Purification of neuronal inclusions of patients with Huntington's disease reveals a broad range of N-terminal fragments of expanded huntingtin and insoluble polymers

Huntington's disease resulting from huntingtin containing an expanded polyglutamine is associated with aggregates largely confined to neuronal inclusions, and with neuronal death. Inclusions are thought to originate from discrete N-terminal fragments of expanded huntingtin produced by specific endopeptidases. We have now purified the neuronal inclusions of Huntington's disease brain. When incubated in concentrated formic acid, purified inclusions release a polymer, an oligomer and a broad range of N-terminal fragments of expanded huntingtin. The fragments and the polymeric forms are linked to each other by non-covalent bonds as they are both released by formic acid, whereas the polymeric forms themselves are presumably stabilized by covalent bonds, as they are resistant to formic acid. We also demonstrate the presence in affected areas of the brain but not in unaffected areas of a broad range of soluble N-terminal fragments of expanded huntingtin not yet associated with the inclusions and which are likely to be the precursors of the inclusions. Fragmentation of expanded huntingtin in Huntington's disease must result from the operation of multiple proteolytic activities with little specificity and not from that of a specific endopeptidase; subsequent aggregation of the fragments by covalent and non-covalent bonds leads to the formation of the inclusions.     

Does tissue transglutaminase play a role in Huntington's disease?

Tissue transglutaminase (tTG) likely plays a role in numerous processes in the nervous system. tTG posttranslationally modifies proteins by transamidation of specific polypeptide bound glutamines (Glns). This reaction results in the incorporation of polyamines into substrate proteins or the formation of protein crosslinks, modifications that likely have significant effects on neural function. Huntington's disease is a genetic disorder caused by an expansion of the polyglutamine domain in the huntingtin protein. Because a polypeptide bound Gln is the determining factor for a tTG substrate, and mutant huntingtin aggregates have been found in Huntington's disease brain, it has been hypothesized that tTG may contribute to the pathogenesis of Huntington's disease. In vitro, polyglutamine constructs and huntingtin are substrates of tTG. Further, the levels of tTG and TG activity are elevated in Huntington's disease brain and immunohistochemical studies have demonstrated that there is an increase in tTG reactivity in affected neurons in Huntington's disease. These findings suggest that tTG may play a role in Huntington's disease. However in situ, neither wild type nor mutant huntingtin is modified by tTG. Further, immunocytochemical analysis revealed that tTG is totally excluded from the huntingtin aggregates, and modulation of the expression level of tTG had no effect on the frequency of the aggregates in the cells. Therefore, tTG is not required for the formation of huntingtin aggregates, and likely does not play a role in this process in Huntington's disease brain. However, tTG interacts with truncated huntingtin, and selectively polyaminates proteins that are associated with mutant truncated huntingtin. Given the fact that the levels of polyamines in cells is in the millimolar range and the crosslinking and polyaminating reactions catalyzed by tTG are competing reactions, intracellularly polyamination is likely to be the predominant reaction. Polyamination of proteins is likely to effect their function, and therefore it can be hypothesized that tTG may play a role in the pathogenesis of Huntington's disease by modifying specific proteins and altering their function and/or localization. Further research is required to define the specific role of tTG in Huntington's disease.     

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.     

Huntingtin aggregation kinetics and their pathological role in a Drosophila Huntington's disease model

Huntington's disease is a neurodegenerative disorder resulting from expansion of a polyglutamine tract in the Huntingtin protein. Mutant Huntingtin forms intracellular aggregates within neurons, although it is unclear whether aggregates or more soluble forms of the protein represent the pathogenic species. To examine the link between aggregation and neurodegeneration, we generated Drosophila melanogaster transgenic strains expressing fluorescently tagged human huntingtin encoding pathogenic (Q138) or nonpathogenic (Q15) proteins, allowing in vivo imaging of Huntingtin expression and aggregation in live animals. Neuronal expression of pathogenic Huntingtin leads to pharate adult lethality, accompanied by formation of large aggregates within the cytoplasm of neuronal cell bodies and neurites. Live imaging and Fluorescence Recovery After Photobleaching (FRAP) analysis of pathogenic Huntingtin demonstrated that new aggregates can form in neurons within 12 hr, while preexisting aggregates rapidly accumulate new Huntingtin protein within minutes. To examine the role of aggregates in pathology, we conducted haplo-insufficiency suppressor screens for Huntingtin-Q138 aggregation or Huntingtin-Q138-induced lethality, using deficiencies covering ~80% of the Drosophila genome. We identified two classes of interacting suppressors in our screen: those that rescue viability while decreasing Huntingtin expression and aggregation and those that rescue viability without disrupting Huntingtin aggregation. The most robust suppressors reduced both soluble and aggregated Huntingtin levels, suggesting toxicity is likely to be associated with both forms of the mutant protein in Huntington's disease.     

Reduced transglutaminase-catalyzed protein aggregation is observed in the presence of creatine using sedimentation velocity

Transglutaminases (TGases) are enzymes that catalyze covalent isopeptide crosslinks between reactive lysine and glutamine residues in proteins. Higher than normal local concentrations of TGase have been correlated with increased protein aggregation in vivo. These insoluble protein aggregates are the hallmark of several neurodegenerative diseases, including Alzheimer's, Parkinson's, and Huntington's diseases, although each aggregating protein involved is disease specific. Because TGase is implicated in protein aggregation, there is evidence that its regulation may retard disease progression. Here we report on a laser light transmission technique as an in vitro tool to gauge the efficacy of creatine, a candidate inhibitor, to regulate aggregation. Sedimentation velocities of protein-coated particles in TGase-containing water-glycerol solutions were tracked with different levels of creatine. Sedimentation velocities were converted to apparent aggregate sizes using Stoke's law of sedimentation. The results indicated that creatine promoted up to a 20% reduction in protein aggregation in vitro. This technique may prove to be useful in identifying other functional TGase inhibitors.     

Intermolecular association and molecular weight of aggregates of a monodisperse homohexapeptide,Nps-[Glu(OBzl)]6-NHEt,in solution

Light scattering from the solutions of Nps-[Glu(OBzl)]₆-NHEt in dioxane or ethylene dichloride has been measured at different concentrations, and a critical concentration of intermolecular association is found to exist, which is equal to the critical concentration of β-form formation. The Debye plot of light scattering leads to the molecular weight of aggregates at the critical concentration, which corresponds to an aggregation number 15 in dioxane and 53 in ethylene dichloride. In the latter solvent the aggregates further associate into a larger aggregate consisting of 330 molecules when the concentration is increased beyond the critical concentration. The content of β-form, which is a measure of number of hydrogen bonds, is derived from the ir data previously obtained. The results on the modes of intermolecular association and hydrogen bonding lead to possible structures of aggregates formed by both hydrogen bonds and other nonbonding side-chain interactions.     

Protein aggregation in Huntington's and Parkinson's disease: implications for therapy

The accumulation of highly insoluble intracellular protein aggregates in neuronal inclusions is a hallmark of Huntington's disease (HD) and Parkinson's disease (PD) as well as several other late-onset neurodegenerative disorders. The aggregates formed in vitro and in vivo generally have a fibrillar morphology, consist of individual beta-strands and are resistant to proteolytic degradation. Although the causal relationship between aggregate formation and disease remains to be proven, the gradual deposition of mutant protein in neurons is consistent with the late-onset and progressive nature of symptoms. Recently, circumstantial evidence from mouse and Drosophila model systems suggests that abnormal protein folding and aggregation play a key role in the pathogenesis of both HD and PD. Therefore, a detailed understanding of the molecular mechanisms of protein aggregation and its effects on neuronal cell death could open new opportunities for therapy.     

Aggregation of some water-soluble derivatives of chitin in aqueous solutions: Role of the degree of acetylation and effect of hydrogen bond breaker

By dynamic light scattering in combination with fluorescence spectroscopy and TEM it was shown that aggregation in aqueous solutions is inherent not only to chitosan, but also to two other water-soluble derivatives of chitin: O-carboxymethylchitin and di-N,N-carboxymethylchitosan. Aggregation is observed even for the samples without N-acetyl-d-glucosamine units, which remain upon incomplete chemical modification of chitin, indicating that specific interactions between residual chitin repeat units cannot be the main reason for the aggregation. At the same time, 7 M urea weakens the aggregation, thus testifying that hydrogen bonding and/or hydrophobic interactions are partially responsible for this phenomenon. The incomplete disruption of aggregates in 7 M urea may arise from crystallization of junction zones between different macromolecules, which makes some hydrogen bonds inaccessible for urea or too stable for breaking by this agent.     

Strong Ionic Hydrogen Bonding Causes a Spectral Isotope Effect in Photoactive Yellow Protein

Standard hydrogen bonds are of great importance for protein structure and function. Ionic hydrogen bonds often are significantly stronger than standard hydrogen bonds and exhibit unique properties, but their role in proteins is not well understood. We report that hydrogen/deuterium exchange causes a redshift in the visible absorbance spectrum of photoactive yellow protein (PYP). We expand the range of interpretable isotope effects by assigning this spectral isotope effect (SIE) to a functionally important hydrogen bond at the active site of PYP. The inverted sign and extent of this SIE is explained by the ionic nature and strength of this hydrogen bond. These results show the relevance of ionic hydrogen bonding for protein active sites, and reveal that the inverted SIE is a novel, to our knowledge, tool to probe ionic hydrogen bonds. Our results support a classification of hydrogen bonds that distinguishes the properties of ionic hydrogen bonds from those of both standard and low barrier hydrogen bonds, and show how this classification helps resolve a recent debate regarding active site hydrogen bonding in PYP.     

Strong Ionic Hydrogen Bonding Causes a Spectral Isotope Effect in Photoactive Yellow Protein

Standard hydrogen bonds are of great importance for protein structure and function. Ionic hydrogen bonds often are significantly stronger than standard hydrogen bonds and exhibit unique properties, but their role in proteins is not well understood. We report that hydrogen/deuterium exchange causes a redshift in the visible absorbance spectrum of photoactive yellow protein (PYP). We expand the range of interpretable isotope effects by assigning this spectral isotope effect (SIE) to a functionally important hydrogen bond at the active site of PYP. The inverted sign and extent of this SIE is explained by the ionic nature and strength of this hydrogen bond. These results show the relevance of ionic hydrogen bonding for protein active sites, and reveal that the inverted SIE is a novel, to our knowledge, tool to probe ionic hydrogen bonds. Our results support a classification of hydrogen bonds that distinguishes the properties of ionic hydrogen bonds from those of both standard and low barrier hydrogen bonds, and show how this classification helps resolve a recent debate regarding active site hydrogen bonding in PYP.     

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.     

Disulphide bond formation in food protein aggregation and gelation

In this short review we discuss the role of cysteine residues and cystine bridges for the functional aggregation of food proteins. We evaluate how formation and cleavage of disulphide bonds proceeds at a molecular level, and how inter- and intramolecular disulfide bonds can be detected and modified. The differences between heat-, high-pressure-, and denaturant-induced unfolding and aggregation are discussed. The effect of disulphide bonding between aggregates of proteins and protein mixtures on the functional macroscopic properties of space filling networks in protein gels is briefly presented.     

Structural investigation of radiation-induced aggregates of ribonuclease

Following irradiation of bovine pancreatic ribonuclease in aqueous solution with 60Co gamma-rays protein aggregates are formed. The nature of the bonds linking these radiation-induced aggregates together has been investigated by chromatographic and electrophoretic methods. Thin-layer gel filtration and polyacrylamide gel electrophoresis, both in the presence of sodium dodecyl sulphate, demonstrated the existence of covalent crosslinks between the aggregates. However, non-covalent crosslinking also plays a role in the radiolysis of ribonuclease. Thin-layer gel filtration with and without 6 M urea and 2 per cent beta-mercaptoethanol added to the gel, revealed that only part of the covalent bonds between the aggregates consisted of disulphide linkages. By separation of the reduced aggregates by thin-layer gel filtration and electrophoresis, both with SDS, this finding was substantiated. Densitometric measurements indicated for example that the percentage of covalently linked dimers held together by disulphide bridges amounted to about 40-45 per cent, whereas the remaining 55-60 per cent of the dimers must be linked by other covalent bonds. The existence of covalent crosslinks other than disulphide bonds was also confirmed by isoelectric focusing. By this method definite differences were established between the proteolytic hydrolysates of the reduced aggregates and the reduced monomer of gamma-irradiated ribonuclease.     

Sensitive biochemical aggregate detection reveals aggregation onset before symptom development in cellular and murine models of Huntington's disease

A CAG-repeat gene expansion translated into a pathogenic polyglutamine stretch at the N-terminus of huntingtin triggers Huntington's Disease. Mutated huntingtin is predicted to adopt toxic properties mainly if aggregation-prone N-terminal fragments are released by proteolysis. Huntingtin-aggregates are indeed a major hallmark of this disorder and could represent useful markers of disease-onset or progression. We designed a simple method for qualitative and quantitative characterization of aggregates. For this, we analyzed samples from in vitro and in vivo Huntington's Disease models by agarose gel electrophoresis and showed that in the brain of transgenic mice huntingtin-aggregates became larger as a function of disease progression. This appears to be a property of cytoplasmic but not nuclear aggregates. In cell cultures, treatment with Congo Red inhibited aggregate growth but not total load. Finally, we showed that in primary striatal neurons and in brains of R6/2 and Hdh Q150 mice, the presence of aggregates preceded initiation of any other functional deficits. This observation argues for a pathogenic role of huntingtin-aggregation in Huntington's Disease. Our results emphasize that thorough analysis of huntingtin metabolism and aggregation is now feasible, thus significantly improving the power of studies assessing therapies designed to lower huntingtin levels or to interfere with its aggregation.     

Arfaptin 2 regulates the aggregation of mutant huntingtin protein

Huntington's disease (HD) is an inherited neurodegenerative disorder. Here we demonstrate that expression of arfaptin 2/POR1 (partner of Rac1) in cultured cells induces the formation of pericentriolar and nuclear aggregates, which morphologically resemble mutant huntingtin aggregates characteristic of HD. Endogenous arfaptin 2 localizes to aggregates induced by expression of an abnormal amino-terminal fragment of huntingtin that contains polyglutamine (polyQ) expansions. A dominant inhibitory mutant of arfaptin 2 inhibits aggregation of mutant huntingtin, but not in the presence of proteasome inhibitors. Using cell-free biochemical assays, we show that arfaptin 2 inhibits proteasome activity. Finally, we show that expression of arfaptin 2 is increased at sites of neurodegeneration and the protein localizes to huntingtin aggregates in HD transgenic mouse brains. Our data suggest that arfaptin 2 is involved in regulating huntingtin protein aggregation, possibly by impairing proteasome function.     

Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide

The distinct protein aggregates that are found in Alzheimer's, Parkinson's, Huntington's and prion diseases seem to cause these disorders. Small intermediates - soluble oligomers - in the aggregation process can confer synaptic dysfunction, whereas large, insoluble deposits might function as reservoirs of the bioactive oligomers. These emerging concepts are exemplified by Alzheimer's disease, in which amyloid beta-protein oligomers adversely affect synaptic structure and plasticity. Findings in other neurodegenerative diseases indicate that a broadly similar process of neuronal dysfunction is induced by diffusible oligomers of misfolded proteins.     

Quality control of the proteins associated with neurodegenerative diseases

Most neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, Huntington's disease and other polyglutamine diseases are associated with degeneration and death of specific neuronal populations due to misfolding or aggregation of certain proteins. These aggregates often contain ubiquitin that is the signal for proteolysis by the ubiquitin-proteasome system, and chaperone proteins that are involved in the assistance of protein folding. Here we review the role of protein quality control systems in the pathogenesis of neurodegenerative diseases, and aim to learn more from the cooperation between molecular chaperones and ubiquitin-proteasome system responding to cellular protein aggregates, in order to find molecular targets for therapeutic intervention.     

Probing the role of backbone hydrogen bonding in beta-amyloid fibrils with inhibitor peptides containing ester bonds at alternate positions

Protein-protein interactions are frequently mediated by stable, intermolecular beta-sheets. A number of cytokines and the HIV Protease, for example, dimerize through beta-sheet motifs. Evidence also suggests that the macromolecular assemblies of peptides and proteins in amyloid fibrils are stabilized by intermolecular beta-sheets. In this paper, we report that interfering with the backbone hydrogen bonding of an amyloidgenic peptide (Abeta16-20) by replacing amide bonds with ester bonds prevents the aggregation of the peptide. The ester bonds were incorporated in an alternating fashion so that the peptide presents two unique hydrogen bonding faces when arrayed in an extended, beta-strand conformation; one face of the peptide has normal hydrogen bonding capabilities, but the other face is missing amide protons and its ability to hydrogen bond is severely limited. Analytical ultracentrifugation experiments demonstrate that this ester peptide, Abeta16-20e, is predominantly monomeric under solution conditions, unlike the fibril-forming Abeta16-20 peptide. Abeta16-20e also inhibits the aggregation of the Abeta1-40 peptide and disassembles preformed Abeta1-40 fibrils. These results suggest that backbone hydrogen bonding is critical for the assembly of amyloid fibrils.