(433d) Poly-Q Peptides and Proteins

Under appropriate conditions, polypeptides and proteins self-associate into large aggregates of ?-sheet structure and fibrillar morphology. Such fibrillar aggregates are observed in a number of neurodegenerative diseases, including Alzheimer's and Parkinson's disease. A particularly interesting collection of aggregation-related diseases are the so-called “trinucleotide repeat diseases”. Most commonly observed are CAG repeats, encoding for expanded polyglutamine (polyQ) inserts. Huntington's disease is the best-known example. In these genetically linked neurodegenerative diseases, the proteins are unrelated but contain a common feature—the presence of a long stretch of glutamines. The disease state is invariably characterized by nuclear and (frequently) cytoplasmic inclusions that contain aggregates of the polyQ protein or its proteolytic fragments. Several hypotheses have been put forth to explain the role of expanded glutamine domains in disease. Some researchers suggest that the misfolded and/or aggregated protein becomes toxic to cells, although there is no firmly established correlation between aggregation and neuronal death. Another hypothesis relies on the observation that components of the transcriptional machinery, including the CREB-binding protein and the TATA-binding protein, are known to bind to polyQ proteins in vitro and are found co-localized with nuclear inclusions in vivo, suggesting that inactivation of transcription factors and subsequent transcriptional down-regulation may be to blame for pathogenesis. Another hypothesis proposes that polyQ-mediated aggregation clogs up the ubiquitin proteasome system. Ubiquitin, chaperones, and proteasomal components are often found in polyQ nuclear inclusions, suggesting that the cell may be attempting to degrade the misfolded proteins but is overwhelmed by the sheer mass of misbehaving proteins.

We are working to develop an appropriate model system for the investigation of polyQ disease-associated protein, and in developing a quantitative description of the kinetics of association of polyQ proteins. Such a framework will facilitate both the identification of the mechanism by which polyQ protein aggregates cause cellular dysfunction, and the development of novel therapeutic strategies to combat the disease.

In this talk we will describe the work we are undertaking with both synthetic polyQ peptides and with recombinant proteins with polyglutamine inserts. We have synthesized polyQ peptides of variable glutamine length and with various flanking residues or imposed structural features. The aggregation properties of these peptides have been tested using a variety of biophysical tools and at several solvent conditions. The data challenge the accepted paradigm that polyQ peptides aggregate via a simple nucleation-elongation mechanism involving a thermodynamically unstable monomeric nucleus. We propose an alternative interpretation of our data along with other published data.

We will also describe ongoing work to develop and characterize a small library of protein mutants containing polyQ inserts. We have chosen apomyoglobin as a model scaffold, and have developed a method for generating a diversity of mutants with polyQ inserts of variable length and insertion location. Currently we are producing these proteins and beginning to characterize their folded structure, stability, and aggregation kinetics. These studies will address questions regarding the role of polyQ in promoting protein aggregation; specifically, is aggregation driven because the polyQ insert disrupts protein folding, or because of specific glutamine-glutamine interactions between proteins?