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Inhibition of polyglutamine aggregate cytotoxicity by a structure-based elongation inhibitor¹

University of Tennessee Graduate School of Medicine, Knoxville, Tennessee, USA

⁴Correspondence: Graduate School of Medicine, R221, University of Tennessee, University of Tennessee Medical Center, 1924 Alcoa Highway, Knoxville, TN 37920, USA. E-mail: rwetzel{at}mc.utmck.edu

SPECIFIC AIMS

Although it is widely accepted that Huntington’s disease and other expanded CAG repeat diseases are caused primarily by gains of function in the expanded polyglutamine (polyGln; polyQ) gene products, the mechanism by which polyGln expansion causes cell dystrophy and/or death remains controversial. A significant body of evidence supports a critical role for polyGln aggregation in cytotoxicity. Previously, as part of a mutational analysis of polyGln aggregate structure, we developed a series of mutated polyGln sequences that fail to aggregate. Our first aim here was to investigate whether these mutated peptides also had the ability to inhibit an ongoing polyGln aggregation reaction. Our second aim was to determine whether these inhibitory peptides could effectively block the cytotoxicity of exogenous, preassembled polyGln aggregates. Successful achievement of these aims has implications for disease mechanism and disease therapy.

PRINCIPAL FINDINGS

1. Certain proline/glycine (PG)-mutated polyGln peptides are effective inhibitors of polyGln elongation reactions in vitro
In most of the expanded CAG repeat diseases, genetic repeat expansions beyond a repeat length of 35 lead to tangible disease risk, and longer repeat lengths are associated with earlier ages of onset. This trend is mirrored in the physical behavior of the polyGln sequence, which becomes much more susceptible to assembly into ß-sheet-rich, amyloid-like aggregates when its repeat length exceeds 35. In a previous study, this laboratory showed that a chemically synthesized polyGln sequence of repeat length 42 could be mutated with regularly spaced PG pairs separated by Q₉ stretches (called the PGQ₉ peptide) without significantly compromising aggregation. The ability of such peptides to aggregate is presumably a result of the PG pairs being localized to turn regions that separate the Q₉-extended chains in the ß-sheet of these amyloid-like aggregates. We also showed, however, that placing single, additional proline residues in the center of one or two of the inner Q₉ stretches (forming, for example, the PGQ₉P² peptide with the additional Pro in the second Q₉ element) totally abrogates aggregation, consistent with the energetic unfavorability of proline in ß-sheets. In the present study, we asked whether such peptides are not only incapable of supporting an aggregation reaction themselves but also might be able to inhibit the aggregation of other, more typical polyGln sequences. Figure 1 shows the results. Although a simple Q₅₀ peptide aggregates readily, as shown in Figure 1A by the time-dependent drop in the amount of peptide remaining in solution, the same peptide mixed with either of two aggregation-incompetent proline mutants, PGQ₉P² or PGQ₉P^2,3, is strongly inhibited from aggregation. In contrast, PGQ₉P¹, an isomeric peptide that contains an additional proline in the first N-terminal Q₉ element, rather than in one of the central Q₉ elements, is not an effective inhibitor of Q₄₂ aggregation. This is consistent with the ability of this PGQ₉P¹ peptide to aggregate itself (Fig. 1B ). Further experiments in an assay that focuses on the ability of pre-existing aggregates to grow by recruiting monomeric, soluble polyGln peptides into the aggregate show that these same PGQ₉P² and PGQ₉P^2,3 peptides are good inhibitors of the elongation phase of the aggregation process, and concentrations that produce 50% inhibition values are in the low µM range. The structural basis of this dramatic effect is not certain but may be a result of the ability of the N- and/or C-terminal portions of the inhibitor peptide to add to the growth point of the polyGln aggregate and, at the same time, block the ability of additional Q₅₀ monomers to dock onto these growth points and continue the elongation process.

View larger version (12K): [in this window] [in a new window]   Figure 1. Inhibition of spontaneous polyGln aggregation in solution by proline-mutated polyGln sequences. A) Aggregation of 9–12 µM Q50 peptide in phosphate-buffered saline (PBS) alone (•) or in the presence of 40–50 µM PGQ9P1 (), PGQ9P2 (), or PGQ9P2,3 (). B) Time-dependent change in soluble concentrations of the peptides PGQ9P1 (), PGQ9P2 (), and PGQ9P2,3 () during the reaction monitored in A. The primary sequences of these peptides are PGQ9P1, K2Q4PQ4PGQ9PGQ9PGQ9K2; PGQ9P2, K2Q9PGQ4PQ4PGQ9PGQ9K2; PGQ9P2,3, K2Q9PGQ4PQ4PGQ4PQ4PGQ9K2.

2. Inhibitors of polyGln elongation protect mammalian cells in culture from the toxic effects of exogenous polyGln aggregates
Most cell and animal models for polyGln toxicity depend on the expression of the expanded, pathological-length polyGln protein, introducing the ambiguity of whether the observed toxicity is a result of the monomeric polyGln protein or its aggregated product. Previously, this laboratory developed a cell model for polyGln toxicity based on the addition of aggregates of synthetic polyGln peptides to culture medium. By mechanisms that are not clear, cells spontaneously take up these aggregates and, if the peptides in the aggregates contain nuclear-localization sequences, also transport the aggregates into the nucleus, an event that triggers rapid, apoptotic cell death in 60–80% of the treated cells. This model avoids the exposure of cells to high levels of monomeric, expanded polyGln proteins, thus unambiguously focusing attention on the role of the aggregates. Using this model, we incubated PC12 cells in culture with the inhibitor peptide PGQ₉P^2,3 before addition of polyGln aggregates. As shown in Figure 2 , the inhibitor peptide protects PC12 cells from polyGln aggregate-induced cytotoxicity and does so in a dose-dependent manner.

View larger version (9K): [in this window] [in a new window]   Figure 2. Protection of PC12 cells from the toxicity of nuclear localization sequence-Q42 aggregates by various concentrations of PGQ9P2,3. A) Percent cell death, assessed by lactate dehydrogenase release assay, of PC12 cells incubated in PBS containing various concentrations of PGQ9P2,3 with () or without () polyGln aggregates (final aggregate concentration equivalent to 10 µM monomeric peptide). B) Percent protection determined as the percentage of aggregate-dependent cell death blocked at each PGQ9P2,3 concentration.

CONCLUSIONS AND SIGNIFICANCE

The previously reported success of the cell model described above already demonstrated that polyGln aggregates are themselves cytotoxic. The results described here and shown in Figure 2 demonstrate that it is a specific, functional activity of these aggregates, namely, their abilities to grow by recruiting additional, cellular polyGln peptides, which confers their ability to kill cells. Previously, in an animal model relying on overexpression of an expanded polyGln protein, coexpression with a polypeptide-based aggregation inhibitor was reported to protect against toxicity. Although demonstrating that aggregation is required for toxicity, these previous results did not address the mechanism of toxicity. As our polypeptide inhibitors specifically have the ability to inhibit aggregate elongation and as our cell model is based on administration of preformed aggregates, the ability of inhibitors such as PGQ₉P^2,3 to protect cells in this model strongly suggests that it is the intracellular elongation process itself that is responsible for aggregate cytotoxicity in our cell model.

These results are thus consistent with the recruitment-sequestration model of expanded polyGln toxicity. In this mechanism, polyGln aggregates that form in response to the presence of the expanded polyGln disease protein can elongate by incorporation of other polyGln-containing proteins in the cell. By sequestering these proteins and/or by stimulating their destruction and removal from the cell after they have added to the aggregate, the recruitment of these proteins leads to their inactivation. The loss to the cell of the activity of these polyGln proteins, many of which are transcription factors, has toxic consequences. This is illustrated in the scheme shown in Figure 3 , which represents the intracellular aggregation and recruitment mechanisms and how these might be affected by the introduction of an elongation inhibitor. The elongation inhibitor does not have to remove the aggregate to block toxicity; it simply has to block the ability of the aggregate to recruit and thereby, inactivate cellular polyGln-containing proteins.

View larger version (16K): [in this window] [in a new window]   Figure 3. The role of an elongation inhibitor in the recruitment-sequestration model for polyGln cytotoxicity. The open trapezoids represent expanded polyGln sequences. Path a shows the formation of polyGln aggregates from expanded polyGln monomers expressed in the cell, and path b represents the introduction of preformed polyGln aggregates into the cell in our cell model. Path c shows how these polyGln aggregates, regardless of their source, can recruit other cellular proteins through their own polyGln sequences (the trapezoidal portions of the shaded symbols) in an ongoing, mixed, polyGln elongation reaction. Such promiscuous polyGln elongation has been demonstrated in vivo and in vitro. Recruitment of these proteins into the aggregate results in their loss to the cell environment. Part d shows how polyGln elongation inhibitors such as PGQ9P2,3 and polyQ enhancer-1 (PQE-1; shown as mottled, right-angle triangles) can bind to the growth points of the polyGln aggregates and disallow further recruitment of other polyGln proteins.

It is particularly interesting that the de novo designed molecule PGQ₉P^2,3 bears significant sequence similarity to the proline–glutamine-rich domain of the Caenorhabditis elegans protein PQE-1. This protein was originally identified in a genetic survey for second site suppressors of polyGln toxicity in C. elegans, and it was subsequently shown that it is specifically the proline–glutamine-rich domain of the protein that is responsible for this blocking effect. Thus, in our design of the peptide PGQ₉P^2,3, originally motivated by fundamental studies on polyGln aggregate structure, we may have arrived at an analog of an evolved modulator of polyGln toxicity. The results described here suggest that peptides that have the ability to block polyGln elongation may have some therapeutic value if it is possible to deliver these sequences to the neurons that need them. Alternatively, small molecules that have the same inhibitory properties may become viable therapeutic agents.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1238fje;

² Present address: Abteilung für Strukturforschung, Max Planck Institute for Biochemistry, Am Klopferspitz 18a, D-82152 Martinsried, Germany.

³ Present address: Center for Immunology and Microbial Disease, Albany Medical College, 47 New Scotland Avenue, Albany, NY 12208, USA.

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