Protein Interactions

Huntingtin protein interactors yield genetic modifiers of the disease in fruitflies.
The search for genetic modifiers of Huntington's Disease is important because it could lead to a treatment. Genetic modifiers are genes which lessen or increase the severity of the disease or advance or delay onset. Treatment could take the form of reducing or enhancing the expression of the modifying gene or the development of a drug which performs the same function as the gene.

Researchers at the Buck Institute along with colleagues from the Baylor College of Medicine, the Fred Hutchinson Cancer Research Center, and Prolexys Pharmaceuticals decided to look for genetic modifiers in proteins which interact with the huntingtin's protein. Using two different types of high tech screens, they identified 234 interacting proteins. Before this study only 50 interacting proteins were known. As hoped, the interacting proteins proved to be a rich source for genetic modifiers of the disease. They arbitrarily selected genes which encode for 60 of these proteins and manipulated their expression in a fruitfly model. Several promising targets were identified.

The next step is to see if these genes modify the disease in mammals. In addition, there may be more modifier genes to discover in the ones not yet selected for testing.

This is important research because we still do not fully understand the functions of the normal huntingtin's protein, nor have we identified all the pathologies caused by the HD protein and their relative importance in the disease. This kind of research promises to shorten the time it takes to develop significant treatments by identifying major targets for drug development or gene therapy.

Marsha L. Miller, Ph.D.
Robert E. Hughes. Ph.D.
Huntingtin Interacting Proteins Are Genetic Modifiers of Neurodegeneration
L. Kaltenbach, E. Romero, R. Becklin, R. Cherrier, R. Bell, A. Phansalkar, A. Strand, C. Torcassi, J. Savage, A. Hurlburt, G. Cha, L. Ukani, C. Chepanoske, Y. Zhen, S. Sahasrabudhe, J. Olson, C. Kurschner, L. Ellerby, J. Peltier, J. Botas, R. Hughes
The Abstract

Huntington's disease (HD) is a fatal neurodegenerative condition caused by expansion of the polyglutamine tract in the huntingtin (Htt) protein. Neuronal toxicity in HD is thought to be, at least in part, a consequence of protein interactions involving mutant Htt. We therefore hypothesized that genetic modifiers of HD neurodegeneration should be enriched among Htt protein interactors. To test this idea, we identified a comprehensive set of Htt interactors using two complementary approaches: high-throughput yeast two-hybrid screening and affinity pull down followed by mass spectrometry. This effort led to the identification of 234 high-confidence Htt-associated proteins, 104 of which were found with the yeast method and 130 with the pull downs. We then tested an arbitrary set of 60 genes encoding interacting proteins for their ability to behave as genetic modifiers of neurodegeneration in a Drosophila model of HD. This high-content validation assay showed that 27 of 60 orthologs tested were high-confidence genetic modifiers, as modification was observed with more than one allele. The 45% hit rate for genetic modifiers seen among the interactors is an order of magnitude higher than the 1%-4% typically observed in unbiased genetic screens. Genetic modifiers were similarly represented among proteins discovered using yeast two-hybrid and pull-down/mass spectrometry methods, supporting the notion that these complementary technologies are equally useful in identifying biologically relevant proteins. Interacting proteins confirmed as modifiers of the neurodegeneration phenotype represent a diverse array of biological functions, including synaptic transmission, cytoskeletal organization, signal transduction, and transcription. Among the modifiers were 17 loss-of-function suppressors of neurodegeneration, which can be considered potential targets for therapeutic intervention. Finally, we show that seven interacting proteins from among 11 tested were able to co-immunoprecipitate with full-length Htt from mouse brain. These studies demonstrate that high-throughput screening for protein interactions combined with genetic validation in a model organism is a powerful approach for identifying novel candidate modifiers of polyglutamine toxicity.

The Press Release

Landmark study identifies large number of new proteins implicated in Huntington's disease

Buck Institute faculty leads large scale screening of protein interactions to identify drug targets for possible treatment of incurable disease

Researchers from four organizations have identified more than 200 new proteins that bind to normal and mutant forms of the protein that causes Huntington’s disease (HD). HD is a fatal inherited disease that affects 30,000 Americans annually by laying waste to their nervous system. The research was led by Buck Institute faculty member Robert E. Hughes, PhD. Results of the study, which may facilitate the discovery of an effective treatment for HD, will be published in the May 11 edition of PLoS Genetics, an online, open-source journal, enabling scientists from around the world to take advantage of the findings immediately.

The work, which involved high-tech screening of the human genome and proteome, was unprecedented both in terms of its scale and in the way the protein interactions were validated in a genetic model of the disease. By conducting additional experiments in fruit flies genetically altered to express features of human HD, scientists showed that changing the expression of these interacting proteins affected the degree of damage seen in the fly neurons. This indicates that a significant number of the proteins might be potential drug targets for HD.

Now that researchers have discovered the interacting proteins using human libraries and human protein extracts and tested them in the fly, Hughes says the next step is to bring the research back into the mammalian world. The new genes and proteins discovered in this study are being screened and analyzed in cultured mammalian cells; the ones that show activity in ongoing experiments will be tested in mouse models of HD.

"Here at the Buck Institute, we’re going to be focusing on a few dozen proteins," said Hughes. "Effective follow-up on any target protein depends, in large part, on how much expertise a scientist has with that target. We are hoping that researchers will look at this study and that those with specific expertise in a particular protein will move forward with their own inquiries."

The work was supported by HD advocacy organizations. "We are very excited about this significant discovery," said Allan Tobin, PhD, Senior Scientific Advisor to the High Q Foundation and CHDI, Inc. "This work helps define and refine possible therapeutic targets for a disease that lacks thorough understanding." Tobin added, "We are pleased this study is being published in an open-access journal, which makes it easier for scientists at other organizations to get to work on following up on this landmark discovery." Traditional peer-reviewed journals usually require scientists to pay a fee to access study results.

Tobin added that the need for further scientific inquiry is urgent. There is currently no effective treatment or cure for HD, which is typically characterized by involuntary movements and dementia. The disease slowly diminishes a person’s ability to move, think and communicate. Those affected eventually become totally dependent on others for their care and usually die from complications such as choking, heart failure or infection. The disease is hereditary; each child of a person with HD has a 50/50 chance of inheriting the fatal gene. Approximately 200,000 Americans are believed to be at risk of developing HD, a disease that affects as many people as hemophilia, cystic fibrosis or muscular dystrophy. The symptoms of HD typically begin to appear in mid-life, although the progression of the disease varies among individuals and within the same family. ###

Joining Hughes as co-authors of the paper are Buck Institute scientists Cameron Torcassi, and Lisa Ellerby; along with Eliana Romano and Juan Botas from the Baylor College of Medicine in Houston; Andrew Strand, and James Olson from the Fred Hutchinson Cancer Research Center in Seattle; and Linda Kaltenbach, Sudhir Sahasrabudhe, Cornelia Kurschner, and John M. Peltier of Prolexys Pharmaceuticals in Salt Lake City. The work was supported by grants from the HighQ Foundation, CCHI Inc, the Hereditary Disease Foundation and National Institutes of Health.

The Buck Institute is an independent non-profit organization dedicated to extending the healthspan, the healthy years of each individual’s life. The National Institute of Aging designated the Buck a Nathan Shock Center of Excellence in the Biology of Aging, one of just five centers in the country. Buck Institute scientists work in an innovative, interdisciplinary setting to understand the mechanisms of aging and to discover new ways of detecting, preventing and treating age-related diseases such as Alzheimer’s and Parkinson’s disease, cancer, stroke, and arthritis. Collaborative research at the Institute is supported by genomics, proteomics and bioinformatics technology. For more information: www.buckinstitute.org.

PLos Genetics 2007 May 11;3(5):e82