Peptide models for inherited neurodegenerative disorders: conformation and aggregation properties of long polyglutamine peptides with and without interruptions.

Several neurodegenerative diseases are caused by expansion of polyglutamine repeats in the affected proteins. In spino-cerebellar ataxia type 1 (SCA1), histidine interruptions have been reported to mitigate the pathological effects of long glutamine stretches. To understand this phenomenon, we investigated the conformational preferences of peptides containing both the uninterrupted polyglutamine stretches and those with histidine interruption(s) as seen in SCA1 normals. Our study suggests that substitution of histidines by glutamines induces a conformational change which results in decreased solubility and increased aggregation. Our findings also suggest that all the polyglutamine peptides with and without interruption(s) adopt a beta-structure and not random coil.     

Appearance and propagation of polyglutamine-based amyloids in yeast: tyrosine residues enable polymer fragmentation

In yeast, fragmentation of amyloid polymers by the Hsp104 chaperone allows them to propagate as prions. The prion-forming domain of the yeast Sup35 protein is rich in glutamine, asparagine, tyrosine, and glycine residues, which may define its prion properties. Long polyglutamine stretches can also drive amyloid polymerization in yeast, but these polymers are unable to propagate because of poor fragmentation and exist through constant seeding with the Rnq1 prion polymers. We proposed that fragmentation of polyglutamine amyloids may be improved by incorporation of hydrophobic amino acid residues into polyglutamine stretches. To investigate this, we constructed sets of polyglutamine with or without tyrosine stretches fused to the non-prion domains of Sup35. Polymerization of these chimeras started rapidly, and its efficiency increased with stretch size. Polymerization of proteins with polyglutamine stretches shorter than 70 residues required Rnq1 prion seeds. Proteins with longer stretches polymerized independently of Rnq1 and thus could propagate. The presence of tyrosines within polyglutamine stretches dramatically enhanced polymer fragmentation and allowed polymer propagation in the absence of Rnq1 and, in some cases, of Hsp104.     

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.     

Screening of sugar converting enzymes using quantitative MALDI-ToF mass spectrometry

Quantitative matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-ToF MS) was applied for the screening of ten pyranose oxidase variants. Quantitative MALDI-ToF MS using isotopic labeled internal standards and ionic liquid matrices was performed using aliquots of enzyme reaction mixtures without prepurification steps. The results obtained were in good agreement with HPLC measurements. Analysis time was approx. 3.5 min for a five-fold determination. Thus, quantitative MALDI-ToF MS can be used as a tool for screening of sugar converting enzymes.     

Puromycin-sensitive aminopeptidase is the major peptidase responsible for digesting polyglutamine sequences released by proteasomes during protein degradation

Long stretches of glutamine (Q) residues are found in many cellular proteins. Expansion of these polyglutamine (polyQ) sequences is the underlying cause of several neurodegenerative diseases (e.g. Huntington's disease). Eukaryotic proteasomes have been found to digest polyQ sequences in proteins very slowly, or not at all, and to release such potentially toxic sequences for degradation by other peptidases. To identify these key peptidases, we investigated the degradation in cell extracts of model Q-rich fluorescent substrates and peptides containing 10-30 Q's. Their degradation at neutral pH was due to a single aminopeptidase, the puromycin-sensitive aminopeptidase (PSA, cytosol alanyl aminopeptidase). No other known cytosolic aminopeptidase or endopeptidase was found to digest these polyQ peptides. Although tripeptidyl peptidase II (TPPII) exhibited limited activity, studies with specific inhibitors, pure enzymes and extracts of cells treated with siRNA for TPPII or PSA showed PSA to be the rate-limiting activity against polyQ peptides up to 30 residues long. (PSA digests such Q sequences, shorter ones and typical (non-repeating) peptides at similar rates.) Thus, PSA, which is induced in neurons expressing mutant huntingtin, appears critical in preventing the accumulation of polyQ peptides in normal cells, and its activity may influence susceptibility to polyQ diseases.     

A structural model of polyglutamine determined from a host-guest method combining experiments and landscape theory

Modeling the structure of natively disordered peptides has proved difficult due to the lack of structural information on these peptides. In this work, we use a novel application of the host-guest method, combining folding theory with experiments, to model the structure of natively disordered polyglutamine peptides. Initially, a minimalist molecular model (C(alpha)C(beta)) of CI2 is developed with a structurally based potential and captures many of the folding properties of CI2 determined from experiments. Next, polyglutamine "guest" inserts of increasing length are introduced into the CI2 "host" model and the polyglutamine is modeled to match the resultant change in CI2 thermodynamic stability between simulations and experiments. The polyglutamine model that best mimics the experimental changes in CI2 thermodynamic stability has 1), a beta-strand dihedral preference and 2), an attractive energy between polyglutamine atoms 0.75-times the attractive energy between the CI2 host Go-contacts. When free-energy differences in the CI2 host-guest system are correctly modeled at varying lengths of polyglutamine guest inserts, the kinetic folding rates and structural perturbation of these CI2 insert mutants are also correctly captured in simulations without any additional parameter adjustment. In agreement with experiments, the residues showing structural perturbation are located in the immediate vicinity of the loop insert. The simulated polyglutamine loop insert predominantly adopts extended random coil conformations, a structural model consistent with low resolution experimental methods. The agreement between simulation and experimental CI2 folding rates, CI2 structural perturbation, and polyglutamine insert structure show that this host-guest method can select a physically realistic model for inserted polyglutamine. If other amyloid peptides can be inserted into stable protein hosts and the stabilities of these host-guest mutants determined, this novel host-guest method may prove useful to determine structural preferences of these intractable but biologically relevant protein fragments.     

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.     

Characterizing the conformational ensemble of monomeric polyglutamine

Studies of synthetic polyglutamine peptides in vitro have established that polyglutamine peptides aggregate via a classic nucleation and growth mechanism. Chen and colleagues [Proc Natl Acad Sci U S A 2002;99:11884-11889] have found that monomeric polyglutamine, which is a disordered statistical coil in solution, is the critical nucleus for aggregation. Therefore, nucleation of beta-sheet-rich aggregates requires an initial disorder to order transition, which is a highly unfavorable thermodynamic reaction. The questions of interest to us are as follows: What are the statistical fluctuations that drive beta-sheet formation in monomeric polyglutamine? How do these fluctuations vary with chain length? And why is this process thermodynamically unfavorable, that is, why is monomeric polyglutamine disordered? To answer these questions we use multiple molecular dynamics simulations to provide quantitative characterization of conformational ensembles for two short polyglutamine peptides. We find that the ensemble for polyglutamine is indeed disordered. However, the disorder is inherently different from that of denatured proteins and the average compactness and magnitude of conformational fluctuations increase with chain length. Most importantly, the effective concentration of sidechain primary amides around backbone units is inherently high and peptide units are solvated either by hydrogen bonds to sidechains or surrounding water molecules. Due to the multiplicity of backbone solvation modes the probability associated with any specific backbone conformation is small, resulting in a conformational entropy bottleneck which makes beta-sheet formation in monomeric polyglutamine thermodynamically unfavorable.     

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.     

The Effects of Amino Acid Composition of Glutamine-Rich Domains on Amyloid Formation and Fragmentation

Fragmentation of amyloid polymers by the chaperone Hsp104 allows them to propagate as prions in yeast. The factors which determine the frequency of fragmentation are unclear, though it is often presumed to depend on the physical strength of prion polymers. Proteins with long polyglutamine stretches represent a tractable model for revealing sequence elements required for polymer fragmentation in yeast, since they form poorly fragmented amyloids. Here we show that interspersion of polyglutamine stretches with various amino acid residues differentially affects the in vivo formation and fragmentation of the respective amyloids. Aromatic residues tyrosine, tryptophan and phenylalanine strongly stimulated polymer fragmentation, leading to the appearance of oligomers as small as dimers. Alanine, methionine, cysteine, serine, threonine and histidine also enhanced fragmentation, while charged residues, proline, glycine and leucine inhibited polymerization. Our data indicate that fragmentation frequency primarily depends on the recognition of fragmentation-promoting residues by Hsp104 and/or its co-chaperones, rather than on the physical stability of polymers. This suggests that differential exposure of such residues to chaperones defines prion variant-specific differences in polymer fragmentation efficiency.     

Proteolysis in miniature cheddar-type cheeses manufactured using extracts from the crustacean Munida as coagulant

Miniature (20 g) Cheddar-type cheeses were manufactured using enzymes extracted from the crustacean Munida or chymosin as coagulant. Cheeses were ripened at 8 degrees C and samples were collected for analysis after 2, 6 and 12 weeks. Proteolysis was assessed by urea-polyacrylamide gel electrophoresis, which showed that cheeses manufactured with the Munida extracts had a higher extent of degradation of beta-casein than cheeses made using chymosin as coagulant. Patterns of proteolysis were also obtained by reverse-phase high-performance liquid chromatography (RP-HPLC) and matrix assisted laser desorption ionisation-time of flight (MALDI-ToF) mass spectrometry. In general, the products of proteolysis were more complex in cheese made using the Munida extracts than in cheese made by chymosin as coagulant. Statistical analysis of results clearly discriminated the cheeses on the basis of coagulant used. Molecular mass of peptides found in cheese made using Munida extracts were similar to those of peptides commonly detected in cheeses made using chymosin as coagulant.     

Solubilization and disaggregation of polyglutamine peptides

A method is described for dissolving and disaggregating chemically synthesized polyglutamine peptides. Polyglutamine peptides longer than about Q20 have been reported to be insoluble in water, but dissolution in--and evaporation from--a mixture of trifluoroacetic acid and hexafluoroisopropanol converts polyglutamine peptides up to at least Q44 to a form readily soluble in aqueous buffers. This procedure also has a dramatic effect on peptides which appear to be completely soluble in water, by removing traces of aggregate that seed aggregation. The protocol makes possible solution studies-including in vitro aggregation experiments--on polyglutamine peptides with repeat lengths associated with increased risk of Huntington's Disease and other expanded CAG repeat diseases. It may also be useful in conducting reproducible, quantitative aggregation studies on other polypeptides.     

Combination of a SAW-biosensor with MALDI mass spectrometric analysis

A S-sens K5 surface acoustic wave biosensor was coupled with mass spectrometry (SAW-MS) for the analysis of a protein complex consisting of human blood clotting cascade factor alpha-thrombin and human antithrombin III, a specific blood plasma inhibitor of thrombin. Specific binding of antithrombin III to thrombin was recorded as a function of time with a S-sens K5 biosensor. Two out of five elements of the sensor chip were used as references. To the remaining three elements coated with RNA anti-thrombin aptamers, thrombin and antithrombin III were bound consecutively. The biosensor measures mass changes on the chip surface showing that 20% of about 400fmol/cm2 thrombin formed a complex with the 1.7-times larger antithrombin III. Mass spectrometry (MS) was applied to identify the bound proteins. Sensor chips with aptamer-captured (1) thrombin and (2) thrombin-antithrombin III complex (TAT-complex) were digested with proteases on the sensor element and subsequently identified by peptide mass fingerprint (PMF) with matrix assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry. A significant identification of thrombin was achieved by measuring the entire digest with MALDI-ToF MS directly from the sensor chip surface. For the significant identification of both proteins in the TAT-complex, the proteolytic peptides had to be separated by nano-capillary-HPLC prior to MALDI-ToF MS. SAW-MS is applicable to protein interaction analysis as in functional proteomics and to miniaturized diagnostics.     

Reduced Levels of Proteasome Products in a Mouse Striatal Cell Model of Huntington’s Disease

Huntington’s disease is the result of a long polyglutamine tract in the gene encoding huntingtin protein, which in turn causes a large number of cellular changes and ultimately results in neurodegeneration of striatal neurons. Although many theories have been proposed, the precise mechanism by which the polyglutamine expansion causes cellular changes is not certain. Some evidence supports the hypothesis that the long polyglutamine tract inhibits the proteasome, a multiprotein complex involved in protein degradation. However, other studies report normal proteasome function in cells expressing long polyglutamine tracts. The controversy may be due to the methods used to examine proteasome activity in each of the previous studies. In the present study, we measured proteasome function by examining levels of endogenous peptides that are products of proteasome cleavage. Peptide levels were compared among mouse striatal cell lines expressing either 7 glutamines (STHdh ^Q7/Q7) or 111 glutamines in the huntingtin protein, either heterozygous (STHdh ^Q7/Q111) or homozygous (STHdh ^Q111/Q111). Both of the cell lines expressing huntingtin with 111 glutamines showed a large reduction in nearly all of the peptides detected in the cells, relative to levels of these peptides in cells homozygous for 7 glutamines. Treatment of STHdh ^Q7/Q7 cells with proteasome inhibitors epoxomicin or bortezomib also caused a large reduction in most of these peptides, suggesting that they are products of proteasome-mediated cleavage of cellular proteins. Taken together, these results support the hypothesis that proteasome function is impaired by the expression of huntingtin protein containing long polyglutamine tracts.     

Ubiquitin conjugating enzymes participate in polyglutamine protein aggregation

Background  

Protein aggregation is a hallmark of several neurodegenerative diseases including Huntington's disease and Parkinson's disease. Proteins containing long, homopolymeric stretches of glutamine are especially prone to form aggregates. It has long been known that the small protein modifier, ubiquitin, localizes to these aggregates. In this report, nematode and cell culture models for polyglutamine aggregation are used to investigate the role of the ubiquitin pathway in protein aggregation.     

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.     

Quantitative matrix-assisted laser desorption/ionization mass spectrometry for the determination of enzyme activities

Quantitative matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF) mass spectrometry (MS) was applied for the determination of concentrations of low-molecular-weight (< 400Da) substrates and products of enzyme-catalyzed reactions. Isotope-labeled and fluorinated internal standards were used for the quantification. Automated quantitative MALDI-ToF MS analysis of quenched samples allowed the direct and simultaneous observation of time-dependent decrease of substrate concentration and increase of product concentration without any need for prepurification or desalting steps. The results showed good agreement with established but more elaborate analytical methods. MALDI-ToF MS thus is an interesting alternative tool for the determination of enzyme activities. Due to automated and miniaturized measurement it is especially suitable for the screening of biocatalysts.     

Study of solid-phase synthesis and purification strategies for the preparation of polyglutamine peptides

Many neurodegenerative diseases are related to an abnormal expansion of the CAG trinucleotide that produces polyglutamine segments in several proteins. However, the pathogenesis of these neurodegenerative states is not yet well understood. Thus, to evaluate the molecular mechanisms leading to those diseases, suitable research tools such as synthetic polyglutamine peptides are required. The synthesis and purification of such peptides are usually difficult because of poor solubility, which leads to low coupling and/or deblocking reactivity. After exploring many synthesis, solubilization and purification approaches, a protocol allowing the production of polyglutamines in good yield and high purity was developed. With this protocol, peptides of 10-30 glutamine residues were synthesized using a linear solid-phase strategy combined with a maximal side-chain protection scheme using fluorenylmethyloxycarbonyl (Fmoc) chemistry. After cleavage of the peptide from the polymeric support, the crude material was treated with glacial acetic acid and lyophilized. This treatment significantly improved the solubility of the polyglutamine peptides thus allowing their dissolution in aqueous conditions and purification through reverse-phase high performance liquid chromatography. These solubilization and purification conditions led to the formation of N-pyroglutamyl peptide derivatives that were easily isolated. These N-pyroglutamylated compounds also appear as useful research tools because data from the literature suggest that N-terminal modification of polyglutamine segments might play a role in their pathogenic properties.     

Stable Polyglutamine Dimers Can Contain β-Hairpins with Interdigitated Side Chains—But Not α-Helices, β-Nanotubes, β-Pseudohelices,or Steric Zippers

A common thread connecting nine fatal neurodegenerative protein aggregation diseases is an abnormally expanded polyglutamine tract found in the respective proteins. Although the structure of this tract in the large mature aggregates is increasingly well described, its structure in the small early aggregates remains largely unknown. As experimental evidence suggests that the most toxic species along the aggregation pathway are the small early ones, developing strategies to alleviate disease pathology calls for understanding the structure of polyglutamine peptides in the early stages of aggregation. Here, we present a criterion, grounded in available experimental data, that allows for using kinetic stability of dimers to assess whether a given polyglutamine conformer can be on the aggregation path. We then demonstrate that this criterion can be assessed using present-day molecular dynamics simulations. We find that although the α-helical conformer of polyglutamine is very stable, dimers of α-helices lack the kinetic stability necessary to support further oligomerization. Dimers of steric zipper, β-nanotube, and β-pseudohelix conformers are also too short-lived to initiate aggregation. The β-hairpin-containing conformers, instead, invariably form very stable dimers when their side chains are interdigitated. Combining these findings with the implications of recent solid-state NMR data on mature fibrils, we propose a possible pathway for the initial stages of polyglutamine aggregation, in which β-hairpin-containing conformers act as templates for fibril formation.