Research Projects
Our lab is interested in the assembly, structure, and cytotoxicity of protein aggregates such as amyloid fibrils and their assembly intermediates. We attack these problems working mainly in two systems, Aß amyloid fibril formation in Alzheimer's disease and polyglutamine aggregate formation in the expanded CAG repeat diseases such as Huntington's Disease.
Alzheimer's Disease Projects
Aß is a 39-42 amino acid long peptide found in amyloid fibrils and plaques in the Alzheimer's Disease (AD) brain. Fibrils or their assembly precursors seem to possess a cytotoxicity that may be pivotal in the development of AD, but we know little about fibril structure at the molecular level. In fact, this is true for all amyloid fibrils.
Fibril structure
One of the few things we know is that amyloid is rich in beta sheet structure. But, even for fibrils composed of small peptides like Aß, we don't know whether the whole peptide, or only a portion of it, is involved in H-bonded ß-sheet structure. To begin to learn about how the Aß peptide “threads” into the amyloid folding motif, we are mapping gross aspects of the fibril structure using a variety of techniques. Using mass spectrometry, we have already determined that only about 50% of the 39 backbone amide hydrogens of Aß(1-40) are involved in H-bonded structures that protect them from hydrogen-deuterium exchange. We now want to map the exchange protection on a residue-by-residue basis. We are also using limited proteolysis to map exposed regions of the polypeptide in the fibril.
Protein binding to fibrils and protein inhibitors of fibril growth
A number of proteins are known to associate with fibrils in vivo, and several globular proteins are known to inhibit amyloid fibril growth in vitro. If we can develop a better understanding of what kind of protein folding motifs work particularly well at binding to fibrils, it will tell us something about fibril structure. To do this, we are studying the ability of globular proteins to bind to and inhibit the extension of Aß fibrils. In addition, we are attempting to generate proteins with enhanced binding to fibrils by screening for anti-amyloid monoclonal antibodies that are conformation specific.
Minor protein constituents of brain amyloid plaques
We are interested in a hypothesis for aggregate cytotoxicity that has been referred to as “recruitment” or “sequestration”. In this model, aggregates capture unrelated proteins from the medium as they grow and deplete the local environment of key proteins, the loss of which leads to cell dysfunction and/or death. To develop this hypothesis, we are looking for new protein components of amyloid plaques.
Huntington's Disease Projects
Genetic expansion of polyglutamine (polyGln) sequences in one of a number of proteins with a repeat length of 35 to 37 or higher can lead to development of one of a series of neurodegenerative disorders. Recent work with animal and cellular models for these diseases indicates that the polyGln proteins make aggregates, particularly when the pathological length cutoff of about 35 is exceeded.
Although aggregates have been found in the cytoplasm as well as outside the cell, they predominate in cell nuclei. Although the protein context of the polyGln sequence may well play a role in modulating the effect, we believe that it is the polyGln sequence and its tendency to aggregate that is responsible for disease. This premise drives our research in this area.
Aggregate Assembly
Using chemically synthesized polyGln peptides, we are mapping the repeat length dependence of the kinetics and thermodynamics of aggregation. In solution phase polyGln aggregation exhibits kinetics similar to Aß aggregation, featuring a lag phase consistent with a nucleation dependence. In the case of polyGln aggregation, the lag phase gets shorter as the repeat length gets longer, consistent with the critical importance of aggregation in the disease mechanism.
We are also mapping the length dependence of the ability of relatively short polyGln peptides to co-aggregate when longer polyGln peptides spontaneously aggregate. We think this property is important in the disease mechanism.
Aggregate Structure
In our preliminary studies, we find that polyGln aggregates exhibit many of the properties of amyloid fibrils. We are looking more closely at this possible relationship, in hopes that such a comparative analysis will instruct us about aggregates of both Aß and polyGln. We are using circular dichroism, electron microscopy, Fourier transform infrared spectroscopy, light scattering, vibrational CD, monoclonal antibodies, dye binding, and other methods to analyze the repeat length dependence, growth condition dependence, and other aspects of polyGln aggregate structure.
Aggregate Toxicity
In order to build further support for the hypothesis that polyGln aggregate toxicity plays a key role in the development of disease pathology, we are developing improved ways of quantifying the aggregate content of material from cell models, animal models, and patients. Success in this endeavor may also provide new diagnostic approaches to monitoring disease onset and progress. To learn more about how aggregates influence cell viability, we are attempting to directly introduce aggregates made in vitro into cells in culture.
Interfering with Aggregate Assembly
We are also developing high throughput screening assays for discovery of inhibitors of polyGln aggregation that might have therapeutic potential.
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