Expansion of polyglutamine induces the formation of quasi-aggregate in the early stage of protein fibrillization

We examined the effects of the expansion of glutamine repeats on the early stage of protein fibrillization. Small-angle x-ray scattering (SAXS) and electron microscopic studies revealed that the elongation of polyglutamine from 35 to 50 repeats in protein induced a large assembly of the protein upon incubation at 37 degrees C and that its formation was completed in approximately 3 h. A bead modeling procedure based on SAXS spectra indicated that the largely assembled species of the protein, quasi-aggregate, is composed of 80 to approximately 90 monomers and a bowl-like structure with long and short axes of 400 and 190 A, respectively. Contrary to fibril, the quasi-aggregate did not show a peak at S = 0.21 A-1 corresponding to the 4.8-A spacing of beta-pleated sheets in SAXS spectra, and reacted with a monoclonal antibody specific to expanded polyglutamine. These results imply that beta-sheets of expanded polyglutamines in the quasi-aggregate are not orderly aligned and are partially exposed, in contrast to regularly oriented and buried beta-pleated sheets in fibril. The formation of non-fibrillary quasi-aggregate in the early phase of fibril formation would be one of the major characteristics of the protein containing an expanded polyglutamine.     

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.     

Extended polyglutamine tracts cause aggregation and structural perturbation of an adjacent beta barrel protein

Formation of fibrillar intranuclear inclusions and related neuropathologies of the CAG-repeat disorders are linked to the expansion of a polyglutamine tract. Despite considerable effort, the etiology of these devastating diseases remains unclear. Although polypeptides with glutamine tracts recapitulate many of the observed characteristics of the gene products with CAG repeats, such as in vitro and in vivo aggregation and toxicity in model organisms, extended polyglutamine segments have also been reported to structurally perturb proteins into which they are inserted. Additionally, the sequence context of a polyglutamine tract has recently been shown to modulate its propensity to aggregate. These findings raise the possibility that indirect influences of the repeat tract on adjacent protein domains are contributory to pathologies. Destabilization of an adjacent domain may lead to loss of function, as well as favoring non-native structures in the neighboring domain causing them to be prone to intermolecular association and consequent aggregation. To explore these phenomena, we have used chimeras of a well studied globular protein and exon 1 of huntingtin. We find that expansion of the polyglutamine segment beyond the pathological threshold (>35 glutamines) results in structural perturbation of the neighboring protein whether the huntingtin exon is N- or C-terminal. Elongation of the polyglutamine region also substantially increases the propensity of the chimera to aggregate, both in vitro and in vivo, and in vitro aggregation kinetics of a chimera with a 53-glutamine repeat follow a nucleation polymerization mechanism with a monomeric nucleus.     

Recruitment and the Role of Nuclear Localization in Polyglutamine-mediated Aggregation

The inherited neurodegenerative diseases caused by an expanded glutamine repeat share the pathologic feature of intranuclear aggregates or inclusions (NI). Here in cell-based studies of the spinocerebellar ataxia type-3 disease protein, ataxin-3, we address two issues central to aggregation: the role of polyglutamine in recruiting proteins into NI and the role of nuclear localization in promoting aggregation. We demonstrate that full-length ataxin-3 is readily recruited from the cytoplasm into NI seeded either by a pathologic ataxin-3 fragment or by a second unrelated glutamine-repeat disease protein, ataxin-1. Experiments with green fluorescence protein/polyglutamine fusion proteins show that a glutamine repeat is sufficient to recruit an otherwise irrelevant protein into NI, and studies of human disease tissue and a Drosophila transgenic model provide evidence that specific glutamine-repeat–containing proteins, including TATA-binding protein and Eyes Absent protein, are recruited into NI in vivo. Finally, we show that nuclear localization promotes aggregation: an ataxin-3 fragment containing a nonpathologic repeat of 27 glutamines forms inclusions only when targeted to the nucleus. Our findings establish the importance of the polyglutamine domain in mediating recruitment and suggest that pathogenesis may be linked in part to the sequestering of glutamine-containing cellular proteins. In addition, we demonstrate that the nuclear environment may be critical for seeding polyglutamine aggregates.     

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.     

Structural Characterization of Polyglutamine Fibrils by Solid-State NMR Spectroscopy

Protein aggregation via polyglutamine stretches occurs in a number of severe neurodegenerative diseases such as Huntington's disease. We have investigated fibrillar aggregates of polyglutamine peptides below, at, and above the toxicity limit of around 37 glutamine residues using solid-state NMR and electron microscopy. Experimental data are consistent with a dry fibril core of at least 70-80 Å in width for all constructs. Solid-state NMR dipolar correlation experiments reveal a largely β-strand character of all samples and point to tight interdigitation of hydrogen-bonded glutamine side chains from different sheets. Two approximately equally frequent populations of glutamine residues with distinct sets of chemical shifts are found, consistent with local backbone dihedral angles compensating for β-strand twist or with two distinct sets of side-chain conformations. Peptides comprising 15 glutamine residues are present as single extended β-strands. Data obtained for longer constructs are most compatible with a superpleated arrangement with individual molecules contributing β-strands to more than one sheet and an antiparallel assembly of strands within β-sheets.     

Deconvolution of the circular dichroism spectra of proteins: the circular dichroism spectra of the antiparallel beta-sheet in proteins.

A recently developed algorithm, called Convex Constraint Analysis (CCA), was successfully applied to determine the circular dichroism (CD) spectra of the pure beta-pleated sheet in globular proteins. On the basis of X-ray diffraction determined secondary structures, the original data set used (Perczel, A., Hollosi, M., Tusnady, G. Fasman, G.D. Convex constraint analysis: A natural deconvolution of circular dichroism curves of proteins, Prot. Eng., 4:669-679, 1991), was improved by the addition of proteins with high beta-pleated sheet content. The analysis yielded CD curves of the pure components of the main secondary structural elements (alpha-helix, antiparallel beta-pleated sheet, beta-turns, and unordered conformation), as well as a curve attributed to the "aromatic contribution" in the wavelength range of 195-240 nm. Upon deconvolution the curves obtained were assigned to various secondary structures. The calculated weights (percentages determining the contributions of each pure component curve in the measured CD spectra of a given protein) were correlated with the X-ray diffraction determined percentages in an assignment procedure and were evaluated. The Pearson product correlation coefficients (R) are significant for all five components. The new pure component curves, which were obtained through deconvolution of the protein CD spectra alone, are promising candidates for determining the percentages of the secondary structural components in globular proteins without the necessity of adopting an X-ray database. The CD spectrum of the CheY protein was interesting because it has the characteristic shape associated with the alpha-helical structure, but upon analysis yielded a considerable amount of beta-sheet in agreement with the X-ray structure.     

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.     

Polyglutamine toxicity is controlled by prion composition and gene dosage in yeast

Polyglutamine expansion causes diseases in humans and other mammals. One example is Huntington's disease. Fragments of human huntingtin protein having an expanded polyglutamine stretch form aggregates and cause cytotoxicity in yeast cells bearing endogenous QN-rich proteins in the aggregated (prion) form. Attachment of the proline(P)-rich region targets polyglutamines to the large perinuclear deposit (aggresome). Aggresome formation ameliorates polyglutamine cytotoxicity in cells containing only the prion form of Rnq1 protein. Here we show that expanded polyglutamines both with (poly-QP) or without (poly-Q) a P-rich stretch remain toxic in the presence of the prion form of translation termination (release) factor Sup35 (eRF3). A Sup35 derivative that lacks the QN-rich domain and is unable to be incorporated into aggregates counteracts cytotoxicity, suggesting that toxicity is due to Sup35 sequestration. Increase in the levels of another release factor, Sup45 (eRF1), due to either disomy by chromosome II containing the SUP45 gene or to introduction of the SUP45-bearing plasmid counteracts poly-Q or poly-QP toxicity in the presence of the Sup35 prion. Protein analysis confirms that polyglutamines alter aggregation patterns of Sup35 and promote aggregation of Sup45, while excess Sup45 counteracts these effects. Our data show that one and the same mode of polyglutamine aggregation could be cytoprotective or cytotoxic, depending on the composition of other aggregates in a eukaryotic cell, and demonstrate that other aggregates expand the range of proteins that are susceptible to sequestration by polyglutamines.     

Dissection and design of yeast prions

Many proteins can misfold into β-sheet-rich, self-seeding polymers (amyloids). Prions are exceptional among such aggregates in that they are also infectious. In fungi, prions are not pathogenic but rather act as epigenetic regulators of cell physiology, providing a powerful model for studying the mechanism of prion replication. We used prion-forming domains from two budding yeast proteins (Sup35p and New1p) to examine the requirements for prion formation and inheritance. In both proteins, a glutamine/asparagine-rich (Q/N-rich) tract mediates sequence-specific aggregation, while an adjacent motif, the oligopeptide repeat, is required for the replication and stable inheritance of these aggregates. Our findings help to explain why although Q/N-rich proteins are relatively common, few form heritable aggregates: prion inheritance requires both an aggregation sequence responsible for self-seeded growth and an element that permits chaperone-dependent replication of the aggregate. Using this knowledge, we have designed novel artificial prions by fusing the replication element of Sup35p to aggregation-prone sequences from other proteins, including pathogenically expanded polyglutamine.     

Polyglutamine pathogenesis.

An increasing number of neurodegenerative disorders have been found to be caused by expanding CAG triplet repeats that code for polyglutamine. Huntington's disease (HD) is the most common of these disorders and dentatorubral-pallidoluysian atrophy (DRPLA) is very similar to HD, but is caused by mutation in a different gene, making them good models to study. In this review, we will concentrate on the roles of protein aggregation, nuclear localization and proteolytic processing in disease pathogenesis. In cell model studies of HD, we have found that truncated N-terminal portions of huntingtin (the HD gene product) with expanded repeats form more aggregates than longer or full length huntingtin polypeptides. These shorter fragments are also more prone to aggregate in the nucleus and cause more cell toxicity. Further experiments with huntingtin constructs harbouring exogenous nuclear import and nuclear export signals have implicated the nucleus in direct cell toxicity. We have made mouse models of HD and DRPLA using an N-terminal truncation of huntingtin (N171) and full-length atrophin-1 (the DRPLA gene product), respectively. In both models, diffuse neuronal nuclear staining and nuclear inclusion bodies are observed in animals expressing the expanded glutamine repeat protein, further implicating the nucleus as a primary site of neuronal dysfunction. Neuritic pathology is also observed in the HD mice. In the DRPLA mouse model, we have found that truncated fragments of atrophin-1 containing the glutamine repeat accumulate in the nucleus, suggesting that proteolysis may be critical for disease progression. Taken together, these data lead towards a model whereby proteolytic processing, nuclear localization and protein aggregation all contribute to pathogenesis.     

Unique features of the structural model of 'hard' cuticle proteins: implications for chitin-protein interactions and cross-linking in cuticle

Cuticular proteins are one of the determinants of the physical properties of cuticle. A common consensus region (extended R&R Consensus) in these proteins binds to chitin, the other major component of cuticle. We previously predicted the preponderance of beta-pleated sheet in the consensus region and proposed its responsibility for the formation of helicoidal cuticle (Iconomidou et al., Insect Biochem. Mol. Biol. 29 (1999) 285). Subsequently, we verified experimentally the abundance of antiparallel beta-pleated sheet in the structure of cuticle proteins (Iconomidou et al., Insect Biochem. Mol. Biol. 31 (2001) 877). Homology modelling of soft (RR-1) cuticular proteins using bovine plasma retinol binding protein (RBP) as a template revealed an antiparallel beta-sheet half-barrel structure as the basic folding motif (Hamodrakas et al., Insect Biochem. Molec. Biol. 32 (2002) 1577). The RR-2 proteins characteristic of hard cuticle, have a far more conserved consensus and frequently more histidine residues. Extension of modelling to this class of consensus, in this work, reveals in detail several unique features of the proposed structural model to serve as a chitin binding structural motif, thus providing the basis for elucidating cuticle's overall architecture and chitin-protein interactions in cuticle.     

Effects of chain length on the aggregation of model polyglutamine peptides: molecular dynamics simulations

Aggregation in the brain of polyglutamine-containing proteins is either a cause or an associated symptom of nine hereditary neurodegenerative disorders including Huntington's disease. The molecular level mechanisms by which these proteins aggregate are still unclear. In an effort to shed light on this important phenomenon, we are investigating the aggregation of model polyglutamine peptides using molecular-level computer simulation with a simplified model of polyglutamine that we have developed. This model accounts for the most important types of intra- and inter-molecular interactions-hydrogen bonding and hydrophobic interactions-while allowing the folding process to be simulated in a reasonable time frame. The model is used to examine the folding of isolated polyglutamine peptides 16, 32, and 48 residues long and the folding and aggregation of systems of 24 model polyglutamine peptides 16, 24, 32, 36, 40, and 48 residues long. Although the isolated polyglutamine peptides did form some alpha and beta backbone-backbone hydrogen bonds they did not have as many of these bonds as they would have if they had folded into a complete alpha helix or beta sheet. In one of the simulations on the isolated polyglutamine peptide 48 residues long, we observed a structure that resembles a beta helix. In the multi-chain simulations we observed amorphous aggregates at low temperatures, ordered aggregates with significant beta sheet character at intermediate temperatures, and random coils at high temperatures. We have found that the temperature at which the model peptides undergo the transition from amorphous aggregates to ordered aggregates and the temperature at which the model peptides undergo the transition from ordered aggregates to random coils increase with increasing chain length. Our finding that the stability of the ordered aggregates increases as the peptide chain length increases may help to explain the experimentally observed relation between polyglutamine tract length and aggregation in vitro and disease progression in vivo. We have also observed in our simulations that the optimal temperature for the formation of beta sheets increases with chain length up to 36 glutamine residues but not beyond. Equivalently, at fixed temperature we find a transition from a region dominated by random coils at chain lengths less than 36 to a region dominated by relatively ordered beta sheet structures at chain lengths greater than 36. Our finding of this critical chain length of 36 glutamine residues is interesting because a critical chain length of 37 glutamine residues has been observed experimentally.     

The effects of aggregation-inducing motifs on amyloid formation of model proteins related to neurodegenerative diseases

To examine the effects of aggregation-inducing motifs related to neurodegenerative diseases on amyloid formation of host protein, we prepared several chimera myoglobins, in which various aggregation-inducing motifs were inserted. The focused aggregation-inducing motifs included five (R5) or two (R2) oligopeptide repeats in yeast Sup35p, five octapeptide repeats (OPR) in the human prion protein, a nonamyloid beta component (NAC) in alpha-synuclein, and tandem repeats of 50 glutamines (Q50). Circular dichroism and infrared spectroscopies suggested that the OPR, R5, and Q50 motifs formed an antiparallel beta sheet as well as a random coil, whereas the R2 and NAC motifs mainly formed random coils. The OPR, R5, and Q50 mutants, but not the R2 and NAC mutants, readily formed the SDS-resistant aggregates under physiological condition, and electron microscopy revealed that the aggregates contained amyloid fibrils. The destabilization and increase in gyration radius of the OPR, R5, and Q50 mutants correlated with the tendency to form amyloid fibrils. A control mutant bearing a nonamyloidgenic sequence was also moderately destabilized but did not form amyloid fibrils. Therefore, we concluded that the OPR, R5, and Q50 motifs, even in a quite stable protein such as myoglobin, led the host protein to formation of amyloid fibrils under physiological condition.     

Modulation of prion-dependent polyglutamine aggregation and toxicity by chaperone proteins in the yeast model

In yeast, aggregation and toxicity of the expanded polyglutamine fragment of human huntingtin strictly depend on the presence of the endogenous self-perpetuating aggregated proteins (prions), which contain glutamine/asparagine-rich domains. Some chaperones of the Hsp100/70/40 complex, modulating propagation of yeast prions, were also reported to influence polyglutamine aggregation in yeast, but it was not clear whether they do it directly or via affecting prions. Our data show that although some chaperone alterations indeed act on polyglutamines via curing endogenous prions, other alterations decrease size and ameliorate toxicity of polyglutamine aggregates without affecting prion propagation. Therefore, the role of yeast chaperones in polyglutamine aggregation and toxicity is not restricted only to their effects on the endogenous prions. Moreover, chaperone interactions with prion and polyglutamine aggregates appear to be of a highly specific nature. One and the same chaperone alteration, substitution A503V in the middle region of the chaperone Hsp104, exhibited opposite effects on one of the endogenous prions ([PSI(+)], the prion form of Sup35) and on polyglutamines, increasing aggregate size and toxicity in the former case and decreasing them in the latter case. On the other hand, different members of a single chaperone family exhibited opposite effects on one and the same type of aggregates: excess of the Hsp40 chaperone Ydj1 increased polyglutamine aggregate size and toxicity, whereas excess of the other Hsp40 chaperone, Sis1, decreased them. As many stress-defense proteins are conserved between yeast and mammals, these data shed light on possible mechanisms modulating polyglutamine aggregation and toxicity in mammalian cells.     

A structural model of the chitin-binding domain of cuticle proteins

The nature of the interaction of insect cuticular proteins and chitin is unknown even though about half of the cuticular proteins sequenced thus far share a consensus region that has been predicted to be the site of chitin binding. We previously predicted the preponderance of beta-pleated sheet in the consensus region and proposed its responsibility for the formation of helicoidal cuticle (Iconomidou et al., Insect Biochem. Mol. Biol. 29 (1999) 285). Consequently, we have also verified experimentally the abundance of antiparallel beta-pleated sheet in the structure of cuticle proteins (Iconomidou et al., Insect Biochem. Mol. Biol. 31 (2001) 877). In this work, based on sequence and secondary structure similarity of cuticle proteins, and especially that of the consensus motif, to that of bovine plasma retinol binding protein (RBP), we propose by homology modelling an antiparallel beta-sheet half-barrel structure as the basic folding motif of cuticle proteins. This folding motif may provide the template for elucidating cuticle protein-chitin interactions in detail and reveal the precise geometrical formation of cuticle's helicoidal architecture. This predicted motif is another example where nature utilizes an almost flat protein surface covered by aromatic side chains to interact with the polysaccharide chains of chitin.     

The Relationship between Aggregation and Toxicity of Polyglutamine-Containing Ataxin-3 in the Intracellular Environment of Escherichia coli

Several neurodegenerative diseases are triggered by proteins containing a polyglutamine (polyQ) stretch expanded beyond a critical threshold. Among these, ataxin-3 (AT3) is the causative agent of spinocerebellar ataxia type-3. We expressed three authentic AT3 variants in Escherichia coli: one normal (AT3-Q24), one expanded (AT3-Q55) and one truncated immediately upstream of the polyQ (AT3-291Δ). Then, based on growth rate reduction, we quantified protein toxicity. We show that AT3-Q55 and -291Δ strongly reduced the growth rate in the early stages (2–4 h), unlike AT3-Q24. This correlated well with the appearance of soluble cytosolic oligomers, but not with the amount of insoluble protein in inclusion bodies (IBs). The impact of AT3-291Δ on cell growth suggests an intrinsic toxicity of the AT3 fragment. Besides the typical Fourier Transform Infrared Spectroscopy (FTIR) signal for intermolecular β-sheets, the expanded form displayed an additional infrared signature, which was assigned to glutamine side-chain hydrogen bonding and associated with SDS-insoluble fibrils. The elongation of the latter was monitored by Atomic Force Microscopy (AFM). This mirrors the well-known in vitro two-step aggregation pattern of expanded AT3. We also demonstrated that final aggregates of strains expressing expanded or truncated AT3 play a protective role against toxicity. Furthermore, our findings suggest that the mechanisms of toxicity are evolutionarily conserved.