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.     

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.     

E6-AP promotes misfolded polyglutamine proteins for proteasomal degradation and suppresses polyglutamine protein aggregation and toxicity

The accumulation of intracellular protein deposits as inclusion bodies is the common pathological hallmark of most age-related neurodegenerative disorders including polyglutamine diseases. Appearance of aggregates of the misfolded mutant disease proteins suggest that cells are unable to efficiently degrade them, and failure of clearance leads to the severe disturbances of the cellular quality control system. Recently, the quality control ubiquitin ligase CHIP has been shown to suppress the polyglutamine protein aggregation and toxicity. Here we have identified another ubiquitin ligase, called E6-AP, which is able to promote the proteasomal degradation of misfolded polyglutamine proteins and suppress the polyglutamine protein aggregation and polyglutamine protein-induced cell death. E6-AP interacts with the soluble misfolded polyglutamine protein and associates with their aggregates in both cellular and transgenic mouse models. Partial knockdown of E6-AP enhances the rate of aggregate formation and cell death mediated by the polyglutamine protein. Finally, we have demonstrated the up-regulation of E6-AP in the expanded polyglutamine protein-expressing cells as well as cells exposed to proteasomal stress. These findings suggest that E6-AP is a critical mediator of the neuronal response to misfolded polyglutamine proteins and represents a potential therapeutic target in the polyglutamine diseases.     

Conformational Behavior and Aggregation of Ataxin-3 in SDS

Spinocerebellar ataxia type 3 (SCA3) is one of nine polyglutamine (polyQ) diseases all characterized by the presence of intraneuronal inclusions that contain aggregated protein. Aggregation of ataxin-3, the causative protein of SCA3, has been well characterized in vitro, with both pathogenic and non-pathogenic length ataxin-3 undergoing fibrillogenesis. However, only ataxin-3 containing an expanded polyQ tract leads to SCA3. Therefore other cellular factors, not present in previous in vitro studies, may modulate aggregation during disease. The interactions between fibrillar species and cell membranes have been characterized in a number of amyloid diseases, including Huntington’s Disease, and these interactions affect aggregation and toxicity. We have characterized the effects of the membrane mimetic sodium dodecyl sulfate (SDS) on ataxin-3 structure and aggregation, to show that both micellar and non-micellar SDS have differing effects on the two stages of ataxin-3 aggregation. We also demonstrate that fibrillar ataxin-3 binds phospholipids, in particular phosphorylated phosphotidylinositols. These results highlight the effect of intracellular factors on the ataxin-3 misfolding landscape and their implications in SCA3 and polyQ diseases in general are discussed.     

The neuropathogenic contributions of lysosomal dysfunction

Multiple lines of evidence implicate lysosomes in a variety of pathogenic events that produce neurodegeneration. Genetic mutations that cause specific enzyme deficiencies account for more than 40 lysosomal storage disorders. These mostly pre-adult diseases are associated with abnormal brain development and mental retardation. Such disorders are characterized by intracellular deposition and protein aggregation, events also found in age-related neurodegenerative diseases including (i) Alzheimer's disease and related tauopathies (ii) Lewy body disorders and synucleinopathies such as Parkinson's disease, and (iii) Huntington's disease and other polyglutamine expansion disorders. Of particular interest for this review is evidence that alterations to the lysosomal system contribute to protein deposits associated with different types of age-related neurodegeneration. Lysosomes are in fact highly susceptible to free radical oxidative stress in the aging brain, leading to the gradual loss of their processing capacity over the lifespan of an individual. Several studies point to this lysosomal disturbance as being involved in amyloidogenic processing, formation of paired helical filaments, and the aggregation of alpha-synuclein and mutant huntingtin proteins. Most notably, experimentally induced lysosomal dysfunction, both in vitro and in vivo, recapitulates important pathological features of age-related diseases including the link between protein deposition and synaptic loss.     

In vitro and in vivo aggregation of a fragment of huntingtin protein directly causes free radical production

Neurodegenerative diseases are characterized by intra- and/or extracellular protein aggregation and oxidative stress. Intense attention has been paid to whether protein aggregation itself contributes to abnormal production of free radicals and ensuing cellular oxidative damage. Although this question has been investigated in the context of extracellular protein aggregation, it remains unclear whether protein aggregation inside cells alters the redox homeostasis. To address this, we have used in vitro and in vivo (cellular) models of Huntington disease, one of nine polyglutamine (poly(Q)) disorders, and examined the causal relationship among intracellular protein aggregation, reactive oxygen species (ROS) production, and toxicity. Live imaging of cells expressing a fragment of huntingtin (httExon1) with a poly(Q) expansion shows increased ROS production preceding cell death. ROS production is poly(Q) length-dependent and not due to the httExon 1 flanking sequence. Aggregation inhibition by the MW7 intrabody and Pgl-135 treatment abolishes ROS production, showing that increased ROS is caused by poly(Q) aggregation itself. To examine this hypothesis further, we determined whether aggregation of poly(Q) peptides in vitro generated free radicals. Monitoring poly(Q) protein aggregation using atomic force microscopy and hydrogen peroxide (H(2)O(2)) production over time in parallel we show that oligomerization of httEx1Q53 results in early generation of H(2)O(2). Inhibition of poly(Q) oligomerization by the single chain antibody MW7 abrogates H(2)O(2) formation. These results demonstrate that intracellular protein aggregation directly causes free radical production, and targeting potentially toxic poly(Q) oligomers may constitute a therapeutic target to counteract oxidative stress in poly(Q) diseases.     

Aggregation Formation in the Polyglutamine Diseases: Protection at a Cost?

Mutant protein aggregation is a hallmark of many neurodegenerative diseases, including the polyglutamine disorders. Although the correlation between aggregation formation and disease pathology originally suggested that the visible inclusions seen in patient tissue might directly contribute to pathology, additional studies failed to confirm this hypothesis. Current opinion in the field of polyglutamine disease research now favors a model in which large inclusions are cytoprotective and smaller oligomers or misfolded monomers underlie pathogenesis. Nonetheless, therapies aimed at reducing or preventing aggregation show promise. This review outlines the debate about the role of aggregation in the polyglutamine diseases as it has unfolded in the literature and concludes with a brief discussion on the manipulation of aggregation formation and clearance mechanisms as a means of therapeutic intervention.     

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.     

Trehalose alleviates polyglutamine-mediated pathology in a mouse model of Huntington disease

Inhibition of polyglutamine-induced protein aggregation could provide treatment options for polyglutamine diseases such as Huntington disease. Here we showed through in vitro screening studies that various disaccharides can inhibit polyglutamine-mediated protein aggregation. We also found that various disaccharides reduced polyglutamine aggregates and increased survival in a cellular model of Huntington disease. Oral administration of trehalose, the most effective of these disaccharides, decreased polyglutamine aggregates in cerebrum and liver, improved motor dysfunction and extended lifespan in a transgenic mouse model of Huntington disease. We suggest that these beneficial effects are the result of trehalose binding to expanded polyglutamines and stabilizing the partially unfolded polyglutamine-containing protein. Lack of toxicity and high solubility, coupled with efficacy upon oral administration, make trehalose promising as a therapeutic drug or lead compound for the treatment of polyglutamine diseases. The saccharide-polyglutamine interaction identified here thus provides a new therapeutic strategy for polyglutamine diseases.     

Polyglutamine misfolding in yeast: Toxic and protective aggregation

Protein misfolding is associated with many human diseases, including neurodegenerative diseases, such as Alzheimer disease, Parkinson disease and Huntington disease. Protein misfolding often results in the formation of intracellular or extracellular inclusions or aggregates. Even though deciphering the role of these aggregates has been the object of intense research activity, their role in protein misfolding diseases is unclear. Here, I discuss the implications of studies on polyglutamine aggregation and toxicity in yeast and other model organisms. These studies provide an excellent experimental and conceptual paradigm that contributes to understanding the differences between toxic and protective trajectories of protein misfolding. Future studies like the ones discussed here have the potential to transform basic concepts of protein misfolding in human diseases and may thus help to identify new therapeutic strategies for their treatment.Key words: polyglutamine proteins, neurodegeneration, aggresome, Huntington disease, yeast models     

HSP70 interacting protein prevents the accumulation of inclusions in polyglutamine disease1

Heat shock proteins (HSPs) are associated with the proteinaceous inclusions that characterise many neurodegenerative diseases. This suggests they may be associated with disease aetiology and/or represents an attempt to remove abnormal protein aggregates. In this study the adenoviral mediated over‐expression of HSP70 interacting protein (HIP) alone was shown to significantly reduce inclusion formation in both an in vitro model of Spinal Bulbar Muscular Atrophy and a primary neuronal model of polyglutamine disease. Experiments to determine the mechanism of action showed that: denatured luciferase activity (a measure of protein refolding) was not increased in the presence of HIP alone but was increased when HIP was co‐expressed with HSP70 or Heat Shock cognate protein 70 (HSC70); the expression of polyglutamine inclusions in cortical neurons mediated an increase in the levels of HSC70 but not HSP70. Our data suggest that HIP may prevent inclusion formation by facilitating the constitutive HSC70 refolding cycle and possibly by preventing aggregation. HIP expression is not increased following stress and its over‐expression may therefore reduce toxic polyglutamine aggregation events and contribute to an effective therapeutic strategy.     

ROS production and apoptosis induction by formation of Gts1p-mediated protein aggregates

GTS1 of Saccharomyces cerevisiae is a pleiotropic gene. Its induction leads to a variety of biological phenomena represented by cell aggregation. The C-terminal polyglutamine sequence in Gts1p is indispensable for its pleiotropy and nuclear localization. This sequence is often observed in polyglutamine diseases, such as Huntington disease, and is believed to induce protein aggregation, leading to cell death. In this study, protein aggregates were formed in a polyglutamine-dependent manner in cells inducing GTS1, and heat-shock protein family, translation elongation factor, and mitochondrial proteins were trapped in Gts1p-mediated protein aggregates. Moreover, the polyglutamine sequence of Gts1p was indispensable to the induction of reactive oxygen species (ROS) production and apoptosis. Deletion of the genes encoding Por1p and Yhb1p altered the profiles of ROS production and apoptosis caused by GTS1 induction, suggesting that the trapping of these proteins in Gts1p-mediated protein aggregates inhibits the intrinsic functions of these proteins.     

F-Actin Binding Regions on the Androgen Receptor and Huntingtin Increase Aggregation and Alter Aggregate Characteristics

Protein aggregation is associated with neurodegeneration. Polyglutamine expansion diseases such as spinobulbar muscular atrophy and Huntington disease feature proteins that are destabilized by an expanded polyglutamine tract in their N-termini. It has previously been reported that intracellular aggregation of these target proteins, the androgen receptor (AR) and huntingtin (Htt), is modulated by actin-regulatory pathways. Sequences that flank the polyglutamine tract of AR and Htt might influence protein aggregation and toxicity through protein-protein interactions, but this has not been studied in detail. Here we have evaluated an N-terminal 127 amino acid fragment of AR and Htt exon 1. The first 50 amino acids of ARN127 and the first 14 amino acids of Htt exon 1 mediate binding to filamentous actin in vitro. Deletion of these actin-binding regions renders the polyglutamine-expanded forms of ARN127 and Htt exon 1 less aggregation-prone, and increases the SDS-solubility of aggregates that do form. These regions thus appear to alter the aggregation frequency and type of polyglutamine-induced aggregation. These findings highlight the importance of flanking sequences in determining the propensity of unstable proteins to misfold.     

Polyglutamine misfolding in yeast: Toxic and protective aggregation

Protein misfolding is associated with many human diseases, including neurodegenerative diseases, such as Alzheimer disease, Parkinson disease and Huntington disease. Protein misfolding often results in the formation of intracellular or extracellular inclusions or aggregates. Even though deciphering the role of these aggregates has been the object of intense research activity, their role in protein misfolding diseases is unclear. Here, I discuss the implications of studies on polyglutamine aggregation and toxicity in yeast and other model organisms. These studies provide an excellent experimental and conceptual paradigm that contributes to understanding the differences between toxic and protective trajectories of protein misfolding. Future studies like the ones discussed here have the potential to transform basic concepts of protein misfolding in human diseases and may thus help to identify new therapeutic strategies for their treatment.     

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.     

Structure–activity relationship study on polyglutamine binding peptide QBP1

Aggregation and deposition of expanded polyglutamine proteins in the brain cause neurodegenerative diseases including Huntington disease. This pathogenic process is suppressed and delayed in the presence of polyglutamine binding peptide 1 (QBP1), which we previously identified as an undecapeptide binding to pathogenic polyglutamine proteins from phage display peptide libraries. In this paper, a structure–activity relationship study on QBP1 was conducted to determine the pharmacophores for inhibition of polyglutamine aggregation. Furthermore, a truncation study identified an octapeptide as the minimum structure for suppressing aggregation of polyglutamine proteins, which is equipotent to the parent undecapeptide QBP1.     

ROCK and PRK-2 mediate the inhibitory effect of Y-27632 on polyglutamine aggregation

Polyglutamine expansion in huntingtin (Htt) and the androgen receptor (AR) causes untreatable neurodegenerative diseases. Y-27632, a therapeutic lead, reduces Htt and AR aggregation in cultured cells, and Htt-induced neurodegeneration in Drosophila. Y-27632 inhibits both Rho-associated kinases ROCK and PRK-2, making its precise intracellular target uncertain. Over-expression of either kinase increases Htt and AR aggregation. Three ROCK inhibitors (Y-27632, HA-1077, and H-1152P), and a specific ROCK inhibitory peptide reduce polyglutamine protein aggregation, as does knockdown of ROCK or PRK-2 by RNAi. RNAi also indicates that each kinase is required for the inhibitory effects of Y-27632 to manifest fully. These two actin regulatory kinases are thus involved in polyglutamine aggregation, and their simultaneous inhibition may be an important therapeutic goal.     

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.