Characterization of the interaction between a stable G-quartet forming oligonucleotide and huntingtin with an expanded poly-glutamine region
Date
2009
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
Huntington’s Disease is a progressive neurodegenerative disorder that is
inherited in an autosomal dominant fashion. The disease is the result of an expanded
CAG repeat in exon 1 of the HD gene which encodes an elongated poly-glutamine
tract in the mutant form of the protein, huntingtin. The expanded poly-glutamine
region causes the mutant protein to mis-fold and oligomerize, initiating a complex
aggregation process. Disease pathogenesis is linked to the formation of these
intracellular aggregates. It has been proposed that the aggregates themselves are toxic
due to their physical presence which may block intracellular trafficking and signaling
processes. A concurrent possibility is the sequestration of important proteins such as
transcription factors during the aggregation process, disrupting their function resulting
in cellular stress and death. In addition, the mechanistic steps that lead to aggregate
formation may be central to HD pathology. A viable approach for HD therapeutics is
to block the aggregation process of mutant huntingtin.
It has been previously reported that guanosine rich oligonucleotides(ODN) that
fold into a G-quartet formation are effective inhibitors of the aggregation process of a
huntingtin protein fragment with an elongated polyglutatmine tract, Htt 1-171
(Q58)(Skogen, Roth, Yerkes, Parekh-Olmedo, & Kmiec, 2006). The most robust ODN
inhibitor of aggregation is a single stranded oligonucleotide composed of 20 guanosine residues termed, G20. It is unique in its propensity to form a secondary structure
known as a G-quadruplex. For this thesis, analysis was done on the G-quadruplex
forming G20 to determine if its structure is central to its aggregation inhibition
activity. Circular dichroism and Atomic Force Microscopy(AFM) experiments were
carried out to investigate the secondary structure and it was determined that G20 most
likely forms a “G-wire” formation of a G-quadruplex and that this structure is
important for the interaction necessary for inhibition of mutant huntingtin aggregation.
The interaction between G20 and huntingtin was investigated in several aggregation
models of huntingtin by both in vitro and in vivo assays. The results indicate that G20
acts by directly binding to huntingtin, and that this interaction is dependent on the
unique structure formed by G20 and on the process of mutant huntingtin aggregation.
We hypothesize that G20 inhibits mutant huntingtin aggregation through a direct and
structurally specific interaction.