathway, with some oligomers serving as the pool from within which nuclei emerge, while those that fail to undergo amyloid nucleation serve as a reservoir for release of monomers to support fibril elongation. Based on a regular pattern of multimers observed in analytical ultracentrifugation, and a concentration dependence of α-helix formation in CD spectroscopy, it is likely that these oligomers assemble via a four-helix assembly unit. PolyQ expansion in these peptides appears to enhance the rates of both oligomer formation and nucleation from within the oligomer population, by structural mechanisms that remain unclear.     

Flanking polyproline sequences inhibit beta-sheet structure in polyglutamine segments by inducing PPII-like helix structure

Polyglutamine (poly(Q)) expansion is associated with protein aggregation into β-sheet amyloid fibrils and neuronal cytotoxicity. In the mutant poly(Q) protein huntingtin, associated with Huntington's disease, both aggregation and cytotoxicity may be abrogated by a polyproline (poly(P)) domain flanking the C terminus of the poly(Q) region. To understand structural changes that may occur with the addition of the poly(P) sequence, we synthesized poly(Q) peptides with 3-15 glutamine residues and a corresponding set of poly(Q) peptides flanked on the C terminus by 11 proline residues (poly(Q)-poly(P)), as occurs in the huntingtin sequence. The shorter soluble poly(Q) peptides (three or six glutamine residues) showed polyproline type II-like (PPII)-like helix conformation when examined by circular dichroism spectroscopy and were monomers as judged by size-exclusion chromatography (SEC), while the longer poly(Q) peptides (nine or 15 glutamine residues) showed a β-sheet conformation by CD and defined oligomers by SEC. Soluble poly(Q)-poly(P) peptides showed PPII-like content but SEC showed poorly defined, overlapping oligomeric peaks, and as judged by CD these peptides retained significant PPII-like structure with increasing poly(Q) length. More importantly, addition of the poly(P) domain increased the threshold for fibril formation to ≈ 15 glutamine residues. X-ray diffraction, electron microscopy, and film CD showed that, while poly(Q) peptides with ≥ 6 glutamine residues formed β-sheet-rich fibrils, only the longest poly(Q)-poly(P) peptide (15 glutamine residues) did so. From these and other observations, we propose that poly(Q) domains exist in a “tug-of-war” between two conformations, a PPII-like helix and a β-sheet, while the poly(P) domain is conformationally constrained into a proline type II helix (PPII). Addition of poly(P) to the C terminus of a poly(Q) domain induces a PPII-like structure, which opposes the aggregation-prone β-sheet. These structural observations may shed light on the threshold phenomenon of poly(Q) aggregation, and support the hypothesized evolution of “protective” poly(P) tracts adjacent to poly(Q) aggregation domains.     

Modulation of Polyglutamine Conformations and Dimer Formation by the N-Terminus of Huntingtin

Polyglutamine expansions within different proteins are associated with nine different neurodegenerative diseases. There is growing interest in understanding the roles of flanking sequences from disease-relevant proteins in the intrinsic conformational and aggregation properties of polyglutamine. We report results from atomistic simulations and circular dichroism experiments that quantify the effect of the N-terminal 17-residue (Nt17) segment of the huntingtin protein on polyglutamine conformations and intermolecular interactions. We show that the Nt17 segment and polyglutamine domains become increasingly disordered as polyglutamine length (N) increases in Nt17-Q_N constructs. Hydrophobic groups within Nt17 become sequestered in intramolecular interdomain interfaces. We also show that the Nt17 segment suppresses the intrinsic propensity of polyglutamine aggregation. This inhibition arises from the incipient micellar structures adopted by monomeric forms of the peptides with Nt17 segments. The degree of intermolecular association increases with increasing polyglutamine length and is governed mainly by associations between polyglutamine domains. Comparative analysis of intermolecular associations for different polyglutamine-containing constructs leads to clearer interpretations of recently published experimental data. Our results suggest a framework for fibril formation and identify roles for flanking sequences in the modulation of polyglutamine aggregation.     

Towards a structural understanding of the fibrillization pathway in Machado-Joseph's disease: trapping early oligomers of non-expanded ataxin-3

Machado-Joseph's disease is caused by a CAG trinucleotide repeat expansion that is translated into an abnormally long polyglutamine tract in the protein ataxin-3. Except for the polyglutamine region, proteins associated with polyglutamine diseases are unrelated, and for all of these diseases aggregates containing these proteins are the major components of the nuclear proteinaceous deposits found in the brain. Aggregates of the expanded proteins display amyloid-like morphological and biophysical properties. Human ataxin-3 containing a non-pathological number of glutamine residues (14Q), as well as its Caenorhabditis elegans (1Q) orthologue, showed a high tendency towards self-interaction and aggregation, under near-physiological conditions. In order to understand the discrete steps in the assembly process leading to ataxin-3 oligomerization, we have separated chromatographically high molecular mass oligomers as well as medium mass multimers of non-expanded ataxin-3. We show that: (a) oligomerization occurs independently of the poly(Q)-repeat and it is accompanied by an increase in beta-structure; and (b) the first intermediate in the oligomerization pathway is a Josephin domain-mediated dimer of ataxin-3. Furthermore, non-expanded ataxin-3 oligomers are recognized by a specific antibody that targets a conformational epitope present in soluble cytotoxic species found in the fibrillization pathway of expanded polyglutamine proteins and other amyloid-forming proteins. Imaging of the oligomeric forms of the non-pathological protein using electron microscopy reveals globular particles, as well as short chains of such particles that likely mimic the initial stages in the fibrillogenesis pathway occurring in the polyglutamine-expanded protein. Thus, they constitute potential targets for therapeutic approaches in Machado-Joseph's disease, as well as valuable diagnostic markers in disease settings.     

Misfolded Polyglutamine,Polyalanine, and Superoxide Dismutase 1 Aggregate via Distinct Pathways in the Cell

Protein aggregation into intracellular inclusions is a key feature of many neurodegenerative disorders. A common theme has emerged that inappropriate self-aggregation of misfolded or mutant polypeptide sequences is detrimental to cell health. Yet protein quality control mechanisms may also deliberately cluster them together into distinct inclusion subtypes, including the insoluble protein deposit (IPOD) and the juxtanuclear quality control (JUNQ). Here we investigated how the intrinsic oligomeric state of three model systems of disease-relevant mutant protein and peptide sequences relates to the IPOD and JUNQ patterns of aggregation using sedimentation velocity analysis. Two of the models (polyalanine (37A) and superoxide dismutase 1 (SOD1) mutants A4V and G85R) accumulated into the same JUNQ-like inclusion whereas the other, polyglutamine (72Q), formed spatially distinct IPOD-like inclusions. Using flow cytometry pulse shape analysis (PulSA) to separate cells with inclusions from those without revealed the SOD1 mutants and 37A to have abruptly altered oligomeric states with respect to the nonaggregating forms, regardless of whether cells had inclusions or not, whereas 72Q was almost exclusively monomeric until inclusions formed. We propose that mutations leading to JUNQ inclusions induce a constitutively “misfolded” state exposing hydrophobic side chains that attract and ultimately overextend protein quality capacity, which leads to aggregation into JUNQ inclusions. Poly(Q) is not misfolded in this same sense due to universal polar side chains, but is highly prone to forming amyloid fibrils that we propose invoke a different engagement mechanism with quality control.     

A microtiter plate assay for polyglutamine aggregate extension

Polyglutamine (polyGln) aggregates are neuropathological markers of expanded CAG repeat disorders, and may also play a critical role in the development of these diseases. We have established a highly sensitive, fast, reproducible, and specific assay capable of monitoring aggregate-dependent deposition of polyglutamine peptides. This assay allows detailed studies on various aspects of aggregation kinetics, and also makes possible the detection and quantitation of low levels of "extension-competent" aggregates. In the simplest form of this assay, polyGln aggregates are made from chemically synthesized peptides and immobilized onto microplate wells. These wells are incubated for different times with low concentrations of a soluble biotinylated polyGln peptide. Europium-streptavidin complexation of the immobilized biotin, followed by time-resolved fluorescence detection of the deposited europium, allows us to calculate the rate (fmol/h) of incorporation of polyGln peptides into polyGln aggregates. This assay will make possible basic studies on the assembly mechanism of polyGln aggregates and on critical features of the reaction, such as polyGln length dependence. The assay also will be a valuable tool for screening and characterizing anti-aggregation inhibitors. It will also be useful for detection and quantitation of aggregation-competent polyGln aggregates in biological materials, which may prove to be of critical importance in understanding the disease mechanism.     

Structural characterization of transglutaminase-catalyzed cross-linking between glyceraldehyde 3-phosphate dehydrogenase and polyglutamine repeats

The accumulation of abnormal polyglutamine-containing protein aggregates within the cytosol and nuclei of affected neurons is a hallmark of the progressive neurodegenerative disorders caused by an elongated (CAG)(n) repeat in the genome. The polyglutamine domains are excellent substrates for the enzyme transglutaminase type 2 (tissue), resulting in the formation of cross-links with polypeptides containing lysyl groups. Enzymatic activity toward the Q(n) domains increases greatly upon lengthening of such Q(n) stretches (n > 40). Among the possible amine donors, the glycolytic enzyme glyceraldehyde-3-phosphate-dehydrogenase was shown to tightly bind several proteins involved in polyglutamine expansion diseases. Recently, the authors have shown that K191, K268, and K331, out of the 26 lysines present in glyceraldehyde-3-phosphate-dehydrogenase, are the reactive amine-donor sites forming cross-links with substance P, which bears the simplest Q(n) domain (n = 2). The present study reports that synthetic peptides of both pathological and nonpathological length (n = 43 and 17, respectively) form cross-links with the same K residues located in the C-terminal region of glyceraldehyde-3-phosphate-dehydrogenase. In addition, it is shown that extra K residues present in the C termini of glyceraldehyde-3-phosphate-dehydrogenase are susceptible to cross-linking in the presence of transglutaminase. The present results indicate a possible modulating effect of Q(n) stretches on tissue transglutaminase substrate specificity and mechanism of recognition.