DISTURBING INTERACTIONS WITHIN: A large-scale study uncovers downstream proteins acted on by a mutated huntingtin protein, which has been linked to Huntington's disease. The new findings could lead to therapeutic targets for battling the debilitating illness. Image: © ISTOCKPHOTO/SEBASTIAN KAULITZKI
A high-throughput screening of the human genome and proteome (the proteins expressed in a cell) has allowed scientists to identify more than 200 proteins impacted directly by a mutation to the huntingtin protein, which is known to be the cause of Huntington's disease.
The actual function of the normal huntingtin protein is unknown, but it is believed to be involved in embryonic development. In its mutated form, however, its amino acid elongates, causing the protein to improperly fold, likely resulting in abnormal interactions with other proteins in the nervous system cells it inhabits.
This cellular disruption results in the incurable Huntington's disease, which may affect as many as 200,000 people in the U.S. The neurodegenerative illness typically onsets in middle age and is characterized by dementia, loss of movement control and eventually death by heart failure, infection or choking—usually about 10 to 15 years after the first symptoms appear. Children of Huntington's sufferers have a 50 percent probability to inherit the gene that codes for the abnormally configured protein.
A team led by Robert Hughes, an assistant professor specializing in the genetics of aging at the Buck Institute for Age Research in Novato, Calif., used two different screening methods to determine which proteins might be directly affected by mutated huntingtin. The first, called yeast two-hybrid screening, uses a yeast cell as an apparatus in which two different proteins are expressed—huntingtin and another, taken from brain and muscle tissues from both humans and mice. If there is an interaction, it will be indicated by growth of the yeast cell. A reporter gene, also expressed in the yeast cell, can also help quantify the relative strength of their dialogue. The second process, affinity pull-down followed by mass spectrometry, employed purified huntingtin protein as bait. When incubated with other proteins, those that interact with the marked bait are separated out and identified by the mass of their ionized fragments.
In all, more than 3,500 proteins were screened and 234 proteins turned out to be associated with huntingtin. "It really does seem to be a protein that, when mutated, can gum up a lot of different parts of the cell," Hughes says. He notes that the 234 proteins identified control everything from neurotransmitter release and storage to cellular structure and the amount of protein synthesis in a cell.
"We were then left with the task of finding out whether these proteins had something to do with Huntington's disease," Hughes explains.
So, the team arbitrarily selected 60 of these proteins for testing in a Drosophila fruit fly model. In the flies, the protein corresponding to huntingtin in humans, when mutated, causes neurodegeneration in the eyes. Flies expressing the mutated huntingtin analogue were mated with flies carrying mutations in one of the 60 chosen proteins, in order to determine if the change in the second protein exacerbated or relieved the function of the eye. Using this method, Hughes says, he and his colleagues determined that "30 percent of the genes we tested could act as enhancers or suppressors." (Normally, he notes, a random screen would ensure about a 1 or 2 percent likelihood of finding a genetic association.) "In particular, we report 17 loss-of-function suppressors," he continues—a suppressor being "a gene or protein that if you knock down it's activity, that will cause an improvement in the disease phenotype."
Al La Spada, an associate professor of laboratory medicine at the University of Washington, says the study, published in this week's issue of PLoS Genetics, "is the starting point of embarking on a series of investigations to get to that ever important goal of coming up with meaningful therapy" to help treat the damage inflicted by Huntington's. This data set, he adds, can be factored into other ongoing investigations, such as one extensive endeavor attempting to tie modifiers of disease progression to chromosomal regions in the genome. For his part, Hughes says, the research "provides a list of candidate proteins that need to be further examined to develop candidate therapeutic targets."